Sperm miR-142-3p reprogramming mediates paternal stress-induced non-alcoholic steatohepatitis in offspring rats

Sperm miR-142-3p reprogramming mediates paternal stress-induced non-alcoholic steatohepatitis in offspring rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Article Sperm miR-142-3p reprogramming mediates paternal stress-induced non-alcoholic steatohepatitis in offspring rats Hui Wang, Cong Zhang, Yu Guo, Yi Liu, Kexin Liu, Wen Hu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3833743/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Although epidemiological data suggest a strong association between paternal adverse environmental exposure and susceptibility to multiple diseases in offspring, the sperm-to-liver pathway involved in offspring disease is complicated and worthy of further exploration. Caffeine contained in many beverages is regarded as a chronic stressor, and exerts reproductive and developmental toxicity. Effects of paternal pre-pregnant caffeine exposure (PPCE) on the long-term health of offspring and the underlying mechanisms remain unclear. This study innovatively reported the occurrence and transgenerational inheritance of PPCE-induced non-alcoholic steatohepatitis (NASH) in offspring, and aimed to elucidate its sperm reprogramming mechanism and the potential intervention targets. Male rats were administrated with caffeine (15 ~ 60 mg/kg/d) by gavage for 8 weeks before mating. Symptoms of NASH were found in two successive generations of male rats (F1 and F2) whose fathers or grandfathers (F0) were exposed to caffeine. RNA-seq was employed to screen out a novel miRNA mediating equilibrant of liver fatty metabolism: miR-142-3p. Role of sperm miR-142-3p in PPCE-induced offspring NAFLD was validated by in vitro fertilization of the sperm of PPCE or miR-142-3p KO sperm with normal oocytes. Overexpression of miR-142-3p in offspring liver reversed NASH manifestation in PPCE male offspring. We further proved that caffeine-induced paternal chronic stress (high glucocorticoid level) but not caffeine itself is the main cause of methylation changes in sperm and offspring NAFLD, via experiments in vitro and glucocorticoid receptor blockade. Moreover, the linkage between serum high glucocorticoids and sperm miR-142-3p low programming was also verified in clinical samples. Overall, we demonstrated for the first time that PPCE induced NASH in offspring with transgenerational inheritance, confirmed the reprogramming mechanism of sperm miR-142-3p, and identified miR-142-3p as a potential intervention target for paternal-derived NASH. Health sciences/Gastroenterology/Hepatology/Liver diseases Biological sciences/Developmental biology/Epigenetic memory paternal pre-pregnant caffeine exposure non-alcoholic steatohepatitis miR-142-3p ACSL4 sperm reprogramming Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Non-alcoholic fatty liver disease (NAFLD) has become the most common chronic liver metabolic disease in the world, with a spectrum of simple steatosis, nonalcoholic steatohepatitis (NASH), cirrhosis, and even hepatocellular carcinoma 1 . NASH is a severe form of NAFLD progression and its current understanding of pathogenesis reported up to now has mostly involved the elaborate reciprocation among genetic susceptibility, environmental factors, and metabolic stress 2 . The theory of “Developmental Origins of Health and Disease (DOHaD)" holds that many adult metabolic diseases have fetal origins 3 , whereby the impact of adverse environmental factors during fetal development can cause metabolic adaptations and developmental programming changes in the offspring, resulting in susceptibility to a variety of metabolic diseases (such as NAFLD and diabetes) after birth 4, 5, 6 . However, gametes (sperm and oocytes) take longer to develop and mature than embryos (fetuses) and are thereby more vulnerable to adverse environmental exposures 7 . In recent years, with the proposition of the “Paternal Origins of Health and Disease (POHaD), substantial studies confirmed paternal adverse lifestyle and environmental exposures as important independent risk factors for the increased incidence of abnormal development and developmental diseases in the offspring 8, 9 . Epidemiological surveys showed that men with experience of famine have a significantly higher morbidity of obesity and diabetes in their offspring 10 . Clinical and laboratory studies have also found that parental pre-pregnancy adverse environmental exposures (such as smoking, high-fat diet, exogenous exposure, chronic stress, etc.) can cause intrauterine growth retardation (IUGR) and postnatal lipid metabolism dysfunction in the offspring 11, 12, 13, 14 . All these studies indicate that paternal adverse pre-pregnancy environmental exposure is an important contributor leading to multi-disease susceptibility (such as NAFLD) in the offspring. Chronic stress refers to long-term exposure to adverse events (such as poor lifestyles, exogenous exposure, psychological stress, etc.), closely related to human health 15 . Studies have shown that paternal chronic stress can lead to poor pregnancy outcomes (such as IUGR) in the offspring and multi-disease susceptibility after birth 16, 17 . Caffeine is a central stimulant as well as a common chronic stressor widely found in coffee, tea, soft drinks, and some compounded drugs in daily life 18, 19 , and has been demonstrated to have reproductive and developmental toxicity 20 . Our previous series of studies confirmed that maternal caffeine exposure during pregnancy could assign the adverse impact on the long-term health of the offspring 21, 22, 23, 24 . However, the increasing global average caffeine intake, especially among men of reproductive age (240 mg/d), surpasses that in women 25, 26 , emphasizing the need for heightened awareness regarding the potential risks associated with pre-pregnancy caffeine intake in men and its impact on the health of future generations. As the most perceptive hormone in stress response, glucocorticoid is the key to germ cell formation, embryo (fetus) development and its fate after birth 27 . Studies have found that caffeine intake can significantly increase serum glucocorticoid levels 28 , whereas paternal pre-pregnancy caffeine intake can affect the early brain development of offspring and is associated with glucocorticoid receptor (GR) activation 29 . These studies suggest that serum-high glucocorticoids may be involved in the effects of pre-pregnancy caffeine exposure on the long-term health of the offspring. Epigenetic modification (such as DNA methylation and non-coding RNA) plays a key role in sperm-mediated inheritance of acquired traits 30 . It has been shown that epigenetic changes caused by paternal adverse pre-pregnancy environmental exposure can transmit these “marks” to the offspring through sperm reprogramming changes, resulting in developmental programming, even over multiple generations 31 . MicroRNAs (miRNAs) are important components of non-coding RNAs that can participate in a variety of cellular activities by modulating multiple gene expressions and protein translation, and they are strongly associated with gametogenesis and embryonic (fetal) development 32, 33 . Clinical and experimental studies have shown that sperm miRNAs, important marker and regulators of epigenetic modifications, can carry paternal epigenetic information and transmit it to the offspring to affect the long-term health 34, 35 . Poor paternal pre-pregnancy lifestyle (such as a high-fat diet) and chronic stress can alter multiple sperm miRNAs, influencing offspring and resulting in altered hepatic lipid metabolism 16, 36, 37 . Meanwhile, elevated serum glucocorticoid under paternal chronic stress were associated with changed sperm miRNAs expression 16, 38 . These results suggest that paternal serum high glucocorticoids may disrupt hepatic lipid metabolic homeostasis in offspring by altering miRNAs expression in sperm. In this study, a paternal pre-pregnant caffeine exposure (PPCE) rat model was established to simulate daily caffeine intake in the population, so as to reveal that PPCE causes alterations in sperm epigenetic programming through high glucocorticoid exposure in the parental generation, thus leading to hepatic lipid metabolism dysfunction in the offspring and the occurrence of adult NASH. This study aims to clarify the developmental origins of paternal NASH, confirm intervention targets, and provide theoretical and practical significance for early prevention and treatment strategies, guiding healthy lifestyles in men of reproductive age. 2. Results 2.1 . PPCE causes adult NASH in male offspring To confirm that paternal caffeine intake can cause the occurrence of NASH in adult offspring, we established a PPCE rat model. First, we observed the effects of PPCE on liver pathology, NAFLD activity score (NAS), and triglyceride metabolism in male offspring before and after birth. H&E and ORO staining results showed that compared to the respective CON groups, there were significant manifestations of fat vacuole-like degeneration and lipid accumulation in the liver pathology of the PPCE group at both GD20 and PW12 (Fig. 1A, B), as well as significantly higher liver steatosis scores (Fig. 1C, D) and triglyceride content (Fig. 1E, F). At PW32, the activities of serum liver function enzymes (AST and ALT) were significantly increased in the PPCE group (Fig. 1G, H), liver appearance was light brown, and liver weight and liver index were significantly increased (Fig. 1I-K). H&E, Masson, and Sirius red staining showed that the liver of PPCE male offspring at PW32 exhibited typical NASH histological features, manifesting as significant vacuolar-like steatosis, inflammatory infiltration (green arrows), and collagen fiber deposition (black and red arrows) (Fig. 1L). Meanwhile, liver NAS and triglyceride content were significantly increased (Fig. 1M-R). It is suggested that PPCE can lead to hepatic lipid accumulation in male offspring and the occurrence of NASH in adulthood. In this study, we also found that although PPCE could cause the occurrence of NAFLD in female offspring in adulthood, the liver pathological changes were milder than that of males, and no obvious inflammatory infiltration and fibrosis were observed (Fig. S1). In summary, PPCE can cause the occurrence of adult NAFLD/NASH in the offspring, and there are significant gender differences, especially evident in males. Therefore, this study subsequently focused on the programming mechanisms by which PPCE led to the occurrence of NASH in male offspring. 2.2. PPCE induces hepatic lipid metabolism disorders and chronic inflammatory activation in male offspring before and after birth We further examined the changes in hepatic lipid metabolism and inflammatory response in PPCE male offspring rats before and after birth. First, sequencing analysis results suggested that the expression of multiple genes was changed in the liver of fetal rats in the PPCE group compared with the CON group (Fig. 2A). KEGG functional enrichment analysis revealed that most differentially expressed genes in the liver of fetal rats in the PPCE group were closely associated with lipid metabolic signaling during the development of NAFLD (Fig. 2B), specifically manifested as that the expression of lipid synthesis-related genes was elevated while that of β-oxidation-related genes was decreased (Fig. 2C). The key factors of hepatic lipid synthesis and β-oxidation were further detected using RT-qPCR and Western blot for expression. The results showed that compared with the CON group, the mRNA and protein expression of key transcription factors and functional enzymes (such as SREBP1, FASN, ACC, and ACLY) of hepatic lipogenesis were significantly increased in PPCE group at GD20 and PW32, while the mRNA and protein expression of key factors of fatty acid β-oxidation (such as PPARα and CPT1α) were significantly decreased (Fig. 2D-I). It was suggested that PPCE could enhance hepatic fatty acid synthesis and weaken β-oxidation in male offspring rats before and after birth. Studies have suggested that lipotoxicity from excessive lipid accumulation in hepatocytes could damage mitochondria and produce excessive reactive oxygen species (ROS), thus promoting inflammatory response and NAFLD progression 39 . Therefore, we also measured the changes in mitochondrial function and inflammatory response in hepatocytes at PW32. The results showed that hepatic ROS levels were significantly increased in the PPCE group (Fig. 2J, K), mitochondrial damage in hepatocytes was severe (mitochondria swelling, rupture, and loss of mitochondrial cristae) (Fig. 2L), and mtDNA copy number was significantly increased (Fig. 2M), indicating that PPCE could cause mitochondrial damage and oxidative stress in hepatocytes of male offspring. Since the release of relevant molecular patterns such as mtDNA from this oxidative stress injury in hepatocytes is capable to activate STING-mediated inflammatory responses 40 . Thus, we examined the changes in the expression of STING-NF-κB inflammatory signaling in the liver. The results showed that macrophages in PPCE group were activated and STING expression in macrophages was significantly increased (Fig. 2N-P). Meanwhile, the protein expressions of cGAS, STING and p-NF-κB p65 (p-p65) and the mRNA expression and content of pro-inflammatory factors (TNF-α, IL–6 and IL–1β) in the liver were significantly increased (Fig. 2Q-T). STING-related inflammatory activation was also observed in the fetal liver of PPCE male offspring (Fig. S2). These results indicated that PPCE can cause hepatic lipid metabolism dysfunction, lipid accumulation and chronic inflammatory in male offspring before and after birth. 2.3. miR–142–3p is a critical target mediating PPCE-induced hepatic lipid metabolism disorders and chronic inflammation in male offspring Sperm miRNAs play a key role in the inheritance of acquired traits as a carrier of paternal genetic information and one of the important markers of epigenetic modifications 41 . Therefore, we first examined the changes in liver miRNAs expression profiles of paternal sperm and male offspring rats before and after birth to select co-differentially expressed miRNAs as potential toxicity targets. Sequencing analysis results showed that compared with the CON group, the expression of multiple miRNAs in paternal sperm and fetal liver of offspring in the PPCE group was changed (Fig. 3A), among which 10 kinds of miRNAs were co-differentially expressed (Fig. 3B), especially miR–142–3p was most significantly changed (Fig. 3C). The results of fluorescence in situ hybridization experiments showed that the expression of miR–142–3p in fetal liver was much higher than that in other organs, showing good organ specificity (Fig. 3D). Meanwhile, sequencing analysis suggested that the differential change of miR–142–3p was most significant in the liver of the PPCE group at PW32 (Fig. 3E). RT-qPCR results showed that hepatic miR–142–3p expression was significantly reduced in the PPCE group at GD20 and PW32 (Fig. 3F, G). These results suggest that PPCE reduce miR–142–3p expression in paternal sperm that can continue to the liver of male offspring, resulting in decreased hepatic miR–142–3p expression before and after birth. Next, to confirm the regulatory effects of miR–142–3p on hepatic lipid metabolism and inflammatory responses, we used hydrodynamic injection to establish a liver-specific miR–142–3p silencing (miR–142–3p -/- ) mouse model and observed after 8W. The results showed that compared with the CON group, the liver of the miR–142–3p -/- group was pale yellow in appearance and showed severe histological macro-vesicular-like steatosis with significant lipid accumulation, inflammatory infiltration (green arrows) and collagen fiber deposition (black and red arrows) (Fig. 3H), and the liver NAS and triglyceride content were significantly elevated (Fig. 3I, J). RT-qPCR results showed that the expressions of hepatic lipogenesis genes ( Srebp1, Fasn, and Acc) and pro-inflammatory factor-related genes ( Tnf-α, Il–6, and Il–1β) were significantly increased in the miR–142–3p -/- group (Fig. 3K), whereas the expressions of Pparα and Cpt1α were significantly decreased (Fig. 3K). Meanwhile, miR–142–3p -/- was able to promote hepatic STING-related inflammatory signaling activation (Fig. S3). These results reveal that miR–142–3p -/- could induce hepatic lipid metabolism dysfunction and inflammatory activation in mice, ultimately leading to the occurrence of NASH. To further validate that miR–142–3p low expression programming mediated alterations in hepatic lipid metabolism in PPCE male offspring, we performed reverse intervention by hydrodynamic injection of liver-targeted AAV8-miR–142–3p (miR–142–3p) overexpressing adeno-associated virus through the tail vein at PW8. The results showed that the liver lipogenesis genes ( Srebp1, Fasn, and Acc) expression was significantly decreased, β-oxidation genes ( Pparα and Cpt1α) expression was remarkably increased in miR–142–3p overexpressed PPCE offspring at PW32 (Fig. 3L), and the content and mRNA expression of liver pro-inflammatory factors (TNF-α, IL–6, and IL–1β) were significantly decreased (Fig. 3M, N). Histological results demonstrated that no obvious steatosis, lipid accumulation, and inflammatory infiltration were observed in the liver of miR–142–3p overexpressed PPCE offspring (Fig. 3O), and both liver NAS and triglyceride content were significantly reduced (Fig. 3P, Q). These results indicate that overexpression of miR–142–3p can reverse hepatic lipid metabolism dysfunction and inflammatory response in PPCE male offspring rats and inhibit adult NASH occurrence. 2.4. miR–142–3p targets ACSL4 to regulate hepatic lipid metabolism and inflammatory responses in PPCE male offspring mice It’s known that miRNAs can specifically bind to target gene mRNAs to regulate the expression of target genes and play important roles in a variety of life activities such as embryonic development, cellular metabolism, and signal transduction 42 . To elucidate the molecular mechanism by which miR–142–3p low expression mediates lipid metabolism disorder in the liver of PPCE male offspring rats, we first predicted the target genes of miR–142–3p using target databases ( TargetScan, miRDB, and miRcode). . The results showed that long-chain acyl-CoA synthetase 4 (ACSL4) had an interaction with miR–142–3p (Fig. 4A). Subsequently, we examined the changes in hepatic ACSL4 expression before and after birth. RT-qPCR and Western blot results showed that compared with the CON group, both mRNA and protein expression of liver ACSL4 were significantly increased in PPCE group at GD20 and PW32 (Figu. 4B-E). Immunofluorescence staining results also demonstrated that hepatic ACSL4 protein expression was significantly increased in PPCE group before and after birth (Fig. 4H-J). Meanwhile, the expression of Acsl4 mRNA in the liver of miR–142–3p -/- mice was significantly elevated (Fig. 4K), while miR–142–3p overexpression markedly reduced the expression of Acsl4 mRNA in the liver of PPCE male offspring (Fig. 4L). These results suggest that miR–142–3p may mediate changes in hepatic lipid metabolism in PPCE male offspring by targeting ACSL4. Further, we confirmed the molecular mechanism by which miR–142–3p low expression mediated lipid metabolism dysfunction in the liver of PPCE male offspring using BMSCs hepatoid differentiated cells. First, miR–142–3p inhibitor and miR–142–3p mimics transfection were used to confirm the regulatory effect of miR–142–3p on lipid metabolism function in hepatocytes. The results showed that in comparison with the CON group, miR–142–3p inhibitor significantly increased the expression of lipogenesis genes (SREBP1 and FASN) and decreased the expression of β oxidation-related genes (PPARα and CPT1α) (Fig. 4M-O), while miR–142–3p mimics produced the opposite effect. Further, we confirmed that miR–142–3p exerted its regulatory effect on lipid metabolism by targeting ACSL4. The results showed that overexpression of ACSL4 could significantly increase the expression of the above lipid synthesis genes and decrease the expression of β-oxidation-related genes (Fig. 4P-R), while miR–142–3p mimics could reverse the above changes caused by ACSL4 overexpression (Fig. 4P-R). Meanwhile, similar results were observed by immunofluorescence analysis (Fig. S4). The regulatory effect of miR–142–3p by targeting ACSL4 on lipid metabolism was also observed in a rat primary hepatocyte model (data not shown). Finally, we confirmed the interaction between miR–142–3p and ACSL4 using a dual luciferase reporter assay (Fig.4T). These results indicate that miR–142–3p regulate hepatic lipid metabolism by targeting ACSL4. 2.5. High glucocorticoid-induced sperm miR–142–3p methylation reprogramming mediates hepatic lipid metabolism dysfunction in PPCE male offspring Studies have shown that sperm “imprinting” paternal epigenetic information from adverse environmental exposures is an important manner to influence the long-term health of the offspring 43 . miRNAs and their methylation modifications are key epigenetic marks in the reprogramming of germ cells, transmitting the “imprint” of the parent’s adverse environmental experiences to the offspring 16, 44 . It was found that paternal chronic stress could lead to epigenetic modification alterations in sperm via high glucocorticoids, thereby programming abnormal liver glucose metabolism in the offspring 16 , suggesting that high glucocorticoids might be an important mechanism for the occurrence of parental metabolic diseases. Therefore, in this study, we first detected the serum corticosterone level, testicular GR expression level, and methylation level of sperm miR–142–3p promoter region in the parental male rats. The results showed that compared with the CON group, serum corticosterone levels, testicular GR mRNA expression level, and sperm methylation levels of miR–142–3p promoter region were significantly increased in PPCE group (Fig. 5A-C). Further, we treated mouse spermatogonia with different concentrations of caffeine (0.1~10 µM) or corticosterone (125~500 nM) in vitro to determine the cause of reduced sperm miR–142–3p expression. The results showed that different concentrations of caffeine treatment had no obvious effect on miR–142–3p expression in spermatogonia (Fig. 5D), while different concentrations of corticosterone treatment were able to significantly decrease miR–142–3p expression level in spermatogonia (Fig. 5E) and significantly increase the methylation level of miR–142–3p promoter region (Fig. 5F, G). To investigate whether a strong relationship between high glucocorticoid exposure and sperm miR–142–3p promoter region methylation also existed in the population, we collected a set of human samples and performed assays and correlation analyses. The results showed that serum corticosterone levels in fertile males were significantly and positively correlated with their sperm miR–142–3p promoter region methylation levels (Fig. S4), and sperm miR–142–3p promoter region methylation levels were significantly and positively correlated with sperm viability concentration/aberration rate (Fig. S4). It is suggested that parental corticosterone (but not caffeine) programs the hypermethylation of miR–142–3p in PPCE paternal sperm. To corroborate the involvement of parental high glucocorticoid exposure and sperm abnormalities in PPCE-induced NAFLD susceptibility in male offspring, we further conducted a series of intervention experiments. First, GR antagonist RU486 was used to synchronize interventions in PPCE rats to confirm the programming mechanism of paternal hyper glucocorticoid-mediated alterations in hepatic lipid metabolism. The results showed that compared with the CON group, the methylation rate of miR–142 promoter region in liver was increased and the expression was significantly decreased in PPCE group at GD20 and PW32 (Fig. 5H-K), and the expressions of hepatic lipogenesis function genes were significantly increased and β oxidation function genes were decreased (Fig. 5L, M), accompanied by significantly increased hepatic lipid accumulation and triglyceride content (Fig. 5N-P). However, RU486 intervention was able to markedly reverse PPCE-induced above changes in male offspring before and after birth (Fig. 5H-P). It is indicated that parental high glucocorticoid exposure mediates the alteration of hepatic lipid metabolism function in PPCE male offspring. Next, we explored the effect of fertilization between PPCE mouse sperm and normal oocytes on changes in liver lipid metabolism function in male offspring using in vitro fertilization technique (Fig. 6A). The results showed that compared with the CON group, the expression of miR–142–3p was decreased and the expressions of lipogenesis genes ( Srebp1, Fasn, and Acc) were significantly increased while the expressions of β-oxidation genes ( Pparα and Cpt1α) were significantly decreased in the fetal liver of the offspring in the PPCE group (Fig. 6B, C). Histological results showed that a large number of fat-like vacuoles, lipid accumulation, and significantly elevated liver triglyceride content were observed in the fetal liver of PPCE group (Fig. 6D, E). Further, to confirm that low expression of sperm miR–142–3p could mediate changes in liver lipid metabolism function in male offspring, we investigated the effect of miR–142–3p KO sperm on changes in liver lipid metabolism function in male offspring after fertilization with normal oocytes (Figu. 6F). The results showed that the expressions of lipogenesis genes were significantly increased in the fetal liver of the miR–142–3p KO group (Fig. 6G), while the mRNA expressions of fatty acid β-oxidation function genes were decreased (Fig. 6G), at the same time, the fetal liver showed obvious steatosis and significantly increased triglyceride content (Fig. 6H, I). At PW12, the liver lipogenesis was enhanced and β-oxidation was diminished in the miR–142–3p KO group, and a large amount of vacuolar-like steatosis along with excessive lipid accumulation was observed in the liver and NAFLD occurred (Fig. 6J-L). It was suggested that low expression of sperm miR–142–3p could mediate hepatic lipid metabolism dysfunction and the occurrence of NAFLD in male offspring. 2.6. PPCE induces NASH occurrence in male offspring with transgenerational inheritance To clarify whether there is a transgenerational genetic effect of PPCE-caused adult NASH in male offspring, we observed changes in liver histology, lipid metabolism function, and inflammatory response in F2 generation of the offspring. The results showed that compared with the CON group, there were no obvious changes in liver pathology and triglyceride content in F2 generation of PPCE offspring at PW12 (Fig. 7A, B), but miR–142–3p expression was significantly decreased (Fig. 7C), Acsl4 and lipogenesis genes ( Srebp1 and Fasn) expressions were significantly increased whereas β-oxidation genes ( Pparα and Cpt1α) expressions were decreased (Fig. 7D). However, at PW34, a large number of hepatocytes in the liver of F2 generation in PPCE offspring showed steatosis with lipid accumulation, inflammatory infiltration (green arrows), and collagen fibril deposition (black and red arrows) (Fig. 7E), and liver triglyceride content was significantly elevated (Fig. 7F). RT-qPCR and Western bolt results showed that at PW34, liver miR–142–3p expression was significantly decreased in F2 generation of PPCE offspring (Fig. 7G), the expression of ACSL4 and lipogenesis genes were significantly increased, while the expression of β-oxidation genes were decreased (Fig. 7H-J). Meanwhile, liver pro-inflammatory factor content (Fig. 7K-M) and mRNA expression (Fig. 7N-P) were significantly increased in F2 generation of PPCE offspring at PW34. It was suggested that PPCE could cause hepatic lipid metabolism dysfunction, inflammatory response activation, and adult NASH occurrence in the F2 generation of male offspring. Taken together, these findings demonstrate that PPCE-induced adult NASH occurrence in male offspring with transgenerational inheritance. 3. Discussion Both epidemiological surveys and laboratory studies have confirmed that caffeine has developmental toxicity 20, 45 , and special populations such as pregnant women, children, and adolescents are more susceptible to its adverse effects 46, 47, 48 . According to the European Food Safety Authority’s report on safe daily doses of caffeine for different populations, the total daily caffeine intake of pregnant women should not exceed 200 mg, while healthy adults should not exceed 400 mg 48 . Although the global average of adult caffeine intake is below the recommended safe dose (<400 mg/d), there is still a rather large population consuming overdose, especially in Europe and the United States 26 . Although our previous studies have systematically revealed the impact of maternal caffeine exposure during pregnancy on the long-term health of the offspring 21, 22, 23, 24 , the effect of PPCE on the long-term metabolic health of the offspring and the programming mechanisms have not been reported so far. In this regard, we established a PPCE rat model, in which male rats were given caffeine dose of 15, 30, and 60 mg/kg/d, equivalent to approximately half, one, and two times of the recommended safe dose for the population. Our main findings highlight that PPCE can lead to the occurrence of NASH in adult male offspring rats with transgenerational inheritance, which is associated with paternal high glucocorticoid-programmed sperm miR–142–3p hypermethylation to mediate hepatic lipid metabolic disorder and inflammatory activation, and confirm that miR–142–3p is a key regulator of lipid metabolism to serve as an early interventional target for paternal NASH. In this study, we first observed the changes in liver histology and lipid metabolism function in F1 and F2 generation rats at different times before and after birth, and found that PPCE could lead to hepatic lipid metabolism dysfunction in offspring and NASH occurrence in adulthood (PW32), with transgenerational inheritance and gender differences, especially in males. Transgenerational inheritance refers to the transmission of epigenetic information through germ cells to the next generation or even to multiple generations, which has important implications for biological evolution and disease occurrence 49 . Studies have previously found that paternal pre-pregnant exposure to adverse environmental factors can have impacts on the health of offspring and even multiple generations 50 . In the present study, we also investigated the transgenerational genetic effects of PPCE-induced NASH occurrence in male offspring and found that similar to the F1 generation of male offspring, PPCE also caused hepatic lipid metabolism dysfunction and steatosis along with inflammatory infiltration and collagen fibril deposition in the F2 male generation. It is suggested that PPCE-induced adult NASH occurrence in male offspring (F1 generation) with transgenerational inheritance. Interestingly, the degree of liver pathological alterations in male offspring of the PPCE-F2 generation was significantly reduced compared to the PPCE-F1 generation. It is suggested that PPCE-induced adult NASH occurrence in male offspring with transgenerational inheritance. We hypothesized that, although the changes in liver pathology in the F2 generation are likely to derive from the intergenerational transmission of PPCE-induced sperm reprogramming in the F0 generation and programming effect in F2 generation due to low glucocorticoid exposure in the F1 generation 23 , further insights into the mechanisms underlying the histopathological changes in the liver of the F2 generation are needed in the future. Other related studies have shown that developmental-derived diseases often have significant gender differences, which might be related to differences in maternal compensatory regulation on fetal development of different genders during intrauterine development of the embryo 51 . In this study, we compared PPCE-induced altered hepatic lipid metabolism in offspring before and after birth and the gender differences in the occurrence of adult NASH. It was found that PPCE could lead to the occurrence of adult NAFLD in female offspring, but the degree of histological alterations was less typical than that of males, no significant inflammatory infiltration and collagen fibril deposition were observed in the liver and hepatic miR–142–3p expression levels were significantly reduced in PPCE male offspring before and after birth. In contrast, there was no significant change in liver miR–142–3p expression in PPCE female offspring (Fig. S1). In conclusion, it is suggested that PPCE-caused occurrence of NASH in adult offspring has gender differences, and the disease characteristics of NASH in female offspring are atypical. Interestingly, we also found that the PPCE-induced abnormal development of multiple organs (including liver, adrenal glands, bone, cartilage, etc.) in female offspring was significantly weaker than that in male offspring, which may be attributed to the active, compensatory regulation of intrauterine offspring development by the maternal parent. We will delve into the gender-specific functional abnormalities of multiple organs in the offspring due to PPCE and the pathogenesis in our following studies. As important epigenetic modification markers, miRNAs are able to mediate the effects of paternal adverse factor exposure on the long-term health of offspring 16, 34, 52 . In this study, we found through sequencing analysis that miR–142–3p was the miRNA with the most significant change in co-expression in the liver of PPCE parental rat sperm and male offspring before and after birth. It was further confirmed that PPCE could cause a significant reduction in hepatic miR–142–3p expression in male offspring before and after birth, and liver-specific silencing of miR–142–3p could induce NASH occurrence in mice. It is suggested that miR–142–3p low expression mediates PPCE-induced NASH occurrence in male offspring. ACSLs are key enzymes that catalyze the synthesis of acyl-CoA and are essential in the regulation of body fatty acid metabolism 53 . ACSL4, one of the subtypes of the ACSLs family that is most closely involved in fatty acid metabolism, is able to catalyze the reaction between long-chain fatty acids and CoA to form acyl-CoA, thus playing an important role in the development of liver metabolic diseases 54 . It was found that ACSL4 can promote fatty acid de novo synthesis process in hepatocytes through activation of transcription factor SREBP1 55, 56 , and inhibit fatty acid β-oxidation by suppressing PPARα transcription through endogenous ligands 54, 56 . In this study, we found that PPCE significantly increased the expression of ACSL4 in the liver of male offspring before and after birth; overexpression of miR–142–3p suppressed the expression of ACSL4 in hepatocytes and reversed the occurrence of long-term NASH in PPCE male offspring rats. We further found that miR–142–3p was able to bind to ACSL4 through target gene prediction and dual luciferase reporter gene experiments, and confirmed that miR–142–3p regulated lipogenesis and β-oxidation function in BMSCs hepatoid differentiated cells by targeting ACSL4. Excessive lipid accumulation in the liver has been demonstrated to result in lipotoxicity, triggering mitochondrial oxidative stress, inflammatory responses, and the progression of NAFLD 57 . STING, a pivotal factor in innate immunity located on the endoplasmic reticulum, becomes activated upon recognizing abnormal double-stranded DNA in the cytoplasm. It regulates inflammatory responses by activating transcription factors like NF-κB and IRF3 58 . In the present study, our findings reveal that PPCE induces an increase in oxidative stress and activates STING-mediated inflammatory signaling in liver macrophages of male offspring. This activation leads to heightened production of pro-inflammatory factors, including TNF-α, IL–6, and IL–1β, in the liver. Additionally, the absence of miR–142–3p (miR–142–3p -/- ) is associated with the activation of STING-mediated inflammatory responses in the livers of mice. Collectively, these results suggest that low expression of miR–142–3p contributes to hepatic lipid metabolism disorder and activation of inflammatory responses through the upregulation of ACSL4. This, in turn, mediates the occurrence of non-alcoholic steatohepatitis (NASH) in male offspring exposed to PPCE. Clinical studies have also found reduced miR–142–3p expression in peripheral blood mononuclear cells (PBMCs) of children with metabolic-associated fatty liver disease (MAFLD) that was potentially correlated with serum triglyceride levels 59 , suggesting that miR–142–3p may be closely associated with the occurrence of MAFLD. Although a growing number of studies confirm that NAFLD/NASH has a paternal origin, the programming mechanisms remain unelucidated, leading to difficulties in early prevention and treatment. Other related research has shown that improving paternal pre-pregnancy lifestyle (such as proper exercise and antioxidant supplementation) has a positive effect on the prevention of paternal NAFLD 60, 61, 62 , but due to individual differences and lack of specificity, these measures are difficult to be effective prevention and treatment strategies for paternal NAFLD/NASH. In the present study, we found that miR–142–3p was a toxic target of PPCE-induced NASH occurrence in male offspring, and confirmed that overexpression of miR–142–3p was effective in reversing PPCE-induced NASH occurrence in male offspring. Intriguingly, we recently also observed hepatic miR–142–3p low expression and abnormal lipid metabolism function in male offspring on a rat model of low-dose mixed paternal pre-pregnancy nicotine/ethanol/caffeine exposure (Fig. S5). It was suggested that miR–142–3p could serve as a potential, common target for early prevention and treatment of paternal NAFLD/NASH. This finding offers valuable insights for developing nucleic acid drugs to prevent and treat paternal NAFLD/NASH. Glucocorticoids are known to be the most important regulatory hormone in body stress response and are crucial in determining germ cell formation, fetal maturation, and postnatal fate. Studies have shown that paternal adverse environmental (such as caffeine, nicotine, and ethanol) exposure can provoke HPA axis activation, resulting in the body being in a chronic stress state 63, 64, 65 , and affecting sperm epigenetic modifications through elevated serum glucocorticoid levels, leading to offspring dysplasia 16, 66 . It is suggested that serum-high glucocorticoid reprogrammed sperm epigenetic modifications may be an important mechanism mediating the transmission of paternally acquired traits to the offspring. DNA methylation, one of the most important forms of epigenetic inheritance in eukaryotes, mediates the inheritance of paternally acquired traits 67 . Clinical evidence suggests that poor paternal dietary habits can lead to altered DNA methylation status during sperm reprogramming, resulting in an increased risk of metabolic disease in the offspring after birth 68, 69 . In this study, we found that serum corticosterone level was significantly elevated, and the methylation level of miR–142–3p promoter region was significantly elevated in sperm and liver of PPCE-F0 generation, and we confirm that it was the corticosterone (but not caffeine) caused the elevated methylation level of miR–142–3p promoter region. To clarify the glucocorticoid programming mechanism of PPCE-induced imbalance of lipid metabolism homeostasis in the liver of offspring and NASH occurrence, we used the GR antagonist RU486 to intervene in PPCE rats and found that it could reduce the methylation level of the miR–142–3p promoter region in the sperm of PPCE-F0 generation and liver of male F1 generation, and reverse the occurrence of long-term NASH. In vitro fertilization experiments further confirmed that miR–142–3p low expression could program the alteration of hepatic lipid metabolism function in PPCE male offspring. Interestingly, we found a significant positive correlation in a population of fertile men between serum corticosterone levels and sperm miR–142–3p promoter region methylation, as well as between sperm miR–142–3p promoter region methylation and sperm concentration/aberration rate in adult men (Fig. S4). It is suggested that high glucocorticoid-programmed sperm miR–142–3p hypermethylation resulting from parental chronic stress mediates the occurrence of PPCE-induced NASH in male offspring, where hypermethylation of sperm miR–142–3p promoter region is expected to be early warning targets for clinical paternal NASH susceptibility. The “DOHaD” theory suggests that many metabolic diseases in adults have a developmental origin and that adverse environmental exposures in the paternal preconception or maternal pregnancy can cause alterations in metabolic adaptations and developmental programming in the offspring, which is an important risk factor leading to an increased risk of multiple disorders (such as NAFLD) in the offspring. Previous systematic studies in our laboratory have confirmed that maternal PCE can lead to the occurrence of IUGR, changes in hepatic lipid metabolism function, and NAFLD susceptibility, along with gender differences in the offspring 70, 71, 72 . To elucidate the effects of (paternal and maternal) parental caffeine exposure on developmental programming and disease susceptibility in offspring, we compared and analyzed the similarities and differences in PPCE and PCE-induced NAFLD susceptibility. The details are shown in Table 1.Our outcomes showed that PPCE-induced and PCE-induced NAFLD susceptibility is different in caffeine exposure dose and duration, disease state, and gender difference. It manifested that PPCE male rats were exposed to caffeine (15~60 mg/kg/d) for 8 weeks (a complete spermatogenic cycle), and PCE female rats were exposed to caffeine (30~120 mg/kg/d) in middle and late pregnancy (GD9~GD20); PPCE could cause the occurrence of NAFLD in the offspring, and there were significant inflammatory response and fibrosis (NASH occurrence) with no need for a “second hit “, while PCE could lead to NAFLD susceptibility in offspring with no obvious inflammatory response and fibrosis and it needs a “second hit (such as high-fat diet)" to develop NAFLD. In conclusion, compared to PCE, the disease course of PPCE-induced NAFLD was shorter and more severe, which may be associated with the fact that paternal effects on the offspring are earlier than maternal. In summary, this study marks the pioneering demonstration of the association between PPCE and disrupted hepatic lipid metabolism in male offspring, both prenatally and postnatally, leading to the development of NASH in adulthood. The observed effects include transgenerational inheritance impacts and notable gender differences. The underlying mechanism involves elevated glucocorticoid levels, specifically arising from PPCE, inducing hypermethylation in the sperm miR–142–3p promoter region. This epigenetic modification persists in the livers of male offspring, contributing to diminished miR–142–3p expression, subsequent dysfunction in hepatic lipid metabolism, and the activation of inflammatory pathways, ultimately culminating in adult-onset NASH (Fig. 8). Importantly, hepatic overexpression of miR–142–3p has been shown to effectively reverse the long-term occurrence of NASH in PPCE-exposed male offspring, providing a potential intervention strategy for high glucocorticoid-mediated programming. The established PPCE rat model faithfully reproduces the adverse lifestyle habits and chronic stress prevalent in the daily lives of contemporary reproductive-age men. This research not only elucidates the developmental origins and programming mechanisms of paternal NASH but also confirms a viable intervention target. The findings hold significant practical implications for understanding the impact of paternal pre-pregnancy chronic stress on the long-term metabolic health of offspring and for guiding the adoption of healthy lifestyles among men of reproductive age. 4. Methods 4.1. Reagents Caffeine (purity ≥ 99.0%) and corticosterone were purchased from Sigma-Aldrich (St Louis, MO, USA). Mifepristone (RU486) was obtained from Hubei Gedian humanwell Pharmaceutical Co. LTD (Ezhou, China). Rat-miR–142–3p inhibitor oligo, Rat-miR–142–3p mimics oligo, Rat-ACSL4 plasmid, and mus-miR–142–3p siRNA were obtained from GenePharma (Shanghai, China). AAV8 adenovirus delivery-miR–142–3p was purchased from Tsingke Biotechnology (Beijing, China). miScript SYBR Green PCR Kit and SYBR Green qPCR Master Mix were purchased from Vazyme (Nanjing, China). All other reagents were of analytical grade. 4.2. Animal treatment Specific pathogen-free (SPF) Wistar rats were purchased from the Experimental Center of the Hubei Medical Scientific Academy and accommodated to standard nutritional conditions with a 12 h light/dark cycle. Animal experiments were performed in the Center for Animal Experiment of Wuhan University (Wuhan, China), which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). All animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee at the Wuhan University (WHEF–2022–0286). Male Wistar rats (F0) were randomly ranged into the control and paternal pre-pregnant caffeine exposure (PPCE) groups ( n = 12). Animals in the PPCE group were administrated with different concentrations of caffeine (15, 30, and 60 mg/kg/d) for consecutive 8 weeks, and rats of the control group were treated with the same volume of normal saline. After caffeine administration, male rats were mated with untreated females in a joint cage. The day when sperm appeared in vaginal smear was designated as gestational day (GD) 0. Half of the pregnant rats were euthanized on GD20 with 3% isoflurane, liver was collected for further analysis. The rest pregnant rats were kept to obtain the F1 generation. One day after birth, the number of pups was standardized to 12 pups per litter (6 males and 6 females). The offspring were fed with normal diet after weaning at postnatal week 4 (PW4), and sacrificed under isoflurane anesthesia at PW12 and PW32, respectively ( n = 12, randomly chose one from each litter). Liver tissues were collected for subsequent analysis. A subset of F1 male rats were randomly selected on PW8, and divided into vector and AAV8-miR–142–3p group ( n = 20). AAV8-miR–142–3p and vectors were injected respectively into rats by hydrodynamic injection via tail vein. These rats were then sacrificed at PW12 ( n = 10) and PW32 ( n = 10). To further investigate the underlying mechanism, F0 male rats were randomly divided into the control (normal saline), PPCE (60 mg/kg/d caffeine), RU486 (1 mg/kg/d) 16, 73 , and RU486 (1 mg/kg/d) + PPCE (60 mg/kg/d caffeine) group ( n = 12). Rats in each group were administrated with corresponding reagents daily for continuous 8 weeks, and then mated with normal female rats at the end of administration. Half of the pregnant rats were killed at GD20, and the rest pregnant rats delivered F1 generation naturally. The F1 rats were sacrificed at PW12 and PW32 ( n = 12). Moreover, another part of F1 male rats was mated with normal female rats at PW12 to produce the F2 generation and F2 rats were sacrificed at PW12 and PW34 ( n = 12), respectively. 4.3. Human plasma and sperm samples Human plasma and sperm samples were collected from 28 male patients who underwent assisted reproductive tests in the Reproductive Medicine Center of Zhongnan Hospital, Wuhan University. The plasma cortisol concentration, sperm general characteristics (sperm concentration, sperm number, sperm malformation rate, etc.) and sperm miR–142–3p promoter region methylation rate were detected, and the correlation between the above results was analyzed. The study was approved by the Institutional Ethics Committee of the Zhongnan Hospital of Wuhan University (No. 2020188). Informed consent was obtained from the patients before the collection of plasma and sperm samples. All the research was performed in accordance with government policies and the Helsinki declaration. 4.4. Hydrodynamic injection The miR–142–3p lentiviral plasmid (miR–142–3p -/- ) was synthesized by Genepharma (Shanghai, China). Male FVB/N mice (20~22 g) were obtained from Charles River Laboratories (Beijing, China), and randomly divided into control and miR–142–3p -/- group ( n = 8). The hydrodynamic injection was performed to conduct a hepatic-specific miR–142–3p -/- mouse model as previously described 74 . In detail, 20 µg miR–142–3p -/- plasmids were diluted in 2 mL normal saline, then plasmids solution with a volume of 10% of the animal’s body weight was transiently injected into the tail-vein of mice in miR–142–3p -/- group within 6~7s. Mice in the control group were also injected with the same volume of the vehicle. After 8 weeks, all mice were euthanized with 3% isoflurane to collect serum and liver samples for the following analysis. 4.5. In vitro fertilization and embryo culture Firstly, the sperm of mice exposed to chronic treatment of HCG or vehicle ( n = 20) was extracted from epididymis and the spermatozoa were diluted at ∼2 × 10 6 of final concentration in human tubal fluid (HTF) medium to be evaluated after 15 min of capacitation. Next, male ICR mice were administrated with a gavage of caffeine (120 mg/kg/d) for continuous 8 weeks to establish a PPCE mice model ( n = 12). The control group was also administrated the same amount of saline. Sperm were isolated from the epididymis after finished the administration and were diluted to a concentration of 2 × 10 6 /mL in the human tubal fluid medium, followed by further investigation after 15 min capacitation. Female ICR mice ( n = 24) were injected with 5 IU equine chorionic gonadotropin and 5 IU HCG for superovulation, followed by collecting mature oocytes from the fallopian tube. In vitro fertilization was performed as previously described 75 . Briefly, sperms of mice in the control or PPCE group and normal oocytes were transferred into the embryo medium for further culture and fertilization to get fertilized eggs. Fertilized zygotes were cultured in vitro for 48 h, then the surviving embryos were transferred into the oviduct of pseudopregnant recipient female mice. All pregnant mice were anesthetized using 3% isoflurane to strip fetal mice 20 days after embryo transfer and collect fetal blood and liver samples for further investigations. Male miR–142–3p KO ICR mice ( n = 5) were generated by Cyagen Biosciences (Nanjing, China) using CRISPR/Cas9 technology, and miR–142–3p KO sperm were collected from the epididymis. Meanwhile, sperm and mature oocytes of normal male and female mice were also extracted. Normal sperm or miR–142–3p KO sperm were fertilized with normal oocytes to generate fertilized oocytes, which were then transferred to pseudopregnant recipient female mice. Fetal blood and liver samples were harvested 20 days after embryo transfer as the above method. The rest of the pregnant mice were fed till natural birth. The offspring were raised to adulthood to determine the effect of miR–142–3p KO on hepatic lipid metabolic function in male offspring. 4.6 Cell culture Bone marrow mesenchymal stem cells (BMSCs) hepatoid differentiated cells were extracted as previously described 76 . In brief, BMSCs hepatoid differentiated cells were extracted from 3-week-old male rats and cultured using α-MEM medium with 10% fetal bovine serum, 100 mg/mL streptomycin, and 100 U/mL penicillin. When the cells reached 80% confluence, they were incubated with hepatocyte differentiation medium (DMEM medium with 1% fetal bovine serum, 100 mg/mL streptomycin, 100 U/mL penicillin, 20 ng/mL HGF, 2 ng/mL EGF, 0.1 μM dexamethasone, and 50 mg/mL ITS) for 14 days 77 . 20 nM miR–142–3p inhibitor, 20 nM miR–142–3p mimics, or 2.5 μg ACSL4 plasmid were transfected into cells using lipofectamine 3000. In detail, the plasmids and transfection reagent were incubated in DMEM for 20 min at room temperature to perform transfection into BMSCs hepatoid differentiated cells, and harvested for further assessment after 24 h. Mouse spermatogonial cell line GC–1 cells (Procell Life Science &Technology, Wuhan, China) were also cultured under the above conditions. GC–1 cells were treated with corticosterone (125, 250, 500 nM) or caffeine (0.1, 1, 10 µM) for 24 h, respectively. Cells were then collected to detect the mRNA expression level of miR–142–3p and the rate of promoter region methylation. All in vitro data were from triplicate independent experiments. 4.7. DNA methylation analysis The bisulfite pyrosequencing method was performed for DNA methylation analyses in sperm and liver samples ( n = 3) as previously reported 78 . DNA was extracted from F0 rat sperm and male F1 liver tissues using a Mammalian genomic DNA extraction kit (Beyotime, Shanghai, China). To measure the DNA permethylation status, EZ DNA Methylation-Gold Kit (ZYMO, CA, USA) was used for sample processing to convert unmethylated cytosine into uracil. After building the MethylTarget library, high-throughput sequencing was employed with the Illumina HiSeq platform. 4.8. Whole transcriptome sequencing Sperm and liver samples of male rats were lysed with Trizol reagent for further analysis. The sequencing libraries were generated using the TruSeq Stranded Total RNA Library Prep Kit (Illumina, USA). Then, the libraries analysis was performed using the online platform of Majorbio Cloud Platform (www.majorbio.com ) (Shanghai Majorbio Bio-pharm Technology Co., Ltd). The analyses of mRNA and miRNA expression were conducted using the DESeq R package, and the transcripts with P 2 were selected as differentially expressed mRNAs and miRNAs. The enrichment analysis was plotted using the R software. All sequencing was analyzed on the Whole Transcriptome Cloud Platform of Shanghai Meggie Biotechnology (https://cloud.majorbio.com) . 4.9. Hematoxylin-eosin (H&E), oil red O (ORO) staining, Masson staining, Sirius red staining, and Transmission electron microscopy (TEM) For histopathological analysis, fresh liver tissues were fixed within 10% paraformaldehyde and then embedded with paraffin to slice into liver sections (5 µm). Liver slides were dewaxed with xylene and rehydrated with ethanol at decreasing concentration gradient to perform H&E staining, Masson staining and Sirius red staining. For ORO staining, frozen liver slices were incubated within ORO solution, rinsed with 60% isopropanol, and nucleus were redyed with hematoxylin. All slides were then mounted under coverslips for further observation under a microscope. The pathological grading and staging of non-alcoholic steatohepatitis (NASH) rats were scored according to the non-alcoholic fatty liver diseases (NAFLD) activity score (NAS) system 79 . The scoring system consisted of a semi-quantitative assessment of three histological features: steatosis (0–3), lobular inflammation (0–3), and hepatocellular ballooning (0–2), in which NAS 4 (NASH), and NAS between 3 and 4 (possibly NASH). For TEM analysis, fresh liver tissues from mice were fixed in 2.5% glutaraldehyde, post-fixed in 1% OsO4, and dehydrated with gradient ethanol and acetone. The tissues were embedded in epoxy resin, cut into serial TEM ultrathin sections, and stained with uranyl acetate and lead citrate. The sections were photographed using a JEM 1400 JEOL instrument (Tokyo, Japan). 4.10. Real-time quantitative polymerase chain reaction (RT-qPCR) Total RNA was extracted from liver homogenate and BMSCs hepatoid differentiated cells using TRIzol™ Reagent, and then converted into complementary DNA (cDNA). The RT-qPCR assay was subsequently processed to determine the quantity of specific DNA sequences by FastStart Universal SYBR Green Master Mix (Roche, Basel, Switzerland) on a 384-well ABI StepOnePlus cycler (Applied Biosystems, Foster City, CA, USA). Glyceraldehyde–3-phosphate dehydrogenase (GAPDH) was taken as an internal reference for quantitative assay. The identified primers were listed in Table S1. NADH dehydrogenase subunit 1 (ND1) gene encoded by mtDNA and GAPDH encoded by nuclear DNA (nDNA) were determined by RT-qPCR analysis as previously described 80 . The mtDNA/nDNA ratio was used to assess the relative mtDNA copy number. The primers are listed in Table S2. 4.11. Western blotting and immunofluorescence Western blotting was performed to determine protein expressions in liver tissues and BMSCs hepatoid differentiated cells. Briefly, the concentrations of extracted cytosol protein were determined using the BCA protein assay kit (Thermo Fisher Scientific, OH, USA) Proteins were then separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto PVDF membranes. After blocking with 5% skim milk powder for at least 1h, membranes were incubated with specific primary antibodies overnight, followed by incubating with corresponding second antibodies for 1h. The bands were visualized by chemiluminescence using an electro-chemiluminescent detection kit (Thermo Fisher Scientific, OH, USA). The involved primary antibodies were listed in Table S3. For immunofluorescence, BMSCs hepatoid differentiated cell climbing slices and fixed liver slides were incubated with primary antibodies overnight at 4℃. After that, slides were washed three times with PBS, incubated with DAPI and FITC-conjugated second antibodies for 1.5 h at 37°C, and observed under a fluorescence microscope. The involved primary antibodies were listed in Table S3. 4.12. Fluorescence in situ hybridization (FISH) FISH assay was performed as previously described 81 . The whole fetal rat was fixed within 10% paraformaldehyde and then embedded with paraffin to slice. Paraffin sections were dewaxed with xylene and rehydrated with ethanol at decreasing concentration gradient to perform FISH. Fluorescent-labeled probe for miR–142–3p (Genepharma, Shanghai, China) was applied during hybridization. FISH was performed using the RNAscope Multiplex Fluorescent Multiplex kit (Advanced Cell Diagnostics, California, USA) according to the manufacturer’s protocol and observed under a fluorescence microscope. 4.13. Dual-luciferase reporter gene assay After 50~70% of confluence, cells were co-transfected with 2 μg pMiR-report vector-RAB22 A/SNHG3 3′UTR and miR–142–3p using Lipofectamine 2000 for 6 h, then lysed for luciferase activity determination using Dual-luciferase Reporter Assay System. Luciferase reporter vectors were provided by GenePharma (Shanghai, China). All experiments above were repeated three times. 4.14. Statistical analysis Prism 8.0 (GraphPad Software Inc., San Diego, CA, USA) was employed for statistical analysis. All data were expressed as Mean ± S. E. M. values. Student’s t- test and one-way ANOVA were used between two groups and among three or more groups, respectively. P < 0.05 represented statistical significance. Abbreviations AAV, adeno-associated virus; ACC, acetyl CoA carboxylase; ACLY, ATP-citrate lyase; ACSL4, acyl-CoA synthetase long chain family member 4; ALT, alanine transaminase; AST, aspartate transaminase; BMSCs, bone mesenchymal stem cells; cGAS, cyclic GMP-AMP synthase; CORT, corticosterone; CPT1α, carnitine palmitoyltransferase 1α; DOHaD, Developmental Origins of Health and Disease; FASN, fatty acid synthase; FISH, fluorescence in situ hybridization; GAPDH, glyceraldehyde–3-phosphate dehydrogenase; GD, gestational day; GR, glucocorticoid receptor; H&E, hematoxylin-eosin; HPA, hypothalamic pituitary adrenal; IL–1β, interleukin–1β; IL–6, interleukin–6; IUGR, intrauterine growth retardation; NAFLD, non-alcoholic fatty liver diseases; NAS, NAFLD activity score; NASH, non-alcoholic steatohepatitis; NF-κB, nuclear factor-k-gene binding; ORO, oil red O; PCE, prenatal caffeine exposure; POHaD, Paternal Origins of Health and Disease; PPARα, peroxisome proliferators activated receptor α; PPCE, paternal pre-pregnancy caffeine exposure; PW, postnatal week; ROS, reactive oxygen species; RT-qPCR, real-time polymerase chain reaction; RU, RU486 (mifepristone); SREBP1, sterol-regulatory element binding protein–1; STING, stimulator of interferon genes; TNF-α, tumor necrosis factor α. Declarations Funding information This work was supported by the National Natural Science Foundation of China (No. U22A20362, No. U23A21019) and the National Key Research and Development Program of China (No. 2020YFA0803900). Conflicts of interest The authors declared no conflict of interest. Authors’ contributions Cong Zhang: conceptualization, data curation, and writing - original draft; Yu Guo: methodology and investigation; Yi Liu: software and visualization; Kexin Liu and Wen Hu: formal analysis; Hui Wang: project administration, funding acquisition, and writing - review & editing. All authors approved the manuscript for submission. Data availability statement The data presented in this study are available on request from the corresponding author. References Younossi ZM, Koenig AB, Abdelatif D, Fazel Y, Henry L, Wymer M. Global epidemiology of nonalcoholic fatty liver disease-Meta-analytic assessment of prevalence, incidence, and outcomes. Hepatology 64 , 73-84 (2016). Eslam M, Valenti L, Romeo S. Genetics and epigenetics of NAFLD and NASH: Clinical impact. J Hepatol 68 , 268-279 (2018). Barker DJ. Developmental origins of adult health and disease. J Epidemiol Community Health 58 , 114-115 (2004). McMillen IC, Robinson JS. Developmental origins of the metabolic syndrome: prediction, plasticity, and programming. Physiol Rev 85 , 571-633 (2005). Zou KX, Ding GL, Huang HF. Advances in research into gamete and embryo-fetal origins of adult diseases. Sci China Life Sci 62 , 360-368 (2019). Monti M. Gamete and embryo-fetal origins of adult diseases. Eur J Histochem 60 , 2696 (2016). Donkin I, Barres R. Sperm epigenetics and influence of environmental factors. Mol Metab 14 , 1-11 (2018). Braun JM, Messerlian C, Hauser R. Fathers Matter: Why It's Time to Consider the Impact of Paternal Environmental Exposures on Children's Health. Curr Epidemiol Rep 4 , 46-55 (2017). Godschalk R , et al. Paternal Exposure to Environmental Chemical Stress Affects Male Offspring's Hepatic Mitochondria. Toxicol Sci 162 , 241-250 (2018). Yan S , et al. Prenatal exposure to the Chinese famine and the risk of metabolic syndrome in adulthood across consecutive generations. Eur J Clin Nutr 74 , 1229-1236 (2020). Chang RC, Thomas KN, Bedi YS, Golding MC. Programmed increases in LXRalpha induced by paternal alcohol use enhance offspring metabolic adaptation to high-fat diet induced obesity. Mol Metab 30 , 161-172 (2019). Chang RC, Wang H, Bedi Y, Golding MC. Preconception paternal alcohol exposure exerts sex-specific effects on offspring growth and long-term metabolic programming. Epigenetics Chromatin 12 , 1-17 (2019). De Jesus DF , et al. Parental metabolic syndrome epigenetically reprograms offspring hepatic lipid metabolism in mice. J Clin Invest 130 , 2391-2407 (2020). Watkins AJ , et al. Paternal diet programs offspring health through sperm- and seminal plasma-specific pathways in mice. P Natl Acad Sci USA 115 , 10064-10069 (2018). Clow A, Hamer M. The iceberg of social disadvantage and chronic stress: implications for public health. Neurosci Biobehav Rev 35 , 1 (2010). Wu L , et al. Paternal Psychological Stress Reprograms Hepatic Gluconeogenesis in Offspring. Cell Metab 23 , 735-743 (2016). Zheng X , et al. Sperm epigenetic alterations contribute to inter- and transgenerational effects of paternal exposure to long-term psychological stress via evading offspring embryonic reprogramming. Cell Discov 7 , 101 (2021). Buerge, II, Poiger T, Muller MD, Buser HR. Caffeine, an anthropogenic marker for wastewater comtamination of surface waters. Environ Sci Technol 37 , 691-700 (2003). Diogo JS, Silva LS, Pena A, Lino CM. Risk assessment of additives through soft drinks and nectars consumption on Portuguese population: a 2010 survey. Food Chem Toxicol 62 , 548-553 (2013). Jensen TK, Swan SH, Skakkebaek NE, Rasmussen S, Jorgensen N. Caffeine intake and semen quality in a population of 2,554 young Danish men. Am J Epidemiol 171 , 883-891 (2010). Shangguan Y , et al. Glucocorticoid mediates prenatal caffeine exposure-induced endochondral ossification retardation and its molecular mechanism in female fetal rats. Cell Death Dis 8 , e3157 (2017). He B , et al. Prenatal caffeine exposure induces liver developmental dysfunction in offspring rats. J Endocrinol 242 , 211-226 (2019). He Z , et al. H19/let-7 axis mediates caffeine exposure during pregnancy induced adrenal dysfunction and its multi-generation inheritance. Sci Total Environ 792 , 148440 (2021). Pei LG , et al. The GC-IGF1 axis-mediated testicular dysplasia caused by prenatal caffeine exposure. J Endocrinol 242 , M17-M32 (2019). Gonzalez de Mejia E, Ramirez-Mares MV. Impact of caffeine and coffee on our health. Trends Endocrinol Metab 25 , 489-492 (2014). Quadra GR , et al. A global trend of caffeine consumption over time and related-environmental impacts. Environ Pollut 256 , 113343 (2020). Busada JT, Cidlowski JA. Mechanisms of Glucocorticoid Action During Development. Curr Top Dev Biol 125 , 147-170 (2017). Pollard I. Increases in plasma concentrations of steroids in the rat after the administration of caffeine: comparison with plasma disposition of caffeine. J Endocrinol 119 , 275-280 (1988). Eyolfson E, Bhatt D, Wang M, Lohman AW, Mychasiuk R. Paternal exposure to exercise and/or caffeine and alcohol modify offspring behavioral and pathophysiological recovery from repetitive mild traumatic brain injury in adolescence. Genes Brain Behav , egbb12736 (2021). Schagdarsurengin U, Steger K. Epigenetics in male reproduction: effect of paternal diet on sperm quality and offspring health. Nat Rev Urol 13 , 584-595 (2016). Chen Q, Yan W, Duan E. Epigenetic inheritance of acquired traits through sperm RNAs and sperm RNA modifications. Nat Rev Genet 17 , 733-743 (2016). Collignon J. miRNA in embryonic development: the taming of Nodal signaling. Dev Cell 13 , 458-460 (2007). Reza A , et al. Roles of microRNAs in mammalian reproduction: from the commitment of germ cells to peri-implantation embryos. Biol Rev Camb Philos Soc 94 , 415-438 (2019). Liang G , et al. microRNAs in aged sperm confer psychiatric symptoms to offspring through causing the dysfunction of estradiol signaling in early embryos. Cell Discov 8 , 63 (2022). Wang Y , et al. Sperm microRNAs confer depression susceptibility to offspring. Sci Adv 7 , (2021). de Castro Barbosa T , et al. High-fat diet reprograms the epigenome of rat spermatozoa and transgenerationally affects metabolism of the offspring. Mol Metab 5 , 184-197 (2016). Rodgers AB, Morgan CP, Leu NA, Bale TL. Transgenerational epigenetic programming via sperm microRNA recapitulates effects of paternal stress. Proc Natl Acad Sci U S A 112 , 13699-13704 (2015). Short AK , et al. Elevated paternal glucocorticoid exposure alters the small noncoding RNA profile in sperm and modifies anxiety and depressive phenotypes in the offspring. Transl Psychiatry 6 , e837 (2016). Mota M, Banini BA, Cazanave SC, Sanyal AJ. Molecular mechanisms of lipotoxicity and glucotoxicity in nonalcoholic fatty liver disease. Metabolism 65 , 1049-1061 (2016). Liu Z , et al. XBP1 deficiency promotes hepatocyte pyroptosis by impairing mitophagy to activate mtDNA-cGAS-STING signaling in macrophages during acute liver injury. Redox Biol 52 , 102305 (2022). Zhang Y, Shi J, Rassoulzadegan M, Tuorto F, Chen Q. Sperm RNA code programmes the metabolic health of offspring. Nat Rev Endocrinol 15 , 489-498 (2019). Yang Q , et al. Highly sensitive sequencing reveals dynamic modifications and activities of small RNAs in mouse oocytes and early embryos. Sci Adv 2 , e1501482 (2016). Immler S. The sperm factor: paternal impact beyond genes. Heredity (Edinb) 121 , 239-247 (2018). Dai J , et al. Paternal nicotine exposure defines different behavior in subsequent generation via hyper-methylation of mmu-miR-15b. Sci Rep 7 , 7286 (2017). Tomaszewski M, Burdan F, Olchowik G, Tomaszewska M. The effects of caffeine administered at different temperatures on foetal development. Ann Agric Environ Med 23 , 148-152 (2016). Qian J, Chen Q, Ward SM, Duan E, Zhang Y. Impacts of Caffeine during Pregnancy. Trends Endocrinol Metab 31 , 218-227 (2020). James JE, Kristjansson AL, Sigfusdottir ID. Adolescent substance use, sleep, and academic achievement: evidence of harm due to caffeine. J Adolesc 34 , 665-673 (2011). Wikoff D , et al. Systematic review of the potential adverse effects of caffeine consumption in healthy adults, pregnant women, adolescents, and children. Food Chem Toxicol 109 , 585-648 (2017). Boskovic A, Rando OJ. Transgenerational Epigenetic Inheritance. Annu Rev Genet 52 , 21-41 (2018). Chen Q , et al. Sperm tsRNAs contribute to intergenerational inheritance of an acquired metabolic disorder. Science 351 , 397-400 (2016). Napso T , et al. Diet-induced maternal obesity impacts feto-placental growth and induces sex-specific alterations in placental morphology, mitochondrial bioenergetics, dynamics, lipid metabolism and oxidative stress in mice. Acta Physiol (Oxf) 234 , e13795 (2022). Labialle S , et al. The miR-379/miR-410 cluster at the imprinted Dlk1-Dio3 domain controls neonatal metabolic adaptation. EMBO J 33 , 2216-2230 (2014). Yan S, Yang XF, Liu HL, Fu N, Ouyang Y, Qing K. Long-chain acyl-CoA synthetase in fatty acid metabolism involved in liver and other diseases: an update. World J Gastroenterol 21 , 3492-3498 (2015). Singh AB, Kan CFK, Kraemer FB, Sobel RA, Liu J. Liver-specific knockdown of long-chain acyl-CoA synthetase 4 reveals its key role in VLDL-TG metabolism and phospholipid synthesis in mice fed a high-fat diet. Am J Physiol Endocrinol Metab 316 , E880-E894 (2019). Chen J , et al. ACSL4 reprograms fatty acid metabolism in hepatocellular carcinoma via c-Myc/SREBP1 pathway. Cancer Lett 502 , 154-165 (2021). Duan J , et al. Therapeutic targeting of hepatic ACSL4 ameliorates NASH in mice. Hepatology 75 , 140-153 (2021). Arroyave-Ospina JC, Wu Z, Geng Y, Moshage H. Role of Oxidative Stress in the Pathogenesis of Non-Alcoholic Fatty Liver Disease: Implications for Prevention and Therapy. Antioxidants (Basel) 10 , 174 (2021). Yu Y, Liu Y, An W, Song J, Zhang Y, Zhao X. STING-mediated inflammation in Kupffer cells contributes to progression of nonalcoholic steatohepatitis. J Clin Invest 129 , 546-555 (2019). Oses M , et al. Peripheral blood mononuclear cells-expressed miRNA profiles derived from children with metabolic-associated fatty liver disease and insulin resistance. Pediatr Obes 17 , e12966 (2022). Stanford KI , et al. Paternal Exercise Improves Glucose Metabolism in Adult Offspring. Diabetes 67 , 2530-2540 (2018). Silva L, Pinheiro-Castro N, Novaes GM, Pascoal GFL, Ong TP. Bioactive food compounds, epigenetics and chronic disease prevention: Focus on early-life interventions with polyphenols. Food Res Int 125 , 108646 (2019). Pataia V , et al. Paternal cholestasis exacerbates obesity-associated hypertension in male offspring but is prevented by paternal ursodeoxycholic acid treatment. Int J Obes (Lond) 43 , 319-330 (2019). al'Absi M, Lovallo WR, McKey B, Sung BH, Whitsett TL, Wilson MF. Hypothalamic-pituitary-adrenocortical responses to psychological stress and caffeine in men at high and low risk for hypertension. Psychosom Med 60 , 521-527 (1998). Soldin OP, Makambi KH, Soldin SJ, O'Mara DM. Steroid hormone levels associated with passive and active smoking. Steroids 76 , 653-659 (2011). Gianoulakis C, Dai X, Brown T. Effect of chronic alcohol consumption on the activity of the hypothalamic-pituitary-adrenal axis and pituitary beta-endorphin as a function of alcohol intake, age, and gender. Alcohol Clin Exp Res 27 , 410-423 (2003). Yokota Y, Yokota H, Yokota M, Araki Y, Araki Y. In vitro fertilization embryo development from caffeine-treated murine sperm. Reprod Med Biol 14 , 117-121 (2015). Zhang W, Yang J, Lv Y, Li S, Qiang M. Paternal benzo[a]pyrene exposure alters the sperm DNA methylation levels of imprinting genes in F0 generation mice and their unexposed F1-2 male offspring. Chemosphere 228 , 586-594 (2019). Carone BR , et al. Paternally induced transgenerational environmental reprogramming of metabolic gene expression in mammals. Cell 143 , 1084-1096 (2010). Soubry A , et al. Obesity-related DNA methylation at imprinted genes in human sperm: Results from the TIEGER study. Clin Epigenetics 8 , 51 (2016). Hu S , et al. Prenatal caffeine exposure increases the susceptibility to non-alcoholic fatty liver disease in female offspring rats via activation of GR-C/EBPalpha-SIRT1 pathway. Toxicology 417 , 23-34 (2019). Wang L , et al. Intrauterine metabolic programming alteration increased susceptibility to non-alcoholic adult fatty liver disease in prenatal caffeine-exposed rat offspring. Toxicol Lett 224 , 311-318 (2014). Hu S , et al. Caffeine programs hepatic SIRT1-related cholesterol synthesis and hypercholesterolemia via A2AR/cAMP/PKA pathway in adult male offspring rats. Toxicology 418 , 11-21 (2019). Hong S, Zheng G, Wu X, Snider NT, Owyang C, Wiley JW. Corticosterone mediates reciprocal changes in CB 1 and TRPV1 receptors in primary sensory neurons in the chronically stressed rat. Gastroenterology 140 , 627-637 e624 (2011). Dalsgaard T , et al. Improved Lentiviral Gene Delivery to Mouse Liver by Hydrodynamic Vector Injection through Tail Vein. Mol Ther-Nucl Acids 12 , 672-683 (2018). Jia YP , et al. Melatonin supplementation in the culture medium rescues impaired glucose metabolism in IVF mice offspring. Journal of Pineal Research 72 , e12778 (2022). Li L , et al. miR-148a/LDLR mediates hypercholesterolemia induced by prenatal dexamethasone exposure in male offspring rats. Toxicol Appl Pharm 395 , 114979 (2020). Kumar A, Kumar V, Rattan V, Jha V, Pal A, Bhattacharyya S. Molecular spectrum of secretome regulates the relative hepatogenic potential of mesenchymal stem cells from bone marrow and dental tissue. Sci Rep-Uk 7 , 15015 (2017). Rattan S , et al. Prenatal and ancestral exposure to di(2-ethylhexyl) phthalate alters gene expression and DNA methylation in mouse ovaries. Toxicol Appl Pharm 379 , 114629 (2019). Kleiner DE , et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 41 , 1313-1321 (2005). Du YZ , et al. Pirfenidone alleviates lipopolysaccharide-induced lung injury by accentuating BAP31 regulation of ER stress and mitochondrial injury. J Autoimmun 112 , 102464 (2020). Fitz NF , et al. Trem2 deficiency differentially affects phenotype and transcriptome of human APOE3 and APOE4 mice. Molecular Neurodegeneration 15 , 1-21 (2020). Table Table 1. Similarities and differences between paternal pre-pregnancy caffeine exposure (PPCE) and prenatal caffeine exposure (PCE) rat models Indicators PPCE PCE Male Female Male Female Caffeine dose 15~60 mg/kg/d 30~120 mg/kg/d Gender with severe disease phenomena Male > Female Male < Female Whether a second strike is needed N N Y Y Lipogenesis ( Srebp1, Fasn, Acc ) ­­↑↑ ­↑ - ­↑ Fatty acid β-oxidation ( Pparα, Cpt1α ) ↓↓ ↓ ↓ - Inflammation ( Tnf-α, Il-6, Il-1β ) ­↑ - - - Fibrosis Y N N N Note: N, no; Y, yes; ↑­, increase; ↓, reduce; -, no change or not detected Additional Declarations There is NO Competing Interest. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3833743","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":266305833,"identity":"49f73bc8-8744-40ea-8f9c-95e7e0089d0b","order_by":0,"name":"Hui Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIiWNgGAWjYJACZgYGCR5+9gY2EIexgVgtMpI9B0jTwmBjcCOBSC0Gx88efl3YZsFjcPON2WMeBhvZDQeYnz3Aq+VMXpr1zDYJHsnbOebGPAxpxhsOsJkb4NNidiDHzJgXqIXvdu42aR6Gw4kbDvCwSeDVcv4NRAvDzbMgLf+J0HIjx/gxSIvADV6QlgOEtdjfeGPGzHMO6Jee/G+ScwySjWceZjPDq0WyP8f4M09ZnT0/+7E0iTcVdrJ9x5uf4dUCBMjOAAUVMwH1ICUfCKsZBaNgFIyCEQ0AaBtDeQ9QuvYAAAAASUVORK5CYII=","orcid":"","institution":"School of Basic Medical Sciences, Wuhan University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Wang","suffix":""},{"id":266305834,"identity":"55b50178-c94c-472c-8231-b53c9d048333","order_by":1,"name":"Cong Zhang","email":"","orcid":"","institution":"School of Basic Medical Sciences, Wuhan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Cong","middleName":"","lastName":"Zhang","suffix":""},{"id":266305835,"identity":"f77f40fd-573a-46c2-9af3-87d737a6c29d","order_by":2,"name":"Yu Guo","email":"","orcid":"","institution":"School of Basic Medical Sciences, Wuhan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Guo","suffix":""},{"id":266305836,"identity":"24e5ce17-79d3-4b0d-8d1c-42b3d972bce2","order_by":3,"name":"Yi Liu","email":"","orcid":"","institution":"School of Basic Medical Sciences, Wuhan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Liu","suffix":""},{"id":266305837,"identity":"f5f84a29-377c-41a2-b948-00f55e779ee1","order_by":4,"name":"Kexin Liu","email":"","orcid":"","institution":"School of Basic Medical Sciences, Wuhan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kexin","middleName":"","lastName":"Liu","suffix":""},{"id":266305838,"identity":"6c55d2cf-2e4b-4929-94bb-4d3947e7ccb4","order_by":5,"name":"Wen Hu","email":"","orcid":"","institution":"Zhongnan Hospital of Wuhan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wen","middleName":"","lastName":"Hu","suffix":""}],"badges":[],"createdAt":"2024-01-04 06:05:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3833743/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3833743/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50755904,"identity":"45a0a914-3272-4050-a0e2-5690f0581109","added_by":"auto","created_at":"2024-02-06 19:17:06","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":22777553,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePPCE causes adult NASH in male offspring.\u003c/strong\u003e (A, B) Liver H\u0026amp;E and ORO staining at GD20 and PW12\u0026nbsp; (400×); (C, D) Liver steatosis score at GD20 and PW12; (E, F) Liver TG contents at GD20 and PW12; (G, H) Serum AST and ALT activities at PW32; (I) Liver appearance morphology at PW32; (J, K) Liver weight and ratio of liver weight to body weight at PW32; (L) Liver H\u0026amp;E, ORO, Masson staining, and Sirius red staining at PW32 (200×, 400×); (M-Q) NAS score and fibrosis score; (R)\u0026nbsp; Liver TG content at PW32. Mean ± S.E.M, \u003cem\u003en \u003c/em\u003e= 12. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs.\u003c/em\u003e CON. Green arrow: inflammatory infiltration. Black and red arrows: deposition of collagen fibers.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3833743/v1/0163e11ea72f5d54c65b7f2d.jpg"},{"id":50756036,"identity":"38d15b0f-3d15-43c5-a378-5a39602d5501","added_by":"auto","created_at":"2024-02-06 19:25:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3836173,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePPCE induces hepatic lipid metabolism disorders and chronic inflammatory activation in male offspring before and after birth. \u003c/strong\u003e(A) Volcanogram of differentially expressed genes at GD20; (B) KEGG analysis at GD20; (C) Differentially expressed lipid metabolic genes at GD20; (D) The mRNA expression of \u003cem\u003eSrebp1\u003c/em\u003e,\u003cem\u003e Fasn\u003c/em\u003e,\u003cem\u003e Acc\u003c/em\u003e,\u003cem\u003e Acly\u003c/em\u003e,\u003cem\u003e Pparα\u003c/em\u003e,\u003cem\u003e \u003c/em\u003eand\u003cem\u003e Cpt1α\u003c/em\u003e at GD20 (\u003cem\u003en = \u003c/em\u003e12); (E, F) Representative immunoblots and quantitative analysis of SREBP1, FASN, ACC, ACLY, PPARα, and CPT1α protein GD20 (\u003cem\u003en = \u003c/em\u003e5); (G) The mRNA expression of \u003cem\u003eSrebp1\u003c/em\u003e, \u003cem\u003eFasn\u003c/em\u003e, \u003cem\u003eAcc\u003c/em\u003e, \u003cem\u003eAcly\u003c/em\u003e, \u003cem\u003ePparα\u003c/em\u003e, and \u003cem\u003eCpt1α\u003c/em\u003e at PW32 (\u003cem\u003en = \u003c/em\u003e12); (H, I) Representative immunoblots and quantitative analysis of SREBP1, FASN, ACC, ACLY, PPARα, and CPT1α protein at PW32 (\u003cem\u003en = \u003c/em\u003e5); (J, K) Liver ROS levels at PW32; (L) TEM images at PW32; (M) mtDNA copy numbers at PW32; (N-P) Typical immunofluorescence images and statistical analysis of fluorescence intensity for F4/80 and STING at PW32; (Q, R) Representative immunoblots and quantitative analysis of cGAS, STING, and p-p65 protein at PW32 (\u003cem\u003en = \u003c/em\u003e5); (S, T) The mRNA expression and contents of TNF-α, IL-6, and IL-1β at PW32 (\u003cem\u003en=\u003c/em\u003e12). Mean ± S.E.M. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP \u0026lt; \u003c/em\u003e0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP \u0026lt; \u003c/em\u003e0.01 \u003cem\u003evs\u003c/em\u003e. CON group.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3833743/v1/38480d97a9c419c4f1de4758.png"},{"id":50755906,"identity":"0f7706f4-c7bd-4fe6-8c08-33c7f1294f77","added_by":"auto","created_at":"2024-02-06 19:17:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":16553655,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-142-3p is a critical target mediating PPCE-induced hepatic lipid metabolism disorders and chronic inflammation in male offspring. \u003c/strong\u003e(A) Heatmap of gene expression in parental sperms and fetal livers; (B) Venn diagram of miRNAs expression in parental sperms and fetal livers; (C) Liver miRNAs expression at GD20; (D) miR-142-3p intensity in fetal rat organs detected by FISH; (E) Liver miRNAs expression at PW32; (F, G) Liver miR-142-3p levels at GD20 and PW32; (H) Liver appearance morphology, H\u0026amp;E, ORO,\u0026nbsp; Masson, and Sirius red staining (200×, 400×); (I, J) NAS score and liver TG content of\u0026nbsp; miR-142-3p\u003csup\u003e-/-\u003c/sup\u003e mice (\u003cem\u003en \u003c/em\u003e= 8); (K) The mRNA expression of \u003cem\u003eSrebp1\u003c/em\u003e, \u003cem\u003eFasn\u003c/em\u003e, \u003cem\u003eAcc\u003c/em\u003e, \u003cem\u003ePparα\u003c/em\u003e, \u003cem\u003eCpt1α\u003c/em\u003e, \u003cem\u003eTnf-α, Il-6, \u003c/em\u003eand\u003cem\u003e Il-1β \u003c/em\u003ein liver of miR-142-3p\u003csup\u003e-/-\u003c/sup\u003e mice (\u003cem\u003en \u003c/em\u003e= 8); (L) The mRNA expression of\u003cem\u003e Srebp1\u003c/em\u003e, \u003cem\u003eFasn\u003c/em\u003e, \u003cem\u003eAcc\u003c/em\u003e, \u003cem\u003ePparα\u003c/em\u003e, and \u003cem\u003eCpt1α\u003c/em\u003e in liver at PW32 (\u003cem\u003en \u003c/em\u003e= 10); (M, N) The content and mRNA expression of TNF-α, IL-6, IL-1β in liver at PW32; (O) Liver H\u0026amp;E, ORO,\u0026nbsp; Masson, and Sirius red staining at PW32 (200×, 400×); (P, Q) NAS score and liver TG content at PW32 (\u003cem\u003en \u003c/em\u003e= 10). Mean ± S.E.M. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP \u0026lt; \u003c/em\u003e0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP \u0026lt; \u003c/em\u003e0.01 \u003cem\u003evs\u003c/em\u003e. CON group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP \u0026lt; \u003c/em\u003e0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP \u0026lt; \u003c/em\u003e0.01 \u003cem\u003evs\u003c/em\u003e. PPCE group. Green arrow: inflammatory infiltration. Black and red arrows: deposition of collagen fibers.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3833743/v1/2a785c71cfd472bdcc1daeb8.png"},{"id":50755898,"identity":"5b5e52c8-4e6a-4305-8841-c06f93097b54","added_by":"auto","created_at":"2024-02-06 19:17:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5902916,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-142-3p targets ACSL4 to regulate hepatic lipid metabolism and inflammatory responses in PPCE male offspring mice. \u003c/strong\u003e(A) miR-142-3p and ACSL4 interactions; (B) The mRNA expression of\u003cem\u003e Acsl4\u003c/em\u003e at GD20 (\u003cem\u003en \u003c/em\u003e= 12); (C, D) Representative immunoblots and quantitative analysis of ACSL4 at GD20 (\u003cem\u003en \u003c/em\u003e= 5); (E) The mRNA expression of \u003cem\u003eAcsl4\u003c/em\u003e at PW32; (F, G) Representative immunoblots and quantitative analysis of ACSL4 at PW32 (\u003cem\u003en \u003c/em\u003e= 5); (H-J) Representative immunofluorescent images and quantitative analysis of ACSL4 at GD20 and PW32 (\u003cem\u003en \u003c/em\u003e= 5); (K) The expression of \u003cem\u003eAcsl4\u003c/em\u003e mRNA in miR-142-3p\u003csup\u003e-/-\u003c/sup\u003e mice (\u003cem\u003en \u003c/em\u003e= 8); (L) The mRNA expression of \u003cem\u003eAcsl4\u003c/em\u003e at PW32 (\u003cem\u003en \u003c/em\u003e= 10); (M) The mRNA expression of \u003cem\u003eSrebp1\u003c/em\u003e,\u003cem\u003e Fasn\u003c/em\u003e,\u003cem\u003e Pparα\u003c/em\u003e,\u003cem\u003e \u003c/em\u003eand\u003cem\u003e Cpt1α\u003c/em\u003e in BMSCs liver-like differentiated cells after miR-142-3p inhibitor or miR-142-3p minics transfection (\u003cem\u003en \u003c/em\u003e= 6); (N, O) Representative immunoblots and quantitative analysis of SREBP1, FASN, PPARα, and CPT1α\u003cem\u003e \u003c/em\u003eprotein in BMSCs liver-like differentiated cells after miR-142-3p inhibitor or miR-142-3p mimics transfection (\u003cem\u003en \u003c/em\u003e= 5); (P) The mRNA expression of \u003cem\u003eSrebp1\u003c/em\u003e,\u003cem\u003e Fasn\u003c/em\u003e,\u003cem\u003e Pparα\u003c/em\u003e,\u003cem\u003e \u003c/em\u003eand\u003cem\u003e Cpt1α\u003c/em\u003e in BMSCs liver-like differentiated cells after miR-142-3p mimics and/or ACSL4 transfection (\u003cem\u003en \u003c/em\u003e= 6); (Q, R) Representative immunoblots and quantitative analysis of SREBP1, FASN, PPARα, and CPT1α\u003cem\u003e \u003c/em\u003eprotein in BMSCs liver-like differentiated cells after miR-142-3p mimics and/or ACSL4 transfection; (S) Representative immunofluorescence images of SREBP1, FASN, PPARα, CPT1α in BMSCs liver-like differentiated cells (\u003cem\u003en \u003c/em\u003e= 5); (T) Dual luciferase reporter gene validation of miR-142-3p target gene (\u003cem\u003en \u003c/em\u003e= 6). Mean ± S.E.M. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP \u0026lt; \u003c/em\u003e0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP \u0026lt; \u003c/em\u003e0.01 \u003cem\u003evs\u003c/em\u003e. CON group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP \u0026lt; \u003c/em\u003e0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP \u0026lt; \u003c/em\u003e0.01 \u003cem\u003evs\u003c/em\u003e. PPCE group.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3833743/v1/fc360f3fde4d7ee0f19583fb.png"},{"id":50755903,"identity":"102e57f6-e090-4d0e-9b66-bb0b162da5a2","added_by":"auto","created_at":"2024-02-06 19:17:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":14248832,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHigh glucocorticoid-induced sperm miR-142-3p methylation reprogramming mediates hepatic lipid metabolism dysfunction in PPCE male offspring. \u003c/strong\u003e(A) Paternal serum CORT content (\u003cem\u003en \u003c/em\u003e= 10); (B) The mRNA expression of GR in paternal testis (\u003cem\u003en \u003c/em\u003e= 10); (C) Methylation levels of miR-142-3p in paternal sperms; (D, E) The mRNA expression of miR-142-3p in spermatogonia after caffeine or CORT treated (\u003cem\u003en \u003c/em\u003e= 6); (F, G) Methylation levels of miR-142-3p in CORT treated spermatogonia (\u003cem\u003en \u003c/em\u003e= 6); (H, I) Methylation levels of miR-142-3p in liver at GD20 (\u003cem\u003en \u003c/em\u003e= 3); (J, K) The mRNA expression of miR-142-3p, \u003cem\u003eAcsl4\u003c/em\u003e, \u003cem\u003eSrebp1\u003c/em\u003e, \u003cem\u003eFasn\u003c/em\u003e, \u003cem\u003ePparα\u003c/em\u003e, \u003cem\u003eCpt1α\u003c/em\u003e in liver at GD20 (\u003cem\u003en \u003c/em\u003e= 10); (L, M) The mRNA expression of miR-142-3p, \u003cem\u003eAcsl4\u003c/em\u003e, \u003cem\u003eSrebp1\u003c/em\u003e, \u003cem\u003eFasn\u003c/em\u003e, \u003cem\u003ePparα\u003c/em\u003e, \u003cem\u003eCpt1α\u003c/em\u003e in liver at PW32; (N) Liver H\u0026amp;E and ORO staining at GD20 and PW32\u0026nbsp; (400×, \u003cem\u003en \u003c/em\u003e= 10); (O, P) Liver TG content in GD20 and PW32 (\u003cem\u003en \u003c/em\u003e= 10). Mean ± S.E.M. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP \u0026lt; \u003c/em\u003e0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP \u0026lt; \u003c/em\u003e0.01 \u003cem\u003evs.\u003c/em\u003e CON group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP \u0026lt; \u003c/em\u003e0.05 \u003cem\u003evs.\u003c/em\u003e PPCE group.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3833743/v1/e43849f72c975764b20d37c7.png"},{"id":50755899,"identity":"5362ca4b-18c7-4726-a470-f661321efb53","added_by":"auto","created_at":"2024-02-06 19:17:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":7401737,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vitro \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003efertilization confirms that low expression of miR-142-3p mediates liver lipid metabolic changes in PPCE male offspring. \u003c/strong\u003e(A) Study design of \u003cem\u003ein vitro\u003c/em\u003e fertilization in sperm of PPCE mice; (B) The mRNA expression of miR-142-3p in GD19 offspring of PPCE mice; (C) The mRNA expression of \u003cem\u003eSrebp1\u003c/em\u003e, \u003cem\u003eFasn\u003c/em\u003e, \u003cem\u003eAcc\u003c/em\u003e, \u003cem\u003ePparα\u003c/em\u003e, \u003cem\u003eCpt1α\u003c/em\u003e in GD19 offspring of PPCE mice; (D) H\u0026amp;E and ORO staining in GD19 offspring of PPCE mice\u0026nbsp; (400×); (E) Liver TG content in GD19 offspring of PPCE mice; (F) Study design of \u003cem\u003ein vitro\u003c/em\u003e fertilization in sperm of miR-142-3p\u003csup\u003eKO\u003c/sup\u003e mice; (G) The mRNA expression of \u003cem\u003eSrebp1\u003c/em\u003e, \u003cem\u003eFasn\u003c/em\u003e, \u003cem\u003eAcc\u003c/em\u003e, \u003cem\u003ePparα\u003c/em\u003e, \u003cem\u003eCpt1α\u003c/em\u003e in GD19 offspring of miR-142-3p\u003csup\u003eKO\u003c/sup\u003e mice, (H) H\u0026amp;E and ORO staining in GD19 offspring of miR-142-3p\u003csup\u003eKO\u003c/sup\u003e mice (400×); (I) liver TG content in GD19 offspring of miR-142-3p\u003csup\u003eKO\u003c/sup\u003e mice; (J) The mRNA expression of \u003cem\u003eSrebp1\u003c/em\u003e, \u003cem\u003eFasn\u003c/em\u003e, \u003cem\u003eAcc\u003c/em\u003e, \u003cem\u003ePparα\u003c/em\u003e, \u003cem\u003eCpt1α\u003c/em\u003e in PW12 offspring of miR-142-3p\u003csup\u003eKO\u003c/sup\u003e mice; (K) H\u0026amp;E and ORO staining in PW12 offspring of miR-142-3p\u003csup\u003eKO\u003c/sup\u003e mice (400×); (L) Liver TG content in PW12 offspring of miR-142-3p\u003csup\u003eKO\u003c/sup\u003e mice. Mean ± S.E.M, \u003cem\u003en \u003c/em\u003e= 6, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP \u0026lt; \u003c/em\u003e0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP \u0026lt; \u003c/em\u003e0.01 \u003cem\u003evs\u003c/em\u003e. CON.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3833743/v1/4ec8a1d9e09b66c8f42bb24a.png"},{"id":50755900,"identity":"ab203dcb-6621-49e4-8b43-80a6662c335e","added_by":"auto","created_at":"2024-02-06 19:17:05","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":7817964,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePPCE-induced NASH occurrence in male offspring with transgenerational inheritance. \u003c/strong\u003e(A) H\u0026amp;E and ORO staining at PW12 (400×); (B) Liver TG content at PW12 (\u003cem\u003en \u003c/em\u003e= 10) ; (C) The mRNA expression of miR-142-3p at PW12 (\u003cem\u003en \u003c/em\u003e= 10); (D) The mRNA expression of \u003cem\u003eAcsl4\u003c/em\u003e, \u003cem\u003eSrebp1\u003c/em\u003e, \u003cem\u003eFasn\u003c/em\u003e, \u003cem\u003ePparα\u003c/em\u003e, and \u003cem\u003eCpt1α\u003c/em\u003e at PW12 (\u003cem\u003en \u003c/em\u003e= 10); (E) H\u0026amp;E, ORO, Masson, and sirius red staining at PW34 (400×); (F) Liver TG content at PW34 (\u003cem\u003en \u003c/em\u003e= 10); (G) The mRNA expression of miR-142-3p at PW34 (\u003cem\u003en \u003c/em\u003e= 10); (H) The mRNA expression of \u003cem\u003eAcsl4\u003c/em\u003e, \u003cem\u003eSrebp1\u003c/em\u003e, \u003cem\u003eFasn\u003c/em\u003e, \u003cem\u003ePparα\u003c/em\u003e, and \u003cem\u003eCpt1α\u003c/em\u003e at PW34 (\u003cem\u003en \u003c/em\u003e= 10); (I, J) Representative immunoblots and quantitative analysis of ACSL4, SREBP1, FASN, PPARα, and CPT1α protein at PW34 (\u003cem\u003en \u003c/em\u003e= 5); (K-M) The contents of TNF-α, IL-6 and IL-1β at PW34 (\u003cem\u003en\u003c/em\u003e=10); (N-P) The mRNA expression of \u003cem\u003eTnf-α\u003c/em\u003e, \u003cem\u003eIl-6\u003c/em\u003e and \u003cem\u003eIl-1β\u003c/em\u003e at PW34 (\u003cem\u003en \u003c/em\u003e= 10). Mean ± S.E.M. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP \u0026lt; \u003c/em\u003e0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP \u0026lt; \u003c/em\u003e0.01 \u003cem\u003evs\u003c/em\u003e. CON.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-3833743/v1/4aa76f3c5a79c331905d72d1.png"},{"id":50756408,"identity":"236eef67-5d9f-46e1-b7ef-24100978c752","added_by":"auto","created_at":"2024-02-06 19:33:05","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":4502441,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram of miR-142-3p low expression-mediated hepatic lipid dysregulation in male offspring of paternal pre-pregnant caffeine exposure.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-3833743/v1/6ae4bb6aee2858c29de59a99.png"},{"id":50756740,"identity":"8e26b537-770b-49b6-9dc8-8f31847e8230","added_by":"auto","created_at":"2024-02-06 19:41:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6733189,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3833743/v1/bf4f7fdb-6133-47ac-afd3-ab0366ef7d74.pdf"},{"id":50755905,"identity":"c828a463-2989-4c00-ad9e-bb000f673179","added_by":"auto","created_at":"2024-02-06 19:17:06","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2854995,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary materials\u003c/p\u003e","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-3833743/v1/8ee2ecd0773b0454214cac4d.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Sperm miR-142-3p reprogramming mediates paternal stress-induced non-alcoholic steatohepatitis in offspring rats","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eNon-alcoholic fatty liver disease (NAFLD) has become the most common chronic liver metabolic disease in the world, with a spectrum of simple steatosis, nonalcoholic steatohepatitis (NASH), cirrhosis, and even hepatocellular carcinoma\u003csup\u003e1\u003c/sup\u003e. NASH is a severe form of NAFLD progression and its current understanding of pathogenesis reported up to now has mostly involved the elaborate reciprocation among genetic susceptibility, environmental factors, and metabolic stress\u003csup\u003e2\u003c/sup\u003e. The theory of “Developmental Origins of Health and Disease (DOHaD)\" holds that many adult metabolic diseases have fetal origins\u003csup\u003e3\u003c/sup\u003e, whereby the impact of adverse environmental factors during fetal development can cause metabolic adaptations and developmental programming changes in the offspring, resulting in susceptibility to a variety of metabolic diseases (such as NAFLD and diabetes) after birth\u003csup\u003e4, 5, 6\u003c/sup\u003e. However, gametes (sperm and oocytes) take longer to develop and mature than embryos (fetuses) and are thereby more vulnerable to adverse environmental exposures\u003csup\u003e7\u003c/sup\u003e. In recent years, with the proposition of the “Paternal Origins of Health and Disease (POHaD), substantial studies confirmed paternal adverse lifestyle and environmental exposures as important independent risk factors for the increased incidence of abnormal development and developmental diseases in the offspring\u003csup\u003e8, 9\u003c/sup\u003e. Epidemiological surveys showed that men with experience of famine have a significantly higher morbidity of obesity and diabetes in their offspring\u003csup\u003e10\u003c/sup\u003e. Clinical and laboratory studies have also found that parental pre-pregnancy adverse environmental exposures (such as smoking, high-fat diet, exogenous exposure, chronic stress, etc.) can cause intrauterine growth retardation (IUGR) and postnatal lipid metabolism dysfunction in the offspring\u003csup\u003e11, 12, 13, 14\u003c/sup\u003e. All these studies indicate that paternal adverse pre-pregnancy environmental exposure is an important contributor leading to multi-disease susceptibility (such as NAFLD) in the offspring.\u003c/p\u003e\u003cp\u003eChronic stress refers to long-term exposure to adverse events (such as poor lifestyles, exogenous exposure, psychological stress, etc.), closely related to human health\u003csup\u003e15\u003c/sup\u003e. Studies have shown that paternal chronic stress can lead to poor pregnancy outcomes (such as IUGR) in the offspring and multi-disease susceptibility after birth\u003csup\u003e16, 17\u003c/sup\u003e. Caffeine is a central stimulant as well as a common chronic stressor widely found in coffee, tea, soft drinks, and some compounded drugs in daily life\u003csup\u003e18, 19\u003c/sup\u003e, and has been demonstrated to have reproductive and developmental toxicity\u003csup\u003e20\u003c/sup\u003e. Our previous series of studies confirmed that maternal caffeine exposure during pregnancy could assign the adverse impact on the long-term health of the offspring\u003csup\u003e21, 22, 23, 24\u003c/sup\u003e. However, the increasing global average caffeine intake, especially among men of reproductive age (240 mg/d), surpasses that in women\u003csup\u003e25, 26\u003c/sup\u003e, emphasizing the need for heightened awareness regarding the potential risks associated with pre-pregnancy caffeine intake in men and its impact on the health of future generations. As the most perceptive hormone in stress response, glucocorticoid is the key to germ cell formation, embryo (fetus) development and its fate after birth\u003csup\u003e27\u003c/sup\u003e. Studies have found that caffeine intake can significantly increase serum glucocorticoid levels\u003csup\u003e28\u003c/sup\u003e, whereas paternal pre-pregnancy caffeine intake can affect the early brain development of offspring and is associated with glucocorticoid receptor (GR) activation\u003csup\u003e29\u003c/sup\u003e. These studies suggest that serum-high glucocorticoids may be involved in the effects of pre-pregnancy caffeine exposure on the long-term health of the offspring.\u003c/p\u003e\u003cp\u003eEpigenetic modification (such as DNA methylation and non-coding RNA) plays a key role in sperm-mediated inheritance of acquired traits\u003csup\u003e30\u003c/sup\u003e. It has been shown that epigenetic changes caused by paternal adverse pre-pregnancy environmental exposure can transmit these “marks” to the offspring through sperm reprogramming changes, resulting in developmental programming, even over multiple generations\u003csup\u003e31\u003c/sup\u003e. MicroRNAs (miRNAs) are important components of non-coding RNAs that can participate in a variety of cellular activities by modulating multiple gene expressions and protein translation, and they are strongly associated with gametogenesis and embryonic (fetal) development\u003csup\u003e32, 33\u003c/sup\u003e. Clinical and experimental studies have shown that sperm miRNAs, important marker and regulators of epigenetic modifications, can carry paternal epigenetic information and transmit it to the offspring to affect the long-term health\u003csup\u003e34, 35\u003c/sup\u003e. Poor paternal pre-pregnancy lifestyle (such as a high-fat diet) and chronic stress can alter multiple sperm miRNAs, influencing offspring and resulting in altered hepatic lipid metabolism\u003csup\u003e16, 36, 37\u003c/sup\u003e. Meanwhile, elevated serum glucocorticoid under paternal chronic stress were associated with changed sperm miRNAs expression\u003csup\u003e16, 38\u003c/sup\u003e. These results suggest that paternal serum high glucocorticoids may disrupt hepatic lipid metabolic homeostasis in offspring by altering miRNAs expression in sperm.\u003c/p\u003e\u003cp\u003eIn this study, a paternal pre-pregnant caffeine exposure (PPCE) rat model was established to simulate daily caffeine intake in the population, so as to reveal that PPCE causes alterations in sperm epigenetic programming through high glucocorticoid exposure in the parental generation, thus leading to hepatic lipid metabolism dysfunction in the offspring and the occurrence of adult NASH. This study aims to clarify the developmental origins of paternal NASH, confirm intervention targets, and provide theoretical and practical significance for early prevention and treatment strategies, guiding healthy lifestyles in men of reproductive age.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cp\u003e\u003cem\u003e2.1\u003c/em\u003e\u003cem\u003e. PPCE causes adult NASH in male offspring\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo confirm that paternal caffeine intake can cause the occurrence of NASH in adult offspring, we established a PPCE rat model. First, we observed the effects of PPCE on liver pathology, NAFLD activity score (NAS), and triglyceride metabolism in male offspring before and after birth. H\u0026amp;E and ORO staining results showed that compared to the respective CON groups, there were significant manifestations of fat vacuole-like degeneration and lipid accumulation in the liver pathology of the PPCE group at both GD20 and PW12 (Fig. 1A, B), as well as significantly higher liver steatosis scores (Fig. 1C, D) and triglyceride content (Fig. 1E, F). At PW32, the activities of serum liver function enzymes (AST and ALT) were significantly increased in the PPCE group (Fig. 1G, H), liver appearance was light brown, and liver weight and liver index were significantly increased (Fig. 1I-K). H\u0026amp;E, Masson, and Sirius red staining showed that the liver of PPCE male offspring at PW32 exhibited typical NASH histological features, manifesting as significant vacuolar-like steatosis, inflammatory infiltration (green arrows), and collagen fiber deposition (black and red arrows) (Fig. 1L). Meanwhile, liver NAS and triglyceride content were significantly increased (Fig. 1M-R). It is suggested that PPCE can lead to hepatic lipid accumulation in male offspring and the occurrence of NASH in adulthood.\u003c/p\u003e\n\u003cp\u003eIn this study, we also found that although PPCE could cause the occurrence of NAFLD in female offspring in adulthood, the liver pathological changes were milder than that of males, and no obvious inflammatory infiltration and fibrosis were observed (Fig. S1). In summary, PPCE can cause the occurrence of adult NAFLD/NASH in the offspring, and there are significant gender differences, especially evident in males. Therefore, this study subsequently focused on the programming mechanisms by which PPCE led to the occurrence of NASH in male offspring.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.2. PPCE induces hepatic lipid metabolism disorders and chronic inflammatory activation in male offspring\u003c/em\u003e \u003cem\u003ebefore and after birth\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe further examined the changes in hepatic lipid metabolism and inflammatory response in PPCE male offspring rats before and after birth. First, sequencing analysis results suggested that the expression of multiple genes was changed in the liver of fetal rats in the PPCE group compared with the CON group (Fig. 2A). KEGG functional enrichment analysis revealed that most differentially expressed genes in the liver of fetal rats in the PPCE group were closely associated with lipid metabolic signaling during the development of NAFLD (Fig. 2B), specifically manifested as that the expression of lipid synthesis-related genes was elevated while that of \u0026beta;-oxidation-related genes was decreased (Fig. 2C). The key factors of hepatic lipid synthesis and \u0026beta;-oxidation were further detected using RT-qPCR and Western blot for expression. The results showed that compared with the CON group, the mRNA and protein expression of key transcription factors and functional enzymes (such as SREBP1, FASN, ACC, and ACLY) of hepatic lipogenesis were significantly increased in PPCE group at GD20 and PW32, while the mRNA and protein expression of key factors of fatty acid \u0026beta;-oxidation (such as PPAR\u0026alpha; and CPT1\u0026alpha;) were significantly decreased (Fig. 2D-I). It was suggested that PPCE could enhance hepatic fatty acid synthesis and weaken \u0026beta;-oxidation in male offspring rats before and after birth. Studies have suggested that lipotoxicity from excessive lipid accumulation in hepatocytes could damage mitochondria and produce excessive reactive oxygen species (ROS), thus promoting inflammatory response and NAFLD progression\u003csup\u003e39\u003c/sup\u003e. Therefore, we also measured the changes in mitochondrial function and inflammatory response in hepatocytes at PW32. The results showed that hepatic ROS levels were significantly increased in the PPCE group (Fig. 2J, K), mitochondrial damage in hepatocytes was severe (mitochondria swelling, rupture, and loss of mitochondrial cristae) (Fig. 2L), and mtDNA copy number was significantly increased (Fig. 2M), indicating that PPCE could cause mitochondrial damage and oxidative stress in hepatocytes of male offspring. Since the release of relevant molecular patterns such as mtDNA from this oxidative stress injury in hepatocytes is capable to activate STING-mediated inflammatory responses\u003csup\u003e40\u003c/sup\u003e. Thus, we examined the changes in the expression of STING-NF-\u0026kappa;B inflammatory signaling in the liver. The results showed that macrophages in PPCE group were activated and STING expression in macrophages was significantly increased (Fig. 2N-P). Meanwhile, the protein expressions of cGAS, STING and p-NF-\u0026kappa;B p65 (p-p65) and the mRNA expression and content of pro-inflammatory factors (TNF-\u0026alpha;, IL\u0026ndash;6 and IL\u0026ndash;1\u0026beta;) in the liver were significantly increased (Fig. 2Q-T). STING-related inflammatory activation was also observed in the fetal liver of PPCE male offspring (Fig. S2). These results indicated that PPCE can cause hepatic lipid metabolism dysfunction, lipid accumulation and chronic inflammatory in male offspring before and after birth.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.3. miR\u0026ndash;142\u0026ndash;3p is a critical target mediating PPCE-induced hepatic lipid metabolism disorders and chronic inflammation in male offspring\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eSperm miRNAs play a key role in the inheritance of acquired traits as a carrier of paternal genetic information and one of the important markers of epigenetic modifications\u003csup\u003e41\u003c/sup\u003e. Therefore, we first examined the changes in liver miRNAs expression profiles of paternal sperm and male offspring rats before and after birth to select co-differentially expressed miRNAs as potential toxicity targets. Sequencing analysis results showed that compared with the CON group, the expression of multiple miRNAs in paternal sperm and fetal liver of offspring in the PPCE group was changed (Fig. 3A), among which 10 kinds of miRNAs were co-differentially expressed (Fig. 3B), especially miR\u0026ndash;142\u0026ndash;3p was most significantly changed (Fig. 3C). The results of fluorescence in situ hybridization experiments showed that the expression of miR\u0026ndash;142\u0026ndash;3p in fetal liver was much higher than that in other organs, showing good organ specificity (Fig. 3D). Meanwhile, sequencing analysis suggested that the differential change of miR\u0026ndash;142\u0026ndash;3p was most significant in the liver of the PPCE group at PW32 (Fig. 3E). RT-qPCR results showed that hepatic miR\u0026ndash;142\u0026ndash;3p expression was significantly reduced in the PPCE group at GD20 and PW32 (Fig. 3F, G). These results suggest that PPCE reduce miR\u0026ndash;142\u0026ndash;3p expression in paternal sperm that can continue to the liver of male offspring, resulting in decreased hepatic miR\u0026ndash;142\u0026ndash;3p expression before and after birth.\u003c/p\u003e\n\u003cp\u003eNext, to confirm the regulatory effects of miR\u0026ndash;142\u0026ndash;3p on hepatic lipid metabolism and inflammatory responses, we used hydrodynamic injection to establish a liver-specific miR\u0026ndash;142\u0026ndash;3p silencing (miR\u0026ndash;142\u0026ndash;3p\u003csup\u003e-/-\u003c/sup\u003e) mouse model and observed after 8W. The results showed that compared with the CON group, the liver of the miR\u0026ndash;142\u0026ndash;3p\u003csup\u003e-/-\u003c/sup\u003e group was pale yellow in appearance and showed severe histological macro-vesicular-like steatosis with significant lipid accumulation, inflammatory infiltration (green arrows) and collagen fiber deposition (black and red arrows) (Fig. 3H), and the liver NAS and triglyceride content were significantly elevated (Fig. 3I, J). RT-qPCR results showed that the expressions of hepatic lipogenesis genes (\u003cem\u003eSrebp1,\u003c/em\u003e \u003cem\u003eFasn,\u003c/em\u003e and \u003cem\u003eAcc)\u003c/em\u003e and pro-inflammatory factor-related genes (\u003cem\u003eTnf-\u0026alpha;,\u003c/em\u003e \u003cem\u003eIl\u0026ndash;6,\u003c/em\u003e and \u003cem\u003eIl\u0026ndash;1\u0026beta;)\u003c/em\u003e were significantly increased in the miR\u0026ndash;142\u0026ndash;3p\u003csup\u003e-/-\u003c/sup\u003e group (Fig. 3K), whereas the expressions of \u003cem\u003ePpar\u0026alpha;\u003c/em\u003e and \u003cem\u003eCpt1\u0026alpha;\u003c/em\u003e were significantly decreased (Fig. 3K). Meanwhile, miR\u0026ndash;142\u0026ndash;3p\u003csup\u003e-/-\u003c/sup\u003e was able to promote hepatic STING-related inflammatory signaling activation (Fig. S3). These results reveal that miR\u0026ndash;142\u0026ndash;3p\u003csup\u003e-/-\u003c/sup\u003e could induce hepatic lipid metabolism dysfunction and inflammatory activation in mice, ultimately leading to the occurrence of NASH.\u003c/p\u003e\n\u003cp\u003eTo further validate that miR\u0026ndash;142\u0026ndash;3p low expression programming mediated alterations in hepatic lipid metabolism in PPCE male offspring, we performed reverse intervention by hydrodynamic injection of liver-targeted AAV8-miR\u0026ndash;142\u0026ndash;3p (miR\u0026ndash;142\u0026ndash;3p) overexpressing adeno-associated virus through the tail vein at PW8. The results showed that the liver lipogenesis genes (\u003cem\u003eSrebp1,\u003c/em\u003e \u003cem\u003eFasn,\u003c/em\u003e and \u003cem\u003eAcc)\u003c/em\u003e expression was significantly decreased, \u0026beta;-oxidation genes (\u003cem\u003ePpar\u0026alpha;\u003c/em\u003e and \u003cem\u003eCpt1\u0026alpha;)\u003c/em\u003e expression was remarkably increased in miR\u0026ndash;142\u0026ndash;3p overexpressed PPCE offspring at PW32 (Fig. 3L), and the content and mRNA expression of liver pro-inflammatory factors (TNF-\u0026alpha;, IL\u0026ndash;6, and IL\u0026ndash;1\u0026beta;) were significantly decreased (Fig. 3M, N). Histological results demonstrated that no obvious steatosis, lipid accumulation, and inflammatory infiltration were observed in the liver of miR\u0026ndash;142\u0026ndash;3p overexpressed PPCE offspring (Fig. 3O), and both liver NAS and triglyceride content were significantly reduced (Fig. 3P, Q). These results indicate that overexpression of miR\u0026ndash;142\u0026ndash;3p can reverse hepatic lipid metabolism dysfunction and inflammatory response in PPCE male offspring rats and inhibit adult NASH occurrence.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.4.\u003c/em\u003e\u003cem\u003e miR\u0026ndash;142\u0026ndash;3p targets ACSL4 to regulate hepatic lipid metabolism\u003c/em\u003e\u003cem\u003eand inflammatory responses\u003c/em\u003e\u003cem\u003e in PPCE male offspring mice\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIt\u0026rsquo;s known that miRNAs can specifically bind to target gene mRNAs to regulate the expression of target genes and play important roles in a variety of life activities such as embryonic development, cellular metabolism, and signal transduction\u003csup\u003e42\u003c/sup\u003e. To elucidate the molecular mechanism by which miR\u0026ndash;142\u0026ndash;3p low expression mediates lipid metabolism disorder in the liver of PPCE male offspring rats, we first predicted the target genes of miR\u0026ndash;142\u0026ndash;3p using target databases (\u003cem\u003eTargetScan,\u003c/em\u003e \u003cem\u003emiRDB,\u003c/em\u003e and \u003cem\u003emiRcode).\u003c/em\u003e. The results showed that long-chain acyl-CoA synthetase 4 (ACSL4) had an interaction with miR\u0026ndash;142\u0026ndash;3p (Fig. 4A). Subsequently, we examined the changes in hepatic ACSL4 expression before and after birth. RT-qPCR and Western blot results showed that compared with the CON group, both mRNA and protein expression of liver ACSL4 were significantly increased in PPCE group at GD20 and PW32 (Figu. 4B-E). Immunofluorescence staining results also demonstrated that hepatic ACSL4 protein expression was significantly increased in PPCE group before and after birth (Fig. 4H-J). Meanwhile, the expression of \u003cem\u003eAcsl4\u003c/em\u003e mRNA in the liver of miR\u0026ndash;142\u0026ndash;3p\u003csup\u003e-/-\u003c/sup\u003e mice was significantly elevated (Fig. 4K), while miR\u0026ndash;142\u0026ndash;3p overexpression markedly reduced the expression of \u003cem\u003eAcsl4 \u003c/em\u003emRNA in the liver of PPCE male offspring (Fig. 4L). These results suggest that miR\u0026ndash;142\u0026ndash;3p may mediate changes in hepatic lipid metabolism in PPCE male offspring by targeting ACSL4.\u003c/p\u003e\n\u003cp\u003eFurther, we confirmed the molecular mechanism by which miR\u0026ndash;142\u0026ndash;3p low expression mediated lipid metabolism dysfunction in the liver of PPCE male offspring using BMSCs hepatoid differentiated cells. First, miR\u0026ndash;142\u0026ndash;3p inhibitor and miR\u0026ndash;142\u0026ndash;3p mimics transfection were used to confirm the regulatory effect of miR\u0026ndash;142\u0026ndash;3p on lipid metabolism function in hepatocytes. The results showed that in comparison with the CON group, miR\u0026ndash;142\u0026ndash;3p inhibitor significantly increased the expression of lipogenesis genes (SREBP1 and FASN) and decreased the expression of \u0026beta; oxidation-related genes (PPAR\u0026alpha; and CPT1\u0026alpha;) (Fig. 4M-O), while miR\u0026ndash;142\u0026ndash;3p mimics produced the opposite effect. Further, we confirmed that miR\u0026ndash;142\u0026ndash;3p exerted its regulatory effect on lipid metabolism by targeting ACSL4. The results showed that overexpression of ACSL4 could significantly increase the expression of the above lipid synthesis genes and decrease the expression of \u0026beta;-oxidation-related genes (Fig. 4P-R), while miR\u0026ndash;142\u0026ndash;3p mimics could reverse the above changes caused by ACSL4 overexpression (Fig. 4P-R). Meanwhile, similar results were observed by immunofluorescence analysis (Fig. S4). The regulatory effect of miR\u0026ndash;142\u0026ndash;3p by targeting ACSL4 on lipid metabolism was also observed in a rat primary hepatocyte model (data not shown). Finally, we confirmed the interaction between miR\u0026ndash;142\u0026ndash;3p and ACSL4 using a dual luciferase reporter assay (Fig.4T). These results indicate that miR\u0026ndash;142\u0026ndash;3p regulate hepatic lipid metabolism by targeting ACSL4.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.5. \u003c/em\u003e\u003cem\u003eHigh glucocorticoid-induced sperm miR\u0026ndash;142\u0026ndash;3p methylation reprogramming mediates hepatic lipid metabolism dysfunction in PPCE male offspring\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eStudies have shown that sperm \u0026ldquo;imprinting\u0026rdquo; paternal epigenetic information from adverse environmental exposures is an important manner to influence the long-term health of the offspring\u003csup\u003e43\u003c/sup\u003e. miRNAs and their methylation modifications are key epigenetic marks in the reprogramming of germ cells, transmitting the \u0026ldquo;imprint\u0026rdquo; of the parent\u0026rsquo;s adverse environmental experiences to the offspring\u003csup\u003e16, 44\u003c/sup\u003e. It was found that paternal chronic stress could lead to epigenetic modification alterations in sperm \u003cem\u003evia\u003c/em\u003e high glucocorticoids, thereby programming abnormal liver glucose metabolism in the offspring\u003csup\u003e16\u003c/sup\u003e, suggesting that high glucocorticoids might be an important mechanism for the occurrence of parental metabolic diseases. Therefore, in this study, we first detected the serum corticosterone level, testicular GR expression level, and methylation level of sperm miR\u0026ndash;142\u0026ndash;3p promoter region in the parental male rats. The results showed that compared with the CON group, serum corticosterone levels, testicular GR mRNA expression level, and sperm methylation levels of miR\u0026ndash;142\u0026ndash;3p promoter region were significantly increased in PPCE group (Fig. 5A-C). Further, we treated mouse spermatogonia with different concentrations of caffeine (0.1~10 \u0026micro;M) or corticosterone (125~500 nM) \u003cem\u003ein vitro\u003c/em\u003e to determine the cause of reduced sperm miR\u0026ndash;142\u0026ndash;3p expression. The results showed that different concentrations of caffeine treatment had no obvious effect on miR\u0026ndash;142\u0026ndash;3p expression in spermatogonia (Fig. 5D), while different concentrations of corticosterone treatment were able to significantly decrease miR\u0026ndash;142\u0026ndash;3p expression level in spermatogonia (Fig. 5E) and significantly increase the methylation level of miR\u0026ndash;142\u0026ndash;3p promoter region (Fig. 5F, G). To investigate whether a strong relationship between high glucocorticoid exposure and sperm miR\u0026ndash;142\u0026ndash;3p promoter region methylation also existed in the population, we collected a set of human samples and performed assays and correlation analyses. The results showed that serum corticosterone levels in fertile males were significantly and positively correlated with their sperm miR\u0026ndash;142\u0026ndash;3p promoter region methylation levels (Fig. S4), and sperm miR\u0026ndash;142\u0026ndash;3p promoter region methylation levels were significantly and positively correlated with sperm viability concentration/aberration rate (Fig. S4). It is suggested that parental corticosterone (but not caffeine) programs the hypermethylation of miR\u0026ndash;142\u0026ndash;3p in PPCE paternal sperm.\u003c/p\u003e\n\u003cp\u003eTo corroborate the involvement of parental high glucocorticoid exposure and sperm abnormalities in PPCE-induced NAFLD susceptibility in male offspring, we further conducted a series of intervention experiments. First, GR antagonist RU486 was used to synchronize interventions in PPCE rats to confirm the programming mechanism of paternal hyper glucocorticoid-mediated alterations in hepatic lipid metabolism. The results showed that compared with the CON group, the methylation rate of miR\u0026ndash;142 promoter region in liver was increased and the expression was significantly decreased in PPCE group at GD20 and PW32 (Fig. 5H-K), and the expressions of hepatic lipogenesis function genes were significantly increased and \u0026beta; oxidation function genes were decreased (Fig. 5L, M), accompanied by significantly increased hepatic lipid accumulation and triglyceride content (Fig. 5N-P). However, RU486 intervention was able to markedly reverse PPCE-induced above changes in male offspring before and after birth (Fig. 5H-P). It is indicated that parental high glucocorticoid exposure mediates the alteration of hepatic lipid metabolism function in PPCE male offspring.\u003c/p\u003e\n\u003cp\u003eNext, we explored the effect of fertilization between PPCE mouse sperm and normal oocytes on changes in liver lipid metabolism function in male offspring using \u003cem\u003ein vitro\u003c/em\u003e fertilization technique (Fig. 6A). The results showed that compared with the CON group, the expression of miR\u0026ndash;142\u0026ndash;3p was decreased and the expressions of lipogenesis genes (\u003cem\u003eSrebp1,\u003c/em\u003e \u003cem\u003eFasn,\u003c/em\u003e and \u003cem\u003eAcc)\u003c/em\u003e were significantly increased while the expressions of \u0026beta;-oxidation genes (\u003cem\u003ePpar\u0026alpha;\u003c/em\u003e and \u003cem\u003eCpt1\u0026alpha;)\u003c/em\u003e were significantly decreased in the fetal liver of the offspring in the PPCE group (Fig. 6B, C). Histological results showed that a large number of fat-like vacuoles, lipid accumulation, and significantly elevated liver triglyceride content were observed in the fetal liver of PPCE group (Fig. 6D, E). Further, to confirm that low expression of sperm miR\u0026ndash;142\u0026ndash;3p could mediate changes in liver lipid metabolism function in male offspring, we investigated the effect of miR\u0026ndash;142\u0026ndash;3p\u003csup\u003eKO\u003c/sup\u003e sperm on changes in liver lipid metabolism function in male offspring after fertilization with normal oocytes (Figu. 6F). The results showed that the expressions of lipogenesis genes were significantly increased in the fetal liver of the miR\u0026ndash;142\u0026ndash;3p\u003csup\u003eKO\u003c/sup\u003e group (Fig. 6G), while the mRNA expressions of fatty acid \u0026beta;-oxidation function genes were decreased (Fig. 6G), at the same time, the fetal liver showed obvious steatosis and significantly increased triglyceride content (Fig. 6H, I). At PW12, the liver lipogenesis was enhanced and \u0026beta;-oxidation was diminished in the miR\u0026ndash;142\u0026ndash;3p\u003csup\u003eKO\u003c/sup\u003e group, and a large amount of vacuolar-like steatosis along with excessive lipid accumulation was observed in the liver and NAFLD occurred (Fig. 6J-L). It was suggested that low expression of sperm miR\u0026ndash;142\u0026ndash;3p could mediate hepatic lipid metabolism dysfunction and the occurrence of NAFLD in male offspring.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.6. PPCE induces NASH occurrence in male offspring with \u003c/em\u003e\u003cem\u003etransgenerational inheritance\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo clarify whether there is a transgenerational genetic effect of PPCE-caused adult NASH in male offspring, we observed changes in liver histology, lipid metabolism function, and inflammatory response in F2 generation of the offspring. The results showed that compared with the CON group, there were no obvious changes in liver pathology and triglyceride content in F2 generation of PPCE offspring at PW12 (Fig. 7A, B), but miR\u0026ndash;142\u0026ndash;3p expression was significantly decreased (Fig. 7C), \u003cem\u003eAcsl4\u003c/em\u003e and lipogenesis genes (\u003cem\u003eSrebp1\u003c/em\u003e and \u003cem\u003eFasn)\u003c/em\u003e expressions were significantly increased whereas \u0026beta;-oxidation genes (\u003cem\u003ePpar\u0026alpha;\u003c/em\u003e and \u003cem\u003eCpt1\u0026alpha;)\u003c/em\u003e expressions were decreased (Fig. 7D). However, at PW34, a large number of hepatocytes in the liver of F2 generation in PPCE offspring showed steatosis with lipid accumulation, inflammatory infiltration (green arrows), and collagen fibril deposition (black and red arrows) (Fig. 7E), and liver triglyceride content was significantly elevated (Fig. 7F). RT-qPCR and Western bolt results showed that at PW34, liver miR\u0026ndash;142\u0026ndash;3p expression was significantly decreased in F2 generation of PPCE offspring (Fig. 7G), the expression of ACSL4 and lipogenesis genes were significantly increased, while the expression of \u0026beta;-oxidation genes were decreased (Fig. 7H-J). Meanwhile, liver pro-inflammatory factor content (Fig. 7K-M) and mRNA expression (Fig. 7N-P) were significantly increased in F2 generation of PPCE offspring at PW34. It was suggested that PPCE could cause hepatic lipid metabolism dysfunction, inflammatory response activation, and adult NASH occurrence in the F2 generation of male offspring. Taken together, these findings demonstrate that PPCE-induced adult NASH occurrence in male offspring with transgenerational inheritance.\u003c/p\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eBoth epidemiological surveys and laboratory studies have confirmed that caffeine has developmental toxicity\u003csup\u003e20, 45\u003c/sup\u003e, and special populations such as pregnant women, children, and adolescents are more susceptible to its adverse effects\u003csup\u003e46, 47, 48\u003c/sup\u003e. According to the European Food Safety Authority’s report on safe daily doses of caffeine for different populations, the total daily caffeine intake of pregnant women should not exceed 200 mg, while healthy adults should not exceed 400 mg\u003csup\u003e48\u003c/sup\u003e. Although the global average of adult caffeine intake is below the recommended safe dose (\u0026lt;400 mg/d), there is still a rather large population consuming overdose, especially in Europe and the United States\u003csup\u003e26\u003c/sup\u003e. Although our previous studies have systematically revealed the impact of maternal caffeine exposure during pregnancy on the long-term health of the offspring\u003csup\u003e21, 22, 23, 24\u003c/sup\u003e, the effect of PPCE on the long-term metabolic health of the offspring and the programming mechanisms have not been reported so far. In this regard, we established a PPCE rat model, in which male rats were given caffeine dose of 15, 30, and 60 mg/kg/d, equivalent to approximately half, one, and two times of the recommended safe dose for the population. Our main findings highlight that PPCE can lead to the occurrence of NASH in adult male offspring rats with transgenerational inheritance, which is associated with paternal high glucocorticoid-programmed sperm miR–142–3p hypermethylation to mediate hepatic lipid metabolic disorder and inflammatory activation, and confirm that miR–142–3p is a key regulator of lipid metabolism to serve as an early interventional target for paternal NASH.\u003c/p\u003e\u003cp\u003eIn this study, we first observed the changes in liver histology and lipid metabolism function in F1 and F2 generation rats at different times before and after birth, and found that PPCE could lead to hepatic lipid metabolism dysfunction in offspring and NASH occurrence in adulthood (PW32), with transgenerational inheritance and gender differences, especially in males. Transgenerational inheritance refers to the transmission of epigenetic information through germ cells to the next generation or even to multiple generations, which has important implications for biological evolution and disease occurrence\u003csup\u003e49\u003c/sup\u003e. Studies have previously found that paternal pre-pregnant exposure to adverse environmental factors can have impacts on the health of offspring and even multiple generations\u003csup\u003e50\u003c/sup\u003e. In the present study, we also investigated the transgenerational genetic effects of PPCE-induced NASH occurrence in male offspring and found that similar to the F1 generation of male offspring, PPCE also caused hepatic lipid metabolism dysfunction and steatosis along with inflammatory infiltration and collagen fibril deposition in the F2 male generation. It is suggested that PPCE-induced adult NASH occurrence in male offspring (F1 generation) with transgenerational inheritance. Interestingly, the degree of liver pathological alterations in male offspring of the PPCE-F2 generation was significantly reduced compared to the PPCE-F1 generation. It is suggested that PPCE-induced adult NASH occurrence in male offspring with transgenerational inheritance. We hypothesized that, although the changes in liver pathology in the F2 generation are likely to derive from the intergenerational transmission of PPCE-induced sperm reprogramming in the F0 generation and programming effect in F2 generation due to low glucocorticoid exposure in the F1 generation\u003csup\u003e23\u003c/sup\u003e, further insights into the mechanisms underlying the histopathological changes in the liver of the F2 generation are needed in the future.\u003c/p\u003e\u003cp\u003eOther related studies have shown that developmental-derived diseases often have significant gender differences, which might be related to differences in maternal compensatory regulation on fetal development of different genders during intrauterine development of the embryo \u003csup\u003e51\u003c/sup\u003e. In this study, we compared PPCE-induced altered hepatic lipid metabolism in offspring before and after birth and the gender differences in the occurrence of adult NASH. It was found that PPCE could lead to the occurrence of adult NAFLD in female offspring, but the degree of histological alterations was less typical than that of males, no significant inflammatory infiltration and collagen fibril deposition were observed in the liver and hepatic miR–142–3p expression levels were significantly reduced in PPCE male offspring before and after birth. In contrast, there was no significant change in liver miR–142–3p expression in PPCE female offspring (Fig. S1). In conclusion, it is suggested that PPCE-caused occurrence of NASH in adult offspring has gender differences, and the disease characteristics of NASH in female offspring are atypical. Interestingly, we also found that the PPCE-induced abnormal development of multiple organs (including liver, adrenal glands, bone, cartilage, etc.) in female offspring was significantly weaker than that in male offspring, which may be attributed to the active, compensatory regulation of intrauterine offspring development by the maternal parent. We will delve into the gender-specific functional abnormalities of multiple organs in the offspring due to PPCE and the pathogenesis in our following studies.\u003c/p\u003e\u003cp\u003eAs important epigenetic modification markers, miRNAs are able to mediate the effects of paternal adverse factor exposure on the long-term health of offspring\u003csup\u003e16, 34, 52\u003c/sup\u003e. In this study, we found through sequencing analysis that miR–142–3p was the miRNA with the most significant change in co-expression in the liver of PPCE parental rat sperm and male offspring before and after birth. It was further confirmed that PPCE could cause a significant reduction in hepatic miR–142–3p expression in male offspring before and after birth, and liver-specific silencing of miR–142–3p could induce NASH occurrence in mice. It is suggested that miR–142–3p low expression mediates PPCE-induced NASH occurrence in male offspring. ACSLs are key enzymes that catalyze the synthesis of acyl-CoA and are essential in the regulation of body fatty acid metabolism\u003csup\u003e53\u003c/sup\u003e. ACSL4, one of the subtypes of the ACSLs family that is most closely involved in fatty acid metabolism, is able to catalyze the reaction between long-chain fatty acids and CoA to form acyl-CoA, thus playing an important role in the development of liver metabolic diseases\u003csup\u003e54\u003c/sup\u003e. It was found that ACSL4 can promote fatty acid\u003cem\u003e de novo\u003c/em\u003e synthesis process in hepatocytes through activation of transcription factor SREBP1\u003csup\u003e55, 56\u003c/sup\u003e, and inhibit fatty acid β-oxidation by suppressing PPARα transcription through endogenous ligands\u003csup\u003e54, 56\u003c/sup\u003e. In this study, we found that PPCE significantly increased the expression of ACSL4 in the liver of male offspring before and after birth; overexpression of miR–142–3p suppressed the expression of ACSL4 in hepatocytes and reversed the occurrence of long-term NASH in PPCE male offspring rats. We further found that miR–142–3p was able to bind to ACSL4 through target gene prediction and dual luciferase reporter gene experiments, and confirmed that miR–142–3p regulated lipogenesis and β-oxidation function in BMSCs hepatoid differentiated cells by targeting ACSL4. Excessive lipid accumulation in the liver has been demonstrated to result in lipotoxicity, triggering mitochondrial oxidative stress, inflammatory responses, and the progression of NAFLD\u003csup\u003e57\u003c/sup\u003e. STING, a pivotal factor in innate immunity located on the endoplasmic reticulum, becomes activated upon recognizing abnormal double-stranded DNA in the cytoplasm. It regulates inflammatory responses by activating transcription factors like NF-κB and IRF3\u003csup\u003e58\u003c/sup\u003e. In the present study, our findings reveal that PPCE induces an increase in oxidative stress and activates STING-mediated inflammatory signaling in liver macrophages of male offspring. This activation leads to heightened production of pro-inflammatory factors, including TNF-α, IL–6, and IL–1β, in the liver. Additionally, the absence of miR–142–3p (miR–142–3p\u003csup\u003e-/-\u003c/sup\u003e) is associated with the activation of STING-mediated inflammatory responses in the livers of mice. Collectively, these results suggest that low expression of miR–142–3p contributes to hepatic lipid metabolism disorder and activation of inflammatory responses through the upregulation of ACSL4. This, in turn, mediates the occurrence of non-alcoholic steatohepatitis (NASH) in male offspring exposed to PPCE.\u003c/p\u003e\u003cp\u003eClinical studies have also found reduced miR–142–3p expression in peripheral blood mononuclear cells (PBMCs) of children with metabolic-associated fatty liver disease (MAFLD) that was potentially correlated with serum triglyceride levels\u003csup\u003e59\u003c/sup\u003e, suggesting that miR–142–3p may be closely associated with the occurrence of MAFLD. Although a growing number of studies confirm that NAFLD/NASH has a paternal origin, the programming mechanisms remain unelucidated, leading to difficulties in early prevention and treatment. Other related research has shown that improving paternal pre-pregnancy lifestyle (such as proper exercise and antioxidant supplementation) has a positive effect on the prevention of paternal NAFLD\u003csup\u003e60, 61, 62\u003c/sup\u003e, but due to individual differences and lack of specificity, these measures are difficult to be effective prevention and treatment strategies for paternal NAFLD/NASH. In the present study, we found that miR–142–3p was a toxic target of PPCE-induced NASH occurrence in male offspring, and confirmed that overexpression of miR–142–3p was effective in reversing PPCE-induced NASH occurrence in male offspring. Intriguingly, we recently also observed hepatic miR–142–3p low expression and abnormal lipid metabolism function in male offspring on a rat model of low-dose mixed paternal pre-pregnancy nicotine/ethanol/caffeine exposure (Fig. S5). It was suggested that miR–142–3p could serve as a potential, common target for early prevention and treatment of paternal NAFLD/NASH. This finding offers valuable insights for developing nucleic acid drugs to prevent and treat paternal NAFLD/NASH. \u003c/p\u003e\u003cp\u003eGlucocorticoids are known to be the most important regulatory hormone in body stress response and are crucial in determining germ cell formation, fetal maturation, and postnatal fate. Studies have shown that paternal adverse environmental (such as caffeine, nicotine, and ethanol) exposure can provoke HPA axis activation, resulting in the body being in a chronic stress state\u003csup\u003e63, 64, 65\u003c/sup\u003e, and affecting sperm epigenetic modifications through elevated serum glucocorticoid levels, leading to offspring dysplasia\u003csup\u003e16, 66\u003c/sup\u003e. It is suggested that serum-high glucocorticoid reprogrammed sperm epigenetic modifications may be an important mechanism mediating the transmission of paternally acquired traits to the offspring. DNA methylation, one of the most important forms of epigenetic inheritance in eukaryotes, mediates the inheritance of paternally acquired traits\u003csup\u003e67\u003c/sup\u003e. Clinical evidence suggests that poor paternal dietary habits can lead to altered DNA methylation status during sperm reprogramming, resulting in an increased risk of metabolic disease in the offspring after birth\u003csup\u003e68, 69\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn this study, we found that serum corticosterone level was significantly elevated, and the methylation level of miR–142–3p promoter region was significantly elevated in sperm and liver of PPCE-F0 generation, and we confirm that it was the corticosterone (but not caffeine) caused the elevated methylation level of miR–142–3p promoter region. To clarify the glucocorticoid programming mechanism of PPCE-induced imbalance of lipid metabolism homeostasis in the liver of offspring and NASH occurrence, we used the GR antagonist RU486 to intervene in PPCE rats and found that it could reduce the methylation level of the miR–142–3p promoter region in the sperm of PPCE-F0 generation and liver of male F1 generation, and reverse the occurrence of long-term NASH. \u003cem\u003eIn vitro\u003c/em\u003e fertilization experiments further confirmed that miR–142–3p low expression could program the alteration of hepatic lipid metabolism function in PPCE male offspring. Interestingly, we found a significant positive correlation in a population of fertile men between serum corticosterone levels and sperm miR–142–3p promoter region methylation, as well as between sperm miR–142–3p promoter region methylation and sperm concentration/aberration rate in adult men (Fig. S4). It is suggested that high glucocorticoid-programmed sperm miR–142–3p hypermethylation resulting from parental chronic stress mediates the occurrence of PPCE-induced NASH in male offspring, where hypermethylation of sperm miR–142–3p promoter region is expected to be early warning targets for clinical paternal NASH susceptibility.\u003c/p\u003e\u003cp\u003eThe “DOHaD” theory suggests that many metabolic diseases in adults have a developmental origin and that adverse environmental exposures in the paternal preconception or maternal pregnancy can cause alterations in metabolic adaptations and developmental programming in the offspring, which is an important risk factor leading to an increased risk of multiple disorders (such as NAFLD) in the offspring. Previous systematic studies in our laboratory have confirmed that maternal PCE can lead to the occurrence of IUGR, changes in hepatic lipid metabolism function, and NAFLD susceptibility, along with gender differences in the offspring\u003csup\u003e70, 71, 72\u003c/sup\u003e. To elucidate the effects of (paternal and maternal) parental caffeine exposure on developmental programming and disease susceptibility in offspring, we compared and analyzed the similarities and differences in PPCE and PCE-induced NAFLD susceptibility. The details are shown in Table 1.Our outcomes showed that PPCE-induced and PCE-induced NAFLD susceptibility is different in caffeine exposure dose and duration, disease state, and gender difference. It manifested that PPCE male rats were exposed to caffeine (15~60 mg/kg/d) for 8 weeks (a complete spermatogenic cycle), and PCE female rats were exposed to caffeine (30~120 mg/kg/d) in middle and late pregnancy (GD9~GD20); PPCE could cause the occurrence of NAFLD in the offspring, and there were significant inflammatory response and fibrosis (NASH occurrence) with no need for a “second hit “, while PCE could lead to NAFLD susceptibility in offspring with no obvious inflammatory response and fibrosis and it needs a “second hit (such as high-fat diet)\" to develop NAFLD. In conclusion, compared to PCE, the disease course of PPCE-induced NAFLD was shorter and more severe, which may be associated with the fact that paternal effects on the offspring are earlier than maternal.\u003c/p\u003e\n\u003cp\u003eIn summary, this study marks the pioneering demonstration of the association between PPCE and disrupted hepatic lipid metabolism in male offspring, both prenatally and postnatally, leading to the development of NASH in adulthood. The observed effects include transgenerational inheritance impacts and notable gender differences. The underlying mechanism involves elevated glucocorticoid levels, specifically arising from PPCE, inducing hypermethylation in the sperm miR–142–3p promoter region. This epigenetic modification persists in the livers of male offspring, contributing to diminished miR–142–3p expression, subsequent dysfunction in hepatic lipid metabolism, and the activation of inflammatory pathways, ultimately culminating in adult-onset NASH (Fig. 8). Importantly, hepatic overexpression of miR–142–3p has been shown to effectively reverse the long-term occurrence of NASH in PPCE-exposed male offspring, providing a potential intervention strategy for high glucocorticoid-mediated programming. The established PPCE rat model faithfully reproduces the adverse lifestyle habits and chronic stress prevalent in the daily lives of contemporary reproductive-age men. This research not only elucidates the developmental origins and programming mechanisms of paternal NASH but also confirms a viable intervention target. The findings hold significant practical implications for understanding the impact of paternal pre-pregnancy chronic stress on the long-term metabolic health of offspring and for guiding the adoption of healthy lifestyles among men of reproductive age.\u003c/p\u003e"},{"header":"4. Methods","content":"\u003ch2\u003e4.1. Reagents\u003c/h2\u003e\n\u003cp\u003eCaffeine (purity \u0026ge; 99.0%) and corticosterone were purchased from Sigma-Aldrich (St Louis, MO, USA). Mifepristone (RU486) was obtained from Hubei Gedian humanwell Pharmaceutical Co. LTD (Ezhou, China). Rat-miR\u0026ndash;142\u0026ndash;3p inhibitor oligo, Rat-miR\u0026ndash;142\u0026ndash;3p mimics oligo, Rat-ACSL4 plasmid, and mus-miR\u0026ndash;142\u0026ndash;3p siRNA were obtained from GenePharma (Shanghai, China). AAV8 adenovirus delivery-miR\u0026ndash;142\u0026ndash;3p was purchased from Tsingke Biotechnology (Beijing, China). miScript SYBR Green PCR Kit and SYBR Green qPCR Master Mix were purchased from Vazyme (Nanjing, China). All other reagents were of analytical grade.\u003c/p\u003e\n\u003ch2\u003e4.2. Animal treatment\u003c/h2\u003e\n\u003cp\u003eSpecific pathogen-free (SPF) Wistar rats were purchased from the Experimental Center of the Hubei Medical Scientific Academy and accommodated to standard nutritional conditions with a 12 h light/dark cycle. Animal experiments were performed in the Center for Animal Experiment of Wuhan University (Wuhan, China), which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). All animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee at the Wuhan University (WHEF\u0026ndash;2022\u0026ndash;0286).\u003c/p\u003e\n\u003cp\u003eMale Wistar rats (F0) were randomly ranged into the control and paternal pre-pregnant caffeine exposure (PPCE) groups (\u003cem\u003en \u003c/em\u003e = 12). Animals in the PPCE group were administrated with different concentrations of caffeine (15, 30, and 60 mg/kg/d) for consecutive 8 weeks, and rats of the control group were treated with the same volume of normal saline. After caffeine administration, male rats were mated with untreated females in a joint cage. The day when sperm appeared in vaginal smear was designated as gestational day (GD) 0. Half of the pregnant rats were euthanized on GD20 with 3% isoflurane, liver was collected for further analysis. The rest pregnant rats were kept to obtain the F1 generation. One day after birth, the number of pups was standardized to 12 pups per litter (6 males and 6 females). The offspring were fed with normal diet after weaning at postnatal week 4 (PW4), and sacrificed under isoflurane anesthesia at PW12 and PW32, respectively (\u003cem\u003en \u003c/em\u003e = 12, randomly chose one from each litter). Liver tissues were collected for subsequent analysis. A subset of F1 male rats were randomly selected on PW8, and divided into vector and AAV8-miR\u0026ndash;142\u0026ndash;3p group (\u003cem\u003en = \u003c/em\u003e20). AAV8-miR\u0026ndash;142\u0026ndash;3p and vectors were injected respectively into rats by hydrodynamic injection \u003cem\u003evia\u003c/em\u003e tail vein. These rats were then sacrificed at PW12 (\u003cem\u003en = \u003c/em\u003e10) and PW32 (\u003cem\u003en = \u003c/em\u003e10).\u003c/p\u003e\n\u003cp\u003eTo further investigate the underlying mechanism, F0 male rats were randomly divided into the control (normal saline), PPCE (60 mg/kg/d caffeine), RU486 (1 mg/kg/d)\u003csup\u003e16, 73\u003c/sup\u003e, and RU486 (1 mg/kg/d) + PPCE (60 mg/kg/d caffeine) group (\u003cem\u003en \u003c/em\u003e = 12). Rats in each group were administrated with corresponding reagents daily for continuous 8 weeks, and then mated with normal female rats at the end of administration. Half of the pregnant rats were killed at GD20, and the rest pregnant rats delivered F1 generation naturally. The F1 rats were sacrificed at PW12 and PW32 (\u003cem\u003en \u003c/em\u003e = 12). Moreover, another part of F1 male rats was mated with normal female rats at PW12 to produce the F2 generation and F2 rats were sacrificed at PW12 and PW34 (\u003cem\u003en = \u003c/em\u003e12), respectively.\u003c/p\u003e\n\u003ch2\u003e4.3. Human plasma and sperm samples\u003c/h2\u003e\n\u003cp\u003eHuman plasma and sperm samples were collected from 28 male patients who underwent assisted reproductive tests in the Reproductive Medicine Center of Zhongnan Hospital, Wuhan University. The plasma cortisol concentration, sperm general characteristics (sperm concentration, sperm number, sperm malformation rate, etc.) and sperm miR\u0026ndash;142\u0026ndash;3p promoter region methylation rate were detected, and the correlation between the above results was analyzed. The study was approved by the Institutional Ethics Committee of the Zhongnan Hospital of Wuhan University (No. 2020188). Informed consent was obtained from the patients before the collection of plasma and sperm samples. All the research was performed in accordance with government policies and the Helsinki declaration.\u003c/p\u003e\n\u003ch2\u003e4.4. Hydrodynamic injection\u003c/h2\u003e\n\u003cp\u003eThe miR\u0026ndash;142\u0026ndash;3p lentiviral plasmid (miR\u0026ndash;142\u0026ndash;3p\u003csup\u003e-/-\u003c/sup\u003e) was synthesized by Genepharma (Shanghai, China). Male FVB/N mice (20~22 g) were obtained from Charles River Laboratories (Beijing, China), and randomly divided into control and miR\u0026ndash;142\u0026ndash;3p\u003csup\u003e-/-\u003c/sup\u003e group (\u003cem\u003en\u003c/em\u003e = 8). The hydrodynamic injection was performed to conduct a hepatic-specific miR\u0026ndash;142\u0026ndash;3p\u003csup\u003e-/-\u003c/sup\u003e mouse model as previously described \u003csup\u003e74\u003c/sup\u003e. In detail, 20 \u0026micro;g miR\u0026ndash;142\u0026ndash;3p\u003csup\u003e-/- \u003c/sup\u003eplasmids were diluted in 2 mL normal saline, then plasmids solution with a volume of 10% of the animal\u0026rsquo;s body weight was transiently injected into the tail-vein of mice in miR\u0026ndash;142\u0026ndash;3p\u003csup\u003e-/-\u003c/sup\u003e group within 6~7s. Mice in the control group were also injected with the same volume of the vehicle. After 8 weeks, all mice were euthanized with 3% isoflurane to collect serum and liver samples for the following analysis.\u003c/p\u003e\n\u003ch2\u003e4.5. \u003cem\u003eIn vitro\u003c/em\u003e fertilization and embryo culture\u003c/h2\u003e\n\u003cp\u003eFirstly, the sperm of mice exposed to chronic treatment of HCG or vehicle (\u003cem\u003en\u003c/em\u003e = 20) was extracted from epididymis and the spermatozoa were diluted at \u0026sim;2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e of final concentration in human tubal fluid (HTF) medium to be evaluated after 15 min of capacitation. Next, male ICR mice were administrated with a gavage of caffeine (120 mg/kg/d) for continuous 8 weeks to establish a PPCE mice model (\u003cem\u003en\u003c/em\u003e = 12). The control group was also administrated the same amount of saline. Sperm were isolated from the epididymis after finished the administration and were diluted to a concentration of 2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e/mL in the human tubal fluid medium, followed by further investigation after 15 min capacitation. Female ICR mice (\u003cem\u003en\u003c/em\u003e = 24) were injected with 5 IU equine chorionic gonadotropin and 5 IU HCG for superovulation, followed by collecting mature oocytes from the fallopian tube. \u003cem\u003eIn vitro\u003c/em\u003e fertilization was performed as previously described \u003csup\u003e75\u003c/sup\u003e. Briefly, sperms of mice in the control or PPCE group and normal oocytes were transferred into the embryo medium for further culture and fertilization to get fertilized eggs. Fertilized zygotes were cultured in vitro for 48 h, then the surviving embryos were transferred into the oviduct of pseudopregnant recipient female mice. All pregnant mice were anesthetized using 3% isoflurane to strip fetal mice 20 days after embryo transfer and collect fetal blood and liver samples for further investigations.\u003c/p\u003e\n\u003cp\u003eMale miR\u0026ndash;142\u0026ndash;3p\u003csup\u003eKO\u003c/sup\u003e ICR mice (\u003cem\u003en\u003c/em\u003e = 5) were generated by Cyagen Biosciences (Nanjing, China) using CRISPR/Cas9 technology, and miR\u0026ndash;142\u0026ndash;3p\u003csup\u003eKO\u003c/sup\u003e sperm were collected from the epididymis. Meanwhile, sperm and mature oocytes of normal male and female mice were also extracted. Normal sperm or miR\u0026ndash;142\u0026ndash;3p\u003csup\u003eKO\u003c/sup\u003e sperm were fertilized with normal oocytes to generate fertilized oocytes, which were then transferred to pseudopregnant recipient female mice. Fetal blood and liver samples were harvested 20 days after embryo transfer as the above method. The rest of the pregnant mice were fed till natural birth. The offspring were raised to adulthood to determine the effect of miR\u0026ndash;142\u0026ndash;3p\u003csup\u003eKO\u003c/sup\u003e on hepatic lipid metabolic function in male offspring.\u003c/p\u003e\n\u003ch2\u003e4.6 Cell culture\u003c/h2\u003e\n\u003cp\u003eBone marrow mesenchymal stem cells (BMSCs) hepatoid differentiated cells were extracted as previously described\u003csup\u003e76\u003c/sup\u003e. In brief, BMSCs hepatoid differentiated cells were extracted from 3-week-old male rats and cultured using \u0026alpha;-MEM medium with 10% fetal bovine serum, 100 mg/mL streptomycin, and 100 U/mL penicillin. When the cells reached 80% confluence, they were incubated with hepatocyte differentiation medium (DMEM medium with 1% fetal bovine serum, 100 mg/mL streptomycin, 100 U/mL penicillin, 20 ng/mL HGF, 2 ng/mL EGF, 0.1 \u0026mu;M dexamethasone, and 50 mg/mL ITS) for 14 days\u003csup\u003e77\u003c/sup\u003e. 20 nM miR\u0026ndash;142\u0026ndash;3p inhibitor, 20 nM miR\u0026ndash;142\u0026ndash;3p mimics, or 2.5 \u0026mu;g ACSL4 plasmid were transfected into cells using lipofectamine 3000. In detail, the plasmids and transfection reagent were incubated in DMEM for 20 min at room temperature to perform transfection into BMSCs hepatoid differentiated cells, and harvested for further assessment after 24 h. Mouse spermatogonial cell line GC\u0026ndash;1 cells (Procell Life Science \u0026amp;Technology, Wuhan, China) were also cultured under the above conditions. GC\u0026ndash;1 cells were treated with corticosterone (125, 250, 500 nM) or caffeine (0.1, 1, 10 \u0026micro;M) for 24 h, respectively. Cells were then collected to detect the mRNA expression level of miR\u0026ndash;142\u0026ndash;3p and the rate of promoter region methylation. All \u003cem\u003ein vitro\u003c/em\u003e data were from triplicate independent experiments.\u003c/p\u003e\n\u003ch2\u003e4.7. DNA methylation analysis\u003c/h2\u003e\n\u003cp\u003eThe bisulfite pyrosequencing method was performed for DNA methylation analyses in sperm and liver samples (\u003cem\u003en\u003c/em\u003e = 3) as previously reported \u003csup\u003e78\u003c/sup\u003e. DNA was extracted from F0 rat sperm and male F1 liver tissues using a Mammalian genomic DNA extraction kit (Beyotime, Shanghai, China). To measure the DNA permethylation status, EZ DNA Methylation-Gold Kit (ZYMO, CA, USA) was used for sample processing to convert unmethylated cytosine into uracil. After building the MethylTarget library, high-throughput sequencing was employed with the Illumina HiSeq platform.\u003c/p\u003e\n\u003ch2\u003e4.8. Whole transcriptome sequencing\u003c/h2\u003e\n\u003cp\u003eSperm and liver samples of male rats were lysed with Trizol reagent for further analysis. The sequencing libraries were generated using the TruSeq Stranded Total RNA Library Prep Kit (Illumina, USA). Then, the libraries analysis was performed using the online platform of Majorbio Cloud Platform \u003ca href=\"http://(www.majorbio.com\"\u003e(www.majorbio.com\u003c/a\u003e) (Shanghai Majorbio Bio-pharm Technology Co., Ltd). The analyses of mRNA and miRNA expression were conducted using the DESeq R package, and the transcripts with \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and log2 fold change \u0026gt;2 were selected as differentially expressed mRNAs and miRNAs. The enrichment analysis was plotted using the R software. All sequencing was analyzed on the Whole Transcriptome Cloud Platform of Shanghai Meggie Biotechnology \u003ca href=\"http://(https://cloud.majorbio.com)\"\u003e(https://cloud.majorbio.com)\u003c/a\u003e.\u003c/p\u003e\n\u003ch2\u003e4.9. Hematoxylin-eosin (H\u0026amp;E), oil red O (ORO) staining, Masson staining, Sirius red staining, and Transmission electron microscopy (TEM)\u003c/h2\u003e\n\u003cp\u003eFor histopathological analysis, fresh liver tissues were fixed within 10% paraformaldehyde and then embedded with paraffin to slice into liver sections (5 \u0026micro;m). Liver slides were dewaxed with xylene and rehydrated with ethanol at decreasing concentration gradient to perform H\u0026amp;E staining, Masson staining and Sirius red staining. For ORO staining, frozen liver slices were incubated within ORO solution, rinsed with 60% isopropanol, and nucleus were redyed with hematoxylin. All slides were then mounted under coverslips for further observation under a microscope. The pathological grading and staging of non-alcoholic steatohepatitis (NASH) rats were scored according to the non-alcoholic fatty liver diseases (NAFLD) activity score (NAS) system\u003csup\u003e79\u003c/sup\u003e. The scoring system consisted of a semi-quantitative assessment of three histological features: steatosis (0\u0026ndash;3), lobular inflammation (0\u0026ndash;3), and hepatocellular ballooning (0\u0026ndash;2), in which NAS \u0026lt; 3 (non-NASH), NAS \u0026gt; 4 (NASH), and NAS between 3 and 4 (possibly NASH).\u003c/p\u003e\n\u003cp\u003eFor TEM analysis, fresh liver tissues from mice were fixed in 2.5% glutaraldehyde, post-fixed in 1% OsO4, and dehydrated with gradient ethanol and acetone. The tissues were embedded in epoxy resin, cut into serial TEM ultrathin sections, and stained with uranyl acetate and lead citrate. The sections were photographed using a JEM 1400 JEOL instrument (Tokyo, Japan).\u003c/p\u003e\n\u003ch2\u003e4.10. Real-time quantitative polymerase chain reaction (RT-qPCR)\u003c/h2\u003e\n\u003cp\u003eTotal RNA was extracted from liver homogenate and BMSCs hepatoid differentiated cells using TRIzol\u0026trade; Reagent, and then converted into complementary DNA (cDNA). The RT-qPCR assay was subsequently processed to determine the quantity of specific DNA sequences by FastStart Universal SYBR Green Master Mix (Roche, Basel, Switzerland) on a 384-well ABI StepOnePlus cycler (Applied Biosystems, Foster City, CA, USA). Glyceraldehyde\u0026ndash;3-phosphate dehydrogenase (GAPDH) was taken as an internal reference for quantitative assay. The identified primers were listed in \u003cem\u003eTable S1.\u003c/em\u003e NADH dehydrogenase subunit 1 (ND1) gene encoded by mtDNA and GAPDH encoded by nuclear DNA (nDNA) were determined by RT-qPCR analysis as previously described\u003csup\u003e80\u003c/sup\u003e. The mtDNA/nDNA ratio was used to assess the relative mtDNA copy number. The primers are listed in \u003cem\u003eTable S2.\u003c/em\u003e\u003c/p\u003e\n\u003ch2\u003e4.11. Western blotting and immunofluorescence\u003c/h2\u003e\n\u003cp\u003eWestern blotting was performed to determine protein expressions in liver tissues and BMSCs hepatoid differentiated cells. Briefly, the concentrations of extracted cytosol protein were determined using the BCA protein assay kit (Thermo Fisher Scientific, OH, USA) Proteins were then separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto PVDF membranes. After blocking with 5% skim milk powder for at least 1h, membranes were incubated with specific primary antibodies overnight, followed by incubating with corresponding second antibodies for 1h. The bands were visualized by chemiluminescence using an electro-chemiluminescent detection kit (Thermo Fisher Scientific, OH, USA). The involved primary antibodies were listed in \u003cem\u003eTable S3.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFor immunofluorescence, BMSCs hepatoid differentiated cell climbing slices and fixed liver slides were incubated with primary antibodies overnight at 4℃. After that, slides were washed three times with PBS, incubated with DAPI and FITC-conjugated second antibodies for 1.5 h at 37\u0026deg;C, and observed under a fluorescence microscope. The involved primary antibodies were listed in \u003cem\u003eTable S3.\u003c/em\u003e\u003c/p\u003e\n\u003ch2\u003e4.12. Fluorescence in situ hybridization (FISH)\u003c/h2\u003e\n\u003cp\u003eFISH assay was performed as previously described\u003csup\u003e81\u003c/sup\u003e. The whole fetal rat was fixed within 10% paraformaldehyde and then embedded with paraffin to slice. Paraffin sections were dewaxed with xylene and rehydrated with ethanol at decreasing concentration gradient to perform FISH. Fluorescent-labeled probe for miR\u0026ndash;142\u0026ndash;3p (Genepharma, Shanghai, China) was applied during hybridization. FISH was performed using the RNAscope Multiplex Fluorescent Multiplex kit (Advanced Cell Diagnostics, California, USA) according to the manufacturer\u0026rsquo;s protocol and observed under a fluorescence microscope.\u003c/p\u003e\n\u003ch2\u003e4.13. Dual-luciferase reporter gene assay\u003c/h2\u003e\n\u003cp\u003eAfter 50~70% of confluence, cells were co-transfected with 2\u0026thinsp;\u0026mu;g\u0026thinsp;pMiR-report vector-RAB22\u0026thinsp;A/SNHG3 3\u0026prime;UTR and miR\u0026ndash;142\u0026ndash;3p using Lipofectamine 2000 for 6 h, then lysed for luciferase activity determination using Dual-luciferase Reporter Assay System. Luciferase reporter vectors were provided by GenePharma (Shanghai, China). All experiments above were repeated three times.\u003c/p\u003e\n\u003ch2\u003e4.14. Statistical analysis\u003c/h2\u003e\n\u003cp\u003ePrism 8.0 (GraphPad Software Inc., San Diego, CA, USA) was employed for statistical analysis. All data were expressed as Mean \u0026plusmn; S.\u0026nbsp;E.\u0026nbsp;M. values. Student\u0026rsquo;s \u003cem\u003et-\u003c/em\u003etest and one-way ANOVA were used between two groups and among three or more groups, respectively. \u003cem\u003eP \u0026lt; \u003c/em\u003e0.05 represented statistical significance.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAAV, adeno-associated virus; ACC, acetyl CoA carboxylase; ACLY, ATP-citrate lyase; ACSL4, acyl-CoA synthetase long chain family member 4; ALT, alanine transaminase; AST, aspartate transaminase; BMSCs, bone mesenchymal stem cells; cGAS, cyclic GMP-AMP synthase; CORT, corticosterone; CPT1α, carnitine palmitoyltransferase 1α; DOHaD, Developmental Origins of Health and Disease; FASN, fatty acid synthase; FISH, fluorescence in situ hybridization; GAPDH, glyceraldehyde–3-phosphate dehydrogenase; GD, gestational day; GR, glucocorticoid receptor; H\u0026amp;E, hematoxylin-eosin; HPA, hypothalamic pituitary adrenal; IL–1β, interleukin–1β; IL–6, interleukin–6; IUGR, intrauterine growth retardation; NAFLD, non-alcoholic fatty liver diseases; NAS, NAFLD activity score; NASH, non-alcoholic steatohepatitis; NF-κB, nuclear factor-k-gene binding; ORO, oil red O; PCE, prenatal caffeine exposure; POHaD, Paternal Origins of Health and Disease; PPARα, peroxisome proliferators activated receptor α; PPCE, paternal pre-pregnancy caffeine exposure; PW, postnatal week; ROS, reactive oxygen species; RT-qPCR, real-time polymerase chain reaction; RU, RU486 (mifepristone); SREBP1, sterol-regulatory element binding protein–1; STING, stimulator of interferon genes; TNF-α, tumor necrosis factor α.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding information\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (No. U22A20362, No. U23A21019) and the National Key Research and Development Program of China (No. 2020YFA0803900).\u003c/p\u003e\n\u003ch2\u003eConflicts of interest\u003c/h2\u003e\n\u003cp\u003eThe authors declared no conflict of interest.\u003c/p\u003e\n\u003ch2\u003eAuthors\u0026rsquo; contributions\u003c/h2\u003e\n\u003cp\u003eCong Zhang: conceptualization, data curation, and writing - original draft; Yu Guo: methodology and investigation; Yi Liu: software and visualization; Kexin Liu and Wen Hu: formal analysis; Hui Wang: project administration, funding acquisition, and writing - review \u0026amp; editing. All authors approved the manuscript for submission.\u003c/p\u003e\n\u003ch2\u003eData availability statement\u003c/h2\u003e\n\u003cp\u003eThe data presented in this study are available on request from the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eYounossi ZM, Koenig AB, Abdelatif D, Fazel Y, Henry L, Wymer M. Global epidemiology of nonalcoholic fatty liver disease-Meta-analytic assessment of prevalence, incidence, and outcomes. \u003cem\u003eHepatology\u003c/em\u003e \u003cstrong\u003e64\u003c/strong\u003e, 73-84 (2016).\u003c/li\u003e\n\u003cli\u003eEslam M, Valenti L, Romeo S. Genetics and epigenetics of NAFLD and NASH: Clinical impact. \u003cem\u003eJ Hepatol\u003c/em\u003e \u003cstrong\u003e68\u003c/strong\u003e, 268-279 (2018).\u003c/li\u003e\n\u003cli\u003eBarker DJ. Developmental origins of adult health and disease. \u003cem\u003eJ Epidemiol Community Health\u003c/em\u003e \u003cstrong\u003e58\u003c/strong\u003e, 114-115 (2004).\u003c/li\u003e\n\u003cli\u003eMcMillen IC, Robinson JS. Developmental origins of the metabolic syndrome: prediction, plasticity, and programming. \u003cem\u003ePhysiol Rev\u003c/em\u003e \u003cstrong\u003e85\u003c/strong\u003e, 571-633 (2005).\u003c/li\u003e\n\u003cli\u003eZou KX, Ding GL, Huang HF. Advances in research into gamete and embryo-fetal origins of adult diseases. \u003cem\u003eSci China Life Sci\u003c/em\u003e \u003cstrong\u003e62\u003c/strong\u003e, 360-368 (2019).\u003c/li\u003e\n\u003cli\u003eMonti M. Gamete and embryo-fetal origins of adult diseases. \u003cem\u003eEur J Histochem\u003c/em\u003e \u003cstrong\u003e60\u003c/strong\u003e, 2696 (2016).\u003c/li\u003e\n\u003cli\u003eDonkin I, Barres R. Sperm epigenetics and influence of environmental factors. \u003cem\u003eMol Metab\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 1-11 (2018).\u003c/li\u003e\n\u003cli\u003eBraun JM, Messerlian C, Hauser R. Fathers Matter: Why It\u0026apos;s Time to Consider the Impact of Paternal Environmental Exposures on Children\u0026apos;s Health. \u003cem\u003eCurr Epidemiol Rep\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 46-55 (2017).\u003c/li\u003e\n\u003cli\u003eGodschalk R\u003cem\u003e, et al.\u003c/em\u003e Paternal Exposure to Environmental Chemical Stress Affects Male Offspring\u0026apos;s Hepatic Mitochondria. \u003cem\u003eToxicol Sci\u003c/em\u003e \u003cstrong\u003e162\u003c/strong\u003e, 241-250 (2018).\u003c/li\u003e\n\u003cli\u003eYan S\u003cem\u003e, et al.\u003c/em\u003e Prenatal exposure to the Chinese famine and the risk of metabolic syndrome in adulthood across consecutive generations. \u003cem\u003eEur J Clin Nutr\u003c/em\u003e \u003cstrong\u003e74\u003c/strong\u003e, 1229-1236 (2020).\u003c/li\u003e\n\u003cli\u003eChang RC, Thomas KN, Bedi YS, Golding MC. Programmed increases in LXRalpha induced by paternal alcohol use enhance offspring metabolic adaptation to high-fat diet induced obesity. \u003cem\u003eMol Metab\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 161-172 (2019).\u003c/li\u003e\n\u003cli\u003eChang RC, Wang H, Bedi Y, Golding MC. Preconception paternal alcohol exposure exerts sex-specific effects on offspring growth and long-term metabolic programming. \u003cem\u003eEpigenetics Chromatin\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 1-17 (2019).\u003c/li\u003e\n\u003cli\u003eDe Jesus DF\u003cem\u003e, et al.\u003c/em\u003e Parental metabolic syndrome epigenetically reprograms offspring hepatic lipid metabolism in mice. \u003cem\u003eJ Clin Invest\u003c/em\u003e \u003cstrong\u003e130\u003c/strong\u003e, 2391-2407 (2020).\u003c/li\u003e\n\u003cli\u003eWatkins AJ\u003cem\u003e, et al.\u003c/em\u003e Paternal diet programs offspring health through sperm- and seminal plasma-specific pathways in mice. \u003cem\u003eP Natl Acad Sci USA\u003c/em\u003e \u003cstrong\u003e115\u003c/strong\u003e, 10064-10069 (2018).\u003c/li\u003e\n\u003cli\u003eClow A, Hamer M. The iceberg of social disadvantage and chronic stress: implications for public health. \u003cem\u003eNeurosci Biobehav Rev\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, 1 (2010).\u003c/li\u003e\n\u003cli\u003eWu L\u003cem\u003e, et al.\u003c/em\u003e Paternal Psychological Stress Reprograms Hepatic Gluconeogenesis in Offspring. \u003cem\u003eCell Metab\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 735-743 (2016).\u003c/li\u003e\n\u003cli\u003eZheng X\u003cem\u003e, et al.\u003c/em\u003e Sperm epigenetic alterations contribute to inter- and transgenerational effects of paternal exposure to long-term psychological stress via evading offspring embryonic reprogramming. \u003cem\u003eCell Discov\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 101 (2021).\u003c/li\u003e\n\u003cli\u003eBuerge, II, Poiger T, Muller MD, Buser HR. Caffeine, an anthropogenic marker for wastewater comtamination of surface waters. \u003cem\u003eEnviron Sci Technol\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 691-700 (2003).\u003c/li\u003e\n\u003cli\u003eDiogo JS, Silva LS, Pena A, Lino CM. Risk assessment of additives through soft drinks and nectars consumption on Portuguese population: a 2010 survey. \u003cem\u003eFood Chem Toxicol\u003c/em\u003e \u003cstrong\u003e62\u003c/strong\u003e, 548-553 (2013).\u003c/li\u003e\n\u003cli\u003eJensen TK, Swan SH, Skakkebaek NE, Rasmussen S, Jorgensen N. Caffeine intake and semen quality in a population of 2,554 young Danish men. \u003cem\u003eAm J Epidemiol\u003c/em\u003e \u003cstrong\u003e171\u003c/strong\u003e, 883-891 (2010).\u003c/li\u003e\n\u003cli\u003eShangguan Y\u003cem\u003e, et al.\u003c/em\u003e Glucocorticoid mediates prenatal caffeine exposure-induced endochondral ossification retardation and its molecular mechanism in female fetal rats. \u003cem\u003eCell Death Dis\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, e3157 (2017).\u003c/li\u003e\n\u003cli\u003eHe B\u003cem\u003e, et al.\u003c/em\u003e Prenatal caffeine exposure induces liver developmental dysfunction in offspring rats. \u003cem\u003eJ Endocrinol\u003c/em\u003e \u003cstrong\u003e242\u003c/strong\u003e, 211-226 (2019).\u003c/li\u003e\n\u003cli\u003eHe Z\u003cem\u003e, et al.\u003c/em\u003e H19/let-7 axis mediates caffeine exposure during pregnancy induced adrenal dysfunction and its multi-generation inheritance. \u003cem\u003eSci Total Environ\u003c/em\u003e \u003cstrong\u003e792\u003c/strong\u003e, 148440 (2021).\u003c/li\u003e\n\u003cli\u003ePei LG\u003cem\u003e, et al.\u003c/em\u003e The GC-IGF1 axis-mediated testicular dysplasia caused by prenatal caffeine exposure. \u003cem\u003eJ Endocrinol\u003c/em\u003e \u003cstrong\u003e242\u003c/strong\u003e, M17-M32 (2019).\u003c/li\u003e\n\u003cli\u003eGonzalez de Mejia E, Ramirez-Mares MV. Impact of caffeine and coffee on our health. \u003cem\u003eTrends Endocrinol Metab\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 489-492 (2014).\u003c/li\u003e\n\u003cli\u003eQuadra GR\u003cem\u003e, et al.\u003c/em\u003e A global trend of caffeine consumption over time and related-environmental impacts. \u003cem\u003eEnviron Pollut\u003c/em\u003e \u003cstrong\u003e256\u003c/strong\u003e, 113343 (2020).\u003c/li\u003e\n\u003cli\u003eBusada JT, Cidlowski JA. Mechanisms of Glucocorticoid Action During Development. \u003cem\u003eCurr Top Dev Biol\u003c/em\u003e \u003cstrong\u003e125\u003c/strong\u003e, 147-170 (2017).\u003c/li\u003e\n\u003cli\u003ePollard I. Increases in plasma concentrations of steroids in the rat after the administration of caffeine: comparison with plasma disposition of caffeine. \u003cem\u003eJ Endocrinol\u003c/em\u003e \u003cstrong\u003e119\u003c/strong\u003e, 275-280 (1988).\u003c/li\u003e\n\u003cli\u003eEyolfson E, Bhatt D, Wang M, Lohman AW, Mychasiuk R. Paternal exposure to exercise and/or caffeine and alcohol modify offspring behavioral and pathophysiological recovery from repetitive mild traumatic brain injury in adolescence. \u003cem\u003eGenes Brain Behav\u003c/em\u003e, egbb12736 (2021).\u003c/li\u003e\n\u003cli\u003eSchagdarsurengin U, Steger K. Epigenetics in male reproduction: effect of paternal diet on sperm quality and offspring health. \u003cem\u003eNat Rev Urol\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 584-595 (2016).\u003c/li\u003e\n\u003cli\u003eChen Q, Yan W, Duan E. Epigenetic inheritance of acquired traits through sperm RNAs and sperm RNA modifications. \u003cem\u003eNat Rev Genet\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 733-743 (2016).\u003c/li\u003e\n\u003cli\u003eCollignon J. miRNA in embryonic development: the taming of Nodal signaling. \u003cem\u003eDev Cell\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 458-460 (2007).\u003c/li\u003e\n\u003cli\u003eReza A\u003cem\u003e, et al.\u003c/em\u003e Roles of microRNAs in mammalian reproduction: from the commitment of germ cells to peri-implantation embryos. \u003cem\u003eBiol Rev Camb Philos Soc\u003c/em\u003e \u003cstrong\u003e94\u003c/strong\u003e, 415-438 (2019).\u003c/li\u003e\n\u003cli\u003eLiang G\u003cem\u003e, et al.\u003c/em\u003e microRNAs in aged sperm confer psychiatric symptoms to offspring through causing the dysfunction of estradiol signaling in early embryos. \u003cem\u003eCell Discov\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 63 (2022).\u003c/li\u003e\n\u003cli\u003eWang Y\u003cem\u003e, et al.\u003c/em\u003e Sperm microRNAs confer depression susceptibility to offspring. \u003cem\u003eSci Adv\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, (2021).\u003c/li\u003e\n\u003cli\u003ede Castro Barbosa T\u003cem\u003e, et al.\u003c/em\u003e High-fat diet reprograms the epigenome of rat spermatozoa and transgenerationally affects metabolism of the offspring. \u003cem\u003eMol Metab\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 184-197 (2016).\u003c/li\u003e\n\u003cli\u003eRodgers AB, Morgan CP, Leu NA, Bale TL. Transgenerational epigenetic programming via sperm microRNA recapitulates effects of paternal stress. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e112\u003c/strong\u003e, 13699-13704 (2015).\u003c/li\u003e\n\u003cli\u003eShort AK\u003cem\u003e, et al.\u003c/em\u003e Elevated paternal glucocorticoid exposure alters the small noncoding RNA profile in sperm and modifies anxiety and depressive phenotypes in the offspring. \u003cem\u003eTransl Psychiatry\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, e837 (2016).\u003c/li\u003e\n\u003cli\u003eMota M, Banini BA, Cazanave SC, Sanyal AJ. Molecular mechanisms of lipotoxicity and glucotoxicity in nonalcoholic fatty liver disease. \u003cem\u003eMetabolism\u003c/em\u003e \u003cstrong\u003e65\u003c/strong\u003e, 1049-1061 (2016).\u003c/li\u003e\n\u003cli\u003eLiu Z\u003cem\u003e, et al.\u003c/em\u003e XBP1 deficiency promotes hepatocyte pyroptosis by impairing mitophagy to activate mtDNA-cGAS-STING signaling in macrophages during acute liver injury. \u003cem\u003eRedox Biol\u003c/em\u003e \u003cstrong\u003e52\u003c/strong\u003e, 102305 (2022).\u003c/li\u003e\n\u003cli\u003eZhang Y, Shi J, Rassoulzadegan M, Tuorto F, Chen Q. Sperm RNA code programmes the metabolic health of offspring. \u003cem\u003eNat Rev Endocrinol\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 489-498 (2019).\u003c/li\u003e\n\u003cli\u003eYang Q\u003cem\u003e, et al.\u003c/em\u003e Highly sensitive sequencing reveals dynamic modifications and activities of small RNAs in mouse oocytes and early embryos. \u003cem\u003eSci Adv\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, e1501482 (2016).\u003c/li\u003e\n\u003cli\u003eImmler S. The sperm factor: paternal impact beyond genes. \u003cem\u003eHeredity (Edinb)\u003c/em\u003e \u003cstrong\u003e121\u003c/strong\u003e, 239-247 (2018).\u003c/li\u003e\n\u003cli\u003eDai J\u003cem\u003e, et al.\u003c/em\u003e Paternal nicotine exposure defines different behavior in subsequent generation via hyper-methylation of mmu-miR-15b. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 7286 (2017).\u003c/li\u003e\n\u003cli\u003eTomaszewski M, Burdan F, Olchowik G, Tomaszewska M. The effects of caffeine administered at different temperatures on foetal development. \u003cem\u003eAnn Agric Environ Med\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 148-152 (2016).\u003c/li\u003e\n\u003cli\u003eQian J, Chen Q, Ward SM, Duan E, Zhang Y. Impacts of Caffeine during Pregnancy. \u003cem\u003eTrends Endocrinol Metab\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e, 218-227 (2020).\u003c/li\u003e\n\u003cli\u003eJames JE, Kristjansson AL, Sigfusdottir ID. Adolescent substance use, sleep, and academic achievement: evidence of harm due to caffeine. \u003cem\u003eJ Adolesc\u003c/em\u003e \u003cstrong\u003e34\u003c/strong\u003e, 665-673 (2011).\u003c/li\u003e\n\u003cli\u003eWikoff D\u003cem\u003e, et al.\u003c/em\u003e Systematic review of the potential adverse effects of caffeine consumption in healthy adults, pregnant women, adolescents, and children. \u003cem\u003eFood Chem Toxicol\u003c/em\u003e \u003cstrong\u003e109\u003c/strong\u003e, 585-648 (2017).\u003c/li\u003e\n\u003cli\u003eBoskovic A, Rando OJ. Transgenerational Epigenetic Inheritance. \u003cem\u003eAnnu Rev Genet\u003c/em\u003e \u003cstrong\u003e52\u003c/strong\u003e, 21-41 (2018).\u003c/li\u003e\n\u003cli\u003eChen Q\u003cem\u003e, et al.\u003c/em\u003e Sperm tsRNAs contribute to intergenerational inheritance of an acquired metabolic disorder. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e351\u003c/strong\u003e, 397-400 (2016).\u003c/li\u003e\n\u003cli\u003eNapso T\u003cem\u003e, et al.\u003c/em\u003e Diet-induced maternal obesity impacts feto-placental growth and induces sex-specific alterations in placental morphology, mitochondrial bioenergetics, dynamics, lipid metabolism and oxidative stress in mice. \u003cem\u003eActa Physiol (Oxf)\u003c/em\u003e \u003cstrong\u003e234\u003c/strong\u003e, e13795 (2022).\u003c/li\u003e\n\u003cli\u003eLabialle S\u003cem\u003e, et al.\u003c/em\u003e The miR-379/miR-410 cluster at the imprinted Dlk1-Dio3 domain controls neonatal metabolic adaptation. \u003cem\u003eEMBO J\u003c/em\u003e \u003cstrong\u003e33\u003c/strong\u003e, 2216-2230 (2014).\u003c/li\u003e\n\u003cli\u003eYan S, Yang XF, Liu HL, Fu N, Ouyang Y, Qing K. Long-chain acyl-CoA synthetase in fatty acid metabolism involved in liver and other diseases: an update. \u003cem\u003eWorld J Gastroenterol\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 3492-3498 (2015).\u003c/li\u003e\n\u003cli\u003eSingh AB, Kan CFK, Kraemer FB, Sobel RA, Liu J. Liver-specific knockdown of long-chain acyl-CoA synthetase 4 reveals its key role in VLDL-TG metabolism and phospholipid synthesis in mice fed a high-fat diet. \u003cem\u003eAm J Physiol Endocrinol Metab\u003c/em\u003e \u003cstrong\u003e316\u003c/strong\u003e, E880-E894 (2019).\u003c/li\u003e\n\u003cli\u003eChen J\u003cem\u003e, et al.\u003c/em\u003e ACSL4 reprograms fatty acid metabolism in hepatocellular carcinoma via c-Myc/SREBP1 pathway. \u003cem\u003eCancer Lett\u003c/em\u003e \u003cstrong\u003e502\u003c/strong\u003e, 154-165 (2021).\u003c/li\u003e\n\u003cli\u003eDuan J\u003cem\u003e, et al.\u003c/em\u003e Therapeutic targeting of hepatic ACSL4 ameliorates NASH in mice. \u003cem\u003eHepatology \u003c/em\u003e\u003cstrong\u003e75\u003c/strong\u003e, 140-153 (2021).\u003c/li\u003e\n\u003cli\u003eArroyave-Ospina JC, Wu Z, Geng Y, Moshage H. Role of Oxidative Stress in the Pathogenesis of Non-Alcoholic Fatty Liver Disease: Implications for Prevention and Therapy. \u003cem\u003eAntioxidants (Basel)\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 174 (2021).\u003c/li\u003e\n\u003cli\u003eYu Y, Liu Y, An W, Song J, Zhang Y, Zhao X. STING-mediated inflammation in Kupffer cells contributes to progression of nonalcoholic steatohepatitis. \u003cem\u003eJ Clin Invest\u003c/em\u003e \u003cstrong\u003e129\u003c/strong\u003e, 546-555 (2019).\u003c/li\u003e\n\u003cli\u003eOses M\u003cem\u003e, et al.\u003c/em\u003e Peripheral blood mononuclear cells-expressed miRNA profiles derived from children with metabolic-associated fatty liver disease and insulin resistance. \u003cem\u003ePediatr Obes\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, e12966 (2022).\u003c/li\u003e\n\u003cli\u003eStanford KI\u003cem\u003e, et al.\u003c/em\u003e Paternal Exercise Improves Glucose Metabolism in Adult Offspring. \u003cem\u003eDiabetes\u003c/em\u003e \u003cstrong\u003e67\u003c/strong\u003e, 2530-2540 (2018).\u003c/li\u003e\n\u003cli\u003eSilva L, Pinheiro-Castro N, Novaes GM, Pascoal GFL, Ong TP. Bioactive food compounds, epigenetics and chronic disease prevention: Focus on early-life interventions with polyphenols. \u003cem\u003eFood Res Int\u003c/em\u003e \u003cstrong\u003e125\u003c/strong\u003e, 108646 (2019).\u003c/li\u003e\n\u003cli\u003ePataia V\u003cem\u003e, et al.\u003c/em\u003e Paternal cholestasis exacerbates obesity-associated hypertension in male offspring but is prevented by paternal ursodeoxycholic acid treatment. \u003cem\u003eInt J Obes (Lond)\u003c/em\u003e \u003cstrong\u003e43\u003c/strong\u003e, 319-330 (2019).\u003c/li\u003e\n\u003cli\u003eal\u0026apos;Absi M, Lovallo WR, McKey B, Sung BH, Whitsett TL, Wilson MF. Hypothalamic-pituitary-adrenocortical responses to psychological stress and caffeine in men at high and low risk for hypertension. \u003cem\u003ePsychosom Med\u003c/em\u003e \u003cstrong\u003e60\u003c/strong\u003e, 521-527 (1998).\u003c/li\u003e\n\u003cli\u003eSoldin OP, Makambi KH, Soldin SJ, O\u0026apos;Mara DM. Steroid hormone levels associated with passive and active smoking. \u003cem\u003eSteroids\u003c/em\u003e \u003cstrong\u003e76\u003c/strong\u003e, 653-659 (2011).\u003c/li\u003e\n\u003cli\u003eGianoulakis C, Dai X, Brown T. Effect of chronic alcohol consumption on the activity of the hypothalamic-pituitary-adrenal axis and pituitary beta-endorphin as a function of alcohol intake, age, and gender. \u003cem\u003eAlcohol Clin Exp Res\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 410-423 (2003).\u003c/li\u003e\n\u003cli\u003eYokota Y, Yokota H, Yokota M, Araki Y, Araki Y. In vitro fertilization embryo development from caffeine-treated murine sperm. \u003cem\u003eReprod Med Biol\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 117-121 (2015).\u003c/li\u003e\n\u003cli\u003eZhang W, Yang J, Lv Y, Li S, Qiang M. Paternal benzo[a]pyrene exposure alters the sperm DNA methylation levels of imprinting genes in F0 generation mice and their unexposed F1-2 male offspring. \u003cem\u003eChemosphere\u003c/em\u003e \u003cstrong\u003e228\u003c/strong\u003e, 586-594 (2019).\u003c/li\u003e\n\u003cli\u003eCarone BR\u003cem\u003e, et al.\u003c/em\u003e Paternally induced transgenerational environmental reprogramming of metabolic gene expression in mammals. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e143\u003c/strong\u003e, 1084-1096 (2010).\u003c/li\u003e\n\u003cli\u003eSoubry A\u003cem\u003e, et al.\u003c/em\u003e Obesity-related DNA methylation at imprinted genes in human sperm: Results from the TIEGER study. \u003cem\u003eClin Epigenetics\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 51 (2016).\u003c/li\u003e\n\u003cli\u003eHu S\u003cem\u003e, et al.\u003c/em\u003e Prenatal caffeine exposure increases the susceptibility to non-alcoholic fatty liver disease in female offspring rats via activation of GR-C/EBPalpha-SIRT1 pathway. \u003cem\u003eToxicology\u003c/em\u003e \u003cstrong\u003e417\u003c/strong\u003e, 23-34 (2019).\u003c/li\u003e\n\u003cli\u003eWang L\u003cem\u003e, et al.\u003c/em\u003e Intrauterine metabolic programming alteration increased susceptibility to non-alcoholic adult fatty liver disease in prenatal caffeine-exposed rat offspring. \u003cem\u003eToxicol Lett\u003c/em\u003e \u003cstrong\u003e224\u003c/strong\u003e, 311-318 (2014).\u003c/li\u003e\n\u003cli\u003eHu S\u003cem\u003e, et al.\u003c/em\u003e Caffeine programs hepatic SIRT1-related cholesterol synthesis and hypercholesterolemia via A2AR/cAMP/PKA pathway in adult male offspring rats. \u003cem\u003eToxicology\u003c/em\u003e \u003cstrong\u003e418\u003c/strong\u003e, 11-21 (2019).\u003c/li\u003e\n\u003cli\u003eHong S, Zheng G, Wu X, Snider NT, Owyang C, Wiley JW. Corticosterone mediates reciprocal changes in CB 1 and TRPV1 receptors in primary sensory neurons in the chronically stressed rat. \u003cem\u003eGastroenterology\u003c/em\u003e \u003cstrong\u003e140\u003c/strong\u003e, 627-637 e624 (2011).\u003c/li\u003e\n\u003cli\u003eDalsgaard T\u003cem\u003e, et al.\u003c/em\u003e Improved Lentiviral Gene Delivery to Mouse Liver by Hydrodynamic Vector Injection through Tail Vein. \u003cem\u003eMol Ther-Nucl Acids\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 672-683 (2018).\u003c/li\u003e\n\u003cli\u003eJia YP\u003cem\u003e, et al.\u003c/em\u003e Melatonin supplementation in the culture medium rescues impaired glucose metabolism in IVF mice offspring. \u003cem\u003eJournal of Pineal Research\u003c/em\u003e \u003cstrong\u003e72\u003c/strong\u003e, e12778 (2022).\u003c/li\u003e\n\u003cli\u003eLi L\u003cem\u003e, et al.\u003c/em\u003e miR-148a/LDLR mediates hypercholesterolemia induced by prenatal dexamethasone exposure in male offspring rats. \u003cem\u003eToxicol Appl Pharm\u003c/em\u003e \u003cstrong\u003e395\u003c/strong\u003e, 114979 (2020).\u003c/li\u003e\n\u003cli\u003eKumar A, Kumar V, Rattan V, Jha V, Pal A, Bhattacharyya S. Molecular spectrum of secretome regulates the relative hepatogenic potential of mesenchymal stem cells from bone marrow and dental tissue. \u003cem\u003eSci Rep-Uk\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 15015 (2017).\u003c/li\u003e\n\u003cli\u003eRattan S\u003cem\u003e, et al.\u003c/em\u003e Prenatal and ancestral exposure to di(2-ethylhexyl) phthalate alters gene expression and DNA methylation in mouse ovaries. \u003cem\u003eToxicol Appl Pharm\u003c/em\u003e \u003cstrong\u003e379\u003c/strong\u003e, 114629 (2019).\u003c/li\u003e\n\u003cli\u003eKleiner DE\u003cem\u003e, et al.\u003c/em\u003e Design and validation of a histological scoring system for nonalcoholic fatty liver disease. \u003cem\u003eHepatology\u003c/em\u003e \u003cstrong\u003e41\u003c/strong\u003e, 1313-1321 (2005).\u003c/li\u003e\n\u003cli\u003eDu YZ\u003cem\u003e, et al.\u003c/em\u003e Pirfenidone alleviates lipopolysaccharide-induced lung injury by accentuating BAP31 regulation of ER stress and mitochondrial injury. \u003cem\u003eJ Autoimmun\u003c/em\u003e \u003cstrong\u003e112\u003c/strong\u003e, 102464 (2020).\u003c/li\u003e\n\u003cli\u003eFitz NF\u003cem\u003e, et al.\u003c/em\u003e Trem2 deficiency differentially affects phenotype and transcriptome of human APOE3 and APOE4 mice. \u003cem\u003eMolecular Neurodegeneration\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 1-21 (2020).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003e\u003cstrong\u003eTable 1. \u003c/strong\u003eSimilarities and differences between paternal pre-pregnancy caffeine exposure (PPCE) and prenatal caffeine exposure (PCE) rat models\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 480px;\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eIndicators\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 224px;\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003ePPCE\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 221.969px;\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003ePCE\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMale\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFemale\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 159px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMale\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.9688px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFemale\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 480px;\"\u003e\n \u003cp\u003eCaffeine dose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 224px;\" colspan=\"2\"\u003e\n \u003cp\u003e15~60 mg/kg/d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 221.969px;\" colspan=\"2\"\u003e\n \u003cp\u003e30~120 mg/kg/d\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 480px;\"\u003e\n \u003cp\u003eGender with severe disease phenomena\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 224px;\" colspan=\"2\"\u003e\n \u003cp\u003eMale\u0026nbsp;\u0026gt;\u0026nbsp;Female\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 221.969px;\" colspan=\"2\"\u003e\n \u003cp\u003eMale\u0026nbsp;\u0026lt;\u0026nbsp;Female\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 480px;\"\u003e\n \u003cp\u003eWhether a second strike is needed\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 159px;\"\u003e\n \u003cp\u003eY\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.9688px;\"\u003e\n \u003cp\u003eY\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 480px;\"\u003e\n \u003cp\u003eLipogenesis (\u003cem\u003eSrebp1, Fasn, Acc\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003e\u0026shy;\u0026shy;\u0026uarr;\u0026uarr;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e\u0026shy;\u0026uarr;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 159px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.9688px;\"\u003e\n \u003cp\u003e\u0026shy;\u0026uarr;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 480px;\"\u003e\n \u003cp\u003eFatty acid \u0026beta;-oxidation (\u003cem\u003ePpar\u0026alpha;, Cpt1\u0026alpha;\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003e\u0026darr;\u0026darr;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e\u0026darr;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 159px;\"\u003e\n \u003cp\u003e\u0026darr;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.9688px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 480px;\"\u003e\n \u003cp\u003eInflammation (\u003cem\u003eTnf-\u0026alpha;, Il-6, Il-1\u0026beta;\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003e\u0026shy;\u0026uarr;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 159px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.9688px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 480px;\"\u003e\n \u003cp\u003eFibrosis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003eY\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 159px;\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.9688px;\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNote: N, no; Y, yes; \u0026uarr;\u0026shy;, increase; \u0026darr;, reduce; -, no change or not detected\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"paternal pre-pregnant caffeine exposure, non-alcoholic steatohepatitis, miR-142-3p, ACSL4, sperm reprogramming","lastPublishedDoi":"10.21203/rs.3.rs-3833743/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3833743/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlthough epidemiological data suggest a strong association between paternal adverse environmental exposure and susceptibility to multiple diseases in offspring, the sperm-to-liver pathway involved in offspring disease is complicated and worthy of further exploration. Caffeine contained in many beverages is regarded as a chronic stressor, and exerts reproductive and developmental toxicity. Effects of paternal pre-pregnant caffeine exposure (PPCE) on the long-term health of offspring and the underlying mechanisms remain unclear. This study innovatively reported the occurrence and transgenerational inheritance of PPCE-induced non-alcoholic steatohepatitis (NASH) in offspring, and aimed to elucidate its sperm reprogramming mechanism and the potential intervention targets. Male rats were administrated with caffeine (15\u0026thinsp;~\u0026thinsp;60 mg/kg/d) by gavage for 8 weeks before mating. Symptoms of NASH were found in two successive generations of male rats (F1 and F2) whose fathers or grandfathers (F0) were exposed to caffeine. RNA-seq was employed to screen out a novel miRNA mediating equilibrant of liver fatty metabolism: miR-142-3p. Role of sperm miR-142-3p in PPCE-induced offspring NAFLD was validated by \u003cem\u003ein vitro\u003c/em\u003e fertilization of the sperm of PPCE or miR-142-3p\u003csup\u003eKO\u003c/sup\u003e sperm with normal oocytes. Overexpression of miR-142-3p in offspring liver reversed NASH manifestation in PPCE male offspring. We further proved that caffeine-induced paternal chronic stress (high glucocorticoid level) but not caffeine itself is the main cause of methylation changes in sperm and offspring NAFLD, \u003cem\u003evia\u003c/em\u003e experiments \u003cem\u003ein vitro\u003c/em\u003e and glucocorticoid receptor blockade. Moreover, the linkage between serum high glucocorticoids and sperm miR-142-3p low programming was also verified in clinical samples. Overall, we demonstrated for the first time that PPCE induced NASH in offspring with transgenerational inheritance, confirmed the reprogramming mechanism of sperm miR-142-3p, and identified miR-142-3p as a potential intervention target for paternal-derived NASH.\u003c/p\u003e","manuscriptTitle":"Sperm miR-142-3p reprogramming mediates paternal stress-induced non-alcoholic steatohepatitis in offspring rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-06 19:17:00","doi":"10.21203/rs.3.rs-3833743/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f524957e-29bd-4bd7-bc04-ecb5df76133b","owner":[],"postedDate":"February 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":28065843,"name":"Health sciences/Gastroenterology/Hepatology/Liver diseases"},{"id":28065844,"name":"Biological sciences/Developmental biology/Epigenetic memory"}],"tags":[],"updatedAt":"2024-02-06T19:17:01+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-06 19:17:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3833743","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3833743","identity":"rs-3833743","version":["v1"]},"buildId":"zQwnuV7TCBrMSSSToR1PI","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}