Smad4 deficiency in hepatocytes attenuates NAFLD progression via inhibition of lipogenesis and macrophage polarization

Smad4 deficiency in hepatocytes attenuates NAFLD progression via inhibition of lipogenesis and macrophage polarization | 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 Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Smad4 deficiency in hepatocytes attenuates NAFLD progression via inhibition of lipogenesis and macrophage polarization Jinhua Zhang, Wei Yang, Xuanxuan Yan, Xin Xin, Shuang Ge, Yongxiang Zhao, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4507474/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 31 Jan, 2025 Read the published version in Cell Death & Disease → Version 1 posted 10 You are reading this latest preprint version Abstract Nonalcoholic fatty liver disease (NAFLD), a major cause of chronic liver disorders, has become a serious public health issue. Although the Smad4 signaling pathway has been implicated in the progression of NAFLD, the specific role of Smad4 in hepatocytes in NAFLD pathogenesis remains unclear. Hepatocyte-specific knockout Smad4 mice (Alb Smad4-/- ) were first constructed using the Cre-Loxp recombinant system to establish a high-fat diet induced NAFLD model. The role of Smad4 in the occurrence and development of NAFLD was determined by monitoring the body weight of mice, detecting triglycerides and free fatty acids in serum and liver tissue homogenates, staining the tissue sections to observe the accumulation of liver fat, and RT-qPCR detecting the expression of genes related to lipogenesis, fatty acid intake and fatty acid β oxidation. The molecular mechanism of Smad4 in hepatocytes affecting NAFLD was therefore investigated through combining in vitro and in vivo experiments. Smad4 deficiency in hepatocytes mitigated NAFLD progression and decreased inflammatory cells infiltration. Moreover, Smad4 deficiency inhibited CXCL1 secretion by suppressing the activation of the ASK1/P38/JNK signaling pathway. Furthermore, targeting CXCL1 using CXCR2 inhibitors diminished hepatocyte lipogenesis and inhibited the polarization of M1-type macrophages. Collectively, these results suggested that Smad4 plays a vital role in exacerbating NAFLD and may be a promising candidate for anti-NAFLD therapy. Biological sciences/Cell biology/Mechanisms of disease Biological sciences/Immunology/Cytokines Samd4 nonalcoholic fatty liver disease hepatocyte lipogenesis CXCL1 macrophage polarization. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Nonalcoholic fatty liver disease (NAFLD) is characterized by the excessive accumulation of lipids within hepatocytes, due to factors other than alcohol consumption and other definitive liver damage sources 1 . NAFLD is a complex, multifactorial condition influenced by both environmental factors and genetic predispositions 2 . It encompasses a spectrum of pathological liver conditions of varying severity, ranging from isolated hepatic steatosis (NAFL) to steatohepatitis (NASH), which can further progress to liver fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) 3 . With an estimated prevalence in nearly one-third of the global adult population, NAFLD has become a public health concern 4 . Hepatocytes, the main functional units of the liver, play a pivotal role in biotransformation, metabolism, and detoxification. During NAFLD progression, there is an excessive accumulation of triglyceride, free fatty acid (FFA), and cholesterol in hepatocytes, a process that is associated with insulin resistance that can lead to dysfunctional triglyceride synthesis and transport 5 . The excessive lipid accumulation induces lipotoxicity, which impairs mitochondrial function, triggers endoplasmic reticulum (ER) stress, and triggers inflammatory responses in hepatocytes due to reactive oxygen species (ROS)-induced inflammation 6 , 7 . Concurrently, FFA accumulation in hepatocytes enhances mitochondrial β-oxidation, sensitizing the liver to oxidative stress and thereby exacerbating liver damage 8 . Moreover, lipotoxicity can disrupt the JNK pathway and Toll-like receptor cellular signaling pathways, thereby affecting hepatocyte metabolism 9 , 10 . Liver macrophages comprise recruited monocyte-derived macrophages and resident Kupffer cells within the liver. Liver macrophages play a central role in the regulation of hepatocyte metabolism and maintenance of hepatic immunological tolerance 11 , 12 . They can promote the progression of metabolic diseases by enhancing insulin resistance, hepatic steatosis, and oxidative stress in obese mice and rats 13 , 14 . Under normal physiological conditions, liver macrophages exhibit a tendency toward the M2 phenotype, which suppresses inflammation by secreting interleukin (IL)-4 and IL-13 15, 16 . However, during NAFLD progression, hepatic stellate cells (HSC) activation triggers the secretion of pro-inflammatory cytokines, which increases the levels of lipopolysaccharide (LPS) in the blood. And high levels of FFAs activate inflammasomes. These changes stimulate the onset of inflammation, with a concomitant increase in the proportion of M1 macrophages secreting pro-inflammatory cytokines 17 , 18 , 19 . The transforming growth factor beta (TGF-β) signaling pathway plays important roles in biological processes of cell growth, apoptosis, migration, and cancer development and progression 19 . Smad acts as a downstream signaling molecule in the TGF-β signaling pathway 20 . In mammals, eight different SMADs are further divided into three distinct classes: R-Smad (Smad1, 2, 3, 5, and 8), Co-Smad (Smad4), and I-Smad (Smad6 and 7) 21 . Smad4 is a central mediator of TGF-β signaling, which binds to nearly all Smad proteins regulated by activated receptors and helps regulate the expression of various downstream genes 22 . Hepatocyte Smad4 expression levels increase progressively as normal liver tissue progresses to NAFLD and finally to NASH 23 , 24 . Smad4 deletion attenuates inflammation, fibrosis, and hepatocyte apoptosis in NASH 20 . After the administration of high-fat diet (HFD), Smad4 deletion in pancreatic β-cells improves blood glucose levels, insulin secretion, and glucose tolerance in obese mice 25 . Although other studies have investigated the role of Smad4 in liver disease, its specific molecular mechanism in hepatocytes in NAFLD remains unclear. In this study, we examined the specific role of Smad4 in NAFLD progression using a mouse model with hepatocyte-specific Smad4 deletion. Our findings revealed that Smad4 deficiency in hepatocytes attenuates NAFLD development. Moreover, hepatocyte Smad4 was found to amplify CXCL1 secretion by facilitating the activation of the ASK1/P38/JNK signaling pathway. CXCL1, in turn, promotes hepatocyte lipogenesis and macrophage M1-type polarization via CXCR2 binding. RESULTS Smad4 expression in hepatocytes is upregulated during NAFLD progression To elucidate the relationship between Smad4 expression and NASH, the expression of Smad4 in healthy liver and NASH tissues from patients was assessed by tissue microarray using immunohistochemistry. We observed that Smad4 expression was significantly upregulated in NASH tissues compared to that in healthy controls (Fig. 1 A, B). Moreover, we analyzed the publicly available Gene Expression Omnibus dataset GSE164760 and compared Smad4 expression between healthy and NASH tissues. The analysis revealed a significant increase in Smad4 mRNA levels in the NASH group compared to healthy controls (Fig. 1 C). To corroborate these findings, we used a short-term HFD mouse model of NAFLD (Fig. 1 D). Immunohistochemical staining demonstrated a significant upregulation of Smad4 expression in the hepatocytes of fatty liver tissues (Fig. 1 E). Double immunofluorescence staining confirmed that Smad4 was expressed in most hepatocytes ( Fig. 1 F). Consistent with this, Western blot analysis indicated a significant increase in Smad4 protein levels in fatty liver tissues of HFD-treated mice (Fig. 1 G). Collectively, these results suggest that Smad4 is activated in hepatocytes during NAFLD, indicating a potentially crucial role for Smad4 in NAFLD pathogenesis. Hepatocyte-specific deletion of Smad4 attenuates high-fat diet-induced non-alcoholic fatty liver disease. To further investigate the role of Smad4 in hepatocytes during NAFLD, we used a conditional Smad4 deletion approach in murine hepatocytes, as previously described 26 . We generated hepatocyte-specific Smad4 knockout mice (Albumin-cre; Smad4 flox/flox , Alb Smad4−/− ) by crossing mice carrying the Loxp-flanked Smad4 allele with Albumin-cre mice. The Alb Smad4−/− mice were born at the expected Mendelian ratio, viable, and fertile. We used Smad4 fl/fl littermates as controls. Smad4 deletion in primary hepatocytes of Alb Smad4−/− mice was confirmed using double immunofluorescence staining (Fig. 2 A). To delineate the role of Smad4 in NAFLD, we established a HFD-induced NAFLD model using Alb Smad4−/− mice and their control littermates. In response to HFD feeding, Alb Smad4−/− mice demonstrated a less pronounced increase in both body and liver weight compared with control littermates (Fig. 2 B, C). After 3 months of HFD, Alb Smad4−/− mice displayed lower serum alanine aminotransferase (ALT), triglyceride (TG), and non-esterified fatty acid (NEFA) levels, while serum aspartate aminotransferase (AST) and total cholesterol (TC) levels were similar between Alb Smad4−/− and control mice (Fig. 2 D). Furthermore, hepatic TG and NEFA levels were notably reduced in Alb Smad4−/− mice compared with Smad4 fl/fl mice (Fig. 2 E). Alb Smad4−/− mice also exhibited impaired glucose tolerance compared with Smad4 fl/fl mice (Fig. 2 F). The absence of Smad4 in hepatocytes in Alb Smad4−/− mice was further validated using double immunofluorescence staining and Western blot (Fig. 2 G, H). Deletion of Smad4 in hepatocytes resulted in decreased fat accumulation, as evidenced by hematoxylin and eosin and Oil Red O staining. No significant differences were observed between Alb Smad4−/− and Smad4 fl/fl mice who were fed a normal diet (Fig. 2 G). Collectively, these data suggest that hepatocyte-specific Smad4 deficiency attenuates the development of HFD-induced NAFLD. Smad4 deficiency in hepatocytes attenuated liver inflammation and CXCL1 secretion To investigate whether Smad4 modulates liver inflammatory cell infiltration and hepatocyte proliferation, we stained liver tissues of Alb Smad4−/− and Smad4 fl/fl mice via immunofluorescence. The infiltration of F4/80 + macrophages and CD11b + monocytes was diminished in Alb Smad4−/− mice compared with that in Smad4 fl/fl mice when fed a HFD. When fed the normal diet, immune cell infiltration was similar between Alb Smad4−/− and control mice (Fig. 3 A). During NAFLD progression, activated chemokines evoke multiple cellular and tissue responses, including hepatocyte proliferation, activation, necrosis, angiogenesis, and immune cell recruitment 27 , 28 . Notably, CXCL1 is a key gene involved in NAFLD progression. Therefore, we examined CXCL1 expression in the livers of Alb Smad4−/− and Smad4 fl/fl mice through double immunofluorescence staining. CXCL1 levels were significantly lower in the livers of Alb Smad4−/− mice than those in Smad4 fl/fl mice (Fig. 3 B). This decrease in CXCL1 levels was further confirmed using quantitative reverse transcription polymerase chain reaction (RT-qPCR) in hepatocytes of NAFLD of Alb Smad4−/− mice (Fig. 3 C). To further clarify the function of Smad4 in hepatocytes, we knocked down Smad4 in AML12 cells using siRNA and verified Smad4 protein levels using Western blot (Fig. 3 D). The cells were then treated with palmitic acid (PA) for 24 h to simulate an in vitro NAFLD environment. RT-qPCR analysis revealed that Smad4 deficiency mitigated the PA-induced CXCL1 expression in hepatocytes (Fig. 3 E). The protein expression level of CXCL1 was further analyzed using enzyme-linked immunosorbent assay (ELISA) with AML12-conditioned medium (CM). Smad4 deficiency partly reduced the secretion of CXCL1 in PA-induced AML12 CM (Fig. 3 F). In addition, AML12 cells were transfected with a lentiviral vector to knock down Smad4, and the expression levels of Smad4 protein were measured using Western blot (Fig. 3 G). In line with previous results, both the expression and secretion of CXCL1 in PA-stimulated Smad4-knockdown AML12 cells were significantly reduced compared to those in control cells (Figs. 3 H, I). Taken together, these results suggest that hepatocyte-specific deletion of Smad4 lessens liver inflammation and CXCL1 secretion. Hepatocyte Smad4 promotes CXCL1 secretion via the ASK1-P38-JNK signaling pathway Previous studies have highlighted the involvement of the JNK and p38 MAPK cascades in the regulation of CXCL1 secretion 29 . Accordingly, we examined the levels of total and phosphorylated proteins involved in ASK1, P38, and JNK signaling. We found that the expression of phosphorylated ASK1 (p-ASK1), p38 (p-p38), and JNK (p-JNK) was diminished in the liver tissue of HFD-treated Alb Smad4−/− mice compared with Smad4 fl/fl mice (Fig. 4 A). In subsequent experiments, we used si-Smad4 and sh-Smad4 to knock down Smad4 in AML12 cells, which were then exposed to PA. The Western blot analysis showed that the ASK1, P38, and JNK signaling pathways were activated in hepatocytes in response to PA administration. However, this activation was remarkably suppressed by Smad4 knockdown, suggesting that Smad4 knockdown considerably inhibited PA-induced activation of the ASK1–P38–JNK pathway in vitro (Figs. 4 B, C). To further elucidate the signaling pathways involved in the induction of CXCL1 secretion by Smad4, we employed the JNK inhibitor (SP600125) and the p38 MAPK inhibitor (SB203580). Both inhibitors suppressed PA-induced CXCL1 secretion and mRNA expression, as evidenced using RT–qPCR and ELISA analysis (Figs. 4 D, E). In conclusion, these results suggest that Smad4 facilitates CXCL1 secretion via the ASK1-P38-JNK signaling pathways during NAFLD progression. CXCL1 promotes fatty acid synthesis in hepatocytes by binding to CXCR2 To further investigate the role of Smad4 in hepatic lipid deposition, RT-qPCR was used to determine the expression levels of genes involved in fatty acid synthesis and consumption. We found that Smad4 deficiency considerably inhibited the expression of genes critical for fatty acid synthesis (ACC1, FASN, and SCD1) and fatty acid binding protein 1 (FABP1). However, we observed no substantial differences in the expression of genes related to fatty acid uptake (FATP1) and fatty acid β-oxidation (CPT1a and ACOX1) (Fig. 5 A). To corroborate these findings, we isolated primary hepatocytes from Alb Smad4−/− and Smad4 fl/fl mice and then exposed them to PA. Following Smad4 knockout, the expression of genes responsible for fatty acid synthesis (ACC1, FASN, and PPAR-γ) was substantially reduced in primary hepatocytes (Fig. 5 B). We further observed decreased lipid deposition in primary hepatocytes of Alb Smad4−/− mice compared with that in Smad4 fl/fl mice following PA treatment, as evidenced by Oil Red O staining ( Fig. 5 C ) . This conclusion was confirmed using si-Smad4 AML12 and sh-Smad4 AML12 cells (Figs. 5 D-G). Taken together, these findings suggest that hepatocyte Smad4 promotes NAFLD development primarily through fatty acid synthesis. CXCL1 binds to specific receptor CXCR2. We then examined whether CXCL1-induced fatty acid synthesis occured in hepatocytes. We assessed the expression levels of CXCR2 in hepatocytes using Western blot (Fig. 5 H). To further elucidate the molecular mechanisms underlying the effects of CXCL1 on fatty acid synthesis, we cultured AML12 cells with CXCL1 recombinant protein and analyzed the expressions of genes related to fatty acid synthesis using RT-qPCR. We observed that ACC1, FASN, and SCD1 were substantially upregulated by CXCL1. However, when CXCR2 activation was inhibited by the CXCR2 inhibitor, SB225002, in AML12 cells, CXCL1-induced ACC1, FASN, and SCD1 expression was reduced (Fig. 5 I). These results suggest that CXCL1 induces fatty acid synthesis in hepatocytes via CXCR2. CXCL1 promotes macrophage M1 polarization Aberrant lipid-mediated hepatic inflammatory-immune dysfunction and chronic low-grade inflammation play important roles in NAFLD pathogenesis. Macrophage polarization is an important mechanism that regulates inflammatory responses 30 . Therefore, we assessed the quantities of CD86 + (M1 marker) and CD206 + (M2 marker) macrophages in NAFLD tissues. Our findings revealed a substantial decrease in M1 macrophages in Alb Smad4−/− mice compared with Smad4 fl/fl mice, while M2-like macrophages remained comparable (Figs. 6 A, B). We further evaluated the expression levels of genes associated with M1-like (IL-6, MCP1, and TNF-α) and M2-like (Arg1, IL-10, and YM1) phenotypes using RT-qPCR and found similar results (Figs. 6 C, D). Given that the absence of hepatocyte Smad4 resulted in diminished CXCL1 secretion, we postulated that hepatocyte Smad4 might facilitate macrophage M1 polarization via CXCL1. We confirmed the expression of CXCR2 in macrophages using western blot (Fig. 6 E). We cultured RAW264.7 cells with LPS and IFN-γ for 24 h to induce M1 polarization. It was found that LPS and IFN-γ activated the expression of CD86 in macrophages in comparison with the control group, CXCL1 recombinant proteins further upregulated CD86. This expression was further augmented by the addition of CXCL1 recombinant proteins, as evidenced by immunofluorescence staining. However, the treatment of RAW264.7 cells with the CXCR2 inhibitor SB225002 eliminated CXCL1-induced expression of CD86 (Fig. 6 F). Following M1 polarization induction, the expression levels of iNOS, MCP1, and TNF-α were significantly increased in RAW264.7 cells compared with those in the control group, and the addition of exogenous CXCL1 recombinant protein further amplified M1-related gene expression (Fig. 6 G). For M2 polarization, RAW264.7 cells were cultured with IL-4 and IL-13 for 48 h. This treatment activated the expression of Arg1, IL-10, and YM1 in macrophages compared with the control group. No significant differences were observed after the administration of CXCL1 and SB225002 (Fig. 6 H). These findings suggest that CXCL1 promotes the M1-type polarization of macrophages via CXCR2. DISCUSSION Smad4 is a general mediator of the TGF-β and bone morphogenetic protein (BMP) signaling pathways, which significantly contribute to intracellular signal transduction and a myriad of cellular processes 19 . However, owing to its ubiquitous expression, the specific role and molecular mechanism of Smad4-mediated signaling in NAFLD progression remain elusive. Our study illustrated that hepatocyte-specific Smad4 expression promoted NAFLD development by enhancing CXCL1 secretion. Targeted deficiency of hepatocyte-specific Smad4 signaling curbed the progression of NAFLD in HFD-fed mice. Hepatocyte-specific genetic deficiency of Smad4 inhibited fatty acid synthesis and macrophage M1 polarization. Moreover, Smad4 in hepatocytes accelerated CXCL1 secretion to enhance fatty acid synthesis and macrophage M1 polarization by activating the ASK-P38-JNK signaling pathway. NAFLD is now recognized as a steatotic liver disease closely associated with metabolic syndrome. The relationship between NAFLD and liver inflammation has been extensively studied. Several studies indicate the pivotal role of TGF β/Smad signaling in metabolic syndrome and related disorders 31 , 32 . Our previous study demonstrated that the targeted knockout of Smad4 in hepatocytes attenuates hepatic inflammatory cell infiltration and fibrosis during the progression of CCl 4 induced liver fibrosis 26 . Disruption of the Smad4 pathway alleviated spontaneous liver injury, hepatic inflammatory cell infiltration, fibrosis, and HCC induced by TAK1 deletion in hepatocytes 33 . Kundu et al. confirmed that the SIRT4/SMAD4 axis played a vital role in HFD-fed induced liver fibrosis. Upregulation of SIRT4 and downregulation of Smad4 can potentially counteract lipid accumulation, inflammation, and fibrosis during NAFLD progression 34 . Hepatocyte-specific deletion of Smad4 markedly reduced the expression of fibrosis, hepatocyte apoptosis-, and inflammation-related genes during NASH progression 20 . Collectively, our results support this conclusion and demonstrate that hepatocyte-specific Smad4 deficiency alleviates HFD-fed induced NAFLD. Hepatocytes comprise the largest number of parenchymal cells in the liver and are the primary undertakers of liver function. With little or no alcohol intake, steatosis in more than 5% of hepatocytes is diagnosed as NAFLD 35 . Increased lipid influx into the liver or reduced lipid disposal precipitates hepatic steatosis, primarily instigated by a HFD, genetic predisposition, gut microbiota, and upregulated expression of lipid transcription factors (e.g, SREBP1c, chREBP and PPAR-γ) 36 . Abnormal accumulation of lipotoxic lipids, including fatty acids, diacylglycerols, and cholesterol in the liver, induces hepatocellular injury, including lipotoxicity, mitochondrial dysfunction, oxidative stress, ER stress, and severe inflammatory responses 37 . Our results revealed that Smad4 deficiency in hepatocytes curtailed the secretion of CXCL1, which consequently mitigated hepatocyte fatty acid synthesis and macrophage M1 polarization. A recent integrative analysis of mild and severe NAFLD identified CXCL1 as one of the five hub genes. In vitro and in vivo experiments demonstrated that high-fat conditions increased CXCL1 levels in both the liver tissue and hepatocytes, which correlated with the duration of HFD feeding and PA concentration, consistent with our findings 28 . CXCL1 is an important chemokine that is implicated in the progression of numerous inflammatory diseases 38 , 39 . In the liver, CXCL1 is predominantly expressed in hepatocytes, with lower level expression in HSCs and liver-sinusoidal endothelial cells 40 . CXCL1 primarily binds to the receptor CXCR2 and recruits neutrophils to inflammation sites. Previous studies have shown that CXCL1 chemokines are induced and released by the P38 MAPK and JNK signaling pathways in human pulmonary epithelial cells and vascular endothelial cells 29 , 41 . In this study, we demonstrated that hepatocyte Smad4 expression stimulated CXCL1 secretion via the ASK1, P38 MAPK, and JNK signaling pathways, thereby promoting the progression of NAFLD. However, the potential role of CXCL1 on hepatocytes via other pathways warrants further investigation. M1 macrophages are key players in chronic inflammatory diseases, such as atherosclerosis, rheumatoid arthritis (RA), and inflammatory bowel disease (IBD) 42 , 43 , 44 .Macrophages play a significant role in NAFLD pathogenesis, as evidenced by the prevention of inflammatory cell recruitment, hepatic steatosis, and hepatic insulin resistance in Kupffer cell -depleted mice 13 , 45 . In the NAFLD environment, macrophages are regulated by various molecular signals to polarize toward the M1 phenotype 46 . Cytokines secreted by M1 liver macrophages are also likely to repress fatty acid oxidation and potentiate triglyceride synthesis 47 , 48 . In line with this, we demonstrated that Smad4 deficiency in hepatocytes inhibits the transition of macrophages to the M1 phenotype in an NAFLD model. Interestingly, the secretion of CXCL1 does not affect macrophage M2 polarization. CXCL1 has been reported to play a crucial role in M1 macrophage polarization during cerebral aneurysm development 49 . Consistent with this, our study showed that hepatocyte Smad4 promoted macrophage M1 polarization by facilitating CXCL1 secretion. Whether Smad4 influences NAFLD via other molecular mechanisms remains unclear. In conclusion, our study revealed that Smad4 expression in hepatocytes plays a crucial role in the development of NAFLD. Smad4 in hepatocytes amplified lipid accumulation and M1 macrophage polarization by stimulating CXCL1 secretion, thereby promoting NAFLD progression. Smad4 in hepatocytes may represent a potential preventive and therapeutic target for NAFLD. MATERIALS AND METHODS Some detailed information was provided in supplementary data. The details of RT-qPCR primers are described in supplementary material, Table S1 . Mice Smad4 flox/flox and Alb Smad4−/− mice on a C57BL/6 background have been described previously 26 , 50 . Mice with a conditional knockout of Smad4 in hepatocytes expressing Albumin (Alb Smad4−/− ) were generated by crossing Alb-Cre and Smad4 flox/flox mice. The mice in the control group are cre-negative littermates. All mice were maintained in specific pathogen-free and humidity- and temperature-controlled microisolator cages with a 12-h light/dark cycle at the Institute of Biophysics, Chinese Academy of Sciences. Alb Smad4−/− mice and their littermate control mice which are used for the experiments were 5 to 6 weeks old. HFD-induced NAFLD model The NAFLD model was administered in mice by feeding an HFD (60% of total energy from fat, Huafukang, Beijing, CN) continuously for 16 weeks. Mice that were administered a normal chow diet (ND, 10% of total energy from fat, Huafukang, Beijing, CN) served as controls. Cell lines and treatment The AML12 and RAW264.7 cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). The AML12 cell was cultured in DMEM/F12 medium (Gibco, Grand Island US) supplemented with 10% fetal bovine serum (FBS, PAN biotech, Adenbach, Germany), 1% penicillin/ streptomycin, 40 ng/mL dexamethasone (Solarbio, Beijing, China), and 1% insulin-transferrin-selenium (ITS, Procell, Wuhan, China). The RAW264.7 cell was cultured in DMEM/ 1640 supplemented with 10% FBS and 1% penicillin/ streptomycin. The cells were cultured at 37°C with 5% CO 2 . AML12 cells were exposed to palmitic acid (500 µM) (Sigma, USA) for 24 h. AML12 cells were treated with inhibitor of P38 MAPK SB203580 (10 µM) (MedChemExpress, Princeton, NJ, USA) and inhibitor of JNK for SP600125 (10 µM) (MedChemExpress, Princeton, NJ, USA) for 2 hours in advance. After incubation, the AML12 cells were challenged with 50 ng/mL CXCL1 recombinant protein (Sino Biological, Beijing, China) for 24 hours for further analysis. AML12 and RAW264.7 cells were stimulated with 50 ng/mL CXCL1 recombinant protein and 500 nM inhibitor of CXCL1 receptor CXCR2 SB225002 (MedChemExpress, Princeton, NJ, USA) for 24 hours for further analysis. Statistical analysis All data were expressed as the mean ± SEM and analyzed using GraphPad Prism V8.0.2 software. Significant differences between mean values were obtained from three independent experiments. Differences between the two groups were compared using two-tailed unpaired Student’s t-test analysis. Two-way ANOVA was used for multiple comparisons. P < 0.05 was considered statistically significant. Declarations AUTHOR CONTRIBUTIONS Jinhua Zhang conceived and supervised the study. Wei Yang, Xuanxuan Yan, Xin Xin and Shuang Ge conducted experiments. Jinhua Zhang, Wei Yang, Xinlong Yan, Yongxiang Zhao and performed data analysis. Jinhua Zhang and Wei Yang wrote the manuscript. COMPETING INTERESTS The authors declare no potential conflicts of interest. FUNDING This work was supported by the National Natural Science Foundation of China (81972689), the Natural Science Foundation of Beijing (7232102) and the R&D program of Beijing Municipal Education Commission (KZ202210005010). ETHICS APPROVAL All animal studies were performed after approval by the Institutional Laboratory Animal Care and Use Committee of the Institute of Biophysics, Chinese Academy of Sciences. DATA AVAILABILITY All data generated or analyzed during this study are included in this article and its online supplementary material. Further inquiries can be directed to the corresponding author. References Azzimato V, Jager J, Chen P, Morgantini C, Levi L, Barreby E , et al. Liver macrophages inhibit the endogenous antioxidant response in obesity-associated insulin resistance. Sci Transl Med 2020, 12 (532). Younossi Z, Anstee QM, Marietti M, Hardy T, Henry L, Eslam M , et al. Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention. 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Yang L, Inokuchi S, Roh YS, Song J, Loomba R, Park EJ , et al. Transforming growth factor-β signaling in hepatocytes promotes hepatic fibrosis and carcinogenesis in mice with hepatocyte-specific deletion of TAK1. Gastroenterology 2013, 144 (5) : 1042-1054.e1044. Kundu A, Dey P, Park JH, Kim IS, Kwack SJ, Kim HS. EX-527 Prevents the Progression of High-Fat Diet-Induced Hepatic Steatosis and Fibrosis by Upregulating SIRT4 in Zucker Rats. Cells 2020, 9 (5). Sanyal AJ, Brunt EM, Kleiner DE, Kowdley KV, Chalasani N, Lavine JE , et al. Endpoints and clinical trial design for nonalcoholic steatohepatitis. Hepatology 2011, 54 (1) : 344-353. Cobbina E, Akhlaghi F. Non-alcoholic fatty liver disease (NAFLD) - pathogenesis, classification, and effect on drug metabolizing enzymes and transporters. Drug Metab Rev 2017, 49 (2) : 197-211. Kumar S, Duan Q, Wu R, Harris EN, Su Q. Pathophysiological communication between hepatocytes and non-parenchymal cells in liver injury from NAFLD to liver fibrosis. Adv Drug Deliv Rev 2021, 176: 113869. Korbecki J, Gąssowska-Dobrowolska M, Wójcik J, Szatkowska I, Barczak K, Chlubek M , et al. The Importance of CXCL1 in Physiology and Noncancerous Diseases of Bone, Bone Marrow, Muscle and the Nervous System. Int J Mol Sci 2022, 23 (8). Korbecki J, Barczak K, Gutowska I, Chlubek D, Baranowska-Bosiacka I. CXCL1: Gene, Promoter, Regulation of Expression, mRNA Stability, Regulation of Activity in the Intercellular Space. Int J Mol Sci 2022, 23 (2). Chang B, Xu MJ, Zhou Z, Cai Y, Li M, Wang W , et al. Short- or long-term high-fat diet feeding plus acute ethanol binge synergistically induce acute liver injury in mice: an important role for CXCL1. Hepatology 2015, 62 (4) : 1070-1085. Shieh JM, Tsai YJ, Tsou CJ, Wu WB. CXCL1 regulation in human pulmonary epithelial cells by tumor necrosis factor. Cell Physiol Biochem 2014, 34 (4) : 1373-1384. Moore KJ, Tabas I. Macrophages in the pathogenesis of atherosclerosis. Cell 2011, 145 (3) : 341-355. Cutolo M, Campitiello R, Gotelli E, Soldano S. The Role of M1/M2 Macrophage Polarization in Rheumatoid Arthritis Synovitis. Front Immunol 2022, 13: 867260. Hunter MM, Wang A, Parhar KS, Johnston MJ, Van Rooijen N, Beck PL , et al. In vitro-derived alternatively activated macrophages reduce colonic inflammation in mice. Gastroenterology 2010, 138 (4) : 1395-1405. Lanthier N, Molendi-Coste O, Cani PD, van Rooijen N, Horsmans Y, Leclercq IA. Kupffer cell depletion prevents but has no therapeutic effect on metabolic and inflammatory changes induced by a high-fat diet. Faseb j 2011, 25 (12) : 4301-4311. Marra F, Tacke F. Roles for chemokines in liver disease. Gastroenterology 2014, 147 (3) : 577-594.e571. Miura K, Kodama Y, Inokuchi S, Schnabl B, Aoyama T, Ohnishi H , et al. Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1beta in mice. Gastroenterology 2010, 139 (1) : 323-334.e327. Stienstra R, Saudale F, Duval C, Keshtkar S, Groener JE, van Rooijen N , et al. Kupffer cells promote hepatic steatosis via interleukin-1beta-dependent suppression of peroxisome proliferator-activated receptor alpha activity. Hepatology 2010, 51 (2) : 511-522. Nowicki KW, Hosaka K, Walch FJ, Scott EW, Hoh BL. M1 macrophages are required for murine cerebral aneurysm formation. J Neurointerv Surg 2018, 10 (1) : 93-97. Postic C, Shiota M, Niswender KD, Jetton TL, Chen Y, Moates JM , et al. Dual roles for glucokinase in glucose homeostasis as determined by liver and pancreatic beta cell-specific gene knock-outs using Cre recombinase. J Biol Chem 1999, 274 (1) : 305-315. Additional Declarations (Not answered) Supplementary Files OriginalWesternblot.docx Supplementalmaterial.docx Cite Share Download PDF Status: Published Journal Publication published 31 Jan, 2025 Read the published version in Cell Death & Disease → Version 1 posted Editorial decision: revise 02 Aug, 2024 Review # 3 received at journal 01 Aug, 2024 Reviewer # 3 agreed at journal 23 Jul, 2024 Review # 1 received at journal 26 Jun, 2024 Reviewer # 2 agreed at journal 19 Jun, 2024 Reviewer # 1 agreed at journal 10 Jun, 2024 Reviewers invited by journal 06 Jun, 2024 Submission checks completed at journal 31 May, 2024 Editor assigned by journal 31 May, 2024 First submitted to journal 31 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board 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-4507474","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":311379143,"identity":"1e5d4496-facb-4f5e-9572-4efef8f8c48c","order_by":0,"name":"Jinhua Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIiWNgGAWjYDACCRDBc0DOgOEAiMVMvBZjA4bDJGlhOJC4AaKaCC3ys5ufPfwicyd9O+P5YxIMFdaJDexnD+DVwjjnmLmxDM+z3J0Nh9kkGM6kJzbw5CXg1cIskWAmLcFzOHfDAaAWxrbDiQ0SPAZ4tbBJpH8DaUk3AGv5R4QWHokcM8kPPIcTIFoaiNAiIZFTJs3Ac9gQ6Bdji4Rj6cZtPDn4tcjPSN8m+bPnsLy5xMGHNz7UWMv2s5/BrwUEmHl7QPYdYGBIAPmOoHogYPzxA0jyNxCjdhSMglEwCkYiAACRKkVZzydAGwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-8957-8893","institution":"Beijing Jiaotong University","correspondingAuthor":true,"prefix":"","firstName":"Jinhua","middleName":"","lastName":"Zhang","suffix":""},{"id":311379144,"identity":"82703991-5109-4c1b-8207-60d972a490a7","order_by":1,"name":"Wei Yang","email":"","orcid":"","institution":"Beijing Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Yang","suffix":""},{"id":311379145,"identity":"369c1ebd-d90b-4f18-9858-46d60e86715f","order_by":2,"name":"Xuanxuan Yan","email":"","orcid":"","institution":"Beijing Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Xuanxuan","middleName":"","lastName":"Yan","suffix":""},{"id":311379146,"identity":"e72102c2-51c3-4935-b18e-89596494322c","order_by":3,"name":"Xin Xin","email":"","orcid":"","institution":"Beijing Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Xin","suffix":""},{"id":311379147,"identity":"235c80f2-aaf3-43ec-a291-2e7dc9ca5a90","order_by":4,"name":"Shuang Ge","email":"","orcid":"","institution":"Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shuang","middleName":"","lastName":"Ge","suffix":""},{"id":311379148,"identity":"2788f014-5af6-4339-b20d-c12ccc7b061a","order_by":5,"name":"Yongxiang Zhao","email":"","orcid":"","institution":"Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yongxiang","middleName":"","lastName":"Zhao","suffix":""},{"id":311379149,"identity":"a2dcc287-2f2c-4cb5-9d76-b9ef45b4a302","order_by":6,"name":"Xinlong Yan","email":"","orcid":"","institution":"Beijing University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Xinlong","middleName":"","lastName":"Yan","suffix":""}],"badges":[],"createdAt":"2024-05-31 08:37:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4507474/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4507474/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41419-025-07376-8","type":"published","date":"2025-01-31T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":58727678,"identity":"d48f4558-53ca-40bd-ab7e-57c2be0392ba","added_by":"auto","created_at":"2024-06-20 10:23:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":610353,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSmad4 expression in hepatocytes is upregulated during NAFLD progression.\u003c/strong\u003e (A-B) Immunohistochemical staining of SMAD4 in a tissue microarray from NASH patients. (A) Representative Smad4 staining is shown. Scale bar, 50 μm. (B) Statistical analysis. *** P \u0026lt; 0.001. (C) Boxplots showing Smad4 expression levels in the GEO dataset GSE164760. Healthy: n=6; NASH: n=74. (D) Schematic illustration of HFD-induced mouse nonalcoholic fatty liver disease (NAFLD) model. (E) Representative staining of Smad4 in mouse liver samples and statistical analysis. Scale bar, 50 μm. *** P \u0026lt; 0.001. (F) Representative double staining and statistical analysis of Albumin (green) and Smad4 (red) in mouse liver tissues. Scale bar, 50 μm. * P \u0026lt; 0.05. (G) The expression levels of Smad4 protein in mouse NAFLD liver tissues were determined using Western blot analysis. Smad4 was normalized to GAPDH. **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Onlinefig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4507474/v1/fbde9a0f8660e73d317f780c.png"},{"id":58727679,"identity":"fd5a71ee-b108-4fb2-a3a0-6bce8852816b","added_by":"auto","created_at":"2024-06-20 10:23:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":472977,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHepatocyte-specific deletion of Smad4 attenuates high-fat diet-induced non-alcoholic fatty liver disease.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSmad4\u003csup\u003efl/fl\u003c/sup\u003e and Alb\u003csup\u003eSmad4-/-\u003c/sup\u003e mice were fed an HFD for 4 months to establish NAFLD model (n=5 per group). Data are representative of at least three independent experiments. (A) Representative double staining of Albumin (red) and Smad4 (green) in primary hepatocytes. Scale bars, 50 μm. (B) Body weight changes in Smad4\u003csup\u003efl/fl \u003c/sup\u003eand Alb\u003csup\u003eSmad4-/-\u003c/sup\u003e mice were monitored during the NAFLD model construction. ** P \u0026lt; 0.01. (C) Representative photographs of the liver specimens and liver weight to body weight ratio. * P \u0026lt; 0.05. (D) Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), triglycerides (TG), and non-esterified fatty acids (NEFA) levels. * P \u0026lt; 0.05. (E) Hepatic TG and NEFA levels. ** P \u0026lt;0.01 and *** P \u0026lt;0.001. (F) Glucose tolerance test (GTT) in Smad4\u003csup\u003efl/fl\u003c/sup\u003e and Alb\u003csup\u003eSmad4-/-\u003c/sup\u003e mice fed an HFD for 4 months. * P \u0026lt; 0.05 and ** P \u0026lt;0.01. \u0026nbsp;(G) Representative staining of hematoxylin and eosin (H\u0026amp;E), Oil Red O staining and double staining of Albumin(red) and Smad4(green) in liver tissues. Scale bar, 50 μm. (H) The protein levels of Smad4 in liver tissues were determined using Western blot analysis.\u003c/p\u003e","description":"","filename":"Onlinefig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4507474/v1/8e213c15d6343a4cb906a466.png"},{"id":58728127,"identity":"94e36068-1db9-491a-9c7f-003d8b39473e","added_by":"auto","created_at":"2024-06-20 10:31:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":407567,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSmad4 deficiency in hepatocytes attenuated liver inflammation and CXCL1 secretion.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSmad4\u003csup\u003efl/fl\u003c/sup\u003e and Alb\u003csup\u003eSmad4-/-\u003c/sup\u003e mice were fed an HFD for 4 months to establish the NAFLD model (n=5 per group). Data are representative of at least three independent experiments. (A) Immunofluorescence detection and statistical analysis of F4/80, CD11b and Gr1 expression in NAFLD liver tissues. Scale bar, 50 µm. ** P \u0026lt;0.01; *** P \u0026lt; 0.001. (B). Double immunofluorescence staining for CXCL1(red) and Albumin (green) in liver tissue. Scale bar, 50 μm. *** P \u0026lt; 0.001. (C). The relative mRNA expression of CXCL1 in liver tissues was measured using RT-qPCR, * P \u0026lt; 0.05. (D-I). AML12 cells were transfected with si-NC or si-Smad4, respectively, cultured with BSA or palmitic acid (PA, 500 μM) for 24 h. (D). The protein levels of Smad4 in control si-NC and si-Smad4 AML12 cells were determined using Western blot. (E). Relative mRNA expression of CXCL1 was measured using RT-qPCR in AML12 cells. * P \u0026lt; 0.05.\u0026nbsp; (F). ELISA verification of culture supernatants from AML12 cells. * P \u0026lt; 0.05. (G-I) AML12 cells transfected with lentiviral vectors for control or Smad4 knockdown were then cultured with BSA or PA (500 μM) for 24 h. (G). Western blot analysis of Smad4 protein expression in sh-GFP and sh-Smad4 AML12 cells. (H). The mRNA levels of CXCL1 were measured by RT-qPCR in AML12 cells *** P \u0026lt;0.001. (I). Secretory protein levels of CXCL1 in AML12 cell medium were determined using ELISA. ** P \u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Onlinefig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4507474/v1/9ae76dc453960e73f4270520.png"},{"id":58727685,"identity":"3a0c1aef-db06-4d26-9925-adf63a983919","added_by":"auto","created_at":"2024-06-20 10:23:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":188880,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHepatocyte Smad4 promotes CXCL1 secretion via the ASK1-P38-JNK signaling pathway.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGroups of Smad4\u003csup\u003efl/fl\u003c/sup\u003e and Alb\u003csup\u003eSmad4-/-\u003c/sup\u003e mice were fed an HFD for 4 months to establish the NAFLD model (n=5 per group). (A). The expression levels of p38, p-p38, JNK, p-JNK, ASK1, and p-ASK1 in the liver were measured using Western blot analysis. Protein densities were quantified using densitometry. Phospho-protein levels were normalized to the total protein levels. * P \u0026lt; 0.05. (B). The expression levels of p38, p-p38, JNK, p-JNK, ASK1, and p-ASK1 proteins were measured using Western blot analysis in si-NC and si-Smad4 AML12 cells treated with PA (500 μM) for 24 h. Phospho-proteins were normalized to total proteins. * P \u0026lt; 0.05. (C). The expression levels of p38, p-p38, JNK, p-JNK, ASK1, and p-ASK1 proteins were measured using Western blot analysis in sh-GFP and sh-Smad4 AML12 cells treated with PA (500 μM) for 24 h. Phospho-protein levels were normalized to total protein levels. * P \u0026lt; 0.05 and ** P \u0026lt;0.01. (D-E). After pretreatment of AML12 cells with SB203580 (P38 MAPK inhibitor) or SP600125 (JNK inhibitor) for 2 h, 500 μM PA was co-incubated for 24 h. (D). The mRNA levels of CXCL1 were measured using RT-qPCR in AML12 cells. *** P \u0026lt;0.001. (E). Secretory protein levels of CXCL1 were measured using ELISA in AML12 cells. * P \u0026lt; 0.05 and *** P \u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Onlinefig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4507474/v1/9f800a392e721b21809efdb1.png"},{"id":58727682,"identity":"7d8ac3ea-ecde-4a42-9c81-fa316d7e9ba6","added_by":"auto","created_at":"2024-06-20 10:23:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":436197,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCXCL1 promotes fatty acid synthesis in hepatocytes by binding to CXCR2.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGroups of Smad4\u003csup\u003efl/fl\u003c/sup\u003e and Alb\u003csup\u003eSmad4-/-\u003c/sup\u003e mice were fed an HFD for 4 months to establish the NAFLD model (n=5 per group). (A). The mRNA levels of ACC1, FASN, SCD1, FABP1, FATP1, ACOX1, and CPT1a in NAFLD liver tissues were measured using RT-qPCR analysis. * P \u0026lt; 0.05 and ** P \u0026lt;0.01. (B-G). Primary hepatocytes and AML12 cells were treated with BSA or PA (500 μM) for 24 h. (B). The mRNA levels of ACC1, FASN, and PPARγ in primary hepatocytes were determined using RT-qPCR analysis. * P \u0026lt; 0.05 and *** P \u0026lt;0.001. (C). Oil Red O staining of primary hepatocytes. Scale bar, 100 μm. (D). The mRNA levels of ACC1, FASN, and SCD1 were determined using RT-qPCR analysis in AML12 cells with si-NC or si-Smad4 transfection. * P \u0026lt; 0.05 and ** P \u0026lt;0.01. (E). Oil Red O staining of AML12 cells transfected with si-NC or si-Smad4. Scale bar, 10 μm. (F). The mRNA levels of ACC1, FASN, and SCD1 were determined using RT-qPCR analysis in AML12 cells transfected with sh-GFP or sh-Smad4. * P \u0026lt; 0.05 and ** P \u0026lt;0.01. (G). Oil Red O staining of AML12 cells transfected with sh-GFP or sh-Smad4, respectively. Scale bar, 10 μm. (H). The protein levels of CXCR2 in AML12 cells treated with 50 ng/mL CXCL1 were detected by Western blot. (I). AML12 cells were cultured with 500 μM PA, 50 ng/mL CXCL1 recombinant protein and 500 nM SB225002 (inhibitor of CXCL1 receptor CXCR2) for 24 h. The mRNA levels of ACC1, FASN, and SCD1 were determined using RT-qPCR analysis in AML12 cells. * P \u0026lt; 0.05, ** P \u0026lt;0.01 and *** P \u0026lt;0.001.\u003c/p\u003e","description":"","filename":"OnlineFig5.png","url":"https://assets-eu.researchsquare.com/files/rs-4507474/v1/69ba96eeb49f4fbbf71431f6.png"},{"id":58727684,"identity":"3fdf1366-2919-42f5-99e7-600ac4ed3b26","added_by":"auto","created_at":"2024-06-20 10:23:06","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":299886,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCXCL1 promotes macrophage M1 polarization.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSmad4\u003csup\u003efl/fl\u003c/sup\u003e and Alb\u003csup\u003eSmad4-/-\u003c/sup\u003e mice were fed an HFD for 4 months to establish the NAFLD model (n=5 per group). Data are representative of at least three independent experiments. (A-B). Representative staining and statistical analysis of CD86 and CD206 expression in liver tissues. Scale bar, 50 μm. (C-D). The mRNA levels of IL-6, MCP1, TNF-α, Arg1, IL-10, and YM1 in liver tissues were determined using RT-qPCR analysis. * P \u0026lt; 0.05 and ** P \u0026lt; 0.01. (E). The protein levels of CXCR2 in Raw264.7 cells treated with 50 ng/mL CXCL1 were detected by Western blot. (F-G). Raw264.7 cells were cultured with 100 ng/mL LPS, 10 ng/mL IFN-γ, 50 ng/mL CXCL1 recombinant protein, and 500 nM SB225002 for 24 h. (F). Immunofluorescence staining of CD86 in RAW264.7 cells. Scale bar, 50 μm. (G). The mRNA levels of iNOS, MCP1, and TNF-α in RAW264.7 cells were determined using RT-qPCR analysis. * P \u0026lt; 0.05, ** P \u0026lt; 0.01 and *** P \u0026lt;0.001. (H). Raw264.7 cells were cultured with 20 ng/mL IL-4, 20 ng/mL IL-13, 50 ng/mL CXCL1 recombinant protein, and 500 nM SB225002 for 48 h. The mRNA levels of Arg1, IL-10, and YM1 in RAW264.7 cells were determined using RT-qPCR analysis. *** P \u0026lt;0.001.\u003c/p\u003e","description":"","filename":"OnlineFig6.png","url":"https://assets-eu.researchsquare.com/files/rs-4507474/v1/b538d78903a1f9df402c84d7.png"},{"id":75217921,"identity":"57627e27-a064-405b-8113-a36a1cb8fa6d","added_by":"auto","created_at":"2025-02-01 08:07:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4182164,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4507474/v1/87b32e55-46eb-4820-8b2b-4b6ad1e460c1.pdf"},{"id":58727683,"identity":"56e486a5-b099-4165-9328-c4dd5aa121ce","added_by":"auto","created_at":"2024-06-20 10:23:06","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":688104,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"OriginalWesternblot.docx","url":"https://assets-eu.researchsquare.com/files/rs-4507474/v1/ab96230551ffd76a87d9cf78.docx"},{"id":58727686,"identity":"933f0d29-44ce-4487-af1c-9227100e8e98","added_by":"auto","created_at":"2024-06-20 10:23:06","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":23446,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalmaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-4507474/v1/3953045d8f2c6a147afe332e.docx"}],"financialInterests":"(Not answered)","formattedTitle":"Smad4 deficiency in hepatocytes\r\nattenuates NAFLD progression via inhibition of lipogenesis and macrophage polarization","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eNonalcoholic fatty liver disease (NAFLD) is characterized by the excessive accumulation of lipids within hepatocytes, due to factors other than alcohol consumption and other definitive liver damage sources\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. NAFLD is a complex, multifactorial condition influenced by both environmental factors and genetic predispositions\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. It encompasses a spectrum of pathological liver conditions of varying severity, ranging from isolated hepatic steatosis (NAFL) to steatohepatitis (NASH), which can further progress to liver fibrosis, cirrhosis, and hepatocellular carcinoma (HCC)\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. With an estimated prevalence in nearly one-third of the global adult population, NAFLD has become a public health concern\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHepatocytes, the main functional units of the liver, play a pivotal role in biotransformation, metabolism, and detoxification. During NAFLD progression, there is an excessive accumulation of triglyceride, free fatty acid (FFA), and cholesterol in hepatocytes, a process that is associated with insulin resistance that can lead to dysfunctional triglyceride synthesis and transport\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The excessive lipid accumulation induces lipotoxicity, which impairs mitochondrial function, triggers endoplasmic reticulum (ER) stress, and triggers inflammatory responses in hepatocytes due to reactive oxygen species (ROS)-induced inflammation\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Concurrently, FFA accumulation in hepatocytes enhances mitochondrial β-oxidation, sensitizing the liver to oxidative stress and thereby exacerbating liver damage\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Moreover, lipotoxicity can disrupt the JNK pathway and Toll-like receptor cellular signaling pathways, thereby affecting hepatocyte metabolism\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eLiver macrophages comprise recruited monocyte-derived macrophages and resident Kupffer cells within the liver. Liver macrophages play a central role in the regulation of hepatocyte metabolism and maintenance of hepatic immunological tolerance\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. They can promote the progression of metabolic diseases by enhancing insulin resistance, hepatic steatosis, and oxidative stress in obese mice and rats\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Under normal physiological conditions, liver macrophages exhibit a tendency toward the M2 phenotype, which suppresses inflammation by secreting interleukin (IL)-4 and IL-13 \u003csup\u003e15, 16\u003c/sup\u003e. However, during NAFLD progression, hepatic stellate cells (HSC) activation triggers the secretion of pro-inflammatory cytokines, which increases the levels of lipopolysaccharide (LPS) in the blood. And high levels of FFAs activate inflammasomes. These changes stimulate the onset of inflammation, with a concomitant increase in the proportion of M1 macrophages secreting pro-inflammatory cytokines\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe transforming growth factor beta (TGF-β) signaling pathway plays important roles in biological processes of cell growth, apoptosis, migration, and cancer development and progression\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Smad acts as a downstream signaling molecule in the TGF-β signaling pathway\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. In mammals, eight different SMADs are further divided into three distinct classes: R-Smad (Smad1, 2, 3, 5, and 8), Co-Smad (Smad4), and I-Smad (Smad6 and 7)\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Smad4 is a central mediator of TGF-β signaling, which binds to nearly all Smad proteins regulated by activated receptors and helps regulate the expression of various downstream genes\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Hepatocyte Smad4 expression levels increase progressively as normal liver tissue progresses to NAFLD and finally to NASH\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Smad4 deletion attenuates inflammation, fibrosis, and hepatocyte apoptosis in NASH\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. After the administration of high-fat diet (HFD), Smad4 deletion in pancreatic β-cells improves blood glucose levels, insulin secretion, and glucose tolerance in obese mice\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Although other studies have investigated the role of Smad4 in liver disease, its specific molecular mechanism in hepatocytes in NAFLD remains unclear.\u003c/p\u003e \u003cp\u003eIn this study, we examined the specific role of Smad4 in NAFLD progression using a mouse model with hepatocyte-specific Smad4 deletion. Our findings revealed that Smad4 deficiency in hepatocytes attenuates NAFLD development. Moreover, hepatocyte Smad4 was found to amplify CXCL1 secretion by facilitating the activation of the ASK1/P38/JNK signaling pathway. CXCL1, in turn, promotes hepatocyte lipogenesis and macrophage M1-type polarization via CXCR2 binding.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSmad4 expression in hepatocytes is upregulated during NAFLD progression\u003c/h2\u003e \u003cp\u003eTo elucidate the relationship between Smad4 expression and NASH, the expression of Smad4 in healthy liver and NASH tissues from patients was assessed by tissue microarray using immunohistochemistry. We observed that Smad4 expression was significantly upregulated in NASH tissues compared to that in healthy controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). Moreover, we analyzed the publicly available Gene Expression Omnibus dataset GSE164760 and compared Smad4 expression between healthy and NASH tissues. The analysis revealed a significant increase in Smad4 mRNA levels in the NASH group compared to healthy controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo corroborate these findings, we used a short-term HFD mouse model of NAFLD (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Immunohistochemical staining demonstrated a significant upregulation of Smad4 expression in the hepatocytes of fatty liver tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Double immunofluorescence staining confirmed that Smad4 was expressed in most hepatocytes \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Consistent with this, Western blot analysis indicated a significant increase in Smad4 protein levels in fatty liver tissues of HFD-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003eCollectively, these results suggest that Smad4 is activated in hepatocytes during NAFLD, indicating a potentially crucial role for Smad4 in NAFLD pathogenesis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHepatocyte-specific deletion of Smad4 attenuates high-fat diet-induced non-alcoholic fatty liver disease.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further investigate the role of Smad4 in hepatocytes during NAFLD, we used a conditional Smad4 deletion approach in murine hepatocytes, as previously described \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. We generated hepatocyte-specific Smad4 knockout mice (Albumin-cre; Smad4\u003csup\u003eflox/flox\u003c/sup\u003e, Alb\u003csup\u003eSmad4\u0026minus;/\u0026minus;\u003c/sup\u003e) by crossing mice carrying the Loxp-flanked Smad4 allele with Albumin-cre mice. The Alb\u003csup\u003eSmad4\u0026minus;/\u0026minus;\u003c/sup\u003e mice were born at the expected Mendelian ratio, viable, and fertile. We used Smad4\u003csup\u003efl/fl\u003c/sup\u003e littermates as controls. Smad4 deletion in primary hepatocytes of Alb\u003csup\u003eSmad4\u0026minus;/\u0026minus;\u003c/sup\u003e mice was confirmed using double immunofluorescence staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo delineate the role of Smad4 in NAFLD, we established a HFD-induced NAFLD model using Alb\u003csup\u003eSmad4\u0026minus;/\u0026minus;\u003c/sup\u003e mice and their control littermates. In response to HFD feeding, Alb\u003csup\u003eSmad4\u0026minus;/\u0026minus;\u003c/sup\u003e mice demonstrated a less pronounced increase in both body and liver weight compared with control littermates (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C). After 3 months of HFD, Alb\u003csup\u003eSmad4\u0026minus;/\u0026minus;\u003c/sup\u003e mice displayed lower serum alanine aminotransferase (ALT), triglyceride (TG), and non-esterified fatty acid (NEFA) levels, while serum aspartate aminotransferase (AST) and total cholesterol (TC) levels were similar between Alb\u003csup\u003eSmad4\u0026minus;/\u0026minus;\u003c/sup\u003e and control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Furthermore, hepatic TG and NEFA levels were notably reduced in Alb\u003csup\u003eSmad4\u0026minus;/\u0026minus;\u003c/sup\u003e mice compared with Smad4\u003csup\u003efl/fl\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Alb\u003csup\u003eSmad4\u0026minus;/\u0026minus;\u003c/sup\u003e mice also exhibited impaired glucose tolerance compared with Smad4\u003csup\u003efl/fl\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). The absence of Smad4 in hepatocytes in Alb\u003csup\u003eSmad4\u0026minus;/\u0026minus;\u003c/sup\u003e mice was further validated using double immunofluorescence staining and Western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, H). Deletion of Smad4 in hepatocytes resulted in decreased fat accumulation, as evidenced by hematoxylin and eosin and Oil Red O staining. No significant differences were observed between Alb\u003csup\u003eSmad4\u0026minus;/\u0026minus;\u003c/sup\u003e and Smad4\u003csup\u003efl/fl\u003c/sup\u003e mice who were fed a normal diet (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Collectively, these data suggest that hepatocyte-specific Smad4 deficiency attenuates the development of HFD-induced NAFLD.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSmad4 deficiency in hepatocytes attenuated liver inflammation and CXCL1 secretion\u003c/h2\u003e \u003cp\u003eTo investigate whether Smad4 modulates liver inflammatory cell infiltration and hepatocyte proliferation, we stained liver tissues of Alb\u003csup\u003eSmad4\u0026minus;/\u0026minus;\u003c/sup\u003e and Smad4\u003csup\u003efl/fl\u003c/sup\u003e mice via immunofluorescence. The infiltration of F4/80\u003csup\u003e+\u003c/sup\u003e macrophages and CD11b\u003csup\u003e+\u003c/sup\u003e monocytes was diminished in Alb\u003csup\u003eSmad4\u0026minus;/\u0026minus;\u003c/sup\u003e mice compared with that in Smad4\u003csup\u003efl/fl\u003c/sup\u003e mice when fed a HFD. When fed the normal diet, immune cell infiltration was similar between Alb\u003csup\u003eSmad4\u0026minus;/\u0026minus;\u003c/sup\u003e and control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). During NAFLD progression, activated chemokines evoke multiple cellular and tissue responses, including hepatocyte proliferation, activation, necrosis, angiogenesis, and immune cell recruitment\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Notably, CXCL1 is a key gene involved in NAFLD progression. Therefore, we examined CXCL1 expression in the livers of Alb\u003csup\u003eSmad4\u0026minus;/\u0026minus;\u003c/sup\u003e and Smad4\u003csup\u003efl/fl\u003c/sup\u003e mice through double immunofluorescence staining. CXCL1 levels were significantly lower in the livers of Alb\u003csup\u003eSmad4\u0026minus;/\u0026minus;\u003c/sup\u003e mice than those in Smad4\u003csup\u003efl/fl\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). This decrease in CXCL1 levels was further confirmed using quantitative reverse transcription polymerase chain reaction (RT-qPCR) in hepatocytes of NAFLD of Alb\u003csup\u003eSmad4\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further clarify the function of Smad4 in hepatocytes, we knocked down Smad4 in AML12 cells using siRNA and verified Smad4 protein levels using Western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). The cells were then treated with palmitic acid (PA) for 24 h to simulate an in vitro NAFLD environment. RT-qPCR analysis revealed that Smad4 deficiency mitigated the PA-induced CXCL1 expression in hepatocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). The protein expression level of CXCL1 was further analyzed using enzyme-linked immunosorbent assay (ELISA) with AML12-conditioned medium (CM). Smad4 deficiency partly reduced the secretion of CXCL1 in PA-induced AML12 CM (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eIn addition, AML12 cells were transfected with a lentiviral vector to knock down Smad4, and the expression levels of Smad4 protein were measured using Western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). In line with previous results, both the expression and secretion of CXCL1 in PA-stimulated Smad4-knockdown AML12 cells were significantly reduced compared to those in control cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH, I). Taken together, these results suggest that hepatocyte-specific deletion of Smad4 lessens liver inflammation and CXCL1 secretion.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eHepatocyte Smad4 promotes CXCL1 secretion via the ASK1-P38-JNK signaling pathway\u003c/h2\u003e \u003cp\u003ePrevious studies have highlighted the involvement of the JNK and p38 MAPK cascades in the regulation of CXCL1 secretion\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Accordingly, we examined the levels of total and phosphorylated proteins involved in ASK1, P38, and JNK signaling. We found that the expression of phosphorylated ASK1 (p-ASK1), p38 (p-p38), and JNK (p-JNK) was diminished in the liver tissue of HFD-treated Alb\u003csup\u003eSmad4\u0026minus;/\u0026minus;\u003c/sup\u003e mice compared with Smad4\u003csup\u003efl/fl\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn subsequent experiments, we used si-Smad4 and sh-Smad4 to knock down Smad4 in AML12 cells, which were then exposed to PA. The Western blot analysis showed that the ASK1, P38, and JNK signaling pathways were activated in hepatocytes in response to PA administration. However, this activation was remarkably suppressed by Smad4 knockdown, suggesting that Smad4 knockdown considerably inhibited PA-induced activation of the ASK1\u0026ndash;P38\u0026ndash;JNK pathway \u003cem\u003ein vitro\u003c/em\u003e (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C).\u003c/p\u003e \u003cp\u003eTo further elucidate the signaling pathways involved in the induction of CXCL1 secretion by Smad4, we employed the JNK inhibitor (SP600125) and the p38 MAPK inhibitor (SB203580). Both inhibitors suppressed PA-induced CXCL1 secretion and mRNA expression, as evidenced using RT\u0026ndash;qPCR and ELISA analysis (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, E). In conclusion, these results suggest that Smad4 facilitates CXCL1 secretion via the ASK1-P38-JNK signaling pathways during NAFLD progression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCXCL1 promotes fatty acid synthesis in hepatocytes by binding to CXCR2\u003c/h2\u003e \u003cp\u003eTo further investigate the role of Smad4 in hepatic lipid deposition, RT-qPCR was used to determine the expression levels of genes involved in fatty acid synthesis and consumption. We found that Smad4 deficiency considerably inhibited the expression of genes critical for fatty acid synthesis (ACC1, FASN, and SCD1) and fatty acid binding protein 1 (FABP1). However, we observed no substantial differences in the expression of genes related to fatty acid uptake (FATP1) and fatty acid β-oxidation (CPT1a and ACOX1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo corroborate these findings, we isolated primary hepatocytes from Alb\u003csup\u003eSmad4\u0026minus;/\u0026minus;\u003c/sup\u003e and Smad4\u003csup\u003efl/fl\u003c/sup\u003e mice and then exposed them to PA. Following Smad4 knockout, the expression of genes responsible for fatty acid synthesis (ACC1, FASN, and PPAR-γ) was substantially reduced in primary hepatocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). We further observed decreased lipid deposition in primary hepatocytes of Alb\u003csup\u003eSmad4\u0026minus;/\u0026minus;\u003c/sup\u003e mice compared with that in Smad4\u003csup\u003efl/fl\u003c/sup\u003e mice following PA treatment, as evidenced by Oil Red O staining \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. This conclusion was confirmed using si-Smad4 AML12 and sh-Smad4 AML12 cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-G). Taken together, these findings suggest that hepatocyte Smad4 promotes NAFLD development primarily through fatty acid synthesis.\u003c/p\u003e \u003cp\u003eCXCL1 binds to specific receptor CXCR2. We then examined whether CXCL1-induced fatty acid synthesis occured in hepatocytes. We assessed the expression levels of CXCR2 in hepatocytes using Western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). To further elucidate the molecular mechanisms underlying the effects of CXCL1 on fatty acid synthesis, we cultured AML12 cells with CXCL1 recombinant protein and analyzed the expressions of genes related to fatty acid synthesis using RT-qPCR. We observed that ACC1, FASN, and SCD1 were substantially upregulated by CXCL1. However, when CXCR2 activation was inhibited by the CXCR2 inhibitor, SB225002, in AML12 cells, CXCL1-induced ACC1, FASN, and SCD1 expression was reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). These results suggest that CXCL1 induces fatty acid synthesis in hepatocytes via CXCR2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCXCL1 promotes macrophage M1 polarization\u003c/h2\u003e \u003cp\u003eAberrant lipid-mediated hepatic inflammatory-immune dysfunction and chronic low-grade inflammation play important roles in NAFLD pathogenesis. Macrophage polarization is an important mechanism that regulates inflammatory responses\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Therefore, we assessed the quantities of CD86\u003csup\u003e+\u003c/sup\u003e (M1 marker) and CD206\u003csup\u003e+\u003c/sup\u003e (M2 marker) macrophages in NAFLD tissues. Our findings revealed a substantial decrease in M1 macrophages in Alb\u003csup\u003eSmad4\u0026minus;/\u0026minus;\u003c/sup\u003e mice compared with Smad4\u003csup\u003efl/fl\u003c/sup\u003e mice, while M2-like macrophages remained comparable (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). We further evaluated the expression levels of genes associated with M1-like (IL-6, MCP1, and TNF-α) and M2-like (Arg1, IL-10, and YM1) phenotypes using RT-qPCR and found similar results (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, D). Given that the absence of hepatocyte Smad4 resulted in diminished CXCL1 secretion, we postulated that hepatocyte Smad4 might facilitate macrophage M1 polarization via CXCL1. We confirmed the expression of CXCR2 in macrophages using western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). We cultured RAW264.7 cells with LPS and IFN-γ for 24 h to induce M1 polarization. It was found that LPS and IFN-γ activated the expression of CD86 in macrophages in comparison with the control group, CXCL1 recombinant proteins further upregulated CD86. This expression was further augmented by the addition of CXCL1 recombinant proteins, as evidenced by immunofluorescence staining. However, the treatment of RAW264.7 cells with the CXCR2 inhibitor SB225002 eliminated CXCL1-induced expression of CD86 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Following M1 polarization induction, the expression levels of iNOS, MCP1, and TNF-α were significantly increased in RAW264.7 cells compared with those in the control group, and the addition of exogenous CXCL1 recombinant protein further amplified M1-related gene expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor M2 polarization, RAW264.7 cells were cultured with IL-4 and IL-13 for 48 h. This treatment activated the expression of Arg1, IL-10, and YM1 in macrophages compared with the control group. No significant differences were observed after the administration of CXCL1 and SB225002 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). These findings suggest that CXCL1 promotes the M1-type polarization of macrophages via CXCR2.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eSmad4 is a general mediator of the TGF-β and bone morphogenetic protein (BMP) signaling pathways, which significantly contribute to intracellular signal transduction and a myriad of cellular processes\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. However, owing to its ubiquitous expression, the specific role and molecular mechanism of Smad4-mediated signaling in NAFLD progression remain elusive. Our study illustrated that hepatocyte-specific Smad4 expression promoted NAFLD development by enhancing CXCL1 secretion. Targeted deficiency of hepatocyte-specific Smad4 signaling curbed the progression of NAFLD in HFD-fed mice. Hepatocyte-specific genetic deficiency of Smad4 inhibited fatty acid synthesis and macrophage M1 polarization. Moreover, Smad4 in hepatocytes accelerated CXCL1 secretion to enhance fatty acid synthesis and macrophage M1 polarization by activating the ASK-P38-JNK signaling pathway.\u003c/p\u003e \u003cp\u003eNAFLD is now recognized as a steatotic liver disease closely associated with metabolic syndrome. The relationship between NAFLD and liver inflammation has been extensively studied. Several studies indicate the pivotal role of TGF β/Smad signaling in metabolic syndrome and related disorders\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Our previous study demonstrated that the targeted knockout of Smad4 in hepatocytes attenuates hepatic inflammatory cell infiltration and fibrosis during the progression of CCl\u003csub\u003e4\u003c/sub\u003e induced liver fibrosis\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Disruption of the Smad4 pathway alleviated spontaneous liver injury, hepatic inflammatory cell infiltration, fibrosis, and HCC induced by TAK1 deletion in hepatocytes\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Kundu et al. confirmed that the SIRT4/SMAD4 axis played a vital role in HFD-fed induced liver fibrosis. Upregulation of SIRT4 and downregulation of Smad4 can potentially counteract lipid accumulation, inflammation, and fibrosis during NAFLD progression\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Hepatocyte-specific deletion of Smad4 markedly reduced the expression of fibrosis, hepatocyte apoptosis-, and inflammation-related genes during NASH progression\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Collectively, our results support this conclusion and demonstrate that hepatocyte-specific Smad4 deficiency alleviates HFD-fed induced NAFLD.\u003c/p\u003e \u003cp\u003eHepatocytes comprise the largest number of parenchymal cells in the liver and are the primary undertakers of liver function. With little or no alcohol intake, steatosis in more than 5% of hepatocytes is diagnosed as NAFLD\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Increased lipid influx into the liver or reduced lipid disposal precipitates hepatic steatosis, primarily instigated by a HFD, genetic predisposition, gut microbiota, and upregulated expression of lipid transcription factors (e.g, SREBP1c, chREBP and PPAR-γ)\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Abnormal accumulation of lipotoxic lipids, including fatty acids, diacylglycerols, and cholesterol in the liver, induces hepatocellular injury, including lipotoxicity, mitochondrial dysfunction, oxidative stress, ER stress, and severe inflammatory responses\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Our results revealed that Smad4 deficiency in hepatocytes curtailed the secretion of CXCL1, which consequently mitigated hepatocyte fatty acid synthesis and macrophage M1 polarization.\u003c/p\u003e \u003cp\u003eA recent integrative analysis of mild and severe NAFLD identified CXCL1 as one of the five hub genes. \u003cem\u003eIn vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments demonstrated that high-fat conditions increased CXCL1 levels in both the liver tissue and hepatocytes, which correlated with the duration of HFD feeding and PA concentration, consistent with our findings\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. CXCL1 is an important chemokine that is implicated in the progression of numerous inflammatory diseases\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. In the liver, CXCL1 is predominantly expressed in hepatocytes, with lower level expression in HSCs and liver-sinusoidal endothelial cells\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. CXCL1 primarily binds to the receptor CXCR2 and recruits neutrophils to inflammation sites. Previous studies have shown that CXCL1 chemokines are induced and released by the P38 MAPK and JNK signaling pathways in human pulmonary epithelial cells and vascular endothelial cells\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. In this study, we demonstrated that hepatocyte Smad4 expression stimulated CXCL1 secretion via the ASK1, P38 MAPK, and JNK signaling pathways, thereby promoting the progression of NAFLD. However, the potential role of CXCL1 on hepatocytes via other pathways warrants further investigation.\u003c/p\u003e \u003cp\u003eM1 macrophages are key players in chronic inflammatory diseases, such as atherosclerosis, rheumatoid arthritis (RA), and inflammatory bowel disease (IBD)\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e.Macrophages play a significant role in NAFLD pathogenesis, as evidenced by the prevention of inflammatory cell recruitment, hepatic steatosis, and hepatic insulin resistance in Kupffer cell -depleted mice\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. In the NAFLD environment, macrophages are regulated by various molecular signals to polarize toward the M1 phenotype\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Cytokines secreted by M1 liver macrophages are also likely to repress fatty acid oxidation and potentiate triglyceride synthesis\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. In line with this, we demonstrated that Smad4 deficiency in hepatocytes inhibits the transition of macrophages to the M1 phenotype in an NAFLD model. Interestingly, the secretion of CXCL1 does not affect macrophage M2 polarization. CXCL1 has been reported to play a crucial role in M1 macrophage polarization during cerebral aneurysm development\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Consistent with this, our study showed that hepatocyte Smad4 promoted macrophage M1 polarization by facilitating CXCL1 secretion. Whether Smad4 influences NAFLD via other molecular mechanisms remains unclear.\u003c/p\u003e \u003cp\u003eIn conclusion, our study revealed that Smad4 expression in hepatocytes plays a crucial role in the development of NAFLD. Smad4 in hepatocytes amplified lipid accumulation and M1 macrophage polarization by stimulating CXCL1 secretion, thereby promoting NAFLD progression. Smad4 in hepatocytes may represent a potential preventive and therapeutic target for NAFLD.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003eSome detailed information was provided in supplementary data. The details of RT-qPCR primers are described in supplementary material, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eMice\u003c/h2\u003e \u003cp\u003eSmad4\u003csup\u003eflox/flox\u003c/sup\u003e and Alb\u003csup\u003eSmad4\u0026minus;/\u0026minus;\u003c/sup\u003e mice on a C57BL/6 background have been described previously\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Mice with a conditional knockout of Smad4 in hepatocytes expressing Albumin (Alb\u003csup\u003eSmad4\u0026minus;/\u0026minus;\u003c/sup\u003e) were generated by crossing Alb-Cre and Smad4\u003csup\u003eflox/flox\u003c/sup\u003e mice. The mice in the control group are cre-negative littermates. All mice were maintained in specific pathogen-free and humidity- and temperature-controlled microisolator cages with a 12-h light/dark cycle at the Institute of Biophysics, Chinese Academy of Sciences. Alb\u003csup\u003eSmad4\u0026minus;/\u0026minus;\u003c/sup\u003e mice and their littermate control mice which are used for the experiments were 5 to 6 weeks old.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eHFD-induced NAFLD model\u003c/h2\u003e \u003cp\u003eThe NAFLD model was administered in mice by feeding an HFD (60% of total energy from fat, Huafukang, Beijing, CN) continuously for 16 weeks. Mice that were administered a normal chow diet (ND, 10% of total energy from fat, Huafukang, Beijing, CN) served as controls.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCell lines and treatment\u003c/h2\u003e \u003cp\u003eThe AML12 and RAW264.7 cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). The AML12 cell was cultured in DMEM/F12 medium (Gibco, Grand Island US) supplemented with 10% fetal bovine serum (FBS, PAN biotech, Adenbach, Germany), 1% penicillin/ streptomycin, 40 ng/mL dexamethasone (Solarbio, Beijing, China), and 1% insulin-transferrin-selenium (ITS, Procell, Wuhan, China). The RAW264.7 cell was cultured in DMEM/ 1640 supplemented with 10% FBS and 1% penicillin/ streptomycin. The cells were cultured at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. AML12 cells were exposed to palmitic acid (500 \u0026micro;M) (Sigma, USA) for 24 h. AML12 cells were treated with inhibitor of P38 MAPK SB203580 (10 \u0026micro;M) (MedChemExpress, Princeton, NJ, USA) and inhibitor of JNK for SP600125 (10 \u0026micro;M) (MedChemExpress, Princeton, NJ, USA) for 2 hours in advance. After incubation, the AML12 cells were challenged with 50 ng/mL CXCL1 recombinant protein (Sino Biological, Beijing, China) for 24 hours for further analysis. AML12 and RAW264.7 cells were stimulated with 50 ng/mL CXCL1 recombinant protein and 500 nM inhibitor of CXCL1 receptor CXCR2 SB225002 (MedChemExpress, Princeton, NJ, USA) for 24 hours for further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM and analyzed using GraphPad Prism V8.0.2 software. Significant differences between mean values were obtained from three independent experiments. Differences between the two groups were compared using two-tailed unpaired Student\u0026rsquo;s t-test analysis. Two-way ANOVA was used for multiple comparisons. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJinhua Zhang\u0026nbsp;conceived and supervised the study.\u0026nbsp;Wei Yang, Xuanxuan Yan, Xin Xin and Shuang Ge conducted experiments. Jinhua Zhang, Wei Yang, Xinlong Yan,\u0026nbsp;Yongxiang Zhao\u0026nbsp;and performed data analysis. Jinhua Zhang and Wei Yang wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no potential conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (81972689), the Natural Science Foundation of Beijing (7232102) and the R\u0026amp;D program of Beijing Municipal Education Commission (KZ202210005010).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eETHICS APPROVAL\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal studies were performed after approval by the Institutional Laboratory Animal Care and Use Committee of the Institute of Biophysics, Chinese Academy of Sciences.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this article and its online supplementary material. Further inquiries can be directed to the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAzzimato V, Jager J, Chen P, Morgantini C, Levi L, Barreby E\u003cem\u003e, et al.\u003c/em\u003e Liver macrophages inhibit the endogenous antioxidant response in obesity-associated insulin resistance. \u003cem\u003eSci Transl Med\u003c/em\u003e 2020, \u003cstrong\u003e12\u003c/strong\u003e(532).\u003c/li\u003e\n\u003cli\u003eYounossi Z, Anstee QM, Marietti M, Hardy T, Henry L, Eslam M\u003cem\u003e, et al.\u003c/em\u003e Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention. \u003cem\u003eNat Rev Gastroenterol Hepatol\u003c/em\u003e 2018, \u003cstrong\u003e15\u003c/strong\u003e(1)\u003cstrong\u003e:\u003c/strong\u003e 11-20.\u003c/li\u003e\n\u003cli\u003eFriedman SL, Neuschwander-Tetri BA, Rinella M, Sanyal AJ. 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Although the Smad4 signaling pathway has been implicated in the progression of NAFLD, the specific role of Smad4 in hepatocytes in NAFLD pathogenesis remains unclear.\u003cstrong\u003e \u003c/strong\u003eHepatocyte-specific knockout Smad4 mice (Alb\u003csup\u003eSmad4-/-\u003c/sup\u003e) were first constructed using the Cre-Loxp recombinant system to establish a high-fat diet induced NAFLD model. The role of Smad4 in the occurrence and development of NAFLD was determined by monitoring the body weight of mice, detecting triglycerides and free fatty acids in serum and liver tissue homogenates, staining the tissue sections to observe the accumulation of liver fat, and RT-qPCR detecting the expression of genes related to lipogenesis, fatty acid intake and fatty acid β oxidation. The molecular mechanism of Smad4 in hepatocytes affecting NAFLD was therefore investigated through combining in vitro and in vivo experiments.\u003cstrong\u003e \u003c/strong\u003eSmad4 deficiency in hepatocytes mitigated NAFLD progression and decreased inflammatory cells infiltration. Moreover, Smad4 deficiency inhibited CXCL1 secretion by suppressing the activation of the ASK1/P38/JNK signaling pathway. Furthermore, targeting CXCL1 using CXCR2 inhibitors diminished hepatocyte lipogenesis and inhibited the polarization of M1-type macrophages.\u003cstrong\u003e \u003c/strong\u003eCollectively, these results suggested that Smad4 plays a vital role in exacerbating NAFLD and may be a promising candidate for anti-NAFLD therapy.\u003c/p\u003e","manuscriptTitle":"Smad4 deficiency in hepatocytes\nattenuates NAFLD progression via inhibition of lipogenesis and macrophage polarization","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-20 10:23:01","doi":"10.21203/rs.3.rs-4507474/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2024-08-02T10:55:18+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-08-01T10:01:23+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-07-23T23:54:28+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-06-26T08:42:58+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-06-19T11:44:05+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-06-10T08:17:40+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-06-06T13:25:20+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-31T09:47:45+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-31T08:34:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Disease","date":"2024-05-31T08:34:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"434eb1ec-7270-4fff-8698-57e419f7068b","owner":[],"postedDate":"June 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":32910511,"name":"Biological sciences/Cell biology/Mechanisms of disease"},{"id":32910512,"name":"Biological sciences/Immunology/Cytokines"}],"tags":[],"updatedAt":"2025-02-01T08:07:18+00:00","versionOfRecord":{"articleIdentity":"rs-4507474","link":"https://doi.org/10.1038/s41419-025-07376-8","journal":{"identity":"cell-death-and-disease","isVorOnly":false,"title":"Cell Death \u0026 Disease"},"publishedOn":"2025-01-31 05:00:00","publishedOnDateReadable":"January 31st, 2025"},"versionCreatedAt":"2024-06-20 10:23:01","video":"","vorDoi":"10.1038/s41419-025-07376-8","vorDoiUrl":"https://doi.org/10.1038/s41419-025-07376-8","workflowStages":[]},"version":"v1","identity":"rs-4507474","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4507474","identity":"rs-4507474","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}