Circadian disruption aggravates non-alcoholic fatty liver disease by activating RIPK1-RIPK3-MLKL axis in mice

Circadian disruption aggravates non-alcoholic fatty liver disease by activating RIPK1-RIPK3-MLKL axis in mice | 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 Circadian disruption aggravates non-alcoholic fatty liver disease by activating RIPK1-RIPK3-MLKL axis in mice Xiaopeng Li, Liang Wang, Xiaoyu cheng, Ming Li, Jiali Chen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7611399/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Dec, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Circadian disruption represents a significant risk factor for non-alcoholic fatty liver disease (NAFLD); however, the underlying regulatory mechanisms remain poorly understood. This study aims to investigate the impact of circadian disruption on NAFLD development in mice and to elucidate the associated molecular pathways. First, A NAFLD mouse model was established by feeding mice a high-fat diet over a period of 12 weeks, during which an intervention to disrupt the circadian rhythm was also implemented. The experimental groups included: the control group, the NAFLD model group, the circadian disruption group (CCD), and the NAFLD combined with circadian disruption group (NAFLD + CCD). Lipid accumulation and liver function were assessed using biochemical assay kits from each group. Serum levels of inflammatory cytokines were measured via ELISA. Histopathological alterations in liver tissues were evaluated using HE staining, Masson staining, and Sirius Red staining. Cell apoptosis in liver tissues was detected using the TUNEL assay, while the expression levels of fibrosis-related (Collagen IV, Fibronectin, and α-SMA) proteins were determined through immunohistochemical analysis. Western blotting was employed to assess the expression of necroptosis-related (p-RIPK1/RIPK1, p-RIPK3/RIPK3, and p-MLKL/MLKL) proteins. Additionally, immunofluorescence triple staining was performed to detect the co-localization of RIPK3, IBA1, and Clec4F. The results showed that circadian disruption markedly enhanced lipid accumulation in both serum and liver tissue of NAFLD mice, thereby exacerbating hepatic functional impairment. Compared with the NAFLD group, the NAFLD + CCD group exhibited increased collagen fiber deposition and elevated expression levels of fibrosis-related and necroptosis-related proteins. Furthermore, circadian disruption significantly promotes necroptosis of kupffer cells in the liver tissue of NAFLD mice. In conclusion, circadian disruption exacerbates lipid accumulation in the livers of NAFLD mice, impairs hepatic function, and enhances collagen fiber deposition. The potential mechanism may involve the activation of the RIPK1/RIPK3/MLKL signaling pathway, which promotes necroptosis in Kupffer cells. Biological sciences/Biochemistry Biological sciences/Cell biology Health sciences/Diseases Health sciences/Gastroenterology Health sciences/Medical research circadian disruption non-alcoholic fatty liver disease kupffer cells fibrosis RIPK1/RIPK3/MLKL signaling pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Non-alcoholic fatty liver disease (NAFLD) encompasses a spectrum of liver disorders not attributable to excessive alcohol intake. It is strongly associated with metabolic disturbances such as obesity, diabetes, hyperlipidemia, and hypertension, and represents a hepatic manifestation of multi-system metabolic dysfunction 1 , 2 . If left unmanaged, NAFLD may progress to non-alcoholic steatohepatitis (NASH), which is recognized as a critical precursor to end-stage liver diseases, including cirrhosis, hepatocellular carcinoma, and liver failure 3 , 4 . In recent years, the global prevalence of NAFLD has shown a continuous upward trend. Epidemiological data estimate that approximately 25.24% of the global population is affected, imposing a substantial burden on healthcare systems worldwide 5 – 7 . Currently, there are no approved pharmacological therapies specifically for NAFLD, and the primary management strategy remains lifestyle modification. Therefore, further investigation into its underlying pathogenic mechanisms and the identification of potential therapeutic targets are of critical importance. The circadian rhythm is an adaptive mechanism that enables organisms to respond to periodic changes in their environment and is considered a fundamental characteristic of life. For example, sleep-wake cycles, rest-activity patterns, body temperature, hormone secretion, and cognitive functions all demonstrate rhythmic fluctuations within a 24-hour period 8 – 10 . As a key component of the peripheral biological clock system and the primary metabolic organ in the body, the liver plays a central role in linking circadian regulation with metabolic processes 11 , 12 . The liver's biological clock orchestrates the temporal organization of physiological activities across the day-night cycle, thereby maintaining metabolic homeostasis in response to environmental changes 13 . However, due to modern societal developments and lifestyle changes, disruptions of the circadian rhythm have become increasingly common. Numerous studies have established a significant association between circadian rhythm disruption and the onset and progression of NAFLD 14 – 16 . The CLOCKΔ19 mouse model provides evidence that disruption of circadian rhythms can independently contribute to the development of obesity and metabolic syndrome conditions that closely resemble the pathophysiological progression of NAFLD 17 . Furthermore, an analysis of 282,303 participants from the UK Biobank revealed that shift work patterns and individual chronotypes were significantly correlated with increased liver fat fraction and the presence of NAFLD 18 . Clinical observations further support the hypothesis that the irregular circadian rhythms induced by modern lifestyle patterns may serve as a key contributing factor to the rising global prevalence of NAFLD 19 . Howerver, the molecular mechanisms underlying how circadian disruptions contribute to the progression of NAFLD remain poorly understood. Therefore, in this study, a NAFLD model was established by administering a high-fat diet to C57BL/6J mice, followed by the induction of circadian disruption as an intervention. The impact of circadian disruption on liver function and liver fibrosis in NAFLD mice was evaluated, and the potential molecular mechanisms were also investigated. MATERIALS AND METHODS Materials. The total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDH-C), high-density lipoprotein cholesterol (HDH-C), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) assay kit were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Mouse TNF-α and IL-6 ELISA kit were purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd. (Shanghai, China). HE, Masson and Sirius Red stain kit were purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). CF640 Tunel Cell Apoptosis Detection Kit was purchased from Servicebio Biotechnology Co., Ltd (Wuhan, China). Antibodies against RIPK1, p-RIPK1, RIPK3, p-RIPK3, MLKL, p-MLKL, IBA1 and GAPDH were purchased from Affinity Biosciences (Cincinnati, USA). Antibodies against Collagen IV, Fibronectin, Clec4F and α-SMA were purchased from Abcam (Cambridge, United Kingdom). Mice and experimental design. Forty SPF male C57BL/6J mice aged 6 weeks were purchased from Jiangsu Jicui Yaoke Biotechnology Co., LTD (license No. SCXK (Su) 2023-00099). All experiments followed ethical guidelines approved by the Medical Ethics Committee (No. 20241110). Modeling commenced for all mice after a 7-day adaptive rearing period. A mouse model of non-alcoholic fatty liver disease (NAFLD) was established through the administration of a high-fat diet over a 12-week period. As for circadian disruption (CCD), the light-dark cycle for the mice is as follows: week 1: light from 8 a.m. to 8 p.m., and darkness from 8 p.m. to 8 a.m.; week 2: darkness from 8 a.m. to 8 p.m., and light from 8 p.m. to 8 a.m.; week 3: light from 8 a.m. to 8 p.m., and darkness from 8 p.m. to 8 a.m.; week 4: darkness from 8 a.m. to 8 p.m., and light from 8 p.m. to 8 a.m.; ... and so forth, continuing in this pattern until week 12: Darkness from 8 a.m. to 8 p.m., and light from 8 p.m. to 8 a.m. The mice exhibiting normal circadian rhythms were subjected to a 12-week light-dark cycle, with light exposure from 8:00 a.m. to 8:00 p.m. and darkness from 8:00 p.m. to 8:00 a.m. The experimental design was divided into four groups: the Control group (n = 10), the NAFLD group (n = 10), the CCD group (n = 10) and the NAFLD + CCD group (n = 10). The Control group and CCD group were maintained on a standard diet, whereas the NAFLD group and NAFLD + CCD group were administered a high-fat diet. Besides, The mice in the Control and NAFLD group exhibited normal circadian rhythms, while those in the CCD and NAFLD + CCD group displayed disrupted circadian patterns. Following the intervention, the mice in each group were humanely euthanized under isoflurane inhalation anesthesia at week 13. Blood and liver tissue samples were systematically collected for subsequent analysis. Plasma and tissue biochemical analysis. The concentrations of TC, TG, LDH-C, HDH-C, ALT, and AST in mouse serum were measured in accordance with the protocols outlined in the kit instructions. The concentration of TG in mouse liver tissue was measured in accordance with the protocols outlined in the kit instructions. HE Staining. The liver tissues of mice were first subjected to paraffin-embedded sections. Then, the sections were subjected to baking, dewaxing, hydration, and stained with hematoxylin solution for 3–5 minutes. After rinsing with running water, they were differentiated with 1% hydrochloric acid alcohol, counterblue with counterblue solution, and stained with eosin for 3–5 minutes. The sections were dehydrated, sealed, and observed by taking photos under a microscope. Masson staining. The liver tissues of mice were paraffin-embedded and sectioned. Subsequently, the sections were stained with freshly prepared iron hematoxylin staining solution for 5 minutes, followed by thorough washing with water and differentiation using hydrochloric acid alcohol. Thereafter, the sections were stained with alizarin red safranin staining solution for 10 minutes and briefly rinsed with weak acid working solution. The slides were then treated with 1% phosphomolybdic acid solution for 2 minutes and washed again with weak acid working solution for an additional 2 minutes. Following this, the sections were counterstained with aniline blue staining solution for 2 minutes. Finally, the samples underwent dehydration and were mounted for observation and imaging under a microscope. Sirius Red Staining. The liver tissues of mice were fixed, embedded in paraffin, and sectioned. Subsequently, the tissue sections were stained with hematoxylin for 10 minutes, after which the excess stain was rinsed off using distilled water. This was followed by staining with Sirius Red solution for 15 minutes, with subsequent removal of the excess dye using distilled water. The sections were then dehydrated using a graded alcohol series, cleared in xylene, and mounted with neutral mounting resin. Finally, the prepared sections were examined and imaged under a microscope. Tunel assay. The apoptosis of cells in the liver tissues of mice was detected using the method described in the CF640 tunel cell apoptosis detection kit manual. Enzyme linked immunosorbent assay (ELISA). The concentrations of TNF-α and IL-6 in mouse serum were detected according to ELISA kit instructions. Immunohistochemistry. The liver tissues of mice were initially processed for paraffin-embedded sectioning. Following baking, dewaxing, and hydration, the sections were incubated with specific primary antibodies (Collagen IV, Fibronectin and α-SMA) and horseradish peroxidase-labeled goat anti-rabbit IgG(H + L). Subsequently, color development was performed using DAB. The sections were then dehydrated, mounted, and examined under a microscope after imaging. Western Blotting. Proteins were extracted from mouse liver tissue and the protein concentration was measured using a bicinchoninic acid assay. Separated proteins were transferred to PVDF membranes. After blocking with 5% milk, the membranes were incubated with the corresponding primary antibodies at 4°C overnight. They were then incubated with secondary antibodies at room temperature for 1 hour. Densitometric analysis was performed using image acquisition and analysis software. Images were processed with Image J software and normalized to GAPDH. Immunofluorescence triple staining. The liver tissues of mice were fixed, embedded in paraffin, and sectioned. Subsequently, the tissue sections were deparaffinized, and antigen retrieval was performed using citrate buffer. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide solution, followed by blocking with serum to reduce nonspecific binding. The sections were then incubated with IBA1 primary antibody, followed by HRP-labeled secondary antibody and TSA (tyramide signal amplification). After thorough washing to remove unbound antibodies, serum blocking was repeated. Next, RIPK1 primary antibody, HRP-labeled secondary antibody, and TSA were sequentially applied. Following another washing step, serum blocking was performed once more before the addition of Clec4F primary antibody, HRP-labeled secondary antibody, and TSA. After counterstaining nuclei with DAPI, the sections were treated with an autofluorescence quenching agent for 5 minutes and subsequently mounted. Finally, the sections were examined and imaged using a fluorescence microscope. Statistical Analyses. Statistical analyses were performed using SPSS 22.0. Data were presented as mean ± SEM. One-way ANOVA was used to compare differences between groups. P-values < 0.05 were considered statistically significant. RESULTS Circadian disruption exacerbates lipid accumulation and impairs liver function in mice with NAFLD. As shown in Fig. 1 A, the flowchart outlines the procedure of the animal experiment. As shown in Fig. 1 B-E and Fig. 1 G, compared to the control group, the NAFLD group and CCD group exhibited significantly higher liver-to-body weight ratios, serum TC, serum TG, hepatic TG, and serum LDH-C levels in mice. In NAFLD mice, circadian disruption intervention further elevated these parameters. As depicted in Fig. 1 F, serum HDL-C levels in the NAFLD and CCD groups were significantly lower than those in the control group. Moreover, circadian disruption intervention in NAFLD mice led to a further reduction in serum HDL-C levels. The above results indicate that circadian disruption further aggravates lipid accumulation in NAFLD mice. Subsequently, we further measured the serum ALT and AST levels in each group of mice (Fig. 1 H-I). Compared with the control group, the serum ALT and AST levels in the NAFLD group and the CCD group were significantly elevated. Moreover, compared with the NAFLD group, the NAFLD + CCD group exhibited a further significant increase in serum ALT and AST levels. These results mean circadian disruption has a damage to liver in NAFLD mice. Effect of circadian disruption on the hepatic histopathological structure in NAFLD Mice. As shown in Fig. 2 A, Histological analysis via HE staining revealed that in the control group, the hepatic lobules and portal areas of mice exhibited well-preserved structures, with orderly arranged hepatic cords and normal hepatic sinusoids. In contrast, the NAFLD group displayed hepatocellular swelling, characterized by the presence of cytoplasmic lipid droplets of varying sizes, both intracellularly and extracellularly, diffusely distributed as a mixture of macrovesicular and microvesicular patterns. The cellular boundaries were indistinct, and the nuclei were peripherally displaced. Inflammatory cell infiltration was observed within both the hepatic lobules and portal areas. Compared with the NAFLD group, the NAFLD + CCD group exhibited a more pronounced degree of hepatic steatosis and inflammatory infiltration. Subsequently, we further used Masson staining (Fig. 2 B) and Sirius Red staining (Fig. 2 C) to observe the effect of circadian disruption on the distribution and extent of collagen fiber deposition in the liver of NAFLD mice. As shown in Fig. 2 B, Collagen fibers are stained blue, whereas muscle fibers are stained red. There were no collagen fibers observed in the liver tissues of the control group mice, whereas a small amount of collagen fiber deposition was detected in the liver tissues of both the NAFLD group and the CCD group. Compared with the NAFLD group, the NAFLD + CCD group exhibited an increased distribution of collagen fibers in the liver tissue. As shown in Fig. 2 C, Collagen fibers are stained red, whereas muscle fibers are stained yellow. There were no collagen fibers observed in the liver tissues of the control group mice, whereas a small amount of collagen fiber deposition was detected in the liver tissues of both the NAFLD group and the CCD group. Compared with the NAFLD group, the NAFLD + CCD group exhibited a huge amount of collagen fibers in the liver tissue. Circadian disruption aggravates apoptosis and inflammation levels in mice with NAFLD. As illustrated in Fig. 3 A-B, the Tunel assay was employed to evaluate the extent of apoptosis in liver tissues across different groups of mice. The results demonstrated that the level of apoptosis in the liver tissues of the NAFLD group was significantly elevated compared to that of the Control group. Furthermore, the NAFLD + CCD group exhibited a higher level of apoptosis than the NAFLD group alone. As presented in Fig. 3 C-D, ELISA was conducted to measure the serum levels of inflammatory cytokines in each group. The findings revealed that the concentrations of TNF-α and IL-6 in both the NAFLD group and the CCD group were markedly higher than those in the control group. Notably, the NAFLD + CCD group showed a further increase in TNF-α and IL-6 levels compared to the NAFLD group. Circadian disruption increase the expression of fibrosis-related proteins in the liver tissue of NAFLD mice. As shown in Fig. 4 A-C, immunohistochemistry analysis was employed to assess the expression of fibrosis-related proteins (Collagen IV, Fibronectin, and α-SMA) in the liver tissues of mice across the experimental groups. The results of this analysis are presented in Fig. 4 D-F. Compared with the Control group, significantly elevated expression levels of Collagen IV, Fibronectin, and α-SMA were observed in the liver tissues of mice in both the NAFLD group and the CCD group. Furthermore, in comparison to the NAFLD group, the NAFLD + CCD group exhibited a further increase in the expression levels of these fibrosis-related proteins. Circadian disruption increase the expression of necroptosis-related proteins in the liver tissue of NAFLD mice. As shown in Fig. 5 A, WB was employed to assess the expression of necroptosis-related proteins (p-RIPK1/RIPK1, p-RIPK3/RIPK3, and p-MLKL/MLKL) in the liver tissues of mice across the experimental groups. The results of this analysis are presented in Fig. 5 B-D. Compared with the Control group, significantly elevated expression levels of p-RIPK1/RIPK1, p-RIPK3/RIPK3, and p-MLKL/MLKL were observed in the liver tissues of mice in both the NAFLD group and the CCD group. Furthermore, in comparison to the NAFLD group, the NAFLD + CCD group exhibited a further increase in the expression levels of these necroptosis-related proteins. Circadian disruption enhance the expression of RIPK1 in hepatic macrophages of mice with NAFLD. To further investigate which cell types in the mouse liver are affected by circadian disruption with respect to necroptosis, we employed triple immunofluorescence staining to examine the co-expression of RIPK1, IBA1, and Clec4F in liver tissues from different experimental groups (Fig. 6 ). Notably, IBA1 serves as a marker for macrophages, while Clec4F specifically identifies Kupffer cells. The results indicated that, compared with the NAFLD group, circadian disruption primarily enhanced the co-expression of Clec4F and RIPK1, rather than that of IBA1 and RIPK1. These findings suggest that circadian rhythm disturbance may predominantly influence the necroptosis of hepatic Kupffer cells, thereby exacerbating disease progression. DISCUSSION Non-alcoholic fatty liver disease (NAFLD) has emerged as the most prevalent chronic liver condition in industrialized nations, with its incidence continually increasing and imposing a substantial burden on public health 20 , 21 . At present, no pharmacological treatments have been specifically approved for NAFLD. The circadian rhythm, an intrinsic oscillatory system, governs various physiological and metabolic processes through a roughly 24-hour cycle, allowing organisms to synchronize with daily environmental changes 22 , 23 . As a central organ for metabolic regulation, the liver's physiological functions are profoundly modulated by the circadian rhythm. Accumulating evidence suggests that disruption of the circadian rhythm not only contributes to the development of NAFLD but may also be exacerbated by the disease itself via the liver-brain axis feedback loop, thereby creating a self-perpetuating pathophysiological cycle 24 , 25 . Our findings demonstrate that, compared with NAFLD mice, those subjected to circadian disruption exhibit significantly increased hepatic lipid accumulation, impaired liver function, and elevated serum levels of pro-inflammatory cytokines. These results are consistent with those reported by Zhao et al 26 . The continuous accumulation of fat in the liver and steatosis can lead to non-alcoholic steatohepatitis (NASH). 35% to 50% of NASH cases will cause liver fibrosis, eventually developing into liver cirrhosis and even liver cancer 27 , 28 . Therefore, liver fibrosis is an important stage in the progression of NAFLD. Our research results show that compared with NAFLD mice, the intervention of NAFLD mice with circadian disruption can lead to the deposition of collagen fibers in their liver tissues and significantly increase the expression of fibrosis-related proteins (Collagen IV, Fibronectin, and α-SMA) in their liver tissues. Notably, Fu et al. demonstrated that saikosaponin D has the potential to ameliorate liver fibrosis in mice through the restoration of circadian rhythm 29 . Therefore, restoring the host's circadian rhythm may be an important intervention measure to reverse NAFLD. A substantial body of research has confirmed that hepatocyte apoptosis plays a pivotal role in the progression of NAFLD to liver fibrosis, cirrhosis, and hepatocellular carcinoma 30 – 32 . Our study also demonstrates that disruption of the circadian rhythm in NAFLD mice is associated with elevated levels of apoptosis in hepatic tissues. Furthermore, recent studies have indicated that the modes of cell death in NAFLD are not confined to apoptosis. Necroptosis, a significant form of regulated necrosis, also plays a role in the progression of NAFLD. Unlike classical apoptosis and necrosis, necroptosis does not depend on caspases activity and is characterized as a programmed form of necrotic cell death. When caspases activity are inhibited, the apoptotic pathway is blocked, thereby triggering necroptosis. This process relies on the formation of necrosomes by RIPK1, RIPK3, and MLKL, which lead to cell membrane permeabilization, disruption of membrane integrity, and the subsequent release of inflammatory mediators 33 – 35 . RIPK1, RIPK3, and MLKL are key regulatory proteins involved in necroptosis. P-RIPK1, p-RIPK3, and p-MLKL are necessary conditions for the activation of the RIPK1/RIPK3/MLKL pathway and the mediation of programmed necrosis in cells 36 . Our findings demonstrate that, compared with NAFLD model mice, those with induced circadian disruption exhibit increased expression levels of necroptosis-related proteins (p-RIPK1/RIPK1, p-RIPK3/RIPK3, and p-MLKL/MLKL) in liver tissues. Macrophages, as one of the most critical components of the innate immune system, contribute to immune homeostasis by recruiting other immune cells, eliminating pathogens, and presenting antigens 37 . They also exert significant regulatory functions in various liver diseases through the secretion of inflammatory mediators 38 . Kupffer cells, also referred to as liver-resident macrophages, constitute approximately 20% to 35% of all non-parenchymal cells in the liver 39 , 40 . These cells efficiently phagocytize pathogens that enter via the portal vein or arterial circulation and serve as the primary defense against bacteria and associated toxins originating from the gastrointestinal tract. Additionally, the cytokines secreted by Kupffer cells modulate immune and inflammatory responses, thereby maintaining hepatic homeostasis 41 , 42 . In recent years, accumulating evidence has demonstrated that KCs play a crucial role in the initiation and progression of NAFLD 43 – 45 . In this study, triple immunofluorescence staining revealed that disruption of the circadian rhythm specifically induces necroptosis in Kupffer cells rather than in other macrophage populations within the liver tissue of NAFLD mice. In conclusion, our findings indicate that disruption of the circadian rhythm exacerbates lipid accumulation in the livers of NAFLD mice, impairs hepatic function, and enhances collagen fiber deposition. The potential mechanism underlying these effects may involve the activation of the RIPK1/RIPK3/MLKL signaling pathway, which promotes necroptosis in Kupffer cells. However, this study also has some limitations. Although the results of this study suggest that circadian disruption may promote the progression of NAFLD in mice by inducing necroptosis in Kupffer cells, no in vitro cellular validation was performed. In our future research, we aim to further investigate the underlying molecular mechanisms through which circadian disruption influences necroptosis in Kupffer cells at the in vitro cellular level. CONCLUSION Based upon the results of the present study, we conclude that circadian disruption exacerbates lipid accumulation in the livers of NAFLD mice, impairs hepatic function, and enhances collagen fiber deposition. The potential mechanism underlying these effects may involve the activation of the RIPK1/RIPK3/MLKL signaling pathway, which promotes necroptosis in Kupffer cells. Declarations Author contributions ： Xiaopeng Li drafted the manuscript and conducted the experiments. Liang Wang, Xiaoyu cheng and Ming Li conducted the experiments and performed data analysis. Jiali Chen designed the experiments and revised the manuscript. All authors reviewed the manuscript. Funding: Not applicable. Data availability: Data is provided within the manuscript or supplementary information files. Ethical Approval ： This article does not contain any studies with human participants (Clinical trial number: not applicable). Besides, animals were housed and operated in strict compliance with the ethical principles of animal experimentation, and the operations were ratified by the animal ethics committee (approval number: 20241110). All animal experiments complied with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Besides, all animal experiments as in accordance with the ARRIVE guidelines. Consent to participate: Not applicable. Consent for publication ： Not applicable. Competing interests ： The authors declare no competing financial interest. References Luo, X. et al. Unveiling the role of disulfidptosis-related genes in the pathogenesis of non-alcoholic fatty liver disease. Front. Immunol. 15 , 1386905. 10.3389/fimmu.2024.1386905 (2024). Wei, S. et al. NAFLD and NASH: etiology, targets and emerging therapies. 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Results Probl. Cell. Differ. 74 , 175–209. 10.1007/978-3-031-65944-7_7 (2024). Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterials.pdf Cite Share Download PDF Status: Published Journal Publication published 15 Dec, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 19 Nov, 2025 Reviews received at journal 19 Oct, 2025 Reviews received at journal 09 Oct, 2025 Reviewers agreed at journal 08 Oct, 2025 Reviewers agreed at journal 26 Sep, 2025 Reviewers invited by journal 26 Sep, 2025 Editor assigned by journal 26 Sep, 2025 Editor invited by journal 26 Sep, 2025 Submission checks completed at journal 25 Sep, 2025 First submitted to journal 25 Sep, 2025 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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17:19:42","extension":"html","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":114008,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7611399/v1/4938c7dd8e673a5259c62ae4.html"},{"id":93068812,"identity":"b17dcc27-097e-4cbc-a014-a168ee23f819","added_by":"auto","created_at":"2025-10-08 17:19:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":128747,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of circadian disruption on lipid metabolism phenotypes and liver function in NAFLD mice. (\u003cstrong\u003eA\u003c/strong\u003e) Experimental flowchart, (\u003cstrong\u003eB\u003c/strong\u003e) The liver to body weight ratio, (\u003cstrong\u003eC\u003c/strong\u003e) The serum TC content, (\u003cstrong\u003eD\u003c/strong\u003e) The serum TG content, (\u003cstrong\u003eE\u003c/strong\u003e) The serum LDL-C content, (\u003cstrong\u003eF\u003c/strong\u003e) The serum HDL-C content, (\u003cstrong\u003eG\u003c/strong\u003e) The liver TG content, (\u003cstrong\u003eH\u003c/strong\u003e) The serum ALT content, (\u003cstrong\u003eI\u003c/strong\u003e) The serum AST content. Data are presented as the mean±SD. *P \u0026lt;0.05 vs. Control, \u003csup\u003e#\u003c/sup\u003eP \u0026lt;0.05 vs. NAFLD.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7611399/v1/05199bbaa2eb3568f72b6ab0.png"},{"id":93068813,"identity":"91d107ff-b0c7-45cb-b417-7c4ea19cb9fa","added_by":"auto","created_at":"2025-10-08 17:19:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":770637,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathological staining of liver tissues from different groups of mice. (\u003cstrong\u003eA\u003c/strong\u003e) HE staining, (\u003cstrong\u003eB\u003c/strong\u003e) Masson staining, (\u003cstrong\u003eC\u003c/strong\u003e) Sirius Red staining.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7611399/v1/d90f62b97a09a47e6ba07c2f.png"},{"id":93068816,"identity":"e2028ecf-0246-4f03-a700-22db23d1fe32","added_by":"auto","created_at":"2025-10-08 17:19:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":223598,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of circadian disruption on apoptosis in liver tissues and serum inflammatory cytokine levels in NAFLD mice. (\u003cstrong\u003eA-B\u003c/strong\u003e) The Tunel assay was employed to assess apoptosis in the liver tissues of different group of mice, (\u003cstrong\u003eC\u003c/strong\u003e) The serum TNF-α content, (\u003cstrong\u003eD\u003c/strong\u003e) The serum IL-6 content. Data are presented as the mean±SD. *P \u0026lt;0.05 vs. Control, \u003csup\u003e#\u003c/sup\u003eP \u0026lt;0.05 vs. NAFLD.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7611399/v1/7f83a020712a28ba0179710a.png"},{"id":93069462,"identity":"4bdfc471-9cde-4323-acce-646f0e968add","added_by":"auto","created_at":"2025-10-08 17:27:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":917070,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of circadian disruption on the protein expression of Collagen IV, Fibronectin and α-SMA in the liver tissues of NAFLD mice. (\u003cstrong\u003eA-C\u003c/strong\u003e) The Collagen IV, Fibronectin and α-SMA protein expression were detected by immunohistochemistry, (\u003cstrong\u003eD-F\u003c/strong\u003e) Quantitative analysis of the (\u003cstrong\u003eD\u003c/strong\u003e) Collagen IV, (\u003cstrong\u003eE\u003c/strong\u003e) Fibronectin and (\u003cstrong\u003eF\u003c/strong\u003e) α-SMA protein expression. Data are presented as the mean±SD. *P \u0026lt;0.05 vs. Control, \u003csup\u003e#\u003c/sup\u003eP \u0026lt;0.05 vs. NAFLD.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7611399/v1/9f1ab63f08f9c49660859ba2.png"},{"id":93068817,"identity":"43fc89bc-c703-4521-a76f-5825e1331e85","added_by":"auto","created_at":"2025-10-08 17:19:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":136602,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of circadian disruption on necroptosis-related protein expression in the liver tissues of NAFLD mice. (\u003cstrong\u003eA\u003c/strong\u003e) The necroptosis-related protein expression in the liver tissues of NAFLD mice were detected by WB, (\u003cstrong\u003eB-D\u003c/strong\u003e) Quantitative analysis of the (\u003cstrong\u003eB\u003c/strong\u003e) p-RIPK1/RIPK1, (\u003cstrong\u003eC\u003c/strong\u003e) p-RIPK3/RIPK3 and (\u003cstrong\u003eD\u003c/strong\u003e) p-MLKL/MLKL protein expression. Data are presented as the mean±SD. *P \u0026lt;0.05 vs. Control, \u003csup\u003e#\u003c/sup\u003eP \u0026lt;0.05 vs. NAFLD.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7611399/v1/ee55e5f076ea1df68f86bab7.png"},{"id":93070518,"identity":"53c71bf0-af9d-4bc6-963c-73613b1bc87c","added_by":"auto","created_at":"2025-10-08 17:54:50","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":573498,"visible":true,"origin":"","legend":"\u003cp\u003eImmunofluorescence triple staining was employed to investigate the co-expression patterns of RIPK1 with IBA-1 and Clec4F in various mouse liver tissues.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7611399/v1/a7cf95fea430733ba61f3902.png"},{"id":98814065,"identity":"d270a571-7be3-4551-bfdf-840e0d6e09b4","added_by":"auto","created_at":"2025-12-22 16:10:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3347058,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7611399/v1/d50e0348-7b74-49a1-8f8b-cffef564b78d.pdf"},{"id":93068814,"identity":"49b7a067-cbb1-4c8e-ad7d-f7c9a19856af","added_by":"auto","created_at":"2025-10-08 17:19:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":577153,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7611399/v1/50ecc7b9770d5e7b4cbccc0c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Circadian disruption aggravates non-alcoholic fatty liver disease by activating RIPK1-RIPK3-MLKL axis in mice","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eNon-alcoholic fatty liver disease (NAFLD) encompasses a spectrum of liver disorders not attributable to excessive alcohol intake. It is strongly associated with metabolic disturbances such as obesity, diabetes, hyperlipidemia, and hypertension, and represents a hepatic manifestation of multi-system metabolic dysfunction\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. If left unmanaged, NAFLD may progress to non-alcoholic steatohepatitis (NASH), which is recognized as a critical precursor to end-stage liver diseases, including cirrhosis, hepatocellular carcinoma, and liver failure\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. In recent years, the global prevalence of NAFLD has shown a continuous upward trend. Epidemiological data estimate that approximately 25.24% of the global population is affected, imposing a substantial burden on healthcare systems worldwide\u003csup\u003e\u003cb\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Currently, there are no approved pharmacological therapies specifically for NAFLD, and the primary management strategy remains lifestyle modification. Therefore, further investigation into its underlying pathogenic mechanisms and the identification of potential therapeutic targets are of critical importance.\u003c/p\u003e\u003cp\u003eThe circadian rhythm is an adaptive mechanism that enables organisms to respond to periodic changes in their environment and is considered a fundamental characteristic of life. For example, sleep-wake cycles, rest-activity patterns, body temperature, hormone secretion, and cognitive functions all demonstrate rhythmic fluctuations within a 24-hour period\u003csup\u003e\u003cb\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. As a key component of the peripheral biological clock system and the primary metabolic organ in the body, the liver plays a central role in linking circadian regulation with metabolic processes\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. The liver's biological clock orchestrates the temporal organization of physiological activities across the day-night cycle, thereby maintaining metabolic homeostasis in response to environmental changes \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. However, due to modern societal developments and lifestyle changes, disruptions of the circadian rhythm have become increasingly common. Numerous studies have established a significant association between circadian rhythm disruption and the onset and progression of NAFLD\u003csup\u003e\u003cb\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. The CLOCKΔ19 mouse model provides evidence that disruption of circadian rhythms can independently contribute to the development of obesity and metabolic syndrome conditions that closely resemble the pathophysiological progression of NAFLD\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Furthermore, an analysis of 282,303 participants from the UK Biobank revealed that shift work patterns and individual chronotypes were significantly correlated with increased liver fat fraction and the presence of NAFLD\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Clinical observations further support the hypothesis that the irregular circadian rhythms induced by modern lifestyle patterns may serve as a key contributing factor to the rising global prevalence of NAFLD\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Howerver, the molecular mechanisms underlying how circadian disruptions contribute to the progression of NAFLD remain poorly understood. Therefore, in this study, a NAFLD model was established by administering a high-fat diet to C57BL/6J mice, followed by the induction of circadian disruption as an intervention. The impact of circadian disruption on liver function and liver fibrosis in NAFLD mice was evaluated, and the potential molecular mechanisms were also investigated.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cb\u003eMaterials.\u003c/b\u003e The total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDH-C), high-density lipoprotein cholesterol (HDH-C), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) assay kit were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Mouse TNF-α and IL-6 ELISA kit were purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd. (Shanghai, China). HE, Masson and Sirius Red stain kit were purchased from Solarbio Science \u0026amp; Technology Co., Ltd. (Beijing, China). CF640 Tunel Cell Apoptosis Detection Kit was purchased from Servicebio Biotechnology Co., Ltd (Wuhan, China). Antibodies against RIPK1, p-RIPK1, RIPK3, p-RIPK3, MLKL, p-MLKL, IBA1 and GAPDH were purchased from Affinity Biosciences (Cincinnati, USA). Antibodies against Collagen IV, Fibronectin, Clec4F and α-SMA were purchased from Abcam (Cambridge, United Kingdom).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMice and experimental design.\u003c/b\u003e Forty SPF male C57BL/6J mice aged 6 weeks were purchased from Jiangsu Jicui Yaoke Biotechnology Co., LTD (license No. SCXK (Su) 2023-00099). All experiments followed ethical guidelines approved by the Medical Ethics Committee (No. 20241110). Modeling commenced for all mice after a 7-day adaptive rearing period. A mouse model of non-alcoholic fatty liver disease (NAFLD) was established through the administration of a high-fat diet over a 12-week period. As for circadian disruption (CCD), the light-dark cycle for the mice is as follows: week 1: light from 8 a.m. to 8 p.m., and darkness from 8 p.m. to 8 a.m.; week 2: darkness from 8 a.m. to 8 p.m., and light from 8 p.m. to 8 a.m.; week 3: light from 8 a.m. to 8 p.m., and darkness from 8 p.m. to 8 a.m.; week 4: darkness from 8 a.m. to 8 p.m., and light from 8 p.m. to 8 a.m.; ... and so forth, continuing in this pattern until week 12: Darkness from 8 a.m. to 8 p.m., and light from 8 p.m. to 8 a.m. The mice exhibiting normal circadian rhythms were subjected to a 12-week light-dark cycle, with light exposure from 8:00 a.m. to 8:00 p.m. and darkness from 8:00 p.m. to 8:00 a.m.\u003c/p\u003e\u003cp\u003eThe experimental design was divided into four groups: the Control group (n\u0026thinsp;=\u0026thinsp;10), the NAFLD group (n\u0026thinsp;=\u0026thinsp;10), the CCD group (n\u0026thinsp;=\u0026thinsp;10) and the NAFLD\u0026thinsp;+\u0026thinsp;CCD group (n\u0026thinsp;=\u0026thinsp;10). The Control group and CCD group were maintained on a standard diet, whereas the NAFLD group and NAFLD\u0026thinsp;+\u0026thinsp;CCD group were administered a high-fat diet. Besides, The mice in the Control and NAFLD group exhibited normal circadian rhythms, while those in the CCD and NAFLD\u0026thinsp;+\u0026thinsp;CCD group displayed disrupted circadian patterns. Following the intervention, the mice in each group were humanely euthanized under isoflurane inhalation anesthesia at week 13. Blood and liver tissue samples were systematically collected for subsequent analysis.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePlasma and tissue biochemical analysis.\u003c/b\u003e The concentrations of TC, TG, LDH-C, HDH-C, ALT, and AST in mouse serum were measured in accordance with the protocols outlined in the kit instructions. The concentration of TG in mouse liver tissue was measured in accordance with the protocols outlined in the kit instructions.\u003c/p\u003e\u003cp\u003e\u003cb\u003eHE Staining.\u003c/b\u003e The liver tissues of mice were first subjected to paraffin-embedded sections. Then, the sections were subjected to baking, dewaxing, hydration, and stained with hematoxylin solution for 3\u0026ndash;5 minutes. After rinsing with running water, they were differentiated with 1% hydrochloric acid alcohol, counterblue with counterblue solution, and stained with eosin for 3\u0026ndash;5 minutes. The sections were dehydrated, sealed, and observed by taking photos under a microscope.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMasson staining.\u003c/b\u003e The liver tissues of mice were paraffin-embedded and sectioned. Subsequently, the sections were stained with freshly prepared iron hematoxylin staining solution for 5 minutes, followed by thorough washing with water and differentiation using hydrochloric acid alcohol. Thereafter, the sections were stained with alizarin red safranin staining solution for 10 minutes and briefly rinsed with weak acid working solution. The slides were then treated with 1% phosphomolybdic acid solution for 2 minutes and washed again with weak acid working solution for an additional 2 minutes. Following this, the sections were counterstained with aniline blue staining solution for 2 minutes. Finally, the samples underwent dehydration and were mounted for observation and imaging under a microscope.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSirius Red Staining.\u003c/b\u003e The liver tissues of mice were fixed, embedded in paraffin, and sectioned. Subsequently, the tissue sections were stained with hematoxylin for 10 minutes, after which the excess stain was rinsed off using distilled water. This was followed by staining with Sirius Red solution for 15 minutes, with subsequent removal of the excess dye using distilled water. The sections were then dehydrated using a graded alcohol series, cleared in xylene, and mounted with neutral mounting resin. Finally, the prepared sections were examined and imaged under a microscope.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTunel assay.\u003c/b\u003e The apoptosis of cells in the liver tissues of mice was detected using the method described in the CF640 tunel cell apoptosis detection kit manual.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEnzyme linked immunosorbent assay (ELISA).\u003c/b\u003e The concentrations of TNF-α and IL-6 in mouse serum were detected according to ELISA kit instructions.\u003c/p\u003e\u003cp\u003e\u003cb\u003eImmunohistochemistry.\u003c/b\u003e The liver tissues of mice were initially processed for paraffin-embedded sectioning. Following baking, dewaxing, and hydration, the sections were incubated with specific primary antibodies (Collagen IV, Fibronectin and α-SMA) and horseradish peroxidase-labeled goat anti-rabbit IgG(H\u0026thinsp;+\u0026thinsp;L). Subsequently, color development was performed using DAB. The sections were then dehydrated, mounted, and examined under a microscope after imaging.\u003c/p\u003e\u003cp\u003e\u003cb\u003eWestern Blotting.\u003c/b\u003e Proteins were extracted from mouse liver tissue and the protein concentration was measured using a bicinchoninic acid assay. Separated proteins were transferred to PVDF membranes. After blocking with 5% milk, the membranes were incubated with the corresponding primary antibodies at 4\u0026deg;C overnight. They were then incubated with secondary antibodies at room temperature for 1 hour. Densitometric analysis was performed using image acquisition and analysis software. Images were processed with Image J software and normalized to GAPDH.\u003c/p\u003e\u003cp\u003e\u003cb\u003eImmunofluorescence triple staining.\u003c/b\u003e The liver tissues of mice were fixed, embedded in paraffin, and sectioned. Subsequently, the tissue sections were deparaffinized, and antigen retrieval was performed using citrate buffer. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide solution, followed by blocking with serum to reduce nonspecific binding. The sections were then incubated with IBA1 primary antibody, followed by HRP-labeled secondary antibody and TSA (tyramide signal amplification). After thorough washing to remove unbound antibodies, serum blocking was repeated. Next, RIPK1 primary antibody, HRP-labeled secondary antibody, and TSA were sequentially applied. Following another washing step, serum blocking was performed once more before the addition of Clec4F primary antibody, HRP-labeled secondary antibody, and TSA. After counterstaining nuclei with DAPI, the sections were treated with an autofluorescence quenching agent for 5 minutes and subsequently mounted. Finally, the sections were examined and imaged using a fluorescence microscope.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistical Analyses.\u003c/b\u003e Statistical analyses were performed using SPSS 22.0. Data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. One-way ANOVA was used to compare differences between groups. P-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cb\u003eCircadian disruption exacerbates lipid accumulation and impairs liver function in mice with NAFLD.\u003c/b\u003e As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, the flowchart outlines the procedure of the animal experiment. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-E and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG, compared to the control group, the NAFLD group and CCD group exhibited significantly higher liver-to-body weight ratios, serum TC, serum TG, hepatic TG, and serum LDH-C levels in mice. In NAFLD mice, circadian disruption intervention further elevated these parameters. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, serum HDL-C levels in the NAFLD and CCD groups were significantly lower than those in the control group. Moreover, circadian disruption intervention in NAFLD mice led to a further reduction in serum HDL-C levels. The above results indicate that circadian disruption further aggravates lipid accumulation in NAFLD mice. Subsequently, we further measured the serum ALT and AST levels in each group of mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH-I). Compared with the control group, the serum ALT and AST levels in the NAFLD group and the CCD group were significantly elevated. Moreover, compared with the NAFLD group, the NAFLD\u0026thinsp;+\u0026thinsp;CCD group exhibited a further significant increase in serum ALT and AST levels. These results mean circadian disruption has a damage to liver in NAFLD mice.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of circadian disruption on the hepatic histopathological structure in NAFLD Mice.\u003c/b\u003e As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, Histological analysis via HE staining revealed that in the control group, the hepatic lobules and portal areas of mice exhibited well-preserved structures, with orderly arranged hepatic cords and normal hepatic sinusoids. In contrast, the NAFLD group displayed hepatocellular swelling, characterized by the presence of cytoplasmic lipid droplets of varying sizes, both intracellularly and extracellularly, diffusely distributed as a mixture of macrovesicular and microvesicular patterns. The cellular boundaries were indistinct, and the nuclei were peripherally displaced. Inflammatory cell infiltration was observed within both the hepatic lobules and portal areas. Compared with the NAFLD group, the NAFLD\u0026thinsp;+\u0026thinsp;CCD group exhibited a more pronounced degree of hepatic steatosis and inflammatory infiltration. Subsequently, we further used Masson staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) and Sirius Red staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) to observe the effect of circadian disruption on the distribution and extent of collagen fiber deposition in the liver of NAFLD mice. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, Collagen fibers are stained blue, whereas muscle fibers are stained red. There were no collagen fibers observed in the liver tissues of the control group mice, whereas a small amount of collagen fiber deposition was detected in the liver tissues of both the NAFLD group and the CCD group. Compared with the NAFLD group, the NAFLD\u0026thinsp;+\u0026thinsp;CCD group exhibited an increased distribution of collagen fibers in the liver tissue. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, Collagen fibers are stained red, whereas muscle fibers are stained yellow. There were no collagen fibers observed in the liver tissues of the control group mice, whereas a small amount of collagen fiber deposition was detected in the liver tissues of both the NAFLD group and the CCD group. Compared with the NAFLD group, the NAFLD\u0026thinsp;+\u0026thinsp;CCD group exhibited a huge amount of collagen fibers in the liver tissue.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eCircadian disruption aggravates apoptosis and inflammation levels in mice with NAFLD.\u003c/b\u003e As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B, the Tunel assay was employed to evaluate the extent of apoptosis in liver tissues across different groups of mice. The results demonstrated that the level of apoptosis in the liver tissues of the NAFLD group was significantly elevated compared to that of the Control group. Furthermore, the NAFLD\u0026thinsp;+\u0026thinsp;CCD group exhibited a higher level of apoptosis than the NAFLD group alone. As presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-D, ELISA was conducted to measure the serum levels of inflammatory cytokines in each group. The findings revealed that the concentrations of TNF-α and IL-6 in both the NAFLD group and the CCD group were markedly higher than those in the control group. Notably, the NAFLD\u0026thinsp;+\u0026thinsp;CCD group showed a further increase in TNF-α and IL-6 levels compared to the NAFLD group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eCircadian disruption increase the expression of fibrosis-related proteins in the liver tissue of NAFLD mice.\u003c/b\u003e As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C, immunohistochemistry analysis was employed to assess the expression of fibrosis-related proteins (Collagen IV, Fibronectin, and α-SMA) in the liver tissues of mice across the experimental groups. The results of this analysis are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-F. Compared with the Control group, significantly elevated expression levels of Collagen IV, Fibronectin, and α-SMA were observed in the liver tissues of mice in both the NAFLD group and the CCD group. Furthermore, in comparison to the NAFLD group, the NAFLD\u0026thinsp;+\u0026thinsp;CCD group exhibited a further increase in the expression levels of these fibrosis-related proteins.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eCircadian disruption increase the expression of necroptosis-related proteins in the liver tissue of NAFLD mice.\u003c/b\u003e As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, WB was employed to assess the expression of necroptosis-related proteins (p-RIPK1/RIPK1, p-RIPK3/RIPK3, and p-MLKL/MLKL) in the liver tissues of mice across the experimental groups. The results of this analysis are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-D. Compared with the Control group, significantly elevated expression levels of p-RIPK1/RIPK1, p-RIPK3/RIPK3, and p-MLKL/MLKL were observed in the liver tissues of mice in both the NAFLD group and the CCD group. Furthermore, in comparison to the NAFLD group, the NAFLD\u0026thinsp;+\u0026thinsp;CCD group exhibited a further increase in the expression levels of these necroptosis-related proteins.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eCircadian disruption enhance the expression of RIPK1 in hepatic macrophages of mice with NAFLD.\u003c/b\u003e To further investigate which cell types in the mouse liver are affected by circadian disruption with respect to necroptosis, we employed triple immunofluorescence staining to examine the co-expression of RIPK1, IBA1, and Clec4F in liver tissues from different experimental groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Notably, IBA1 serves as a marker for macrophages, while Clec4F specifically identifies Kupffer cells. The results indicated that, compared with the NAFLD group, circadian disruption primarily enhanced the co-expression of Clec4F and RIPK1, rather than that of IBA1 and RIPK1. These findings suggest that circadian rhythm disturbance may predominantly influence the necroptosis of hepatic Kupffer cells, thereby exacerbating disease progression.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eNon-alcoholic fatty liver disease (NAFLD) has emerged as the most prevalent chronic liver condition in industrialized nations, with its incidence continually increasing and imposing a substantial burden on public health\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. At present, no pharmacological treatments have been specifically approved for NAFLD. The circadian rhythm, an intrinsic oscillatory system, governs various physiological and metabolic processes through a roughly 24-hour cycle, allowing organisms to synchronize with daily environmental changes\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. As a central organ for metabolic regulation, the liver's physiological functions are profoundly modulated by the circadian rhythm. Accumulating evidence suggests that disruption of the circadian rhythm not only contributes to the development of NAFLD but may also be exacerbated by the disease itself via the liver-brain axis feedback loop, thereby creating a self-perpetuating pathophysiological cycle\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Our findings demonstrate that, compared with NAFLD mice, those subjected to circadian disruption exhibit significantly increased hepatic lipid accumulation, impaired liver function, and elevated serum levels of pro-inflammatory cytokines. These results are consistent with those reported by Zhao et al\u003csup\u003e\u003cb\u003e26\u003c/b\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe continuous accumulation of fat in the liver and steatosis can lead to non-alcoholic steatohepatitis (NASH). 35% to 50% of NASH cases will cause liver fibrosis, eventually developing into liver cirrhosis and even liver cancer\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Therefore, liver fibrosis is an important stage in the progression of NAFLD. Our research results show that compared with NAFLD mice, the intervention of NAFLD mice with circadian disruption can lead to the deposition of collagen fibers in their liver tissues and significantly increase the expression of fibrosis-related proteins (Collagen IV, Fibronectin, and α-SMA) in their liver tissues. Notably, Fu et al. demonstrated that saikosaponin D has the potential to ameliorate liver fibrosis in mice through the restoration of circadian rhythm\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Therefore, restoring the host's circadian rhythm may be an important intervention measure to reverse NAFLD.\u003c/p\u003e\u003cp\u003eA substantial body of research has confirmed that hepatocyte apoptosis plays a pivotal role in the progression of NAFLD to liver fibrosis, cirrhosis, and hepatocellular carcinoma\u003csup\u003e\u003cb\u003e\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Our study also demonstrates that disruption of the circadian rhythm in NAFLD mice is associated with elevated levels of apoptosis in hepatic tissues. Furthermore, recent studies have indicated that the modes of cell death in NAFLD are not confined to apoptosis. Necroptosis, a significant form of regulated necrosis, also plays a role in the progression of NAFLD. Unlike classical apoptosis and necrosis, necroptosis does not depend on caspases activity and is characterized as a programmed form of necrotic cell death. When caspases activity are inhibited, the apoptotic pathway is blocked, thereby triggering necroptosis. This process relies on the formation of necrosomes by RIPK1, RIPK3, and MLKL, which lead to cell membrane permeabilization, disruption of membrane integrity, and the subsequent release of inflammatory mediators\u003csup\u003e\u003cb\u003e\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. RIPK1, RIPK3, and MLKL are key regulatory proteins involved in necroptosis. P-RIPK1, p-RIPK3, and p-MLKL are necessary conditions for the activation of the RIPK1/RIPK3/MLKL pathway and the mediation of programmed necrosis in cells\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Our findings demonstrate that, compared with NAFLD model mice, those with induced circadian disruption exhibit increased expression levels of necroptosis-related proteins (p-RIPK1/RIPK1, p-RIPK3/RIPK3, and p-MLKL/MLKL) in liver tissues.\u003c/p\u003e\u003cp\u003eMacrophages, as one of the most critical components of the innate immune system, contribute to immune homeostasis by recruiting other immune cells, eliminating pathogens, and presenting antigens\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. They also exert significant regulatory functions in various liver diseases through the secretion of inflammatory mediators\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Kupffer cells, also referred to as liver-resident macrophages, constitute approximately 20% to 35% of all non-parenchymal cells in the liver\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. These cells efficiently phagocytize pathogens that enter via the portal vein or arterial circulation and serve as the primary defense against bacteria and associated toxins originating from the gastrointestinal tract. Additionally, the cytokines secreted by Kupffer cells modulate immune and inflammatory responses, thereby maintaining hepatic homeostasis\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. In recent years, accumulating evidence has demonstrated that KCs play a crucial role in the initiation and progression of NAFLD\u003csup\u003e\u003cb\u003e\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. In this study, triple immunofluorescence staining revealed that disruption of the circadian rhythm specifically induces necroptosis in Kupffer cells rather than in other macrophage populations within the liver tissue of NAFLD mice. In conclusion, our findings indicate that disruption of the circadian rhythm exacerbates lipid accumulation in the livers of NAFLD mice, impairs hepatic function, and enhances collagen fiber deposition. The potential mechanism underlying these effects may involve the activation of the RIPK1/RIPK3/MLKL signaling pathway, which promotes necroptosis in Kupffer cells. However, this study also has some limitations. Although the results of this study suggest that circadian disruption may promote the progression of NAFLD in mice by inducing necroptosis in Kupffer cells, no in vitro cellular validation was performed. In our future research, we aim to further investigate the underlying molecular mechanisms through which circadian disruption influences necroptosis in Kupffer cells at the in vitro cellular level.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eBased upon the results of the present study, we conclude that circadian disruption exacerbates lipid accumulation in the livers of NAFLD mice, impairs hepatic function, and enhances collagen fiber deposition. The potential mechanism underlying these effects may involve the activation of the RIPK1/RIPK3/MLKL signaling pathway, which promotes necroptosis in Kupffer cells.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXiaopeng Li drafted the manuscript and conducted the experiments. Liang Wang, Xiaoyu cheng and Ming Li conducted the experiments and performed data analysis. Jiali Chen designed the experiments and revised the manuscript. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is provided within the manuscript or supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies with human participants (Clinical trial number: not applicable). Besides, animals were housed and operated in strict compliance with the ethical principles of animal experimentation, and the operations were ratified by the animal ethics committee (approval number: 20241110). All animal experiments complied with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Besides, all animal experiments as in accordance with the ARRIVE guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLuo, X. et al. Unveiling the role of disulfidptosis-related genes in the pathogenesis of non-alcoholic fatty liver disease. \u003cem\u003eFront. 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Differ.\u003c/em\u003e \u003cb\u003e74\u003c/b\u003e, 175\u0026ndash;209. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/978-3-031-65944-7_7\u003c/span\u003e\u003cspan address=\"10.1007/978-3-031-65944-7_7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"circadian disruption, non-alcoholic fatty liver disease, kupffer cells, fibrosis, RIPK1/RIPK3/MLKL signaling pathway","lastPublishedDoi":"10.21203/rs.3.rs-7611399/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7611399/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCircadian disruption represents a significant risk factor for non-alcoholic fatty liver disease (NAFLD); however, the underlying regulatory mechanisms remain poorly understood. This study aims to investigate the impact of circadian disruption on NAFLD development in mice and to elucidate the associated molecular pathways. First, A NAFLD mouse model was established by feeding mice a high-fat diet over a period of 12 weeks, during which an intervention to disrupt the circadian rhythm was also implemented. The experimental groups included: the control group, the NAFLD model group, the circadian disruption group (CCD), and the NAFLD combined with circadian disruption group (NAFLD\u0026thinsp;+\u0026thinsp;CCD). Lipid accumulation and liver function were assessed using biochemical assay kits from each group. Serum levels of inflammatory cytokines were measured via ELISA. Histopathological alterations in liver tissues were evaluated using HE staining, Masson staining, and Sirius Red staining. Cell apoptosis in liver tissues was detected using the TUNEL assay, while the expression levels of fibrosis-related (Collagen IV, Fibronectin, and α-SMA) proteins were determined through immunohistochemical analysis. Western blotting was employed to assess the expression of necroptosis-related (p-RIPK1/RIPK1, p-RIPK3/RIPK3, and p-MLKL/MLKL) proteins. Additionally, immunofluorescence triple staining was performed to detect the co-localization of RIPK3, IBA1, and Clec4F. The results showed that circadian disruption markedly enhanced lipid accumulation in both serum and liver tissue of NAFLD mice, thereby exacerbating hepatic functional impairment. Compared with the NAFLD group, the NAFLD\u0026thinsp;+\u0026thinsp;CCD group exhibited increased collagen fiber deposition and elevated expression levels of fibrosis-related and necroptosis-related proteins. Furthermore, circadian disruption significantly promotes necroptosis of kupffer cells in the liver tissue of NAFLD mice. In conclusion, circadian disruption exacerbates lipid accumulation in the livers of NAFLD mice, impairs hepatic function, and enhances collagen fiber deposition. The potential mechanism may involve the activation of the RIPK1/RIPK3/MLKL signaling pathway, which promotes necroptosis in Kupffer cells.\u003c/p\u003e","manuscriptTitle":"Circadian disruption aggravates non-alcoholic fatty liver disease by activating RIPK1-RIPK3-MLKL axis in mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-08 17:19:36","doi":"10.21203/rs.3.rs-7611399/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-19T09:59:23+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-20T01:15:50+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-09T06:24:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"339256594520471353086460612330757890934","date":"2025-10-09T03:55:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"34504434346966620423307537459333616591","date":"2025-09-26T10:10:43+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-26T09:54:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-26T09:46:33+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-26T09:19:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-26T01:32:04+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-09-26T01:28:03+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ad9b10da-0ab7-4a11-aaeb-0ca2d584efdd","owner":[],"postedDate":"October 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":55867408,"name":"Biological sciences/Biochemistry"},{"id":55867409,"name":"Biological sciences/Cell biology"},{"id":55867410,"name":"Health sciences/Diseases"},{"id":55867411,"name":"Health sciences/Gastroenterology"},{"id":55867412,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2025-12-22T16:04:58+00:00","versionOfRecord":{"articleIdentity":"rs-7611399","link":"https://doi.org/10.1038/s41598-025-32711-6","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-12-15 15:57:16","publishedOnDateReadable":"December 15th, 2025"},"versionCreatedAt":"2025-10-08 17:19:36","video":"","vorDoi":"10.1038/s41598-025-32711-6","vorDoiUrl":"https://doi.org/10.1038/s41598-025-32711-6","workflowStages":[]},"version":"v1","identity":"rs-7611399","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7611399","identity":"rs-7611399","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}