Fu-Gan-Hua-Xian Decoction Attenuates Liver Fibrosis via Circadian Clock Regulation

Fu-Gan-Hua-Xian Decoction Attenuates Liver Fibrosis via Circadian Clock Regulation | 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 Research Article Fu-Gan-Hua-Xian Decoction Attenuates Liver Fibrosis via Circadian Clock Regulation Zhenghua Xiao This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6212489/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective: This study aimed to explore the antifibrotic effects of Fu-Gan-Hua-Xian decoction (FGHXT) in a CCl4-induced liver fibrosis rat model and to determine whether its therapeutic benefits are associated with the regulation of circadian clock genes Clock and Bmal1. Methods : A liver fibrosis model was established using CCl4 induction in rats, followed by FGHXT intervention. Liver histopathology was assessed by H&E and Masson staining. The expression levels of fibrosis markers (LN, Col IV, and PC III) and circadian clock genes Clock and Bmal1 were analyzed using RT-PCR and Western blot. Results: Compared with the control group, Clock ( p <0.01) and Bmal1 ( p <0.05) expression were significantly downregulated in the model group, indicating circadian rhythm disruption in liver fibrosis. FGHXT administration significantly upregulated Clock and Bmal1 expression, suggesting a restoration of circadian function. Additionally, fibrosis markers (LN, Col IV, and PC III) were markedly reduced in the FGHXT-treated group. Histological analysis revealed a decrease in collagen deposition and inflammatory cell infiltration, further confirming the antifibrotic effects of FGHXT. Conclusion: Our findings suggest that FGHXT alleviates liver fibrosis by modulating circadian clock genes Clock and Bmal1, potentially through the TGF-β1 signaling pathway. These results provide novel insights into the circadian-based mechanisms underlying the antifibrotic effects of FGHXT, highlighting its potential as a therapeutic strategy for liver fibrosis. Hospital Medicine Liver fibrosis circadian clock Fu-Gan-Hua-Xian decoction (FGHXT) Clock Bmal1 TGF-β1 Figures Figure 1 Figure 2 Figure 3 Introduction Liver fibrosis (LF) is a pathological process characterized by excessive extracellular matrix (ECM) deposition, resulting from chronic liver injury due to viral infections, metabolic disorders, or toxin exposure(Li et al., 2023 ; Zhang et al., 2023 ). If left untreated, fibrosis can progress to cirrhosis and even hepatocellular carcinoma (HCC), posing a significant global health burden(Hou et al., 2024 ; Wang et al., 2024 ). Despite advances in understanding the molecular mechanisms of fibrosis, effective pharmacological treatments remain limited. Recent studies have highlighted the critical role of the circadian clock in liver homeostasis.(Ferrell, 2023 ; Mezhnina et al., 2022 ; Mukherji et al., 2019 ; Sato et al., 2020 ; Zhao et al., 2024 ) The liver's intrinsic circadian machinery, primarily governed by Clock and Bmal1, orchestrates metabolic and immune processes(Ferrell, 2023 ). Disruptions in this system have been implicated in liver diseases, including non-alcoholic fatty liver disease (NAFLD) and fibrosis(Fang et al., 2023 ). Experimental models of CCl4-induced liver fibrosis have demonstrated that circadian rhythm dysregulation exacerbates fibrotic progression, likely through altered hepatic stellate cell (HSC) activation(Chen et al., 2023 ) and TGF-β1 signaling(Long et al., 2024 ). Conversely, restoring circadian function has been shown to alleviate fibrosis, suggesting a potential therapeutic target. Fu-Gan-Hua-Xian decoction (FGHXT), a traditional Chinese herbal formula, has been widely used in clinical practice to ameliorate hepatic fibrosis(李珊珊 et al., 2024; 肖政华 & 李婷婷 et al., 2023; 肖政华 & 石以石则 et al., 2023; 肖政华, 杨辉, & 雷伟 et al., 2019; 肖政华, 杨辉, & 杨君 et al., 2019; 肖政华 & 邹艳 et al., 2019; 钟燕 et al., 2023). Previous studies have reported that FGHXT exerts hepatoprotective effects by inhibiting HSC activation, reducing ECM deposition, and modulating TGF-β1/Smad signaling(肖政华, 杨辉, & 雷伟 et al., 2019; 肖政华 & 邹艳 et al., 2019). However, whether FGHXT mitigates fibrosis through circadian clock regulation remains unclear. In this study, we aimed to investigate the potential role of FGHXT in restoring circadian rhythm dysfunction in a CCl4-induced liver fibrosis rat model. We analyzed the expression of core clock genes Clock and Bmal1 and assessed histopathological changes to evaluate its therapeutic effects. This research may provide new insights into the circadian-based mechanisms underlying FGHXT’s hepatoprotective properties, contributing to the development of more effective antifibrotic strategies. Materials and Methods 1.1 Animals and experimental design SPF-grade Sprague-Dawley (SD) rats (6 weeks old) were purchased from Suzhou Xishan Biotechnology Co., Ltd. (License No.: SYXK(Gan)2020-0001). The animals were housed under controlled conditions (temperature: 20–26°C, humidity: 40–70%) with free access to food and water. The rats were randomly divided into three groups: control (CON), model (MOD), and Fu-Gan-Hua-Xian Decoction (FGHXT, 65.16 g/kg/day) treatment group. The control group received no intervention, while the MOD and FGHXT groups were subjected to liver fibrosis induction using carbon tetrachloride (CCl₄). After successful modeling, the CON and MOD groups received daily intragastric administration of normal saline, whereas the FGHXT group was administered the decoction. Following four weeks of treatment, serum and liver tissue samples were collected. Serum samples were treated with protease inhibitors and stored at -20°C until further analysis. Liver tissue samples were rinsed with 0.01% DEPC-treated normal saline and stored at -80°C. Additionally, some liver specimens were fixed in neutral formalin for 12 hours, embedded in paraffin, and sectioned into 3–5 µm thick slices for histopathological examination. 1.2 Main reagents and instruments Reagents : Carbon tetrachloride (Macklin, Lot: C12773729), Olive oil (Shanghai Yuanye Biotechnology Co., Ltd., Lot: S30503), Hematoxylin-eosin (HE) staining solution (Sigma-Aldrich, Lot: H9627), Masson’s trichrome staining solution (Wuhan Servicebio, Lot: G1006), ELISA kits: LN (Jianglai Biology, Lot: 120647005137370107 ), PCIII (Jianglai Biology, Lot: 120647005207990107), COL IV (Jianglai Biology, Lot: 120647005207540107), Trizol reagent (Ambion, Lot: 15596-026), HiScript® II Q Select RT SuperMix for qPCR (Vazyme, Lot: R223), Antibodies: BMAL1 (rabbit polyclonal, Novus, Lot: NB100-2288SS), CLOCK (rabbit polyclonal, Boster, Lot: PB0638), HRP-labeled goat anti-mouse secondary antibody (Wuhan Sanying Biotechnology, Lot: SA00001-1). Instruments : Real-time fluorescence quantitative PCR system (ABI ViiA-7); Cryogenic high-speed centrifuge (Hunan Kechen, Model: H1-16KR); Electrophoresis and electrotransfer system (Beijing Liuyi, Model: DYCZ-40) 1.3 Preparation of FGHXT The FGHXT consists of Astragalus membranaceus (20 g), Atractylodes macrocephala (20 g), Polygonatum sibiricum (15 g), Angelica sinensis (20 g), Trionyx sinensis (20 g), Salvia miltiorrhiza (20 g), Panax notoginseng (5 g), Gynostemma pentaphyllum (10 g), Bupleurum chinense (10 g), Aurantii Fructus Immaturus (15 g), Paeonia lactiflora (20 g), and Glycyrrhiza uralensis Fisch. (6 g). The formulation was provided by the Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine. The herbs were decocted twice with water, filtered, combined, and concentrated to 2 g/mL for further use. 1.4 Measurement of liver fibrosis indicators Serum samples were thawed at 4°C before analysis. The levels of LN, PCIII, and COL IV were measured using an automated biochemical analyzer (BECKMAN, USA) according to the manufacturer’s protocol. 1.5 Pathological analysis of liver tissue Liver pathology was assessed using HE staining and Masson’s trichrome staining. Paraffin-embedded liver sections were subjected to standard deparaffinization, hydration, staining, differentiation, dehydration, clearing, and microscopic examination to evaluate hepatic lesions. 1.6 Western Blotting Analysis Liver tissue was lysed, and total protein was extracted. The protein concentration was determined using a BCA protein assay kit. Proteins were separated using SDS-PAGE (5% stacking gel, 12% separating gel) and transferred onto PVDF membranes via a semi-dry transfer system. Membranes were blocked with 5% skim milk at room temperature for 2 hours, followed by overnight incubation at 4°C with primary antibodies against CLOCK and BMAL1. After washing, membranes were incubated with secondary antibodies at room temperature for 2 hours. Protein bands were visualized using ECL chemiluminescence, and ImageJ software was used for quantitative analysis of band intensities. 1.7 Quantitative Real-Time PCR (RT-qPCR) Analysis Liver tissue samples were homogenized in Trizol reagent, followed by centrifugation and RNA extraction. RNA purity and concentration were determined using Nanodrop 2000. cDNA was synthesized using 2 µg total RNA in a 20 µL reaction volume, and qPCR was performed using cDNA as a template. Primer sequences: Bmal1(Forward:5'-TGAACCAGACAATGAGGGCT,Reverse: 5'-TATGCCAAAATAGCCGTCGC); Clock(Forward: 5'-TGGTCCCGATTCCATCCAGTAT, Reverse: 5'-TGGCAAAGGTAGGATAGGCAGT). 1.8 Statistical methods All data were analyzed using SPSS 23.0 software. Normally distributed data with homogeneity of variance were expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used for comparisons among multiple groups, followed by LSD post hoc tests for pairwise comparisons. A p-value < 0.05 was considered statistically significant. Results 2.1 Effects of Fuganhuaxian Decoction on Liver Fibrosis-Related Indicators in Rats Compared with the normal control group, the levels of liver fibrosis-related indicators, including LN, COL IV, and PC III, were significantly elevated in the model group. In contrast, the FGHXT group exhibited a significant reduction in serum levels of LN ( p < 0.01) (Fig. 1A), COL IV ( p < 0.001) (Fig. 1B), and PC III ( p < 0.01) (Fig. 1C) compared to the model group, with statistically significant differences. These findings suggest that FGHXT has a beneficial effect in alleviating and improving liver fibrosis in rats. 2.2 Histopathological and Staining Results of Rat Liver Tissue HE staining revealed that liver cells in the normal control group (Fig. 2A) displayed a normal morphology, with no inflammatory cell infiltration or fat accumulation in the portal area, and an intact lobular structure. In the model group (Fig. 2B), a large number of inflammatory cells infiltrated the portal area, accompanied by the formation of fat vacuoles, fibrous tissue proliferation, and disorganized and damaged liver lobular architecture. Following FGHXT intervention (Fig. 2C), there was a significant reduction in fibrous tissue proliferation, fewer inflammatory cells in the liver tissue, and no obvious fat infiltration. Masson staining results demonstrated that in the normal control group (Fig. 2D), liver lobules remained intact and clearly defined, with tightly arranged liver cells and no significant deposition of blue collagen fibers. In contrast, the model group (Fig. 2E) exhibited abnormal liver lobule structures, extensive hepatocyte degeneration and necrosis, and irregular accumulation of large amounts of blue collagen fibers, which disrupted normal physiological structures. However, in the FGHXT group (Fig. 2F), the area covered by blue collagen fibers in liver tissue was markedly reduced, indicating an improvement in liver fibrosis. 2.3 RT-PCR and Western Blot Analysis Compared with the normal control group, the mRNA expression levels of Bmal1 (p < 0.01) (Fig. 3A) and Clock (p < 0.001) (Fig. 3B) were significantly decreased in CCl₄-induced liver fibrosis rats. Similarly, the protein expression levels of Bmal1 (p < 0.001) (Fig. 3C, 3E) and Clock (p < 0.001) (Fig. 3D, 3E) were also significantly reduced. However, FGHXT intervention significantly increased the mRNA expression levels of Bmal1 (p < 0.05) (Fig. 3A) and Clock (p < 0.01) (Fig. 3B), along with the corresponding protein expression levels of Bmal1 (p < 0.05) (Fig. 3C, 3E) and Clock (p < 0.01) (Fig. 3D, 3E). Discussion The circadian clock plays a crucial role in regulating various physiological and pathological processes in the liver, including metabolism, inflammation, and fibrosis(Bishehsari et al., 2020 ; Ferrell, 2023 ; Mezhnina et al., 2022 ; Saran et al., 2020 ; Segers & Depoortere, 2021 ). At the molecular level, Clock and Bmal1 are core components of the circadian machinery, orchestrating the transcription of clock-controlled genes that influence hepatic homeostasis(Yang et al., 2020 ). Disruptions in circadian rhythms have been implicated in liver fibrosis, with previous studies reporting that Clock and Bmal1 expression are significantly downregulated in CCl4-induced fibrosis models(Yang et al., 2020 ; Zhao et al., 2020 ), suggesting an association between fibrosis progression and circadian dysfunction. In this study, we observed a similar trend, where Clock and Bmal1 mRNA and protein levels were significantly reduced in the CCl4-induced liver fibrosis model, further supporting the hypothesis that fibrosis is linked to circadian dysregulation(Segers & Depoortere, 2021 ). Notably, treatment with FGHXT restored Clock and Bmal1 expression, suggesting that its anti-fibrotic effects may be partially mediated through circadian clock regulation(Park et al., 2022 ). Given that the circadian clock influences HSC activation and ECM deposition via multiple pathways, our findings highlight a novel mechanism by which FGHXT may exert its therapeutic effects. Histological analyses further confirmed the protective role of FGHXT against liver fibrosis. Compared to the model group, FGHXT-treated rats exhibited significantly lower levels of LN, Col IV, and PC III, key markers of ECM accumulation, suggesting reduced fibrotic burden. H&E staining demonstrated reduced fibrotic tissue proliferation and minimal inflammatory infiltration following FGHXT treatment. Masson staining further revealed a significant reduction in collagen fiber deposition, indicating that FGHXT effectively mitigates fibrosis severity. These findings are consistent with prior studies demonstrating the hepatoprotective effects of FGHXT(李珊珊 et al., 2024; 肖政华, 杨辉, & 雷伟 et al., 2019; 肖政华 & 邹艳 et al., 2019; 钟燕 et al., 2023), including its ability to inhibit TGF-β1/Smad signaling and suppress HSC activation(肖政华, 杨辉, & 雷伟 et al., 2019; 肖政华 & 邹艳 et al., 2019). Given the established link between circadian rhythms and fibrosis, we propose that FGHXT exerts its anti-fibrotic effects not only through direct inhibition of TGF-β1-mediated signaling but also by restoring circadian clock function. Emerging evidence suggests that targeting the liver clock can attenuate fibrosis by modulating key fibrogenic pathways, including TGF-β1, Wnt/β-catenin, and NF-κB. Our study provides preliminary evidence that circadian regulation may represent a novel therapeutic target in hepatic fibrosis treatment. Future Perspectives While our findings suggest a role for circadian clock restoration in FGHXT-mediated fibrosis attenuation, several questions remain: Mechanistic Insights: Further studies are needed to elucidate whether FGHXT directly influences circadian clock genes or acts through upstream regulators such as Rev-Erbα, RORγ, or PER2, which are known to modulate fibrotic pathways. Clinical Relevance: Investigating whether FGHXT treatment in clinical settings can normalize circadian disruptions in patients with liver fibrosis will be essential to validate its translational potential. Chronotherapy Considerations: Given that circadian misalignment exacerbates fibrosis, exploring the optimal timing of FGHXT administration based on circadian rhythms could enhance its therapeutic efficacy. In conclusion, our study provides novel insights into the anti-fibrotic effects of FGHXT, demonstrating its ability to mitigate fibrosis by restoring circadian clock function. These findings underscore the potential of integrating circadian-based interventions into liver fibrosis treatment strategies. Future research should explore the precise molecular interactions between FGHXT and circadian regulators to optimize its therapeutic applications. Declarations All animal experiments in this study were approved by The Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine (Approval No. EC2023008) and conducted in accordance with its guidelines for ethical animal research. References Bishehsari F, Voigt RM, Keshavarzian A (2020) Circadian rhythms and the gut microbiota: from the metabolic syndrome to cancer. Nat Reviews Endocrinol 16(12):731–739 Chen L, Xia S, Wang F, Zhou Y, Wang S, Yang T, Li Y, Xu M, Zhou Y, Kong D (2023) m6A methylation-induced NR1D1 ablation disrupts the HSC circadian clock and promotes hepatic fibrosis. Pharmacol Res 189:106704 Fang Y, Fang Z, Li Z, Yu R, Zhang H, Wang Q, Cheng X, Le G, Wu G (2023) The role of the gut-liver axis in modulating non-alcoholic fatty liver disease through dietary patterns and microecological agents. Food Bioscience, 103335 Ferrell JM (2023) Circadian rhythms and inflammatory diseases of the liver and gut. 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J Pharm Pharmacol 72(12):1854–1864 李珊珊 黄薪竹, 汤韦韦, 杨庆万, 郭冬妮, 孙文华, 肖政华 (2024) 扶肝化纤汤调节Sph K1/S1P信号通路对非酒精性脂肪肝大鼠肝纤维化的影响. 中药材(05), 1260–1264. http://doi.org/10.13863/j.issn1001-4454.2024.05.032 肖政华 李婷婷 (2023) 石以石则, 杨庆万, & 李珊珊. 基于JNK信号通路探讨扶肝化纤汤对肝星状细胞活化增殖的影响 中草药 54(16):5283–5288 肖政华 石以石则 杨庆万, 李婷婷, 李珊珊, & 钟燕. (2023). 基于Wnt/β-catenin信号通路研究扶肝化纤汤抑制HSC-T6活化的作用. 山东大学学报(医学版), 61(06), 41–46 肖政华 杨辉, 雷伟, 杨君, 易旭, & 谭敏敏. (2019). 扶肝化纤汤对肝纤维化大鼠TGF-β1/Smad信号通路的影响. 山东大学学报(医学版), 57(06), 51–60 肖政华 杨辉 (2019) 杨君, 雷伟, 易旭, & 谭敏敏. 扶肝化纤汤对肝纤维化模型大鼠MAPK信号通路的影响 中草药 50(14):3374–3381 肖政华 邹艳, 杨辉, 谭芊任, 崔峻松, 胡芳 (2019) 扶肝化纤汤含药血清对TGF-β1诱导HSC-T6细胞增殖及TGF-β1/Smad信号通路的影响. 中医杂志, 60(19), 1673–1678. http://doi.org/10.13288/j.11-2166/r.2019.19.012 钟燕 谭芊任, 崔峻松, 李青, 卢露星, 肖政华 (2023) 扶肝化纤汤对肝纤维化正虚毒蕴血瘀证模型大鼠Th17/Treg平衡及相关转录因子的影响. 中医杂志, 64(19), 2019–2026. http://doi.org/10.13288/j.11-2166/r.2023.19.013 Additional Declarations The authors declare no competing interests. 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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-6212489","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":427849783,"identity":"d251ce20-d6e1-4db8-9931-593f46eb242b","order_by":0,"name":"Zhenghua Xiao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCUlEQVRIiWNgGAWjYDACZiBmbAASEkCcYFAjx8befIAELR8qjhnz8RxLIGwTTAvjjDPMifMkchTwqjY4zvzw4c8dNnny0T2Gn3nb2NLbGHIYGH5UbMOpRbKZzdhA8kxaseGdM8bSvG0yuW0MZw8w9py5jVMLPzODmYRh2+HEjTNyDIBa2HLbGPsSmBnbcGthY2b/JpHY9h+kxfg3bxtzOhszjwFeLfzMPGYSB9sOJM6XyDGTBHo/gY2NgBbJZp5iw8a25MQNEmllFsBANmzjYUs4iM8vBuePb3z4s80ucf6M5M03gFEpLz//8cEHPypwa0HoPYDEOYBDESqQbyBK2SgYBaNgFIxEAADZs1ZCWSh6pQAAAABJRU5ErkJggg==","orcid":"","institution":"Guizhou University Of Traditional Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Zhenghua","middleName":"","lastName":"Xiao","suffix":""}],"badges":[],"createdAt":"2025-03-12 13:32:25","currentVersionCode":1,"declarations":{"humanSubjects":true,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":true,"humanSubjectConsent":true,"humanSubjectClinicalTrial":true,"humanSubjectCaseReport":true,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-6212489/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6212489/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":78636829,"identity":"9460cfef-6ccb-4377-8708-cf7a39ccba4e","added_by":"auto","created_at":"2025-03-17 05:29:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":188164,"visible":true,"origin":"","legend":"\u003cp\u003eHepatic Fibrosis indicators.(A)laminin (LN).(B).type IV collagen (COL IV) .(C) type III procollagen (PCIII)..CON, Control; MOD, Model; FGHXT, Fu-Gan-Hua-Xian decoction;Data are expressed as the mean ± SD. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6212489/v1/9289b5134dcdb88f4ec08170.png"},{"id":78638998,"identity":"1108b43e-97f7-4678-a038-c7e996c2f513","added_by":"auto","created_at":"2025-03-17 06:02:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1970986,"visible":true,"origin":"","legend":"\u003cp\u003ePathological sections and staining.HE staining（A-C).Masson staining(D-F).\u003cstrong\u003e(× 400）\u003c/strong\u003eCON, Control; MOD, Model; FGHXT, Fu-Gan-Hua-Xian decoction.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6212489/v1/687f8b1c664d17243e636124.png"},{"id":78636827,"identity":"93f6343b-6423-44fd-ac8a-5c272702eb80","added_by":"auto","created_at":"2025-03-17 05:29:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":180341,"visible":true,"origin":"","legend":"\u003cp\u003eBmal1 Clock RT-PCR(A-B) and Western blot(C-E).CON, Control; MOD, Model. Data are expressed as the mean ± SD. \u0026nbsp;*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6212489/v1/d3913ec997a45fa8b87b7140.png"},{"id":78639073,"identity":"decb9bda-1a2e-44d5-ac42-51609ec01118","added_by":"auto","created_at":"2025-03-17 06:02:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3198331,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6212489/v1/9966c637-85cb-4eaf-91dd-4d6f15c0d271.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eFu-Gan-Hua-Xian Decoction Attenuates Liver Fibrosis via Circadian Clock Regulation\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLiver fibrosis (LF) is a pathological process characterized by excessive extracellular matrix (ECM) deposition, resulting from chronic liver injury due to viral infections, metabolic disorders, or toxin exposure(Li et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). If left untreated, fibrosis can progress to cirrhosis and even hepatocellular carcinoma (HCC), posing a significant global health burden(Hou et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Despite advances in understanding the molecular mechanisms of fibrosis, effective pharmacological treatments remain limited.\u003c/p\u003e \u003cp\u003eRecent studies have highlighted the critical role of the circadian clock in liver homeostasis.(Ferrell, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Mezhnina et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Mukherji et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sato et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) The liver's intrinsic circadian machinery, primarily governed by Clock and Bmal1, orchestrates metabolic and immune processes(Ferrell, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Disruptions in this system have been implicated in liver diseases, including non-alcoholic fatty liver disease (NAFLD) and fibrosis(Fang et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Experimental models of CCl4-induced liver fibrosis have demonstrated that circadian rhythm dysregulation exacerbates fibrotic progression, likely through altered hepatic stellate cell (HSC) activation(Chen et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and TGF-β1 signaling(Long et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Conversely, restoring circadian function has been shown to alleviate fibrosis, suggesting a potential therapeutic target.\u003c/p\u003e \u003cp\u003eFu-Gan-Hua-Xian decoction (FGHXT), a traditional Chinese herbal formula, has been widely used in clinical practice to ameliorate hepatic fibrosis(李珊珊 et al., 2024; 肖政华 \u0026amp; 李婷婷 et al., 2023; 肖政华 \u0026amp; 石以石则 et al., 2023; 肖政华, 杨辉, \u0026amp; 雷伟 et al., 2019; 肖政华, 杨辉, \u0026amp; 杨君 et al., 2019; 肖政华 \u0026amp; 邹艳 et al., 2019; 钟燕 et al., 2023). Previous studies have reported that FGHXT exerts hepatoprotective effects by inhibiting HSC activation, reducing ECM deposition, and modulating TGF-β1/Smad signaling(肖政华, 杨辉, \u0026amp; 雷伟 et al., 2019; 肖政华 \u0026amp; 邹艳 et al., 2019). However, whether FGHXT mitigates fibrosis through circadian clock regulation remains unclear.\u003c/p\u003e \u003cp\u003eIn this study, we aimed to investigate the potential role of FGHXT in restoring circadian rhythm dysfunction in a CCl4-induced liver fibrosis rat model. We analyzed the expression of core clock genes Clock and Bmal1 and assessed histopathological changes to evaluate its therapeutic effects. This research may provide new insights into the circadian-based mechanisms underlying FGHXT\u0026rsquo;s hepatoprotective properties, contributing to the development of more effective antifibrotic strategies.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003e1.1 Animals and experimental design\u003c/h2\u003e\n\u003cp\u003eSPF-grade Sprague-Dawley (SD) rats (6 weeks old) were purchased from Suzhou Xishan Biotechnology Co., Ltd. (License No.: SYXK(Gan)2020-0001). The animals were housed under controlled conditions (temperature: 20\u0026ndash;26\u0026deg;C, humidity: 40\u0026ndash;70%) with free access to food and water.\u003c/p\u003e\n\u003cp\u003eThe rats were randomly divided into three groups: control (CON), model (MOD), and Fu-Gan-Hua-Xian Decoction (FGHXT, 65.16 g/kg/day) treatment group. The control group received no intervention, while the MOD and FGHXT groups were subjected to liver fibrosis induction using carbon tetrachloride (CCl₄). After successful modeling, the CON and MOD groups received daily intragastric administration of normal saline, whereas the FGHXT group was administered the decoction. Following four weeks of treatment, serum and liver tissue samples were collected.\u003c/p\u003e\n\u003cp\u003eSerum samples were treated with protease inhibitors and stored at -20\u0026deg;C until further analysis. Liver tissue samples were rinsed with 0.01% DEPC-treated normal saline and stored at -80\u0026deg;C. Additionally, some liver specimens were fixed in neutral formalin for 12 hours, embedded in paraffin, and sectioned into 3\u0026ndash;5 \u0026micro;m thick slices for histopathological examination.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003e1.2 Main reagents and instruments\u003c/h3\u003e\n\u003cp\u003e\u003cstrong\u003eReagents\u003c/strong\u003e: Carbon tetrachloride (Macklin, Lot: C12773729), Olive oil (Shanghai Yuanye Biotechnology Co., Ltd., Lot: S30503), Hematoxylin-eosin (HE) staining solution (Sigma-Aldrich, Lot: H9627), Masson\u0026rsquo;s trichrome staining solution (Wuhan Servicebio, Lot: G1006), ELISA kits: LN (Jianglai Biology, Lot: 120647005137370107 ), PCIII (Jianglai Biology, Lot: 120647005207990107), COL IV (Jianglai Biology, Lot: 120647005207540107), Trizol reagent (Ambion, Lot: 15596-026), HiScript\u0026reg; II Q Select RT SuperMix for qPCR (Vazyme, Lot: R223), Antibodies: BMAL1 (rabbit polyclonal, Novus, Lot: NB100-2288SS), CLOCK (rabbit polyclonal, Boster, Lot: PB0638), HRP-labeled goat anti-mouse secondary antibody (Wuhan Sanying Biotechnology, Lot: SA00001-1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstruments\u003c/strong\u003e: Real-time fluorescence quantitative PCR system (ABI ViiA-7); Cryogenic high-speed centrifuge (Hunan Kechen, Model: H1-16KR); Electrophoresis and electrotransfer system (Beijing Liuyi, Model: DYCZ-40)\u003c/p\u003e\n\u003ch3\u003e1.3 Preparation of FGHXT\u003c/h3\u003e\n\u003cp\u003eThe FGHXT consists of Astragalus membranaceus (20 g), Atractylodes macrocephala (20 g), Polygonatum sibiricum (15 g), Angelica sinensis (20 g), Trionyx sinensis (20 g), Salvia miltiorrhiza (20 g), Panax notoginseng (5 g), Gynostemma pentaphyllum (10 g), Bupleurum chinense (10 g), Aurantii Fructus Immaturus (15 g), Paeonia lactiflora (20 g), and Glycyrrhiza uralensis Fisch. (6 g). The formulation was provided by the Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine. The herbs were decocted twice with water, filtered, combined, and concentrated to 2 g/mL for further use.\u003c/p\u003e\n\u003ch3\u003e1.4 Measurement of liver fibrosis indicators\u003c/h3\u003e\n\u003cp\u003eSerum samples were thawed at 4\u0026deg;C before analysis. The levels of LN, PCIII, and COL IV were measured using an automated biochemical analyzer (BECKMAN, USA) according to the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e\n\u003ch3\u003e1.5 Pathological analysis of liver tissue\u003c/h3\u003e\n\u003cp\u003eLiver pathology was assessed using HE staining and Masson\u0026rsquo;s trichrome staining. Paraffin-embedded liver sections were subjected to standard deparaffinization, hydration, staining, differentiation, dehydration, clearing, and microscopic examination to evaluate hepatic lesions.\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003e1.6 Western Blotting Analysis\u003c/h2\u003e\n\u003cp\u003eLiver tissue was lysed, and total protein was extracted. The protein concentration was determined using a BCA protein assay kit. Proteins were separated using SDS-PAGE (5% stacking gel, 12% separating gel) and transferred onto PVDF membranes via a semi-dry transfer system.\u003c/p\u003e\n\u003cp\u003eMembranes were blocked with 5% skim milk at room temperature for 2 hours, followed by overnight incubation at 4\u0026deg;C with primary antibodies against CLOCK and BMAL1. After washing, membranes were incubated with secondary antibodies at room temperature for 2 hours. Protein bands were visualized using ECL chemiluminescence, and ImageJ software was used for quantitative analysis of band intensities.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003e1.7 Quantitative Real-Time PCR (RT-qPCR) Analysis\u003c/h3\u003e\n\u003cp\u003eLiver tissue samples were homogenized in Trizol reagent, followed by centrifugation and RNA extraction. RNA purity and concentration were determined using Nanodrop 2000. cDNA was synthesized using 2 \u0026micro;g total RNA in a 20 \u0026micro;L reaction volume, and qPCR was performed using cDNA as a template. Primer sequences: Bmal1(Forward:5'-TGAACCAGACAATGAGGGCT,Reverse: 5'-TATGCCAAAATAGCCGTCGC); Clock(Forward: 5'-TGGTCCCGATTCCATCCAGTAT, Reverse: 5'-TGGCAAAGGTAGGATAGGCAGT).\u003c/p\u003e\n\u003ch3\u003e1.8 Statistical methods\u003c/h3\u003e\n\u003cp\u003eAll data were analyzed using SPSS 23.0 software. Normally distributed data with homogeneity of variance were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). One-way analysis of variance (ANOVA) was used for comparisons among multiple groups, followed by LSD post hoc tests for pairwise comparisons. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\"\u003e\n \u003ch2\u003e2.1 Effects of Fuganhuaxian Decoction on Liver Fibrosis-Related Indicators in Rats\u003c/h2\u003e\n \u003cp\u003eCompared with the normal control group, the levels of liver fibrosis-related indicators, including LN, COL IV, and PC III, were significantly elevated in the model group. In contrast, the FGHXT group exhibited a significant reduction in serum levels of LN (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01) (Fig.\u0026nbsp;1A), COL IV (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) (Fig.\u0026nbsp;1B), and PC III (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01) (Fig.\u0026nbsp;1C) compared to the model group, with statistically significant differences. These findings suggest that FGHXT has a beneficial effect in alleviating and improving liver fibrosis in rats.\u003c/p\u003e\n \u003cdiv id=\"Sec13\"\u003e\n \u003ch2\u003e2.2 Histopathological and Staining Results of Rat Liver Tissue\u003c/h2\u003e\n \u003cp\u003eHE staining revealed that liver cells in the normal control group (Fig.\u0026nbsp;2A) displayed a normal morphology, with no inflammatory cell infiltration or fat accumulation in the portal area, and an intact lobular structure. In the model group (Fig.\u0026nbsp;2B), a large number of inflammatory cells infiltrated the portal area, accompanied by the formation of fat vacuoles, fibrous tissue proliferation, and disorganized and damaged liver lobular architecture. Following FGHXT intervention (Fig.\u0026nbsp;2C), there was a significant reduction in fibrous tissue proliferation, fewer inflammatory cells in the liver tissue, and no obvious fat infiltration.\u003c/p\u003e\n \u003cp\u003eMasson staining results demonstrated that in the normal control group (Fig.\u0026nbsp;2D), liver lobules remained intact and clearly defined, with tightly arranged liver cells and no significant deposition of blue collagen fibers. In contrast, the model group (Fig.\u0026nbsp;2E) exhibited abnormal liver lobule structures, extensive hepatocyte degeneration and necrosis, and irregular accumulation of large amounts of blue collagen fibers, which disrupted normal physiological structures. However, in the FGHXT group (Fig.\u0026nbsp;2F), the area covered by blue collagen fibers in liver tissue was markedly reduced, indicating an improvement in liver fibrosis.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec14\"\u003e\n \u003ch2\u003e2.3 RT-PCR and Western Blot Analysis\u003c/h2\u003e\n \u003cp\u003eCompared with the normal control group, the mRNA expression levels of Bmal1 (p \u0026lt; 0.01) (Fig.\u0026nbsp;3A) and Clock (p \u0026lt; 0.001) (Fig.\u0026nbsp;3B) were significantly decreased in CCl₄-induced liver fibrosis rats. Similarly, the protein expression levels of Bmal1 (p \u0026lt; 0.001) (Fig.\u0026nbsp;3C, 3E) and Clock (p \u0026lt; 0.001) (Fig.\u0026nbsp;3D, 3E) were also significantly reduced.\u003c/p\u003e\n \u003cp\u003eHowever, FGHXT intervention significantly increased the mRNA expression levels of Bmal1 (p \u0026lt; 0.05) (Fig.\u0026nbsp;3A) and Clock (p \u0026lt; 0.01) (Fig.\u0026nbsp;3B), along with the corresponding protein expression levels of Bmal1 (p \u0026lt; 0.05) (Fig.\u0026nbsp;3C, 3E) and Clock (p \u0026lt; 0.01) (Fig.\u0026nbsp;3D, 3E).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe circadian clock plays a crucial role in regulating various physiological and pathological processes in the liver, including metabolism, inflammation, and fibrosis(Bishehsari et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ferrell, \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e; Mezhnina et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e; Saran et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e; Segers \u0026amp; Depoortere, \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). At the molecular level, Clock and Bmal1 are core components of the circadian machinery, orchestrating the transcription of clock-controlled genes that influence hepatic homeostasis(Yang et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). Disruptions in circadian rhythms have been implicated in liver fibrosis, with previous studies reporting that Clock and Bmal1 expression are significantly downregulated in CCl4-induced fibrosis models(Yang et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhao et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e), suggesting an association between fibrosis progression and circadian dysfunction.\u003c/p\u003e\n\u003cp\u003eIn this study, we observed a similar trend, where Clock and Bmal1 mRNA and protein levels were significantly reduced in the CCl4-induced liver fibrosis model, further supporting the hypothesis that fibrosis is linked to circadian dysregulation(Segers \u0026amp; Depoortere, \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). Notably, treatment with FGHXT restored Clock and Bmal1 expression, suggesting that its anti-fibrotic effects may be partially mediated through circadian clock regulation(Park et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). Given that the circadian clock influences HSC activation and ECM deposition via multiple pathways, our findings highlight a novel mechanism by which FGHXT may exert its therapeutic effects.\u003c/p\u003e\n\u003cp\u003eHistological analyses further confirmed the protective role of FGHXT against liver fibrosis. Compared to the model group, FGHXT-treated rats exhibited significantly lower levels of LN, Col IV, and PC III, key markers of ECM accumulation, suggesting reduced fibrotic burden. H\u0026amp;E staining demonstrated reduced fibrotic tissue proliferation and minimal inflammatory infiltration following FGHXT treatment. Masson staining further revealed a significant reduction in collagen fiber deposition, indicating that FGHXT effectively mitigates fibrosis severity. These findings are consistent with prior studies demonstrating the hepatoprotective effects of FGHXT(李珊珊 et al., 2024; 肖政华, 杨辉, \u0026amp; 雷伟 et al., 2019; 肖政华 \u0026amp; 邹艳 et al., 2019; 钟燕 et al., 2023), including its ability to inhibit TGF-\u0026beta;1/Smad signaling and suppress HSC activation(肖政华, 杨辉, \u0026amp; 雷伟 et al., 2019; 肖政华 \u0026amp; 邹艳 et al., 2019).\u003c/p\u003e\n\u003cp\u003eGiven the established link between circadian rhythms and fibrosis, we propose that FGHXT exerts its anti-fibrotic effects not only through direct inhibition of TGF-\u0026beta;1-mediated signaling but also by restoring circadian clock function. Emerging evidence suggests that targeting the liver clock can attenuate fibrosis by modulating key fibrogenic pathways, including TGF-\u0026beta;1, Wnt/\u0026beta;-catenin, and NF-\u0026kappa;B. Our study provides preliminary evidence that circadian regulation may represent a novel therapeutic target in hepatic fibrosis treatment.\u003c/p\u003e\n"},{"header":"Future Perspectives","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003cp\u003eWhile our findings suggest a role for circadian clock restoration in FGHXT-mediated fibrosis attenuation, several questions remain:\u003c/p\u003e\n\u003col\u003e\n\u003cli\u003e\n\u003cp\u003eMechanistic Insights: Further studies are needed to elucidate whether FGHXT directly influences circadian clock genes or acts through upstream regulators such as Rev-Erb\u0026alpha;, ROR\u0026gamma;, or PER2, which are known to modulate fibrotic pathways.\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eClinical Relevance: Investigating whether FGHXT treatment in clinical settings can normalize circadian disruptions in patients with liver fibrosis will be essential to validate its translational potential.\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eChronotherapy Considerations: Given that circadian misalignment exacerbates fibrosis, exploring the optimal timing of FGHXT administration based on circadian rhythms could enhance its therapeutic efficacy.\u003c/p\u003e\n\u003c/li\u003e\n\u003c/ol\u003e\n\u003cul\u003e\n\u003cli\u003e\n\u003cp\u003eIn conclusion, our study provides novel insights into the anti-fibrotic effects of FGHXT, demonstrating its ability to mitigate fibrosis by restoring circadian clock function. These findings underscore the potential of integrating circadian-based interventions into liver fibrosis treatment strategies. Future research should explore the precise molecular interactions between FGHXT and circadian regulators to optimize its therapeutic applications.\u003c/p\u003e\n\u003c/li\u003e\n\u003c/ul\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eAll animal experiments in this study were approved by The Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine (Approval No. EC2023008) and conducted in accordance with its guidelines for ethical animal research.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBishehsari F, Voigt RM, Keshavarzian A (2020) Circadian rhythms and the gut microbiota: from the metabolic syndrome to cancer. 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J Pharm Pharmacol 72(12):1854\u0026ndash;1864\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e李珊珊 黄薪竹, 汤韦韦, 杨庆万, 郭冬妮, 孙文华, 肖政华 (2024) 扶肝化纤汤调节Sph K1/S1P信号通路对非酒精性脂肪肝大鼠肝纤维化的影响. 中药材(05), 1260\u0026ndash;1264. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://doi.org/10.13863/j.issn1001-4454.2024.05.032\u003c/span\u003e\u003cspan address=\"10.13863/j.issn1001-4454.2024.05.032\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e肖政华 李婷婷 (2023) 石以石则, 杨庆万, \u0026amp; 李珊珊. 基于JNK信号通路探讨扶肝化纤汤对肝星状细胞活化增殖的影响 中草药 54(16):5283\u0026ndash;5288\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e肖政华 石以石则 杨庆万, 李婷婷, 李珊珊, \u0026amp; 钟燕. (2023). 基于Wnt/β-catenin信号通路研究扶肝化纤汤抑制HSC-T6活化的作用. 山东大学学报(医学版), 61(06), 41\u0026ndash;46\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e肖政华 杨辉, 雷伟, 杨君, 易旭, \u0026amp; 谭敏敏. (2019). 扶肝化纤汤对肝纤维化大鼠TGF-β1/Smad信号通路的影响. 山东大学学报(医学版), 57(06), 51\u0026ndash;60\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e肖政华 杨辉 (2019) 杨君, 雷伟, 易旭, \u0026amp; 谭敏敏. 扶肝化纤汤对肝纤维化模型大鼠MAPK信号通路的影响 中草药 50(14):3374\u0026ndash;3381\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e肖政华 邹艳, 杨辉, 谭芊任, 崔峻松, 胡芳 (2019) 扶肝化纤汤含药血清对TGF-β1诱导HSC-T6细胞增殖及TGF-β1/Smad信号通路的影响. 中医杂志, 60(19), 1673\u0026ndash;1678. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://doi.org/10.13288/j.11-2166/r.2019.19.012\u003c/span\u003e\u003cspan address=\"10.13288/j.11-2166/r.2019.19.012\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e钟燕 谭芊任, 崔峻松, 李青, 卢露星, 肖政华 (2023) 扶肝化纤汤对肝纤维化正虚毒蕴血瘀证模型大鼠Th17/Treg平衡及相关转录因子的影响. 中医杂志, 64(19), 2019\u0026ndash;2026. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://doi.org/10.13288/j.11-2166/r.2023.19.013\u003c/span\u003e\u003cspan address=\"10.13288/j.11-2166/r.2023.19.013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"The Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Liver fibrosis, circadian clock, Fu-Gan-Hua-Xian decoction (FGHXT), Clock, Bmal1, TGF-β1","lastPublishedDoi":"10.21203/rs.3.rs-6212489/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6212489/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eObjective: \u003c/strong\u003e\u003c/em\u003eThis study aimed to explore the antifibrotic effects of Fu-Gan-Hua-Xian decoction (FGHXT) in a CCl4-induced liver fibrosis rat model and to determine whether its therapeutic benefits are associated with the regulation of circadian clock genes Clock and Bmal1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e:\u003c/strong\u003eA liver fibrosis model was established using CCl4 induction in rats, followed by FGHXT intervention. Liver histopathology was assessed by H\u0026amp;E and Masson staining. The expression levels of fibrosis markers (LN, Col IV, and PC III) and circadian clock genes Clock and Bmal1 were analyzed using RT-PCR and Western blot.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eResults:\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eCompared with the control group, Clock (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01) and Bmal1 (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) expression were significantly downregulated in the model group, indicating circadian rhythm disruption in liver fibrosis. FGHXT administration significantly upregulated Clock and Bmal1 expression, suggesting a restoration of circadian function. Additionally, fibrosis markers (LN, Col IV, and PC III) were markedly reduced in the FGHXT-treated group. Histological analysis revealed a decrease in collagen deposition and inflammatory cell infiltration, further confirming the antifibrotic effects of FGHXT.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003e\u003c/em\u003eOur findings suggest that FGHXT alleviates liver fibrosis by modulating circadian clock genes Clock and Bmal1, potentially through the TGF-β1 signaling pathway. These results provide novel insights into the circadian-based mechanisms underlying the antifibrotic effects of FGHXT, highlighting its potential as a therapeutic strategy for liver fibrosis.\u003c/p\u003e","manuscriptTitle":"Fu-Gan-Hua-Xian Decoction Attenuates Liver Fibrosis via Circadian Clock Regulation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-17 05:29:49","doi":"10.21203/rs.3.rs-6212489/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7ebf4c88-875d-4f71-8220-c5b185e415ff","owner":[],"postedDate":"March 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":45584690,"name":"Hospital Medicine"}],"tags":[],"updatedAt":"2025-03-17T05:29:49+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-17 05:29:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6212489","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6212489","identity":"rs-6212489","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}