Network Pharmacological Analysis and Experimental Validation of the Mechanisms of Action of Si-Ni-San Against Liver Fibrosis

Background: Si-Ni-San (SNS), a commonly used traditional Chinese medicine (TCM) formula, has potency against liver diseases, such as hepatitis and non-alcoholic fatty liver disease (NAFLD). However, the therapeutic efficacy and pharmacological mechanisms of action of SNS against liver fibrosis remain largely unclear.Methods: A carbon tetrachloride (CCl4)-induced liver fibrosis mouse model was adopted for the first time to investigate the beneficial effects of SNS on liver fibrosis. The potential mechanisms of action of SNS were explored using the network pharmacology-based strategy and validated with the aid of diverse assays.Results: SNS treatment reduced collagen and ECM deposition, downregulated fibrosis-related factor (hyaluronic acid and laminin) contents in serum, maintained the morphological structure of liver tissue, and improved liver function in the liver fibrosis model. Based on network pharmacology results, apoptosis, inflammation and angiogenesis, together with the associated pathways (including VEGF, TNF, caspase, PPAR-γ and NF-κB), were identified as the mechanisms underlying the effects of SNS on liver fibrosis. Further in vivo experiments validated the significant mitigatory effects of SNS on inflammatory infiltration and pro-inflammatory cytokine contents (IFNγ, IL-1β and TGF-β1) in liver tissues of mice with liver fibrosis. SNS suppressed pathologic neovascularization as well as levels of VEGFR1, VEGF and VEGFR2 in liver tissues. SNS treatment additionally inhibited hepatic parenchyma cell apoptosis in liver tissues of mice with liver fibrosis and regulated apoptin expression while protecting L02 cells against apoptosis induced by TNF-α and Act D in vitro. Activation of hepatic stellate cells was suppressed and the balance between MMP13 and TIMP1 maintained in vitro by SNS. These activities may be associated with SNS-induced NF-κB suppression and PPAR-γ activation.Conclusion: SNS effectively impedes liver fibrosis progression through alleviating inflammation, ECM accumulation, aberrant angiogenesis and apoptosis of hepatic parenchymal cells along with inhibiting activation of hepatic stellate cells through effects on multiple targets and may thus serve as a novel therapeutic regimen for this condition.

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Gli2-regulated activation of hepatic stellate cells and liver fibrosis by TGF-β signaling

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00310.2020 ◽

2021 ◽

Author(s):

Junyan Yan ◽

Baowei Hu ◽

Wenjie Shi ◽

Xiaoyi Wang ◽

Jiayuan Shen ◽

...

Keyword(s):

Liver Fibrosis ◽

Hepatic Stellate Cells ◽

Stellate Cells ◽

Conditional Knockout ◽

Rna Seq ◽

Expression Levels ◽

Liver Fibrogenesis ◽

Hh Signaling ◽

Signaling Activity

The Hedgehog (Hh) signaling pathway is correlated with hepatic stellate cells (HSCs) activation and liver fibrosis. Gli2 is a key transcription effector of Hh signaling. However, the role of Gli2 in HSC-mediated liver fibrosis progression is largely unknown. In the present study, we investigated the effect of Gli2 on liver fibrogenesis and its possible mechanism using conditional knockout (cKO) Gli2 mice and HSC models. Wild-type (WT) and GFAP-CreERT;Gli2flox/flox male mice were exposed to CCl4 for one month to induce liver fibrosis. Primary HSCs were isolated from mice and the transition of HSCs into a myofibroblastic phenotype was evaluated. Livers from mice underwent histological, immunohistochemical, and immunofluorescence analyses. The expression levels of proteins and genes were evaluated by Western blot (WB) analysis and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. RNA-seq was used to screen differentially expressed genes. Results showed that CCl4 treatment induced liver fibrosis, promoted HSCs activation and proliferation, and up-regulated Hh signaling activity. The cKO of Gli2 in GFAP-CreERT;Gli2flox/flox mice decreased liver fibrosis as well as HSC activation and proliferation. In vitro studies showed that KO of Gli2 in HSCs blocked cell proliferation and activation by decrease of cyclin D1/D2 expression. The RNA-seq results revealed that the expression levels TGF-β1 ligands were down-regulated in Gli2 KO HSCs. Furthermore, overexpression of Gli2 rescued proliferation and activation of HSCs by up-regulation of TGF-β signaling activity. Our data demonstrated that Gli2 regulated HSC activation and liver fibrosis by TGF-β signaling, thus providing support for future Gli2-based investigations of liver fibrosis therapy.

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Human placental mesenchymal stem cells ameliorate liver fibrosis in mice by upregulation of Caveolin1 in hepatic stellate cells

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02358-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Yunqi Yao ◽

Zhemin Xia ◽

Fuyi Cheng ◽

Qingyuan Jang ◽

Jiao He ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Liver Fibrosis ◽

Therapeutic Effect ◽

Hepatic Stellate Cells ◽

Time Window ◽

Stellate Cells ◽

Therapeutic Benefits ◽

Placental Mesenchymal Stem Cells

Abstract Background Liver fibrosis (LF) is a common pathological process characterized by the activation of hepatic stellate cells (HSCs) and accumulation of extracellular matrix. Severe LF causes cirrhosis and even liver failure, a major cause of morbidity and mortality worldwide. Transplantation of human placental mesenchymal stem cells (hPMSCs) has been considered as an alternative therapy. However, the underlying mechanisms and the appropriate time window for hPMSC transplantation are not well understood. Methods We established mouse models of CCl4-injured LF and administered hPMSCs at different stages of LF once a week for 2 weeks. The therapeutic effect of hPMSCs on LF was investigated, according to histopathological and blood biochemical analyses. In vitro, the effect of hPMSCs and the secretomes of hPMSCs on the inhibition of activated HSCs was assessed. RNA sequencing (RNA-seq) analysis, real-time PCR array, and western blot were performed to explore possible signaling pathways involved in treatment of LF with hPMSCs. Results hPMSC treatment notably alleviates experimental hepatic fibrosis, restores liver function, and inhibits inflammation. Furthermore, the therapeutic effect of hPMSCs against mild-to-moderate LF was significantly greater than against severe LF. In vitro, we observed that the hPMSCs as well as the secretomes of hPMSCs were able to decrease the activation of HSCs. Mechanistic dissection studies showed that hPMSC treatment downregulated the expression of fibrosis-related genes, and this was accompanied by the upregulation of Caveolin-1 (Cav1) (p < 0.001). This suggested that the amelioration of LF occurred partly due to the restoration of Cav1 expression in activated HSCs. Upregulation of Cav1 can inhibit the TGF-β/Smad signaling pathway, mainly by reducing Smad2 phosphorylation, resulting in the inhibition of activated HSCs, whereas this effect could be abated if Cav1 was silenced in advance by siRNAs. Conclusions Our findings suggest that hPMSCs could provide multifaceted therapeutic benefits for the treatment of LF, and the TGF-β/Cav1 pathway might act as a therapeutic target for hPMSCs in the treatment of LF.

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Transcriptional factor ATF3 promotes liver fibrosis via activating hepatic stellate cells

Cell Death and Disease ◽

10.1038/s41419-020-03271-6 ◽

2020 ◽

Vol 11 (12) ◽

Author(s):

Zhemin Shi ◽

Kun Zhang ◽

Ting Chen ◽

Yu Zhang ◽

Xiaoxiao Du ◽

...

Keyword(s):

Liver Fibrosis ◽

Hepatic Stellate Cells ◽

In Vitro Study ◽

Stellate Cells ◽

Protective Role ◽

Activating Transcription Factor 3 ◽

Dependent Manner ◽

Liver Fibrogenesis

AbstractThe excessive accumulation of extracellular matrix (ECM) is a key feature of liver fibrosis and the activated hepatic stellate cells (HSCs) are the major producer of ECM proteins. However, the precise mechanisms and target molecules that are involved in liver fibrosis remain unclear. In this study, we reported that activating transcription factor 3 (ATF3) was over-expressed in mice and human fibrotic livers, in activated HSCs and injured hepatocytes (HCs). Both in vivo and in vitro study have revealed that silencing ATF3 reduced the expression of pro-fibrotic genes and inhibited the activation of HSCs, thus alleviating the extent of liver fibrosis, indicating a potential protective role of ATF3 knockdown. However, ATF3 was not involved in either the apoptosis or proliferation of HCs. In addition, our data illustrated that increased nuclear localization of ATF3 promoted the transcription of fibrogenic genes and lnc-SCARNA10, which functioned as a novel positive regulator of TGF-β signaling in liver fibrogenesis by recruiting SMAD3 to the promoter of these genes. Interestingly, further study also demonstrated that lnc-SCARNA10 promoted the expression of ATF3 in a TGF-β/SMAD3-dependent manner, revealing a TGF-β/ATF3/lnc-SCARNA10 axis that contributed to liver fibrosis by activating HSCs. Taken together, our data provide a molecular mechanism implicating induced ATF3 in liver fibrosis, suggesting that ATF3 may represent a useful target in the development of therapeutic strategies for liver fibrosis.

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Metabolic Hallmarks of Hepatic Stellate Cells in Liver Fibrosis

Cells ◽

10.3390/cells9010024 ◽

2019 ◽

Vol 9 (1) ◽

pp. 24 ◽

Cited By ~ 7

Author(s):

Olga Khomich ◽

Alexander V. Ivanov ◽

Birke Bartosch

Keyword(s):

Liver Disease ◽

Liver Fibrosis ◽

Hepatic Stellate Cells ◽

Current Knowledge ◽

Stellate Cells ◽

Central Carbon Metabolism ◽

Regenerative Process ◽

Therapeutic Interventions ◽

Alcoholic Fatty Liver ◽

Liver Functions

Liver fibrosis is a regenerative process that occurs after injury. It is characterized by the deposition of connective tissue by specialized fibroblasts and concomitant proliferative responses. Chronic damage that stimulates fibrogenic processes in the long-term may result in the deposition of excess matrix tissue and impairment of liver functions. End-stage fibrosis is referred to as cirrhosis and predisposes strongly to the loss of liver functions (decompensation) and hepatocellular carcinoma. Liver fibrosis is a pathology common to a number of different chronic liver diseases, including alcoholic liver disease, non-alcoholic fatty liver disease, and viral hepatitis. The predominant cell type responsible for fibrogenesis is hepatic stellate cells (HSCs). In response to inflammatory stimuli or hepatocyte death, HSCs undergo trans-differentiation to myofibroblast-like cells. Recent evidence shows that metabolic alterations in HSCs are important for the trans-differentiation process and thus offer new possibilities for therapeutic interventions. The aim of this review is to summarize current knowledge of the metabolic changes that occur during HSC activation with a particular focus on the retinol and lipid metabolism, the central carbon metabolism, and associated redox or stress-related signaling pathways.

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Pharmacological Inhibition of Cyclin-Dependent Kinases Triggers Anti-Fibrotic Effects in Hepatic Stellate Cells In Vitro

International Journal of Molecular Sciences ◽

10.3390/ijms21093267 ◽

2020 ◽

Vol 21 (9) ◽

pp. 3267

Author(s):

Anna Hübbers ◽

Julia Hennings ◽

Daniela Lambertz ◽

Ute Haas ◽

Christian Trautwein ◽

...

Keyword(s):

Cell Cycle ◽

Liver Fibrosis ◽

Hepatic Stellate Cells ◽

Healing Process ◽

Stellate Cells ◽

Wound Healing Process ◽

Cdk Inhibitor ◽

Cyclin E1 ◽

Cyclin Dependent Kinases

Liver fibrosis is a wound healing process in response to chronic liver injury, which is characterized by the accumulation of extracellular collagen produced by Hepatic Stellate Cells (HSCs). This process involves cell cycle re-entry and proliferation of normally quiescent HSCs controlled by cyclins and associated cyclin-dependent kinases (Cdks). Cdk2 mediates the entry and progression through S-phase in complex with E-and A-type cyclins. We have demonstrated that cyclin E1 is essential for liver fibrogenesis in mice, but it is not known if this is dependent on Cdk2 or related Cdks. Here, we aimed to evaluate the benefit of the pan-Cdk inhibitor CR8 for treatment of liver fibrosis in vitro. CR8-treatment reduced proliferation and survival in immortalized HSC lines and in addition attenuated pro-fibrotic properties in primary murine HSCs. Importantly, primary murine hepatocytes were much more tolerant against the cytotoxic and anti-proliferative effects of CR8. We identified CR8 dosages mediating anti-fibrotic effects in primary HSCs without affecting cell cycle activity and survival in primary hepatocytes. In conclusion, the pharmacological pan-Cdk inhibitor CR8 restricts the pro-fibrotic properties of HSCs, while preserving proliferation and viability of hepatocytes at least in vitro. Therefore, CR8 and related drugs might be beneficial for the treatment of liver fibrosis.

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Suppression of fibrogenic signaling in hepatic stellate cells by Twist1-dependent microRNA-214 expression: Role of exosomes in horizontal transfer of Twist1

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00140.2015 ◽

2015 ◽

Vol 309 (6) ◽

pp. G491-G499 ◽

Cited By ~ 67

Author(s):

Li Chen ◽

Ruju Chen ◽

Sherri Kemper ◽

Alyssa Charrier ◽

David R. Brigstock

Keyword(s):

Liver Fibrosis ◽

Hepatic Stellate Cells ◽

Stellate Cells ◽

Cell Types ◽

Tissue Growth ◽

Helix Loop Helix ◽

Downstream Effectors ◽

E Box ◽

Induced Changes

A hallmark of liver fibrosis is the activation of hepatic stellate cells (HSC), which results in their production of fibrotic molecules, a process that is largely regulated by connective tissue growth factor (CCN2). CCN2 is increasingly expressed during HSC activation because of diminished expression of microRNA-214 (miR-214), a product of dynamin 3 opposite strand (DNM3os) that directly suppresses CCN2 mRNA. We show that an E-box in the miR-214 promoter binds the basic helix-loop-helix transcription factor, Twist1, which drives miR-214 expression and results in CCN2 suppression. Twist1 expression was suppressed in HSC of fibrotic livers or in cultured HSC undergoing activation in vitro or after treatment with ethanol. Furthermore, Twist1 decreasingly interacted with DNM3os as HSC underwent activation in vitro. Nanovesicular exosomes secreted by quiescent but not activated HSC contained high levels of Twist1, thus reflecting the suppression of cellular Twist1 during HSC activation. Exosomal Twist1 was intercellularly shuttled between HSC and stimulated expression of miR-214 in the recipient cells, causing expression of CCN2 and its downstream effectors to be suppressed. Additionally, the miR-214 E-box in HSC was also regulated by hepatocyte-derived exosomes, showing that functional transfer of exosomal Twist1 occurs between different cell types. Finally, the levels of Twist1, miR-214, or CCN2 in circulating exosomes from fibrotic mice reflected fibrosis-induced changes in the liver itself, highlighting the potential utility of these and other constituents in serum exosomes as novel circulating biomarkers for liver fibrosis. These findings reveal a unique function for cellular or exosomal Twist1 in CCN2-dependent fibrogenesis.

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Inhibition of ASICs reduces rat hepatic stellate cells activity and liver fibrosis: An in vitro and in vivo study

Cell Biology International ◽

10.1002/cbin.10287 ◽

2014 ◽

pp. n/a-n/a ◽

Cited By ~ 1

Author(s):

Chun-xiao Pan ◽

Fan-rong Wu ◽

Xiao-yu Wang ◽

Jie Tang ◽

Wen-fan Gao ◽

...

Keyword(s):

Liver Fibrosis ◽

Hepatic Stellate Cells ◽

Stellate Cells ◽

In Vivo Study

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Reduction in SNAP-23 Alters Microfilament Organization in Myofibrobastic Hepatic Stellate Cells

Gene Expression ◽

10.3727/105221619x15742818049365 ◽

2020 ◽

Vol 20 (1) ◽

pp. 25-37

Author(s):

Haleigh B. Eubanks ◽

Elise G. Lavoie ◽

Jessica Goree ◽

Jeffrey A. Kamykowski ◽

Neriman Gokden ◽

...

Keyword(s):

Liver Fibrosis ◽

Hepatic Stellate Cells ◽

Cell Movement ◽

Stellate Cells ◽

Snare Proteins ◽

Actin Dynamics ◽

Critical Feature ◽

Effector Cells

Hepatic stellate cells (HSC) are critical effector cells of liver fibrosis. In the injured liver, HSC differentiate into a myofibrobastic phenotype. A critical feature distinguishing myofibroblastic from quiescent HSC is cytoskeletal reorganization. Soluble NSF attachment receptor (SNARE) proteins are important in trafficking of newly synthesized proteins to the plasma membrane for release into the extracellular environment. The goals of this project were to determine the expression of specific SNARE proteins in myofibroblastic HSC and to test whether their alteration changed the HSC phenotype in vitro and progression of liver fibrosis in vivo. We found that HSC lack the t-SNARE protein, SNAP-25, but express a homologous protein, SNAP-23. Downregulation of SNAP-23 in HSC induced reduction in polymerization and disorganization of the actin cytoskeleton associated with loss of cell movement. In contrast, reduction in SNAP-23 in mice by monogenic deletion delayed but did not prevent progression of liver fibrosis to cirrhosis. Taken together, these findings suggest that SNAP-23 is an important regular of actin dynamics in myofibroblastic HSC, but that the role of SNAP-23 in the progression of liver fibrosis in vivo is unclear.

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Serum-Derived miR-574-5p-Containing Exosomes Contribute to Liver Fibrosis by Activating Hepatic Stellate Cells

10.21203/rs.3.rs-858448/v1 ◽

2021 ◽

Author(s):

Xia Zhou ◽

Ziyu Liang ◽

Shanyu Qin ◽

Xianxian Ruan ◽

Haixing Jiang

Keyword(s):

Liver Fibrosis ◽

Human Serum ◽

Hepatic Stellate Cells ◽

High Speed ◽

Stellate Cells ◽

Collagen Deposition ◽

Serum Samples ◽

Serum Exosomes ◽

Masson Staining ◽

Liver Tissues

Abstract Aim: To investigate the association of serum exosomes miR-574-5p with liver fibrosis, and explore the effect and mechanism of serum exosomes on HSC activation.Materials and methods: Using serum samples collected from healthy adults and patients with liver cirrhosis, we extracted human serum exosomes via ultra-high-speed centrifugation, and co-cultured them with hepatic stellate cells (HSCs) line LX2.LX-2-mediated intake of human serum exosomes was examined by confocal microscopy. To induce liver fibrosis, we administered 20% CCl4 to mice intraperitoneally and adopted an exoEasy MIDI kit to extract serum exosomes.Liver fibrosis-related molecules were determined via qRT-PCR, Western blot, Masson staining, and Immunohistochemical staining.Results: Significantly high miR-574-5p levels were expressed in serum exosomes and were positively correlated with the expression of miR-574-5p, collagen deposition, and α-SMA expression in liver tissues of mice during liver fibrosis. Compared to healthy subjects, serum exosomes from cirrhosis patients were associated with higher expression of miR-574-5p, α-SMA, and COL1A1 in LX-2. miR-574-5p mimic promoted the expression of α-SMA and COL1A1 mRNA and protein in LX-2, whereas miR-574-5p inhibitor exerted no effect.Conclusion: This article demonstrates that miR-574-5p expression in serum exosomes is positively correlated with collagen deposition and HSC activation in liver tissues during liver fibrosis.Serum exosomes potentially activate HSC through the transfer of miR-574-5p to HSC during liver fibrosis.

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