A novel role of vimentin filaments: binding and stabilization of collagen mRNAs

The stem-loop in the 5' untranslated region (UTR) of collagen α1(I) and α2(I) mRNAs (5'SL) is the key element regulating their stability and translation. Stabilization of collagen mRNAs is the predominant mechanism for high collagen expression in fibrosis. LARP6 binds the 5'SL of α1(I) and α2(I) mRNAs with high affinity. Here, we report that vimentin filaments associate with collagen mRNAs in a 5'SL- and LARP6-dependent manner and stabilize collagen mRNAs. LARP6 interacts with vimentin filaments through its La domain and colocalizes with the filaments in vivo. Knockdown of LARP6 by small interfering RNA (siRNA) or mutation of the 5'SL abrogates the interaction of collagen mRNAs with vimentin filaments. Vimentin knockout fibroblasts produce reduced amounts of type I collagen due to decreased stability of collagen α1(I) and α2(I) mRNAs. Disruption of vimentin filaments using a drug or by expression of dominant-negative desmin reduces type I collagen expression, primarily due to decreased stability of collagen mRNAs. RNA fluorescence in situ hybridization (FISH) experiments show that collagen α1(I) and α2(I) mRNAs are associated with vimentin filaments in vivo. Thus, vimentin filaments may play a role in the development of tissue fibrosis by stabilizing collagen mRNAs. This finding will serve as a rationale for targeting vimentin in the development of novel antifibrotic therapies.     

Pirfenidone inhibits the expression of HSP47 in TGF-beta1-stimulated human lung fibroblasts

Pirfenidone (5-methyl-1-phenyl-2-(1H)-pyridone) is a novel anti-fibrotic and anti-inflammatory agent that inhibits the progression of fibrosis in animal models and patients with idiopathic pulmonary fibrosis (IPF). Heat shock protein (HSP) 47, a collagen-specific molecular chaperone, is involved in the processing and/or secretion of procollagen and plays an important role in the pathogenesis of IPF. The present study evaluated the in vitro effects of pirfenidone on expression of HSP47 and collagen type I in cultured normal human lung fibroblasts (NHLF). Expression levels of HSP47 and collagen type I in NHLF stimulated by transforming growth factor (TGF)-beta1 were evaluated genetically, immunologically and immunocytochemically. Treatment with TGF-beta1 stimulated both mRNA and protein expressions of both HSP47 and collagen type I in NHLF, and pirfenidone significantly inhibited this TGF-beta1-enhanced expression in a dose-dependent manner. We concluded that the anti-fibrotic effect of pirfenidone may be mediated not only through direct inhibition of collagen type I expression but also at least partly through inhibition of HSP47 expression in lung fibroblasts, with a resultant reduction of collagen synthesis in lung fibrosis.     

Sulforaphane attenuates hepatic fibrosis via NF-E2-related factor 2-mediated inhibition of transforming growth factor-β/Smad signaling

Sulforaphane (SFN) is a dietary isothiocyanate that exerts chemopreventive effects via NF-E2-related factor 2 (Nrf2)-mediated induction of antioxidant/phase II enzymes, such as heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO1). This work was undertaken to evaluate the effects of SFN on hepatic fibrosis and profibrotic transforming growth factor (TGF)-β/Smad signaling, which are closely associated with oxidative stress. SFN suppressed TGF-β-enhanced expression of α-smooth muscle actin (α-SMA), a marker of hepatic stellate cell (HSC) activation, and profibrogenic genes such as type I collagen, fibronectin, tissue inhibitor of matrix metalloproteinase (TIMP)-1, and plasminogen activator inhibitor (PAI)-1 in hTERT, an immortalized human HSC line. SFN inhibited TGF-β-stimulated activity of a PAI-1 promoter construct and (CAGA)(9) MLP-Luc, an artificial Smad3/4-specific reporter, in addition to reducing phosphorylation and nuclear translocation of Smad3. Nrf2 overexpression was sufficient to inhibit the TGF-β/Smad signaling and PAI-1 expression. Conversely, knockdown of Nrf2, but not inhibition of HO-1 or NQO1 activity, significantly abolished the inhibitory effect of SFN on (CAGA)(9) MLP-Luc activity. However, inhibition of NQO1 activity reversed repression of TGF-β-stimulated expression of type I collagen by SFN, suggesting the involvement of antioxidant activity of SFN in the suppression of Smad-independent fibrogenic gene expression. Finally, SFN treatment attenuated the development and progression of early stage hepatic fibrosis induced by bile duct ligation in mice, accompanied by reduced expression of type I collagen and α-SMA. Collectively, these results show that SFN elicits an antifibrotic effect on hepatic fibrosis through Nrf2-mediated inhibition of the TGF-β/Smad signaling and subsequent suppression of HSC activation and fibrogenic gene expression.     

Nonmuscle Myosin-Dependent Synthesis of Type I Collagen

Type I collagen, synthesized in all tissues as the heterotrimer of two α1(I) polypeptides and one α2(I) polypeptide, is the most abundant protein in the human body. Here we show that intact nonmuscle myosin filaments are required for the synthesis of heterotrimeric type I collagen. Conserved 5′ stem-loop in collagen α1(I) and α2(I) mRNAs binds the RNA-binding protein LARP6. LARP6 interacts with nonmuscle myosin through its C-terminal domain and associates collagen mRNAs with the filaments. Dissociation of nonmuscle myosin filaments results in secretion of collagen α1(I) homotrimer, diminished intracellular colocalization of collagen α1(I) and α2(I) polypeptides (required for folding of the heterotrimer), and their increased intracellular degradation. Inhibition of the motor function of myosin has similar collagen-specific effects, while disruption of actin filaments has a general effect on protein secretion. Nonmuscle myosin copurifies with polysomes, and there is a subset of polysomes involved in myosin-dependent translation of collagen mRNAs. These results indicate that association of collagen mRNAs with nonmuscle myosin filaments is necessary to coordinately synthesize collagen α1(I) and α2(I) polypeptides. We postulate that LARP6/myosin-dependent mechanism regulates the synthesis of heterotrimeric type I collagen by coordinating the translation of collagen mRNAs.     

The inhibitory effect of ginsan on TGF-β mediated fibrotic process

Transforming growth factor-beta (TGF-β) plays a central role in the development of fibrosis by stimulating extracellular matrix accumulation, and signals either directly or indirectly through types I, II, and III (TβRI, II, and III) TGF-β receptor complexes. Ginsan, a polysaccharide extracted from Panax ginseng, has multiple immunomodulatory effects. Here, we examine whether ginsan regulates the fibrogenic process by interfering with TGF-β signaling pathways. TGF-β treatment of murine or human normal lung fibroblasts enhanced the levels of several fibrotic markers, including smooth muscle alpha actin (α-SMA), collagen-1, and fibronectin. Interestingly, ginsan treatment either before or after TGF-β administration led to significant reductions in all of α-SMA, collagen-1, and fibronectin expression levels. Ginsan not only inhibited phosphorylation of Smad2 and Smad3, but also attenuated pERK and pAKT signaling induced by TGF-β. Moreover, ginsan restored TβRIII protein expression, which was significantly downregulated by TGF-β, but reduced TβRI and TβRII protein levels. In a murine model of bleomycin (BLM)-induced pulmonary fibrosis, ginsan significantly suppressed accumulation of collagen, α-SMA, and TGF-β. These data collectively suggest that ginsan acts as an effective anti-fibrotic agent in the treatment of pulmonary fibrosis by blocking multiple TGF-β signaling pathways.     

Smad, but not MAPK, pathway mediates the expression of type I collagen in radiation induced fibrosis

Radiation induced fibrosis occurs following a therapeutic or accidental radiation exposure in normal tissues. Tissue fibrosis is the excessive accumulation of collagen and other extracellular matrix components. This study investigated how ionizing radiation affects the expression level and signal pathway of type I collagen. Real time RT-RCR showed that both α1 and α2 chain of type I collagen mRNA were elevated from 48 h after irradiation with 10 Gy in NIH3T3 cells. The relative luciferase activities of both genes and type I collagen marker were elevated at 72 h. TGF-β1 mRNA was elevated earlier than those of type I collagen genes. A Western blot analysis showed the elevation of Smad phosphorylation at 72 h. Conversely, treatment with TGF-β receptor inhibitor inhibited the mRNA and relative luciferase activity of type I collagen. The phosphorylation of Smad was repressed with the inhibitor, and the luciferase activity was cancelled using a mutant construct of Smad binding site of α2(I) collagen gene. However, the MAPK pathways, p38, ERK1/2 and JNK, were not affected with specific inhibitors or siRNA. The data showed that the Smad pathway mediated the expression of type I collagen in radiation induced fibrosis.     

Formin mDia1 contributes to migration and epithelial-mesenchymal transition of tubular epithelial cells exposed to TGF-β1

Renal tubular epithelial cells may undergo epithelial-mesenchymal transition (EMT) in response to stimuli, such as transforming growth factor (TGF)-β1, leading to myofibroblast activation and renal fibrosis. The formin mDia1 is required for nucleation and polymerization of actin and the microtubule cytoskeleton. The present study sought to explore the role of mDia1 in EMT of tubular epithelial cells. A rat model of unilateral ureteral obstruction (UUO) was established. The expression of TGF-β1, collagen I, collagen III, and mDia1 in the kidneys was examined at day 7 after surgery. The effect of mDia1 on EMT was explored in NRK-52E cells by exposing them to TGF-β1. Increased expression of TGF-β1, collagen I, collagen III, and mDia1 was found in obstructive kidneys of UUO model rats. Exposing rat tubular epithelial cells to TGF-β1 promoted collagen I and collagen III expression but had no effect on mDia1 expression. Silencing mDia1 expression impeded epithelial cell migration as well as reduced TGF-β1, collagen, and Profilin1 expression, whereas mDia1 overexpression exerted an opposite effect. Furthermore, mDia1 regulated the expression of vimentin, α-smooth muscle actin, and E-cadherin and focal adhesion-kinase (FAK)/Src activation through Profilin1. Inhibition of the mDia1 activator RhoA by fasudil reversed EMT, and FAK/Src activation induced by mDia1. In conclusion, mDia1 regulated tubular epithelial cell migration, collagen expression, and EMT in NRK-52E cells exposed to TGF-β1. Thus, suppression of mDia1 activation might be a strategy to counteract renal fibrosis.     

Epithelial�Cmesenchymal transition of rat peritoneal mesothelial cells via Rhoa/Rock pathway

The objective of this study was to investigate the role of the RhoA/Rock signaling pathway in the epithelial-mesenchymal transition (EMT) of rat peritoneal mesothelial cells (RPMCs). Primary SD rat peritoneal mesothelial cells were cultured in vitro. RPMCs were randomly assigned to four groups: group A (control), group B (TGF-β1, 10?μg/L), group C (10?μg/L TGF-β1?+?10?μmol/L Y-27632, an inhibitor of Rock that was pre-applied for 2?h before TGF-β1 stimulation), and group D (Y-27632 alone, 10?μmol/L). Our results were as follows: (1) TGF-β1 stimulation elicited a robust increase in RhoA activity in a time-dependent manner; the increase was 2.57?±?0.52 times larger than the activity observed for the control group (P?相似文献   

Total extract of Yupingfeng attenuates bleomycin-induced pulmonary fibrosis in rats

Yupingfeng is a Chinese herbal compound used efficaciously to treat respiratory tract diseases. Total glucosides of Yupingfeng have been proven effective in anti-inflammation and immunoregulation. Nevertheless, the role of total extract of Yupingfeng (YTE) in pulmonary fibrosis (PF), a severe lung disease with no substantial therapies, remains unknown. Present study was conducted to elucidate the anti-fibrotic activity of YTE. The rat PF model was induced by intratracheal administration of bleomycin (BLM, 5 mg/kg), and YTE (12 mg/kg/d) was gavaged from the second day. At 14 and 28 days, the lungs were harvested and stained with H&E and Masson's trichrome. The content of hydroxyproline (HYP) and type I collagen (Col-I) were detected, while the protein expression of high-mobility group box 1 (HMGB1), transforming growth factor-beta 1 (TGF-β1), Col-I and α-smooth muscle actin (α-SMA) were analyzed by immunohistochemistry or Western blot. As observed, YTE treatment attenuated the alveolitis and fibrosis induced by BLM, reduced the loss of body weight and increase of lung coefficient. Meanwhile, YTE strongly decreased the levels of HYP and Col-I, and reduced the over-expression of HMGB1, TGF-β1, Col-I and α-SMA. In conclusion, YTE could ameliorate BLM-induced lung fibrosis by alleviating HMGB1 activity and TGF-β1 activation, suggesting therapeutic potential for PF.     

Effects of TGF-β1 on the proliferation and differentiation of human periodontal ligament cells and a human periodontal ligament stem/progenitor cell line

Periodontal ligament (PDL) is a specialized connective tissue that influences the lifespan of the tooth. Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine, but little is known about the effects of TGF-β1 on PDL cells. Our aim has been to demonstrate the expression of TGF-β1 in rat PDL tissues and to evaluate its effects on the proliferation and gene expression in human PDL cells (HPLCs) and a human PDL stem/progenitor cell line, line 1-11, that we have recently developed. The expression of TGF-β1 in the entire PDL tissue was confirmed immunohistochemically, and both HPLCs and cell line 1-11 expressed mRNA from the TGF-β1, TGF-β type I receptor, and TGF-β type II receptor genes. Although exogenous TGF-β1 stimulated the proliferation of HPLCs, it did not upregulate the expression of alpha-smooth muscle actin (α-SMA), type I collagen (Col I), or fibrillin-1 (FBN1) mRNA or of α-SMA protein in HPLCs, whereas expression for these genes was attenuated by an anti-TGF-β1 neutralizing antibody. In contrast, exogenous TGF-β1 reduced the proliferation of cell line 1-11, although it upregulated the expression of α-SMA, Col I, and FBN1 mRNA and of α-SMA protein in this cell line. In addition, interleukin-1 beta stimulation significantly reduced the expression of TGF-β1 mRNA and protein in HPLCs. Thus, TGF-β1 seems to play an important role in inducing fibroblastic differentiation of PDL stem/progenitor cells and in maintaining the PDL apparatus under physiological conditions.     

Alveolar epithelial cells express mesenchymal proteins in patients with idiopathic pulmonary fibrosis

Prior work has shown that transforming growth factor-β (TGF-β) can mediate transition of alveolar type II cells into mesenchymal cells in mice. Evidence this occurs in humans is limited to immunohistochemical studies colocalizing epithelial and mesenchymal proteins in sections of fibrotic lungs. To acquire further evidence that epithelial-to-mesenchymal transition occurs in the lungs of patients with idiopathic pulmonary fibrosis (IPF), we studied alveolar type II cells isolated from fibrotic and normal human lung. Unlike normal type II cells, type II cells isolated from the lungs of patients with IPF express higher levels of mRNA for the mesenchymal proteins type I collagen, α-smooth muscle actin (α-SMA), and calponin. When cultured on Matrigel/collagen, human alveolar type II cells maintain a cellular morphology consistent with epithelial cells and expression of surfactant protein C (SPC) and E-cadherin. In contrast, when cultured on fibronectin, the human type II cells flatten, spread, lose expression of pro- SPC, and increase expression of vimentin, N-cadherin, and α-SMA; markers of mesenchymal cells. Addition of a TGF-β receptor kinase inhibitor (SB431542) to cells cultured on fibronectin inhibited vimentin expression and maintained pro-SPC expression, indicating persistence of an epithelial phenotype. These data suggest that alveolar type II cells can acquire features of mesenchymal cells in IPF lungs and that TGF-β can mediate this process.     

microRNA-206 overexpression inhibits epithelial-mesenchymal transition and glomerulosclerosis in rats with chronic kidney disease by inhibiting JAK/STAT signaling pathway

Chronic kidney disease (CKD) is a traumatic disease with significant psychic consequences to the patient's overall physical condition. microRNA-206 (miR-206) has been reported to play an essential role in the development of various diseases. The purpose of the present study is to investigate the effect of miR-206 through the JAK/STAT signaling pathway on epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells and glomerulosclerosis in rats with CKD. The targeting relationship between miR-206 and ANXA1 was verified. To explore the role of miR-206 in CKD, the model of CKD rats was established to detect glomerular sclerosis index (GSI), contents of interleukin-6 (IL-6) and transforming growth factor-beta1 (TGF-β1), and expression of type IV collagen. Moreover, to further determine the roles of both miR-206 and the JAK/STAT signaling pathway in CKD, the gain- and loss-of function approaches were performed with the expression of ANXA1, α-SMA, E-cadherin, vimentin, N-cadherin, and the JAK/STAT signaling pathway-related genes detected. miR-206 negatively targeted ANXA1. Overexpressed miR-206 inhibited the degeneration and interstitial fibrosis of renal tubular epithelial cells, decreased GSI of rats, and the expression of type IV collagen, TGF-β1 and IL-6. Overexpressed miR-206 inhibited the degeneration of renal tubular epithelial cells, the expression of ANXA1, α-SMA, TGF-β1, p-STAT3, STAT3, p-STAT1, STAT1, p-JAK2, and JAK2, while promoted the expression of E-cadherin. Taken together the results, miR-206 inhibits EMT of renal tubular epithelial cells and glomerulosclerosis by inactivating the JAK/STAT signaling pathway via ANXA1 in CKD.     

Autophagy promotes intracellular degradation of type I collagen induced by transforming growth factor (TGF)-β1

Autophagy is a highly conserved cellular process regulating turnover of cytoplasmic proteins via a lysosome-dependent pathway. Here we show that kidneys from mice deficient in autophagic protein Beclin 1 exhibited profibrotic phenotype, with increased collagen deposition. Reduced Beclin 1 expression, through genetic disruption of beclin 1 or knockdown by specific siRNA in primary mouse mesangial cells (MMC), resulted in increased protein levels of type I collagen (Col-I). Inhibition of autolysosomal protein degradation by bafilomycin A(1) also increased Col-I protein levels and colocalization of Col-I with LC3, an autophagy marker, or LAMP-1, a lysosome marker, whereas treatment with TFP, an inducer of autophagy, resulted in decreased Col-I protein levels induced by TGF-β1, without alterations in Col-I α1 mRNA. Heterozygous deletion of beclin 1 increased accumulation of aggregated Col-I under nonstimulated conditions, and stimulation with TGF-β1 further increased aggregated Col-I. These data indicate that Col-I and aggregated, insoluble procollagen I undergo intracellular degradation via autophagy. A cytoprotective role of autophagy is implicated in kidney injury, and we demonstrate that low-dose carbon monoxide, shown to exert cytoprotection against renal fibrosis, induces autophagy to suppress accumulation of Col-I induced by TGF-β1. We also show that TGF-β1 induces autophagy in MMC via TAK1-MKK3-p38 signaling pathway. The dual functions of TGF-β1, as both an inducer of Col-I synthesis and an inducer of autophagy and Col-I degradation, underscore the multifunctional nature of TGF-β1. Our findings suggest a novel role of autophagy as a cytoprotective mechanism to negatively regulate and prevent excess collagen accumulation in the kidney.     

Activation of Wnt11 by transforming growth factor-β drives mesenchymal gene expression through non-canonical Wnt protein signaling in renal epithelial cells

Transforming growth factor β1 (TGF-β) promotes renal interstitial fibrosis in vivo and the expression of mesenchymal genes in vitro; however, most of its direct targets in epithelial cells are still elusive. In a screen for genes directly activated by TGF-β, we found that components of the Wnt signaling pathway, especially Wnt11, were targets of activation by TGF-β and Smad3 in primary renal epithelial cells. In gain and loss of function experiments, Wnt11 mediated the actions of TGF-β through enhanced activation of mesenchymal marker genes, such as Zeb1, Snail1, Pai1, and αSMA, without affecting Smad3 phosphorylation. Inhibition of Wnt11 by receptor knockdown or treatment with Wnt inhibitors limited the effects of TGF-β on gene expression. We found no evidence that Wnt11 activated the canonical Wnt signaling pathway in renal epithelial cells; rather, the function of Wnt11 was mediated by the c-Jun N-terminal kinase (JNK) pathway. Consistent with the in vitro results, all the TGF-β, Wnt11, and JNK targets were activated in a unilateral ureteral obstruction (UUO) model of renal fibrosis in vivo. Our findings demonstrated cooperativity among the TGF-β, Wnt11, and JNK signaling pathways and suggest new targets for anti-fibrotic therapy in renal tissue.     

Macrophage-stimulated cardiac fibroblast production of IL-6 is essential for TGF β/Smad activation and cardiac fibrosis induced by angiotensin II

Interleukin-6 (IL-6) is an important cytokine participating in multiple biologic activities in immune regulation and inflammation. IL-6 has been associated with cardiovascular remodeling. However, the mechanism of IL-6 in hypertensive cardiac fibrosis is still unclear. Angiotensin II (Ang II) infusion in mice increased IL-6 expression in the heart. IL-6 knockout (IL-6-/-) reduced Ang II-induced cardiac fibrosis: 1) Masson trichrome staining showed that Ang II infusion significantly increased fibrotic areas of the wild-type mouse heart, which was greatly suppressed in IL-6-/- mice and 2) immunohistochemistry staining showed decreased expression of α-smooth muscle actin (α-SMA), transforming growth factor β1 (TGF-β1) and collagen I in IL-6-/- mouse heart. The baseline mRNA expression of IL-6 in cardiac fibroblasts was low and was absent in cardiomyocytes or macrophages; however, co-culture of cardiac fibroblasts with macrophages significantly increased IL-6 production and expression of α-SMA and collagen I in fibroblasts. Moreover, TGF-β1 expression and phosphorylation of TGF-β downstream signal Smad3 was stimulated by co-culture of macrophages with cardiac fibroblasts, while IL-6 neutralizing antibody decreased TGF-β1 expression and Smad3 phosphorylation in co-culture of macrophage and fibroblast. Taken together, our results indicate that macrophages stimulate cardiac fibroblasts to produce IL-6, which leads to TGF-β1 production and Smad3 phosphorylation in cardiac fibroblasts and thus stimulates cardiac fibrosis.     

Ascorbic acid supplementation down-regulates the alcohol induced oxidative stress, hepatic stellate cell activation, cytotoxicity and mRNA levels of selected fibrotic genes in guinea pigs

Both oxidative stress and endotoxins mediated immunological reactions play a major role in the progression of alcoholic hepatic fibrosis. Ascorbic acid has been reported to reduce alcohol-induced toxicity and ascorbic acid levels are reduced in alcoholics. Hence, we investigated the hepatoprotective action of ascorbic acid in the reversal of alcohol-induced hepatic fibrosis in male guinea pigs (n = 36), and it was compared with the animals abstenting from alcohol treatment. In comparison with the alcohol abstention group, there was a reduction in the activities of toxicity markers and levels of lipid and protein peroxidation products, expression of α-SMA, caspase-3 activity and mRNA levels of CYP2E1, TGF-β(1), TNF-α and α(1)(I) collagen in liver of the ascorbic acid-supplemented group. The ascorbic acid content in liver was significantly reduced in the alcohol-treated guinea pigs. But it was reversed to normal level in the ascorbic acid-supplemented group. The anti-fibrotic action of ascorbic acid in the rapid regression of alcoholic liver fibrosis may be attributed to decrease in the oxidative stress, hepatic stellate cells activation, cytotoxicity and mRNA expression of fibrotic genes CYP2E1, TGF-β(1), TNF-α and α(1) (I) collagen in hepatic tissues.     

Germacrone improves liver fibrosis by regulating the PI3K/AKT/mTOR signalling pathway

Liver fibrosis is a primary threat to public health, owing to limited therapeutic options. Germacrone (GM) has been shown to exert various curative effects against human diseases, including liver injury. The aim of this study was to investigate the pharmacological effects of GM in the pathophysiology of hepatic fibrosis and determine its potential mechanisms of action. A liver fibrosis rat model was established via carbon tetrachloride (CCl₄) treatment, and LX-2 cells were stimulated with TGF-β1. The effects of GM on liver fibrosis and its relationship with the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signalling pathway were investigated. In the CCl₄ fibrosis-induced rat model, GM improved histological damage, inhibited the activity of hepatic α-smooth muscle actin and improved serum alanine aminotransferase and aspartate aminotransferase levels in a dose-dependent manner. GM potently inhibited hepatic stellate cells (HSCs) growth and epithelial–mesenchymal transition (EMT) progression, as reflected by the altered expression of proliferative (Ki-67, PCNA and cleaved caspase-3) and EMT-related (E-cadherin and vimentin) proteins. In TGF-β1-stimulated LX-2 cells, GM significantly inhibited the survival and activation of HSCs and induced cell apoptosis. GM also suppressed the migration ability and reversed the EMT process in HSCs. Following GM treatment, the phosphorylation of the PI3K, AKT and mTOR proteins was reduced in the liver of CCl₄-treated rats and TGF-β1-stimulated LX-2 cells, indicating that GM may attenuate hepatic fibrosis via the PI3K/AKT/mTOR signalling pathway. These outcomes highlight the anti-fibrotic effects of GM and suggest that it is a potential therapeutic agent for the treatment of liver fibrosis.