TRPC1 Drives Hepatic Stellate Cell Activation To Promote Liver Fibrogenesis

Background:Liver fibrosis is a process involving pathological changes in liver tissue structure resulted from long-term injury-induced wound healing response with chronic stimulation.It is mainly characterized by excessive deposition of extracellular matrix（ECM）and the formation of fibrous scar tissue.The activation of hepatic stellate cells（HSCs）and the excess secretion of ECM proteins by activated HSCs are key factors in the progression of hepatic fibrosis.Liver fibrosis injury is very common in the progression of various chronic liver diseases.If liver fibrosis continues to progress,it will gradually develop into liver cirrhosis and hepatocellular carcinoma with limited effective clinical therapeutic strategies.Exploring its potential prevention and treatment targets and therapeutic drugs is still an urgent unsolved challenge in the field of medicine.Previous reports indicated that transient receptor potential canonical channels（TRPCs）were closely related to fibroblast transformation and wound healing processes.Besides,they could participate in some fibroproliferative diseases’progression,such as progressive nephropathy,systemic sclerosis and cardiovascular fibrosis.The expression,distribution and function of TRPCs in the liver remain vague.In this study,data showed that,among TRPCs,the high expression of TRPC1 was closely related to the poor survival rate of end-stage liver fibrosis patients with cancer.In addition,the expression level of TRPC1 was positively correlated with levels of liver fibrosis markers in human liver tissues.However,no studies have been carried out to state whether TPRC1 might be involved in the progression of liver fibrosis.Objective:Based on the expression data of TRPC1 in fibrotic liver tissue of patients,this research intends to establish different etiologies-induced experimental mouse models of liver fibrosis and adopt a genetic intervention strategy to identify the role of TRPC1 in liver fibrosis from animal,tissue,cell and molecular levels.Moreover,this research aims to elucidate the contribution of TRPC1 to activation of HSCs during the pathological process of liver fibrosis,providing experimental basis for in-depth understanding of the pathological mechanism of liver fibrosis and research foundation for exploring novel therapeutic targets of liver fibrosis.Methods:1.Expression of TRPC1 in fibrotic liver tissue（1）Clinical sample research:H&E and Sirius red staining methods were conducted for histopathological examination of human liver samples to examine the degree of fibrosis hyperplasia.Immunohistochemical staining was performed to detect the expression ofα-SMA（HSCs activation marker）and TRPC1 in normal and patient fibrotic liver tissue to identify the expression changes and distribution areas.Immunofluorescence staining and confocal observation were conducted to detect the cellular source and distribution of TRPC1 in liver tissue of patients.（2）Animal research:Experimental liver fibrosis model of mice induced by CCl₄injection,common bile duct ligation（BDL）and a progressive model of liver fibrosis induced by DEN combined with long-term CCl₄ stimulation were established.H&E,Sirius red and Masson staining were performed to evaluate the degree of liver fibrosis in mice.Immunohistochemical staining and western blotting were used to detect the expression changes of TRPC,α-SMA and major ECM protein CollagenⅠin liver tissue of experimental model mice.Immunofluorescence staining and confocal mothed were performed to observe distribution of TRPC1 in the liver tissues of mice.Immunofluorescence and western blotting were performed to detect the distribution and expression of TRPC1in in vivo and in vitro-activated primary mouse HSCs.2.The role of TRPC1 in the progression of experimental liver fibrosis（1）Gene knockout studies:HSCs-specific Trpc1 knockout mice were established.Trpc1 knockout and control mice were used to detect the effect of TRPC1 in CCl₄ and BDL-induced mouse liver fibrosis.H&E,Sirius red and Masson staining were used to evaluate the degree of liver fibrosis in mice.Immunohistochemical staining and Western blotting were used to detect the distribution and expression ofα-SMA and CollagenⅠin liver tissues of mice to evaluate the degree of HSCs activation and ECM deposition.Serum of mice was used to detect the level changes of liver function-related indicators（ALT,AST and TBil）.Trpc1 knockout and control mice were challenged by DEN plus long-term CCl₄stimulation to identify the role of TRPC1 in the progression of liver fibrosis,cirrhosis and hepatocellular carcinoma through pathological examination and maker detection.H&E and Sirius red staining methods were used to evaluate the degree of liver fibrosis in mice.Immunohistochemical staining was conducted to detect the distribution and expression ofα-SMA and cell proliferation marker Ki67 in mice liver tissues.Besides,the tumor formation,tumor number and maximum tumor diameter in livers of mice were observed and counted.（2）Gene complementation research:On the basis of clarifying the role of TRPC1,the Trpc1 knockout mice were used as the research objects,the contribution of TRPC1 to CCl₄ and BDL-induced fibrosis progression and activation of HSCs was further validated by the supplementing expression of TRPC1 in HSCs of knockout mice by AAV injection mothed.Expression of TRPC1 in mice liver tissues was detected by immunoblotting method.H&E,Sirius red and Masson staining were performed to evaluate the degree of fibrotic injury in liver tissues of mice.The distribution and expression ofα-SMA and CollagenⅠin mouse liver tissue were detected by immunohistochemical staining.3.The role and associated mechanism of TRPC1 in the regulation of HSCs’activation（1）Functional elucidation:TGF-β1（5 ng/m L）-induced in vitro activation model of human HSCs（Lx-2 cell line）was established,and then the expressions of TRPC1 andα-SMA during the activation of HSCs were detected by Western Blotting.The stable Control Sh RNA and TRPC1 Sh RNA-transfected Lx-2 cells were established.Western Blot assays were performed to detect the effect of TRPC1 knockout on HSCs activation indicators（α-SMA and CollagenⅠ）expressions.Scratch and migration assays were conducted to evaluate the effect of TRPC1 knockout on the migration ability of HSCs.（2）Mechanism exploration:Western blotting and immunofluorescence assays were used to detect the effect of TRPC1 on the signal transduction of TGF-β/SMAD pathway and SMAD2/3 nuclear translocation.Ca²⁺imaging experiments were used to detect intracellular Ca²⁺changes in Lx-2 cells.BAPTA-AM and Ionomycin were used to evaluate the contribution of TRPC1-mediated Ca²⁺to HSCs activation and downstream fibrosis-related gene transcription by western blotting and PCR experiments.IP/MS experiments to screen potential proteins that may directly bind to TRPC1.CO-IP experiments were performed to further validate the protein interactions between TRPC1and vimentin as well as ERK2.Western Blotting was used to detect changes in the phosphorylation level of vimentin.Immunofluorescence and solubility assays were used to detect the contribution of TRPC1 to the vimentin filaments assemble and following cytoskeleton remodeling.Results:1.Expression of TRPC1 in fibrotic liver tissue（1）The expression of TRPC1 was up-regulated in the fibrotic liver tissue of the patients.Compared with the normal group,the positive staining area of TRPC1 in the patient liver tissues was significantly increased,up to about 42%.The location of TRPC1was consistent with the Sirius Red-marked area,indicating that it was mainly distributed in the liver Fibrosis lesion area.Confocal results showed that TRPC1 mainly co-localized with HSCs marker proteins Desmin andα-SMA,indicating that TRPC1 was mainly up-regulated in HSCs.（2）The expression of TRPC1 was significantly increased in fibrotic liver tissues of mice induced by different etiologies.Protein levels of TRPC1 in the liver tissues of CCl₄and BDL-induced mice were increased by about 1.94 and 3.69 times,respectively,compared with the control group.The TRPC1-positive staining areas were significantly increased,with an average value of about 18.29%（P<0.01）and 26.31%（P<0.01）,respectively.TRPC1 mainly co-localized with HSCs marker proteins Desmin andα-SMA in the fibrotic liver tissue of BDL mice.In the DEN combined with CCl₄-induced liver fibrosis-hepatoma progression model,the expression level and distribution area of TRPC1gradually increased with the prolongation of the modeling period.The expression level of TRPC1 was significantly up-regulated by more than 3 times in the activated mouse primary HSCs induced,and displayed wide intracellular distribution.2.The role of TRPC1 in the progression of experimental liver fibrosis（1）TRPC1 knockout significantly inhibited the progression of experimental liver fibrosis of mice.After TRPC1 knockout,the fibrotic hyperplasia area and collagen deposition area induced by CCl₄ injection were significantly reduced,and the positive area of Masson and Sirius Red decreased from 8.37±0.73%and 7.78±0.50%to 1.96±0.17%and 1.99±0.18%.Liver function was significantly improved as ALT and AST levels decreased by about 43%and 47%.CollagenⅠandα-SMA protein expression levels decreased by 45%and 47%,and the positive staining areas decreased from 26.85±1.56%and 21.53±1.51%to 10.43±0.78%and 10.66±0.99%.In the BDL-induced model,compared to model mice,TRPC1 knockout mice showed significantly decreased fibrous hyperplasia and collagen deposition areas,and the positive areas of Masson and Sirius Red staining decreased from 17.64%±1.40%and 13.33%±0.95to 6.95%±0.84%and 4.02%±0.35%,respectively.BDL-upregulated CollagenⅠandα-SMA protein levels and their positive staining areas were significantly reduced by at least40%.BDL-induced serum levels of ALT and AST were reduced by about 58%and 56%,indicating that liver function of mice was significantly improved.In the DEN plus CCl₄ stimulation-induced liver fibrosis-cirrhosis-hepatocellular carcinoma model,the liver tissue damage and fibrosis progression were significantly alleviated in TRPC1 knockout group,and the formation of hepatocellular carcinoma occurred later than that in the control model group.At 6 months,the liver injury and cancer cell infiltration in the liver tissue of mice were significantly reduced in TRPC1knockout group.After TRPC1 knockout,the number and size of tumors were significantly reduced by about 60%and 56%,respectively.Besides,after TRPC1 knockout,the positive staining areas of Sirius Red andα-SMA were all significantly decreased at different stimulation time point,and the numbers of Ki-67-positive cells were also significantly decreased.（2）Specifically supplementing TRPC1 expression in HSCs would exacerbate CCl₄and BDL-induced fibrotic liver injury.Immunoblotting results showed that AAV injection could successfully facilitate the overexpression of TRPC1 in HSCs of knockout mice.Compared to control group,CCl₄ and BDL-induced collagen fiber deposition and fibrotic hyperplasia areas in the mouse liver tissue were significantly increased TRPC1-overexpressed groups.CCl₄-induced CollagenⅠandα-SMA positive distribution increased by 2.05 and 2.58 times,respectively.In the BDL-induced model,the positive areas of Masson and Sirius Red staining increased by 4.08 times and 2.83 times,andα-SMA and type I collagen were up-regulated by 2.89 times and 4.79 times,respectively.3.The role and associated mechanism of TRPC1 in the regulation of HSCs’activation（1）TRPC1 knockdown inhibited the secretion and migration properties of activated HSCs.In TGF-β1-activated Lx-2 cells,the protein expression level of TRPC1 increased by about 3.04 times.TGF-β1-upregulated protein expression levels ofα-SMA and CollagenⅠin decreased in TRPC1 Sh RNA-transfected cells compared with Control Sh RNA-transfected group.In the cell scratch and migration experiments,after 48 h of TGF-β1 stimulation,TRPC1 Sh RNA-transfected group still retained obvious unhealed scratch area,the average healing area of Control Sh RNA-transfected group was78.86%±1.43%,and the average healing area of the TRPC1 Sh RNA transfection group was 39.01%±1.04%with about 50%decrease.In cell migration assays,the number of migrating cells in the TRPC1 Sh RNA transfection group decreased by about 36%compared with the Control Sh RNA transfection group.Moreover,after TRPC1 knockout,the numbers of migrated primary HSCs dropped significantly by about 58%.（2）TRPC1 could promote HSCs activation by regulating Ca²⁺and participating in protein interactions.After TRPC1 knockout,the contents of P-SMAD2/3 in primary HSCs of CCl₄ and BDL-induced mice were reduced by about 50%.Under the stimulation of TGF-β1（5 ng/m L）,compared with the Control Sh RNA group,the expression levels of P-SMAD2 and P-SMAD3 in TRPC1 Sh RNA-transfected cells decreased by about 58%and55%,respectively,and the amount of SMAD2/3 in the cell nucleus was dropped by about40%.After TRPC1 knockdown,peak intensity of TGF-β1（5 ng/m L）-triggered intracellular Ca²⁺rise in Lx-2 cells was significantly decreased by about 53%.Application of Ca²⁺chelator BAPTA-AM in Control Sh RNA-transfected Lx-2 cells inhibited TGF-β1-upregulated expression ofα-SMA,CollagenⅠ,P-SMAD2/3 and transcription of profibrotic genes.BAPTA-AM didn’t show while further stronger inhibitory effects in TRPC1 Sh RNA-transfected Lx-2 cells,while the use of Ionomycin could upregulate TGF-β1-induced m RNA levels of SMAD7,ID-1 and PAI-1 by approximately 1.32-fold,1.49-fold and 2.22-fold,respectively.IP/MS and CO-IP experiments suggested that TRPC1could directly bind to vimentin.After TRPC1 knockout,the formation and distribution of vimentin filaments in mouse primary HSCs and Lx-2 cells were significantly reduced,and the proportion of insoluble vimentin filaments decreased by about 60%and 41%respectively,indicating TRPC1 knockdown inhibited the formation insoluble vimentin filaments.The phosphorylation levels of vimentin Ser39 and Ser56 sites in TRPC1knockout mouse primary HSCs and TRPC1 Sh RNA-transfected Lx-2 cells decreased by about approximately 40%.Further mechanism studies have shown that TRPC1 can form a protein complex with ERK2 and vimentin.The use of ERK2-specific inhibitor VX-11e can inhibit the TGF-β1-induced phosphorylation of vimentin as evidenced by the expression levels of Ser39 and Ser56 sites decreased by 44%and 38%,respectively.Moreover,after TRPC1 knockout,the expression level of ERK2 in the immunoprecipitated complexes decreased significantly by about 50%.Conclusion:TRPC1 is highly expressed in both human and mouse fibrotic liver tissues,and its expression changes are mainly located in HSCs distribution areas.TRPC1 can mediate the activation of HSCs and promote liver fibrosis injury in mice.As for the contribution of TRPC1 in HSCs activation:TRPC1 could mediate TGF-β1-triggered Ca²⁺to facilitate induction of TGF-β/SMAD pathway and transcription of profibrotic genes.In addition,TRPC1 could form a protein complex with vimentin and ERK2 to promote vimentin phosphorylation,thereby increasing vimentin filament assembly and HSCs cytoskeleton remodeling,promoting the activation and migration of HSCs,which indicated that TRPC1might be a potential new therapeutic target of liver fibrosis.