| Liver fibrosis is the repair response of the liver to chronic damage caused by various causes.It is characterized by hyperplasia and deposition of collagen-based extracellular matrix(ECM)in the liver,which is a necessary stage for the development of all chronic liver damage to cirrhosis.Hepatic stellate cells(HSCs)are the main source of ECM in liver fibrosis.Activated HSCs undergo phenotypic transformation,obtain myofibroblast-like characte-ristics,migrate to the liver injury site,proliferate,and synthesize and secrete a large amount of ECM,leading to the occurrence and progression of liver fibrosis.HSCs are the ultimate target cells for various fibrogenic factors.Therefore,studying the biological functions of HSCs and their regulatory mechanisms is essential for the prevention and control of liver fibrosis.The biological axis composed of the chemokine CXCL12 and its receptor CXCR4 is involved in various pathophysiological processes such as inflammation,immunity and injury repair.Studies have shown that the CXCL12/CXCR4 bioaxis is closely related to various tissue and organ fibrosis,such as kidney,lung,prostate,et al.;however,the role of this bioaxis in the pathogenesis of liver fibrosis remains unclear.RhoA,a member of the ras homologous gene family,is mainly involved in the RhoA/ROCK pathway and is a star molecule in recent years,which is closely related to the activation,contraction,migration and adhesion of HSCs.However,the regulatory network of RhoA in liver fibrosis is not yet clear.Therefore,in order to explore the role of the CXCL12/CXCR4 bioaxis in the pathogenesis of liver fibrosis and the regulation of RhoA,we used the rat liver fibrosis model and HSC-T6 cell line as the research object,and administered CXCR4 inhibitor and RhoA inhibitor,and construct a CXCR4 overexpressing stable cell line,and then observe changes in expression of related molecules in liver tissues and cells as well as functional changes of HSCs.This research work was carried out in the following three aspects:Part I CXCL12/CXCR4 axis and RhoA induce hepatic stellate cell activation to promote liver fibrosis in ratsObjective:To observe the degree of liver fibrosis,the expression of CXCR4,a-SMA and COL1,and the activity of RhoA in liver tissue in the rats of each group after the intervention of CXCR4 inhibitor and RhoA inhibitor.Methods:Forty male Sprague-Dawley rats were randomly divided into 4 groups:normal group,model group,CXCR4 inhibitor group,and RhoA inhibitor group(n=10/group).All rats except the normal group were subcutaneously injected with carbon tetrachloride to construct liver fibrosis models for 8 weeks.Intervention is implemented while modeling.The CXCR4 inhibitor group and the RhoA inhibitor group were intraperitoneally injected with CXCR4 inhibitor(AMD3100)and RhoA inhibitor(Rhosin)everyday,respectively.After 8 weeks of intervention,all rats were sacrificed and liver tissue was collected.The mRNA and protein expression levels of CXCR4 and a-SMA in liver tissues of each group were detected by RT-qPCR and Western blot.The protein expression levels of COL1 and a-SMA in liver tissues of each group were detected by immunohistochemistry.The relative activities of RhoA in liver tissues of each group were detected by Western blot.Results:The liver histopathology was normal in the normal group,while the model group showed extensive hepatic bridging fibrosis and massive collagen deposition;however,compared with the model group,the CXCR4 inhibitor group and the RhoA inhibitor group had significantly reduced bridging fibrosis and collagen deposition(P<0.01).Compared with the normal group,the mRNA and protein expression levels of CXCR4 in the model group were significantly increased(P<0.01).Compared with the model group,the mRNA and protein expression levels of CXCR4 in the CXCR4 inhibitor group were significantly lower(P<0.05).However,there was no significant change in the expression level of CXCR4 in the RhoA inhibitor group(P>0.05).Compared with the normal group,the expression levels of a-SMA and COL1 and the relative activities of RhoA in the model group were significantly increased(P<0.05);however,compared with the model group,the expression levels of a-SMA and COL 1 and the relative activities of RhoA were significantly decreased in the CXCR4 inhibitor and RhoA inhibitor group(P<0.05).Conclusions:CXCL12/CXCR4 axis and RhoA are involved in the pathogenesis of liver fibrosis.Inhibition of CXCR4 expression and RhoA activity may reduce liver fibrosis;CXCL12/CXCR4 axis and RhoA may affect activation of HSCs,inhibition of CXCR4 expression and RhoA activity may reduce activation of HSCs;the CXCL12/CXCR4 axis may affect RhoA activity,but RhoA has no significant effect on the CXCL12/CXCR4 axis.Part ? CXCL12/CXCR4 axis and RhoA promote activation,proliferation and migration of hepatic stellate cellsObjective:To observe the expression of CXCR4,a-SMA and COL 1,RhoA activity and the function of cells in each group of cells after intervention of HSC-T6 cells with CXCR4 inhibitors and RhoA inhibitors and transfection with CXCR4 overexpressing lentiviral vectors.Methods:HSC-T6 cells were randomly divided into 6 groups:blank group,solvent control group(PBS),CXCR4 inhibitor group(CXCR4 inhibitor),empty vector group(Empty vector),CXCR4 overexpression group(CXCR4 overexpression,CXCR4-OE)and RhoA inhibitor group(RhoA inhibitor).The CXCR4 inhibitor group and the RhoA inhibitor group were treated with AMD3100 and Rhosin,respectively;the solvent control group was treated with an equal volume of PBS solution;the blank group was given no treatment.The empty vector group and the CXCR4 overexpression group were transfected with an empty vector and a CXCR4 overexpression vector,respectively.The mRNA and protein expression levels of CXCR4 and ?-SMA in each group were detected by RT-qPCR and Western blot;G-LISA Activation Assay was used to detect the RhoA activity of each group of cells;ELISA was used to detect the expression of COL1 in each group of cells;the proliferation rate of each group of cells was detected by CCK-8 method;the migration ability of each group of cells was detected by Transwell test;Flow cytometry was used to determine the apoptotic rate and cell cycle distribution of each group of cells.Results:Compared with the blank group,the expression levels of CXCR4 and a-SMA in the empty vector group and the solvent control group did not change significantly(P>0.05);compared with the solvent control group,the mRNA and protein expression levels of CXCR4 and a-SMA were significantly decreased in the CXCR4 inhibitor group(P<0.05);the mRNA and protein expression of a-SMA was significantly decreased in the RhoA inhibitor group(P<0.05).Compared with the blank group,there was no significant change in RhoA activity and COL1 expression in the solvent control group and the empty vector group(P>0.05);compared with the solvent control group,the RhoA activity and COL1 expression in the CXCR4 inhibitor group and the RhoA inhibitor group were significantly lower(P<0.05);the RhoA activity and COL1 expression were significantly increased in the CXCR4 overexpression group compared with the empty vector group(P<0.01).Compared with the blank group,there was no significant change in cell proliferation rate and migration ability between the empty vector group and the empty vector group(P>0.05);compared with the solvent control group,the cell proliferation rate and migration ability of the CXCR4 inhibitor group and the RhoA inhibitor group were significantly decreased(P<0.05);the cell proliferation rate and migration ability of the CXCR4 overexpression group were significantly increased compared with the empty vector group(P<0.05).Compared with the blank group,the apoptotic rate of cells and the cell ratio of each cycle(GI,S and G2/M)in the empty vector group and the solvent control group did not change significantly(P>0.05);compared with the solvent control group,the apoptotic rate and the proportion of cells in each cycle did not change significantly in the CXCR4 inhibitor group and the RhoA inhibitor group(P>0.05);compared with the empty vector group,the apoptosis rate of the CXCR4 overexpression group and the proportion of cells in each cycle did not change significantly(P>0.05).Conclusions:CXCL12/CXCR4 axis and RhoA can promote the proliferation and migration of HSCs to promote profibrosis.The CXCL12/CXCR4 axis may affect the function of HSCs by regulating RhoA activity.In liver fibrosis,CXCL12/CXCR4 axis and RhoA may have no effect on HSCs apoptosis and cell cycle.Part ? CXCL12/CXCR4 axis promotes hepatic stellate cells activation,proliferation and migration by up-regulating RhoA activityObjective:To clarify the regulatory relationship between CXCL12/CXCR4 axis and RhoA,RhoA inhibitors were used to intervene CXCR4 overexpressing HSC-T6 cells,and the expression of CXCR4,a-SMA and COL1,RhoA activity and Functional changes of cells in each group were observed.Methods:The Rescue experiment was designed to randomly divide CXCR4 overexpressing cells into three groups:CXCR4-OE group(CXCR4 overexpression group),CXCR4-OE+PBS group,CXCR4-OE+RhoA inhibitor group.The CXCR4-OE+RhoA inhibitor group and the CXCR4-OE+PBS group were treated with Rhosin and PBS solution,respectively;the CXCR4-OE group was not given any treatment.The mRNA and protein expression levels of CXCR4 and a-SMA in each group were detected by RT-qPCR and Western blot;G-LISA Activation Assay was used to detect the RhoA activity of each group of cells;ELISA was used to detect the expression of COL1 in each group of cells;the proliferation rate of each group of cells was detected by CCK-8 method;the migration ability of each group of cells was examined by Transwell assay.Results:Compared with CXCR4-OE group,RhoA activity,mRNA and protein expression of a-SMA,and protein expression of COL 1 were not significantly changed in CXCR4-OE+PBS group(P>0.05);However,compared with the CXCR4-OE+PBS group,RhoA activity,mRNA and protein expression of a-SMA,and protein expression of COL1 were significantly decreased in the CXCR4-OE+RhoA inhibitor group(P<0.05).There was no significant change in proliferation rate and migration ability of cells in the CXCR4-OE+PBS group compared with the CXCR4-OE group(P>0.05);However,compared with the CXCR4-OE+PBS group,the proliferation rate and migration ability of cells in the CXCR4-OE+RhoA inhibitor group were significantly decreased(P<0.05).Conclusions:RhoA is a downstream cytokine of the CXCL12/CXCR4 axis in liver fibrosis.The CXCL12/CXCR4 axis can promote activation,proliferation and migration of HSCs by up-regulating RhoA activity,leading to liver fibrosis. |
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