Study On Effects Of NOX4/ROS And Rhoa/ROCK1 Signaling Pathways On Behaviors Of HSC-T6 Cells And Ursolic Acid Intervention Targets

Background:Recent works have suggested imperative roles of NOX4/ROS and RhoA/ROCK1 signaling in inducing the fibrosis:in pulmonary fibrosis,the NOX4/ROS was proved to be the upstream signal molecules to activate RhoA/ROCK1 signaling pathways,while the activation of RhoA/ROCK1 signaling pathway seemed to be a stimulus to upregulate the expressions of NOX4/ROS pathway in renal fibrosis.However,if there also exists such a functioning way similar to above-mentioned or other behaviors between these two signaling pathways in the process of liver fibrosis is still unknown.Our previous study found that ursolic acid?UA?can reverse liver fibrosis by down-regulating the expression of NOX4/ROS in hepatic stellate cells?HSC?,inhibiting the expression of Rac1,a member of Rho GTPase family.However,the effect of UA on RhoA,another important member of the Rho GTPase family,has not been reported yet.Based on these,we hypothesized that RhoA/ROCK1 may be another signaling pathway besides NOX4/ROSS that plays an important role in the development of hepatic fibrosis,and there may be a mutual regulation between the two signaling pathways;UA may be by inhibiting HSC activation of RhoA/ROCK1 signaling pathway,thereby inhibiting the interaction of NOX4/ROS and RhoA/ROCK1 affect the biological behavior of HSC,so as to achieve reversal of hepatic fibrosis.Objective:To investigate the effects of NOX4/ROS and RhoA/ROCK1 signaling pathway on the proliferation,apoptosis,migration and activation of HSC-T6 cells and the interaction between them and the effect of UA on the two signaling pathways to elucidate the effect of UA on hepatic fibrosis.Methods:The recombinant plasmids were transfected into HSC-T6 cells by lentivirus transfection technique.NOX4-shRNA?NOX4i?,NOX4-OE and RhoA-shRNA?RhoAi?cell lines were constructed and the cells were divided into CON group?blank control?,CON+UA?50?mol/L?group,NOX4i group,RhoAi group,NOX4OE group and NOX4OE+UA?50?mol/L?group,and 48 hours after stimulation with TGF-?1?10ng/mL?,the cell proliferation level was measured by using the MTS method.The level of apoptosis was measured by using TUNEL method.Cell migration was detected by cell scratch test and Transwell method.The intracellular ROS levels were measured by using DCFH-DA method.The mRNA expression levels of Nox4,Rhoa,Rock1,Alphasma,Col1a1,Mmp1,and Timp1 were measured by using RT-qPCR.The protein levels of NOX4,RhoA,ROCK1,?-SMA,Collagen-I,MMP1,TIMP1 and F-actin were detected by Western-blotting.The interaction between NOX4 and RhoA was observed by using CoIP?Co-immunoprecipitation?technique in NOX4OE stable cell lines.The interaction between full-length NOX4 and RhoA proteins was explored by Biacore protein-interaction technique.Results:1.NOX4 and RhoA in proliferation and apoptosis of HSC-T6 cells induced by TGF-?11.1 NOX4 and RhoA in proliferation of HSC-T6 cells induced by TGF-?1After treated with TGF-?1?10ng/mL?for 48 h,the proliferation levels of CON+UA?50?mol/L?group?0.674�0.972?,NOX4i group?0.580�0.990?and Rho Ai group?0.617�0.947?decreased?p<0.05?,and the NOX4OE group?1.177�0.102?increased compared with CON group?0.872�0.136??p<0.05?.NOX4OE+UA group?0.765�0.084?decreased compared with NOX4OE group?1.177�0.102??p<0.05?.According to this,NOX4 and RhoA can promote the proliferation of HSC-T6 cells;UA can inhibit the proliferation of HSC-T6 cells.1.2 NOX4 and RhoA in the apoptosis of HSC-T6 cells induced by TGF-?1After treated with TGF-?1?10ng/mL?for 48 h,the percentage of apoptotic cells in CON+UA?50?mol/L?group[?1.980�0.179?%]was significantly higher than that in CON group[?1.044�0.131?%]?p<0.05?,but there was no significant difference between NOX4i group[?1.141�0.011?%],RhoAi group[?0.940�0.106?%]and NOX4OE group[?0.883�0.083?%]when compared with CON group[?1.044�0.131?%].The percentage of apoptotic cells in NOX4OE+UA group[?1.580�0.921?%]was significantly higher than that in NOX4OE group[?0.883�0.083?%]?p<0.01?.The results showed that NOX4 and RhoA had no effect on the apoptosis of HSC-T6 cells.UA could promote the apoptosis of HSC-T6 cells.2.NOX4 and RhoA in migration and invasion of HSC-T6 cells induced by TGF-?12.1 NOX4 and RhoA in TGF-?1-induced HSC-T6 cell scratchesAfter treated with TGF-?1?10ng/mL?for 48 h,the migration index of CON+UA?50?mol/L?group?274.046�12.036?,NOX4i group?166.873�8.624?and RhoAi group?178.837�11.696?decreased?p<0.05?was significantly higher than that in CON group?413.920�10.851??p<0.01?;NOX4OE group?537.433�11.976?was significantly higher than CON group?413.920�10.851??p<0.01?;NOX4OE+UA group?217.964�7.788?decreased compared with NOX4OE group?537.433�11.976??p<0.05?.According to these,NOX4 and RhoA can promote the migration of HSC-T6cells;UA can inhibit the migration of HSC-T6 cells.2.2 NOX4 and RhoA in TGF-?1-induced invasion of HSC-T6 cellsAfter treated with TGF-?1?10ng/mL?for 48 h,the invasion levels of CON+UA group?1.609�0.092??50?mol/L?,NOX4i group?1.577�0.137?and RhoAi group?1.087�0.132?was higher than that of CON group?0.438�0.393??p<0.05?.The RhoAi group?1.087�0.132?was much lower than NOX4i gruop???p<0.05?.NOX4OE+UA group?0.945�0.074?decreased when compared with NOX4OE group?0.632�0.052??p<0.05?.These results suggested that NOX4 can inhibit the invasion of HSC-T6 cells,RhoA can promote HSC-T6 cell invasion;UA can promote HSC-T6cell invasion.3.NOX4 and RhoA in ROS levels in HSC-T6 cells induced by TGF-?1After treated with TGF-?1?10ng/mL?for 48h,the content of ROS in CON+UA group?23068.560�6992.552?and NOX4i group?20428.110�2040.489?was significantly lower than that in CON group?33973.330�11415.949??p<0.01?,and the content of ROS in NOX4OE group?45943.560�2210.026?was significantly higher than that in CON group?33973.330�11415.949??p<0.01?;NOX4OE+UA group?20359.220�1527.088?decreasedcomparedwithNOX4OEgroup?45943.560�2210.026??p<0.01?.It was suggested that NOX4 could promote the ROS content in HSC-T6 cells and RhoA had no effect on ROS.UA could decrease the content of ROS in HSC-T6 cells.4.NOX4 and RhoA in the formation of F-actin in HSC-T6 cells induced by TGF-?1After treated with TGF-?1?10ng/mL?for 48 h,the concentration of F-actin in CON+UA?50?mol/L?group[?2.827�0.223?�10⁶],NOX4i group[?1.532�0.204?�10⁶]and RhoAi group[?1.605�0.115?�10⁶]were lower than those in CON group[?5.124�0.165?�10⁶]?p<0.05?.NOX4OE group[?7.947�0.175?�10⁶]were significantly higher than those in the CON group[?5.124�0.165?�10⁶]?p<0.05?.The F-actin staining intensity and protein content in the NOX4OE+UA group[?3.603�0.215?�10⁶]were significantly higher than those in the NOX4OE group[?7.947�0.175?�10⁶]?p<0.05?.It was suggested that NOX4 and RhoA could promote F-actin polymerization in HSC-T6 cells.UA could decrease F-actin polymerization in HSC-T6 cells.5.Effects of UA intervention in TGF-?1-induced NOX4/ROS and RhoA/ROCK1 signal transduction-related gene mRNA expression in HSC-T6 cellsAfter treated with TGF-?1?10ng/mL?for 48 h,compared with CON group,the expression of NOX4 protein in CON+UA group and NOX4i group decreased?p<0.05?,and the NOX4OE+UA group decreased compared with NOX4OE group?p<0.05?.Compared with CON group,the expression of Rhoa,Rock1,Alphasma,Coll and Timp1 mRNA in CON+UA group,NOX4i group and RhoAi group decreased?p<0.05?,and NOX4OE+UA group decreased compared with NOX4OE group?p<0.05?.Compared with CON group,the expression of Mmp1 mRNA in CON+UA group,NOX4i group and Rho Ai group increased?p<0.05?,and NOX4OE+UA group increased compared with NOX4OE group?p<0.05?.That is to say,Nox4 mRNA and Rhoa mRNA can inhibit the expression of Rock1,Alphasma,Col1a1 and Timp1mRNA but can promote the expression of Mmp1 mRNA.In addition,the inhibition of Nox4 mRNA can inhibit the expression of Rhoa mRNA;the inhibition of Rhoa mRNA cannot inhibit the expression of Nox4 mRNA.UA can inhibit the expression of Nox4,Rhoa,Rock1,Alphasma,Col1a1 and Timp1 mRNA and promote the expression of Mmp1 mRNA.6.Effects of UA intervention on the expression of NOX4/ROS and RhoA/ROCK1 signaling pathway in HSC-T6 cells induced by TGF-?1After treated with TGF-?1?10ng/mL?for 48 h,compared with CON group,the expression of Nox4 mRNA in CON+UA group and NOX4i group decreased?p<0.05?,and the NOX4OE+UA group decreased compared with NOX4OE group?p<0.05?.Compared with CON group,the expression of RhoA,ROCK1,?-SMA,Collagen-I,TIMP1 and F-actin protein in CON+UA group,NOX4i group and RhoAi group decreased?p<0.05?,NOX4OE+UA group?p<0.05?.Compared with CON group,the expression of MMP1 protein in CON+UA group,NOX4i group and RhoAi group increased?p<0.05?,and the expression of NOX4OE+UA group was higher than that of NOX4OE group?p<0.05?.Inhibition of NOX4 protein expression can inhibit RhoA protein expression,but inhibition of RhoA protein expression cannot inhibit the expression of NOX4 protein;inhibition of NOX4 protein and RhoA protein expression can inhibit RhoA,ROCK1,?-SMA,Collagen-I,TIMP1 protein and F-actin promote the expression of MMP1.UA can inhibit the expression of NOX4,RhoA,ROCK1,?-SMA,Collagen-I,TIMP1 and F-actin protein,and promote the expression of MMP1 protein.7.Interaction between NOX4 and RhoA protein7.1 NOX4 and RhoA immunoprecipitation?CoIP?in NOX4OE stable cell linesIn the stable cell line of NOX4OE,the bind of p67^phox and Rac1 protein were detected.The bind of NOX4 and Rac1,p67^phox and RhoA,NOX4 and RhoA were not detected.7.2 Interaction of full-length NOX4 and RhoA pure protein detection by Biacore protein-interaction technique in vitroIn vitro full length NOX4 and RhoA pure protein binding does not show a typical concentration-dependent curve.It was suggested that NOX4 and RhoA protein does not exist a direct interaction.Conclusions:1.The NOX4/ROS and RhoA/ROCK1 pathways can promote the proliferation,migration and activation of HSC-T6 cells.UA may inhibit cell proliferation,migration and activation by inhibiting the activation of two signaling pathways,but its mechanisms for inducing apoptosis have nothing to do with the two pathways;2.The NOX4/ROS pathway in HSC-T6 cells is the upstream signaling pathway of RhoA/ROCK1 pathway and has a positive effect on RhoA/ROCK1 pathway.There is no direct interaction between NOX4 and RhoA protein.