Effects Of NOX4/ROS And RhoA/ROCK1 Signaling Pathways On Liver Fibrosis And Interventional Targets Of Ursolic Acid

BackgroundNADPH oxidase?NOX?,especially NOX4 which is abundant in hepatic stellate cells?HSCs?,with reactive oxygen species?ROS?can induce oxidative stress to promote the development of hepatic fibrosis.The cytokine RhoA which regulates actin cytoskeleton and cell morphological heterogeneity,with its effector molecule ROCK1 plays an important role in the activation of HSCs into fibroblasts.Recent studies have shown that NOX4/ROS can act as the upstream molecule of RhoA/ROCK1 to induce the occurrence and development of pulmonary fibrosis;however,In the process of renal fibrosis,Their relationship is opposite.Whether The two have the above relationship or other mutual regulative mechanisms in liver fibrosis is still unclear.Our previous study have found that ursolic acid?UA?could down-regulate the expression of NOX4/ROS in HSCs and inhibit the activation of Rac1 which is one of the RhoA homologous gene family members to reverse liver fibrosis.But the effects of UA intervention on RhoA/ROCK1 has not been reported.In the topic,we use liver fibrosis mice,NOX4 gene knockout mice with liver fibrosis,liver fibrosis mice with Apocynin?AP?or UA intervention as the object to investigate the effects of NOX4/ROS and RhoA/ROCK1 on liver fibrosis and their relationship,whether UA can suppress NOX4/ROS and RhoA/ROCK1 signaling pathways to reverse liver fibrosis.ObjectiveTo explore the effects of NOX4/ROS and RhoA/ROCK1 signaling pathways on hepatic fibrosis and the relationship between the two;observe the influences of UA on the two signaling pathways to identify the molecular targets of UA against liver fibrosis.Method1.C57BL/6 WT male mice and NOX4 gene knockout(NOX4^-/-)mice were divided into 6 groups:control group,liver fibrosis group,NOX4^-/-group,Apocynin?AP?intervention group,UA intervention group,UA intervention-NOX4^-/-group.The control group were injected intraperitoneally with olive oil 1ml/kg,twice a week for 6 weeks;The liver fibrosis group and NOX4^-/-group were injected intraperitoneally with CCL4?dissolved in olive oil to 20%concentration?1ml/kg,twice a week for 6 weeks;The drug intervention group were injected intraperitoneally with the same dose and frequency of CCl4,and at the last four weeks,were gavaged with AP or UA?50mg/kg/day?.After the end of modeling,fasting for three days,blood and liver samples were obtained.2.Hepatic pathological changes were observed by HE and Masson staining;The content of hydroxyproline in liver tissue was determined by colorimetry;malondialdehyde?MDA?was detected by thiobarbituric acid?TBA?method.3.The levels of serum ALT,AST and TBIL were detected by automatic biochemical analyzer.The degrees of activation of HSCs and hepatocyte apoptosis were observed by double immunofluorescence staining with a-SMA antibody and TUNEL.4.The expression of a-SMA,Collagen I,Mmp9,Timp1,NOX4,RhoA and ROCK1 protein in liver tissues were detected by WB.The expression of NOX4,RhoA and ROCK1 protein in liver tissues were also detected by immunohistochemistry.5.the expression of NOX4,RhoA and ROCK1 mRNA in liver tissues were detected by Real-time quantitative PCRResults1.The expression of NOX4 and its mediated oxidative stress product and RhoA/ROCK1 proteins in liver of hepatic fibrosis mice.1.1 The expression of NOX4 proteins and the content of oxidative stress product MDA in liver of hepatic fibrosis miceCompared with the control group,the expression of NOX4 protein and the content of oxidative stress product MDA in liver tissues of the liver fibrosis group were significantly increased?P<0.001,P<0.01 respectively?.1.2 The expression of RhoA and ROCK1 proteins in liver of hepatic fibrosis miceCompared with the control group,the the expression of RhoA and ROCK1protein in liver tissues of the liver fibrosis group were significantly increased?P<0.01,P<0.001 respectively?.2.Effects of NOX4^-/-and AP intervention on liver fibrosis2.1 The influences of NOX4^-/-and AP intervention on collagen fibers and hydroxyproline in liver tissueThe content of collagen fiber and hydroxyproline in liver tissues of the liver fibrosis group were significantly higher than that in the control group?both P<0.001?;Compared with the liver fibrosis group,The collagen deposition were significantly reduced in NOX4^-/-group and AP intervention group?P<0.01,P<0.001respectively?.The content of hydroxyproline in liver tissues were also significantly decreased?P<0.05,P<0.01 respectively?.2.2 The influences of NOX4^-/-and AP intervention on liver function and oxidative stress in miceCompared with the liver fibrosis group,the levels of serum ALT and TBIL in NOX4^-/-group and the AP intervention group were decreased?both P<0.001?,the levels of serum AST also were decreased significantly?P<0.05,P<0.01 respectively?.the content of oxidative stress product MDA were significantly reduced?both P<0.01?.2.3 The influences of NOX4^-/-and AP intervention on fibrosis-associated proteins in miceCompared with the liver fibrosis group,the expression of a-SMA,Collagen I,Mmp9 and Timp1 proteins in liver of NOX4^-/-group and AP intervention group were significantly lower?P<0.05,P<0.01 respectively?.3.Effects of NOX4^-/-and AP intervention on RhoA/ROCK1 in liver of hepatic fibrosis mice3.1 The influences of NOX4^-/-and AP intervention on the expression of RhoA?ROCK1 mRNA.Compared with the liver fibrosis group,the expression of RhoA mRNA were down-regulated in the NOX4^-/-group and AP intervention group?both P<0.05?.the expression of ROCK1 mRNA were also significantly decreased?P<0.05,P<0.01respectively?.3.2 The influences of NOX4^-/-and AP intervention on the expression of RhoA?ROCK1 proteins.Compared with the liver fibrosis group,the expression of RhoA and ROCK1proteins in liver tissues of NOX4^-/-group and AP intervention group were significantly decreased?both P<0.05 and P<0.01 respectively?.4.Effects of ursolic acid on the apoptosis of hepatocytes and activation of HSCs.The degrees of hepatocyte apoptosis and HSCs activation in the liver fibrosis group were significantly higher than that of the control group?P<0.001,P<0.01,respectively?.However,compared with the liver fibrosis group,the degrees of hepatocyte apoptosis and HSCs activation in UA intervention group were decreased?both P<0.01?.5.Effects of ursolic acid on NOX4 and oxidative stress in liver of hepatic fibrosis mice5.1 The influences of ursolic acid on NOX4 in liver of hepatic fibrosis miceCompared with the liver fibrosis group,the expression of NOX4 protein and mRNA in the UA intervention group were significantly decreased?both P<0.01?.5.2 The influences of ursolic acid on oxidative stress in hepatic fibrosis miceCompared with the liver fibrosis group,the content of oxidative stress product MDA in UA intervention group was significantly decreased?P<0.01?.6.Effects of ursolic acid on liver fibrosis and the expression RhoA/ROCK1in NOX4^-/-mice6.1 The influences of ursolic acid on liver histopathology and the content of hydroxyproline in NOX4^-/-hepatic fibrosis miceIn the NOX4^-/-group,the hepatic lobule structure was slightly disordered,with hepatocyte necrosis,fibrous hyperplasia in the portal area,fiber bridging and occasional nodules.However,All of these pathological changes were improved by UA Intervention.Compared with the NOX4^-/-group,the content of collagen deposition and hydroxyproline in liver tissues in UA intervention-NOX4^-/-group were significantly decreased?P<0.01,P<0.05,respectively?.6.2 The influences of ursolic acid on the expression of RhoA and ROCK1mRNA in NOX4^-/-hepatic fibrosis miceCompared with the NOX4^-/-group,the expression of RhoA and ROCK1 mRNA in UA intervention-NOX4^-/-group were significantly decreased?P<0.05,P<0.01,respectively?.6.3 The influences of ursolic acid on the expression of RhoA and ROCK1protein in NOX4^-/-hepatic fibrosis miceCompared with the NOX4^-/-group,the expression of RhoA and ROCK1 protein in UA intervention-NOX4^-/-group were significantly decreased?P<0.05,P<0.01,respectively?.Conclusion1.Both NOX4/ROS and RhoA/ROCK1 signaling pathways can promote liver fibrosis and NOX4/ROS as upstream factors regulate the expression of RhoA/ROCK1 to aggravate liver fibrosis.2.Ursolic acid can suppress both NOX4/ROS and RhoA/ROCK1 signaling pathways to reverse liver fibrosis.