| Hepatic fibrosis is the common pathological manifestation of various liver diseases,characterized by diffuse fibrous hyperplasia and insufficient degradation in the liver,extensive deposition of extracellular matrix,eventually progresses to cirrhosis.Activation of hepatic stellate cells(HSC)is a key event in the progression of liver fibrosis.Many studies have shown that activation of HSC is closely related to oxidative stress,and elevated intracellular ROS can promote the transduction of TGF-?/SMAD fibrotic signaling pathway.The use of antioxidants to block ROS production can inhibit the activation of HSC and extracellular matrix secretion,delay the process of liver fibrosis.Imidazoline I2 receptor(I2R)is widely distributed in the body.Preliminary experiment shown that I2 R inhibitors had an anti-inflammatory and anti-oxidative stress effects,whether it has anti-fibrotic effect is unclear.In this study,we detected the effets and the underline molecular mechanisms of idazoxan(IDA),the commonly used I2 R inhibitor,in CC14-induced liver fibrosis mouse model and TGF-?-stimulated LX2 cell model,in order to do some exploratory work for the clinical prevention and treatment of liver fibrosis.The first part: Protective effect of imidazole on CCl4-induced liver fibrosis in miceObjective:to explore the anti-fibrosis protective effect of idazoxan on CCl4-induced liver fibrosis mouse model and TGF-?-stimulated LX2 cell model.Methods:1.In vivo experiments: 40-22 g male C57 BL / 6J mice were randomly divided into 4 groups: normal control group(n = 8),idazoxan control group(n = 8),hepatic fibrosis model group(8 rats)and idazoxan treated group(n = 8).CC14 and olive oil were mixed at a volume ratio of 2: 3,which mice were injected intraperitoneally twice a week for 0.8 ml / kg.Idazoxan was injected at the dose of 3mg / kg,simultaneously with CC14.At week 8,all mice were sacrificed and serum and liver specimens were collected.The liver pathological changes and fibrosis were observed by HE,Sirius Red and Masson staining.Alanine aminotransferase(ALT),aspartate aminotransferase(AST)and total bilirubin(TBil)were measured by biochemistry test.The levels of IL-1?,IL-6,TNF-? and TGF-? in serum were detected by enzyme-linked immunosorbent assays(ELISA).The expression of ?-SMA and Col1 in liver tissue were detected by immunohistochemistry.The expression of p-SMAD2,SMAD2,p-SMAD3 and SMAD3 in liver tissue were detected by Western Blot.2.In vitro culture: Human immortalized hepatic stellate cell line LX2 cells were randomly divided into 5 groups: normal control group(PBS),TGF-? stimulation group(5ng/ml TGF-?),low dosage idazoxan treated group(25?M IDA + 5ng/ml TGF-?),middle dosage idazoxan treated group(50?M IDA + 5ng/ml TGF-?)and high dosage idazoxan treated group(100?M IDA + 5ng/ml TGF-?).Western blot was used to detect the expression of p-SMAD2,SMAD2,p-SMAD3 and SMAD3 in the cytoplasm and nucleus,and the expression of ?-SMA and Col1 in the cells.Result:1.In vivo experiments: 8 weeks after CC14 intraperitoneal injection,liver fibrosis model group mice showed symptoms including weight loss,liver weight gained,liver weight ratio significantly increased,while the mice in idazoxan treated group were significantly improved.HE,Sirius Red and Masson staining showed that hepatocellular degeneration and necrosis,hepatic lobular structure disorder,central venous circumference and portal area were observed in liver fibrosis model group,while idazoxan group had significant improvement.The levels of ALT,AST,TBil,IL-1?,IL-6,TNF-? and TGF-? in the serum of liver fibrosis mice were significantly decreased by hematoxylin and ELISA tests.Western blot analysis showed that idazoxan could significantly reduce the phosphorylation of SMAD2 and SMAD3 in liver of mice with hepatic fibrosis.2.In vitro experiments: After TGF-?-induced activation of LX2 cells treated by idazoxan,SMAD2 and SMAD3 phosphorylation was significantly reduced,translocation into the nuclear was restricted and ?-SMA and Col1 expression was significantly reduced.Conclusion:Idazoxan prevented and ameliorated CC14-induced liver fibrosis in mice by inhibiting TGF-?-SMAD signaling pathway in hepatic stellate cells.The second part: Idazoxan suppressed oxidative stress by activating the NRF2 signaling pathway in the liverObjective:To investigate the anti-oxidative effects of idazoxan on the model of liver fibrosis and the LX2 cell model,and to verify whether this effect is related to the activation of NRF2 signaling pathway.Method:1.The levels of superoxide dismutase-2(SOD2),catalase(CAT),heme oxygenase(HO-1),NAD(P)H:quinone oxidoreductase(NQO-1)and Nrf2 in the normal control group,idazoxan control group,hepatic fibrosis model group and idazoxan treated group were detected by Western Blot.The total superoxide dismutase(SOD)and glutathione Oxidase(GPx)were detected by enzyme activity test.SOD2,CAT,HO-1 and NQO-1 expression was detected by Western Blot,as well as the expression of Nrf2 and Keap1 in the cytoplasm and nucleus.Cell immunofluorescence was used to detect the localization of NRF2 in the cell,and the binding activity of NRF2 and DNA was detected by EMSA in LX2 cells of the normal control group,TGF-? stimulated group,low dose idazoxan treated group,middle dose idazoxan treated group and high dose idazoxan treated group,respectively.2.In vivo gene silencing: Thirty-six male C57BL/6J mice,18-22 g,were randomly divided into three groups,normal control group(n = 12),liver fibrosis model group(n = 12)and idazoxan treated group(n = 12).Each group was divided into two groups,which were respectively injected with control adenovirus and NRF2 knockdown adenovirus.CC14 and olive oil were mixed at a volume ratio of 2: 3,and mice were injected intraperitoneally twice a week for 0.8 ml / kg.Idazoxan was injected at the dose of 3mg / kg,simultaneously with CC14.After 8 weeks of injection,all mice were sacrificed and serum and liver specimens were collected.HE and Sirius red staining were used to detect the fibrosis changes in liver tissue.The enzyme activity of GPx and SOD in liver tissue was detected by enzyme activity test.The expression of HO-1,NQO-1,p-SMAD2,SMAD2,p-SMAD3,SMAD3,?-SMA and Col1 were detected by Western Blot.3.In vitro gene silencing: LX2 cells cultured in vitro were randomly divided into 3 groups,normal control group(PBS),TGF-? stimulated group(5ng/ml TGF-?)and idazoxan treated group(100?M IDA + 5ng / ml TGF-?).Each group was divided into two groups,which were respectively stimulated with control lentivirus and NRF2 knockdown lentivirus.The enzyme activity of GPx and SOD in liver tissue was detected by enzyme activity test.The expression of HO-1,NQO-1,p-SMAD2,SMAD2,p-SMAD3,SMAD3,?-SMA and Col1 were detected by Western Blot.The expression of ?-SMA was detected by cell immunofluorescence assay.Results1.The expression of SOD2 and CAT in liver tissue of liver fibrosis mice was significantly lower than that of control group,and the activities of SOD and GPx were significantly decreased.The expression of SOD2 and CAT and the enzyme activity significantly reduced and production of reactive oxygen species ROS increased in TGF-? stimulated LX2 cells,while the mice in idazoxan treated group were significantly improved.2.Idazoxan can help significantly enhance the expression of HO-1,NQO-1 and Nrf2 in liver fibrosis mice,promote the expression of HO-1 and NQO-1 in LX2 cells,and the translocation of Nrf2 into nucleus and DNA binding activity.3.After Nrf2 inhibition in mice,the protect effects of idazoxan on liver of mice with fibrosis were weakened by HE and Sirius red staining,the effects of idazoxan on SOD and GPx activities were decreased by enzyme activity test,the effects of idazoxan on antioxidant enzymes HO-1,NQO-1,SMAD2/3 phosphorylation,?-SMA and Col1 expression were attenuated by western blot.4.After Nrf2 inhibition in LX2 cells,the effects of idazoxan on SOD and GPx activities were significantly attenuated by enzyme activity test.The effects of idazoxan on antioxidant enzymes HO-1,NQO-1,SMAD2/3 phosphorylation,?-SMA and Col1 expression were attenuated by Western Blot and immunofluorescence assays.Conclusion:Idazoxan inhibited oxidative stress in CC14-induced liver fibrosis mice,thereby inhibiting the activation of HSC and the progress of hepatic fibrosis in mice,and its mechanism is related to the activation of NRF2 signaling pathway.The third part: Idazoxan inhibits HSC activation and hepatic fibrosis progression through AKT-NRF2-SMAD signaling pathwayObjective:To investigate the effect of idazoxan on AKT signaling pathway in liver fibrosis mice and TGF-? stimulated LX2 cells,and to verify whether idazoxan can exert anti-fibrosis through AKT-NRF2 signaling pathway.Method:1.The expression of p-AKT and AKT was detected by Western Blot in the mice of normal control group,the idazoxan control group,the hepatic fibrosis model group and the idazoxan treated group.The expression of p-AKT and AKT expression was detected by Western Blot in LX2 cells of the normal control group,the idazoxan control group,TGF-? stimulated group,low dose idazoxan treated group,middle dose idazoxan treated group and high dose idazoxan treated group.2.Thirty-two male C57BL/6J mice,18-22 g,were randomly divided into 4 groups: normal control group(n = 8),hepatic fibrosis model group(n = 8),idazoxan group(n = 8)and Perifosine intervention group(n = 8).CC14 and olive oil were mixed in a volume ratio of 2: 3,and each group of mice was injected intraperitoneally twice a week for 0.8 ml/kg to replicate the liver fibrosis model.Idazoxan was injected at a dose of 3mg / kg,while Perifosine(PE)20mg / kg,simultaneously with CC14.After 8 weeks of injection,all mice were sacrificed and serum and liver specimens were collected.Sirius red staining were used to detect the fibrosis changes in liver tissue.The expression of ?-SMA was detected by immunohistochemical staining.The expression of p-AKT,AKT,NRF2,HO-1,NQO-1,p-SMAD2,SMAD2,p-SMAD3 and SMAD3 in liver tissue were detected by Western Blot.3.LX2 cells were randomly divided into 4 groups: normal control group(PBS),TGF-? stimulated group(5ng/ml TGF-?),idazoxan group(100?M IDA +5ng/ml TGF-?)and Perifosine intervention group(5 ?M PE + 100 ?M IDA + 5 ng / ml TGF-?).The expression of p-AKT,AKT,p-SMAD2,SMAD2,p-SMAD3,SMAD3,?-SMA and Col1 in cytoplasm and NRF2,SMAD2 and SMAD3 in nucleus were detected by Western Blot.Cell immunofluorescence was used to detect the localization of NRF2 in the cell.The binding activity of NRF2 and DNA was examined by EMSA.Results1.Idazoxan significantly promoted the phosphorylation of AKT in the CCl4-induced liver fibrosis mouse model and TGF-?-stimulated LX2 cell model.2.After the inhibition of AKT phosphorylation by Perifosine in mice,the protect effects of idazoxan on liver of mice with fibrosis were weakened by Sirius red staining,the effects of idazoxan on NRF2,HO-1,NQO-1,SMAD2/3 phosphorylation,?-SMA expression were attenuated by western blot and immunohistochemical staining.3.After the inhibition of AKT phosphorylation by Perifosine in LX2 cells,the effect of idazoxan on the translocation of Nrf2 into nucleus and DNA binding activity,the expression of SMAD2/3 phosphorylation,?-SMA,and Col1 was significantly attenuated by Western Blot and immunofluorescent assay.ConclusionIdazoxan inhibited oxidative stress and fibrosis progression in CC14-induced liver fibrosis mice through regulating AKT-NRF2-SMDA signaling pathway in hepatic stellate cells. |
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