Effect Of Nrf2^-/- On The Mechanism Of AS-Ⅳ’ Anti-hepatic Fibrosis Via PSmad3C/3L

Backgrounds and Aim: Various pathogenic factors can lead to acute and chronic inflammation of the liver.Long-term inflammatory status can cause liver cancer to occur,increasing the risk of hepatic carcinoma,and posing a serious threat to human health.The main active ingredient of the natural medicine Astragalus membranaceus is Astragaloside（AS-Ⅳ）.It has been shown to have definite anti-inflammatory and antioxidant biological activities and can inhibit the development of liver fibrosis by modulating the Nrf2/HO-1 and TGF-b1/Smad3 pathways.Nrf2 is subjected to oxidative stress and other stimuli and is then phosphorylated into the nucleus,stimulating the expression of downstream antioxidant genes,which in turn produces hepatoprotective effects;Smad3 is the main signal of the TGF-b1/Smad3 signaling pathway,and its regional phosphorylation can regulate the progression of liver diseases.In the previous study,Nrf2/HO-1 was found to be correlated with TGF-b1/Smad3 pathway in liver fibrosis disease.In this study,we will verify the anti-liver fibrosis effect of AS-Ⅳ through in vivo and ex vivo experiments,and investigate the effect of Nrf2 knockout on the mechanism of anti-liver fibrosis effect of astragaloside via p Smad3C/3L.Method: 1.Vitro experiments demonstrated that AS-Ⅳ inhibits TGF-β1-stimulated HSC-T6 ’s cell proliferation,migration and suppresses ROS levels HSC-T6 was resuscitated and cultured to the exponential growth phase,and the following experimental groups were established: lysate control group,TGF-b1 stimulation group,AS-Ⅳ（5mM）+TGF-b1 stimulation group,AS-Ⅳ（10mM）+TGF-b1 stimulation group and AS-Ⅳ（20mM）+TGF-b1 stimulation group.MTT,cell scratching,and ROS level were measured.2.Analysis of AS-Ⅳ anti-DEN/CCl4/C2H5OH-induced liver fibrosis and Nrf2/HO-1 pathway mechanism by using C57BL/6 mice Male 6-week-old C57BL/6 mice in good condition with SPF class cleanliness,required number 36,weighing 20-23 g;uniformly randomized into 6 groups: control group,model group,AS-Ⅳ low,medium,high dose group（20,40,80 mg/kg/d;ig）,positive drug group（0.1mg/kg/d;ig）.Liver fibrosis modeling methods: intraperitoneal injection of DEN 100 mg/Kg（weeks 1-2,once each at the same time point each week）;gavage of 20% CCl4 olive oil solution 0.05 ml/10g（weeks 3-7,twice a week）;gavage of 20% CCl4 olive oil solution 0.06 ml/10g（weeks 8-12,twice a week）.10% concentration of alcohol Drinking in place of water（weeks 3-12,changed every other day）.From the first to the last day of modeling,the corresponding doses were administered daily by gavage to each dose group of colchicine and AS-Ⅳ,and the corresponding lysis medium was administered by gavage to the control and model groups,which were administered continuously and taken at the end of 12 weeks.Initial visual observation of the liver,observation of pathological changes with HE and Masson test for altered fibrosis levels.Nrf2,p Nrf2,HO-1,and NQO1 protein levels were measured by Western-blot,and p Nrf2 expression and distribution were detected by immunofluorescence staining.Correlation analysis of p Smad3C/3L with p Nrf2 was performed.3.Establishment of Nrf2 knockout mouse model of liver fibrosis and AS-Ⅳ intervention and investigation of its effect on TGF-b1/Smad3 pathway The successfully constructed Nrf2 knockout mice were sent to the SPF-level laboratory for breeding,expansion and genotype identification,and 18 six-week-old homozygous（HO）and C57BL/6 mice（WT）each were screened and randomly grouped as follows: WTcontrol group,HO-control group,WT-model group,HO-model group WT-AS-Ⅳ group and HO-AS-Ⅳ group,and 6 mice were prepared in each group.Except for the control group,the mice in the rest of the groups were established as a whole model of DEN/CCl4/C2H5OH-induced hepatic fibrosis,and AS-Ⅳ was given at 40 mg/kg/d in the treated group,while the rest of the mice were given lysate.A portion of liver tissue was frozen at-80℃ and the other portion was fixed overnight in paraformaldehyde,paraffincoated and sectioned,and stained with HE and Masson.The serum samples were tested according to the instructions of ALT/AST/ALP/ TGF-b1/HA kit.Liver tissue homogenate was taken according to the kit instructions to determine SOD and MDA.Western-blot for Nrf2,p Nrf2,TGF-b1,p Smad3 C,p Smad3 L,a-muscle actin（a-SMA）,p21,fibrinogen activator inhibitor-1（PAI-1）protein levels,and immunofluorescence for p Nrf2,psmad3 C and p Smad3 L.To investigate the effects of AS-Ⅳ’s anti-DEN/CCl4/C2H5OH-induced hepatic fibrosis effect in Nrf2 knockout mice.Results 1.AS-Ⅳ inhibits the proliferation and migration of HSC-T6 cells and reduces ROS levels.MTT,cell scratching and reactive oxygen species assays showed that AS-Ⅳ significantly inhibited the proliferation and migration of HSC-T6 cells and reduced ROS levels.2.AS-Ⅳ increases protein levels of the Nrf2/HO-1 pathway in mice with liver fibrosis.AS-Ⅳ attenuated DEN/CCl4/C2H5OH-induced liver fibrosis significantly.Western-blot showed that AS-Ⅳ increased the level of p Nrf2,HO-1 and NQO1 in the liver tissue significantly.Immunofluorescence staining showed that AS-Ⅳ significantly promoted p Nrf2 expression.Pearson Product-Moment Correlation showed that the expression of p Smad3C/3L and p Nrf2 was a significant correlation in vivo.3.Knockout of Nrf2 gene impairs the therapeutic effect of AS-Ⅳ on DEN/CCl4/C2H5OH-induced hepatic fibrosis in mice The liver index results showed that Nrf2 knockout mice had lower liver weight compared with WT mice significantly,which,combined with the reduced liver size observed at the time of sampling,suggested that Nrf2 knockout might lead to liver atrophy.AST,ALT,ALP and MDA levels were elevated in the wild-type model group and decreased in the drug-administered group;SOD levels were decreased and increased after drug administration.In contrast,the levels of AST,ALT,ALP and MDA were higher in the Nrf2 knockout mouse model group than in the wild-type model group,and were not significantly reduced after drug administration;the levels of SOD were significantly reduced in the Nrf2 knockout mouse model group,and were not significantly increased after drug administration.The results of wild-type HE and Masson showed that fibrous deposition appeared in the model group and was significantly decreased in the AS-Ⅳ group,while fibrous deposition was significantly increased in the HO-Model compared with the WT-Model,and was not reduced in the HO-Model+AS-Ⅳ.Compared with the control group,the a-SMA and HA were increased.At the same time,the above two indicators showed a higher degree of fibrosis in HO mice.With the AS-Ⅳ treatment,the improvement of fibrosis in WT mice was prominent,while the improvement of fibrosis in HO mice was not obvious.4.Nrf2 knockout inhibits the downregulation of TGF-b1 proteins during AS-Ⅳ antiliver fibrosis After 12 weeks of hepatic fibrosis modeling,TGF-b1 protein levels in serum and liver tissue were significantly elevated in both mice,and the elevation of TGF-b1 protein was more prominent in HO mice;TGF-b1 levels were significantly lower in WT mice and insignificantly changed in HO mice after AS-Ⅳ administration.5.Nrf2 knockout inhibits the upregulation of p Smad3 C and p21 proteins during ASⅣ anti-liver fibrosis.The levels of p Smad3 C and p21 were elevated in the model mice,but the two indicators were less elevated in the model group of HO mice than in the WT mice.Levels were significantly elevated in the WT-model+AS-Ⅳ group compared to the corresponding model group,while no significant elevation was observed in the HO-model+AS-Ⅳ group compared to the corresponding model group.6.Nrf2 knockout inhibits the downregulation of p Smad3 L and PAI-1 proteins during AS-Ⅳ anti-liver fibrosis The levels of p Smad3 L and PAI-1 were elevated in the liver tissues of the model group mice,and the levels of p Smad3 L and PAI-1 were higher in the HO model mice compared with the WT model mice.These two indices were significantly lower in the WTmodel+AS-Ⅳ group compared with the corresponding model group,while there was no significant reduction in the HO-model+AS-Ⅳ group compared with the corresponding model group.Conclusion 1.AS-Ⅳ exerts anti-fibrotic effects via the Nrf2/HO-1 pathway,and Nrf2 knockout weakens the anti-fibrotic ability of AS-Ⅳ.2.Nrf2 knockout inhibited the upregulation of p Smad3C/p21 signaling pathway during AS-Ⅳ anti-liver fibrosis.3.Nrf2 knockout inhibited the downregulation of p Smad3L/PAI-1 signaling pathway during AS-Ⅳ anti-liver fibrosis.