Salvianolic Acid B Anti-liver Injury Via Phosphorylation Of Smad 2/3 At Different Sites And NRF2/HO-1 Signal Pathway

As the main place of metabolism,Liver functions include maintaining redoxbalance,regulating blood sugar and protein synthesis.When the liver is subjected to harmful stimulation,highly active molecules such as reactive oxygen species?ROS?are produced in the body,and the imbalance between oxidation system and antioxidant system results in liver free radical damage.Liver fibrosis is a continuous pathological injury-repair process under various liver injuries.And then liver fibrosis develops into liver cirrhosis and HCC,which threat human health.Further study and reveal the pathogenesis of liver injury,especially on the cellular and molecular levels to clarify the acute liver injury-liver fibrosis process,to target intervention of specific cells and molecules,is a new strategy to delay or block the process of liver fibrosis.The factors of liver injury are viral injury,drug-induced injury,and genetic factors,among which drug damage to the liver is the most serious.It is mainly reflected in the occurrence of hepatocyte inflammation,the mechanism of which is the production of electronic products,free radicals etc.,which resulting in lipid peroxidation.Hepatic fibrosis is a pathological process was characterised by the excessive proliferation and abnormal deposition of extracellular matrix?ECM?caused by the activation of?HSC?in hepatic stellate cells.TGF-?₁ is considered to be the most effective activator of HSC,which plays an important role in promoting fibrosis through TGF-?/Smad signaling pathway.Recent studies have shown that pSmad2C?Smad2C terminal phosphorylation?and pSmad2L?Smad2 link phosphorylation?mediate fibrosis and migration promoting signals in TGF-?/Smad signaling pathway.Salvianolic acid B?Sal B?is extracted from the root and rhizome of Salvia miltiorrhiza Bunge,which has strong antioxidant and anti-fibrosis effects,which play a role by regulating Nrf2/HO-1 and TGF-?/Smad signaling pathway,respectively.However,how Sal B regulates pSmad2C and pSmad2L is not clear,and whether the antioxidant effect of Sal B affects Nrf2/HO-1 pathway has not been reported.In this study,the model of acute liver injury and liver fibrosis in mice was inducedby CCl₄ in vivo,and the anti-acute and chronic liver injury effects of Sal B were further observed.And the model of HSC-T6 oxidative damage induced by H₂O₂ in cell experiment to explore the molecular mechanism of Salvianolic acid B intervention.Objective:1.Based on previous proof that Sal B can reduce the incidence and diversity of liver cancer,the histopathological analysis of liver tissue was carried out by hematoxylin eosin staining and VG staining.To evaluate the effect of Sal B on histopathological changes during acute liver injury and hepatic fibrosis induced by CCl₄ in mice.2.The antioxidant effects of Sal B on CCl₄-induced acute liver injury and liver fibrosis in mice were analyzed by detecting liver function and oxidative stress in tissues.3.To observe the changes of Smad2,Smad3 phosphorylated proteins?pSmad2C,pSmad2L,pSmad3C,pSmad3L?and Nrf-2/HO-1 levels in CCl₄-induced acute liver injury,liver fibrosis model and Sal B intervention in mice.The relationship between Sal B's anti-CCl₄-induced acute liver injury,liver fibrosis,pSmad2,pSmad3 signal transduction and Nrf-2/HO-1 signal was analyzed.4.To observe the effect of Sal B on the levels of Smad2 phosphorylated proteins?pSmad2C,pSmad2L?and Nrf-2/HO-1 proteins in HSC-T6 cells induced by H₂O₂.To analyze whether Sal B has direct regulatory effect on pSmad2C and pSmad2L and the role of Nrf-2/HO-1 in this process.Methods:1.In vivo,study of salvianolic acid B against CCl4-induced acute and chronic liver injury and its relationship with Nrf2/HO-1 and TGF-beta/Smad pathwayAcute liver injury model and Sal B intervention:Mice were divided into 6 groups:control group,model group,Sal B group,and colchicine group.The mice in the model group and Sal B group?7.5,15,30 mg/kg?were injected intraperitoneally with?ip?CCl₄?0.1%CCl₄ 10 mg/kg/,twice a week?,while the control group was given the same amount of solvent?olive oil?.Sal B group and colchicine group were given the corresponding dose of drugs intragastrically every day,and the control group and model group were given solvent?normal saline?in parallel for 1 week.Liver Fibrosis Model and Sal B Intervention:Mice were divided into 6 groups:control group,model group,Sal B group?7.5,15,30 mg/kg?,and colchicine group.The mice in the model group,Sal B group and colchicine group were injected intraperitoneally with?ip?CCl₄?20%CCl₄ 1 mg/kg/twice a week?,while the control group was given the same amount of solvent?olive oil?.Sal B group and colchicine group were given the corresponding dose of drugs intragastrically every day,and the control group and model group were given solvent?normal saline?in parallel for 8 weeks.The pathological changes of liver tissue were observed by embedded hematoxylin and HE and VG.The levels of ALT and AST were detected by kits.Superoxide dismutase?SOD?,glutathione?GSH?and malondialdehyde?MDA?kit were used to detect oxidative stress in liver tissue.Immunohistochemical staining was used to detect the distribution of pSmad2C,pSmad2L and Nrf-2.Western blotting was used to detect the changes of pSmad2C,pSmad2L,pSmad3C,pSmad3L,Nrf-2 and HO-1 levels.2.In vitro,the effects of Salvianolic acid B on Smad2 phosphorylated proteins?pSmad2C,pSmad2L?and Nrf-2/HO-1 levels in H2O2-induced HSC-T6 cells were observed.HSC-T6 cells were routinely cultured in logarithmic growth phase in vitro.the cells were divided into three groups:control group,H₂O₂ group and Sal B?25,50,100 mmol/L?.24 hours before the end of culture,the serum-free medium,Sal B group was replaced with the corresponding dose of drugs.One hour before the end of culture.TGF-?1 was co-cultured in H₂O₂ group and Sal B group.At the end of culture,the cells were collected and the total proteins were extracted.The expression of pSmad2C,pSmad2L pSmad3C,pSmad3L,Nrf-2 and HO-1 proteins in the samples was detected by Western blot method.Results:1.Sal B alleviates the pathological process of acute and chronic liver injury induced by CCl4 in mice,interferes with pSmad2L/C signal transduction and enhances Nrf-2/HO-1 signal.1.2 Sal B alleviates pathological changes of acute and chronic liver injury induced by CCl₄ in miceIn acute liver injury stage:The liver of normal mice was stained with HE,the hepatocytes were arranged in strips,the structure of hepatic lobule was intact,and there was almost no degeneration or necrosis of hepatocytes.In the model group,the arrangement of hepatocytes was irregular,the infiltration of inflammatory cells was obvious in the hepatocyte space and portal area,and the vascular lesions of hepatocytes were observed.Compared with the model group,the infiltration of inflammatory cells in the liver of Salvianolic acid B group was significantly lower than of the model group.In hepatic fibrosis stage:In the control group,the surface of the liver was smooth and glossy,the texture was soft,the edge was sharp,and the structure of hepatic lobule was intact.After 8 weeks of CCl₄ modeling,the surface of the liver was rough and granular,the texture was hard,and the edge became obtuse,A large number of hepatic fibrosis cells were observed in the liver tissue.In Sal B treatment group,the liver lesions were significantly improved and the degree of fibrosis was alleviated.1.2 Sal B improves Serum ALT and AST in mice with Acute and chronic liver injury induced by CCl₄In acute liver injury stage:The contents of serum ALT and AST in the model group were significantly higher than those in the normal group.The contents of serum ALT and AST in Salvianolic acid B middle dose group decreased significantly vs model group.The contents of serum ALT and AST in high dose Salvianolic acid B group were significantly lower than those in model group.In hepatic fibrosis stage:The contents of serum ALT and AST in the model group were significantly higher than those in the normal group.The contents of serum ALT and AST in Salvianolic acid B middle dose group decreased significantly vs model group.The contents of serum ALT and AST in high dose Salvianolic acid B group were significantly lower than those in model group.1.3 Sal B improves oxidative stress in acute and chronic liver injury induced by CCl₄in miceIn acute liver injury stage:After CCl₄ stimulation,SOD activity in liver tissue of mice decreased significantly,while that in salvianolic acid B high dose group increased significantly?improved?;GSH level in liver tissue of mice decreased significantly after CCl₄ stimulation,while that in salvianolic acid B medium and high dose group increased significantly;MDA level increased significantly after CCl₄ stimulation,while that in salvianolic acid B medium and high dose group decreased significantly.In the stage of hepatic fibrosis,the activity of SOD in liver tissue of mice was significantly decreased after CCl₄ stimulation,while that in salvianolic acid B medium and high dose groups was significantly increased?improved?;the level of GSH in liver tissue of mice was significantly decreased after CCl₄ stimulation,and that in salvianolic acid B medium and high dose groups was significantly increased;the level of MDA was significantly increased after CCl₄ stimulation,while that in salvianolic acid B low,medium and high dose groups was significantly reduced.1.4 Sal B regulates the expression of pSmad2C,pSmad2L,pSmad3C and pSmad3L in CCl₄-induced acute and chronic liver injury in mice.In CCl₄-induced acute liver injury and hepatic fibrosis in mice,the levels of pSmad2C and pSmad2L proteins were significantly up-regulated,while Salvianolic acid B decreased the expression of pSmad2C and pSmad2L proteins in a concentration-dependent manner.1.5 Sal B up-regulates the expression of Nrf2 and HO-1 in CCl₄-induced acute liver injury and liver fibrosis in mice.In the experiment of acute liver injury induced by CCl₄ in mice,the levels of Nrf2and HO-1 proteins were up-regulated but not significantly,while Salvianolic acid B increased the expression of Nrf2 and HO-1 proteins in a concentration-dependent manner.immunohistochemical detection showed that the expression of Nrf2 was less in liver tissue.Salvianolic acid B enhanced the expression of Nrf2 protein in a concentration-dependent manner.In the experiment of CCl₄-induced hepatic fibrosis in mice,the levels of Nrf2 andHO-1 proteins were significantly up-regulated,while the expression of Nrf2 protein in Salvianolic acid B group was down-regulated compared with the model group.The expression of Nrf2 in liver tissue of mice with liver fibrosis induced by CCl₄ was significantly increased,and Nrf2 was more common around the fibrosis of portal area,while in Salvianolic acid B group,the expression area of Nrf2 around the portal area was reduced due to the decrease of fibrosis.2 Effects of 2 Sal B on Smad2 phosphorylated proteins?pSmad2C,pSmad2L?and Nrf-2/HO-1 levels in HSC-T6 cells induced by H2O22.1 Sal B down-regulates pSmad2C and pSmad2L in H₂O₂-induced HSC-T6 cell injuryUnder the stimulation of H₂O₂,the expression of pSmad2C and pSmad2L proteins was increased in HSC-T6 cells,while Sal B pretreatment group could inhibit or reverse the up-regulation of pSmad2C and pSmad2L induced by H₂O₂ stimulation.2.2 Sal B up-regulates Nrf-2 and HO-1 in HSC-T6 cell injury induced by hydrogen peroxideUnder the stimulation of H₂O₂,the expression of Nrf-2 and HO-1 proteins in HSC-T6 cells did not change significantly,but Salvianolic acid B enhanced the expression of Nrf2 and HO-1 proteins in a concentration-dependent manner in Sal B pretreatment group.Conclusions:1.Sal B has a protective effect on acute and chronic liver injury induced by CCl₄ in mice.2.Sal B inhibited the levels of pSmad2C,pSmad2L and pSmad3L proteins,and enhanced the levels of pSmad3C protein.It is suggested that Sal B's anti-liver injury effect is related to the regulation of Smad3 C terminal and junction phosphorylation of Smad 2;Sal B enhances the expression of Nrf 2 and HO-1 proteins,suggesting that Sal B enhances the signal of Nrf-2/HO-1 to play an anti-oxidative role.