| Objective1.To study the preventive and therapeutic effects of Tanshinone ⅡA(TⅡA)on non-alcoholic steatohepatitis(NASH)in mice.2.To explore the mechanism of TⅡA in preventing and treating NASH mice based on the formation of neutrophil extracellular traps(NETs)and the changes of hepatocyte apoptosis-related parameters.Methods1.Animal grouping and model preparationTwenty-four SPF C57B/L6 mice were randomly divided into normal group,model group and treatment group,with 8 mice in each group.The normal group was fed with normal diet and administration group were induced by methionine choline deficiency(MCD)diet to duplicate the NASH mouse model until the end of the 6th week.2.The dosage and route of administrationAt the same time of modeling,the treatment group was intraperitoneally injected with30 mg/kg/d TⅡA sodium sulfonate injection,and the normal group and model group were intraperitoneally injected with 30 mg/kg/d 0.9% normal saline once daily for 6 weeks.3.Index detectionDuring the modeling period,the body weight,feed intake and other general conditions of mice were observed and recorded every day for 6 weeks;at the end of the sixth week,the serum and liver tissue of mice were collected for index detection,HE staining and Sudan IV staining were used to observe the pathological changes of the liver of the mice;the changes of serum liver function(AST,ALT,TBIL)and blood lipids(TC,TG)of the mice in each group were detected by automatic biochemical analyzer;the expression of TNF-α,IL-6 and TGF-β m RNA in the liver tissues of the mice in each group was detected by RT-q PCR;the expression of MPO in the liver tissues of the mice in each group was detected by immunohistochemistry;and the expression of Cit H3,Caspase-3,and Bax protein in the liver tissues of the mice in each group was detected by Western Bolt.Results1.General observationDuring the modeling period,the weight,intake and mental state of mice in the normal group were good;Compared with the normal group,the weight of mice in the model group had no statistical significance in the first week(P> 0.05),but decreased in the second to sixth weeks(P< 0.001).The dietary intake also decreased with the duration of modeling cycle,which showed that the mice were dispirited,their hair was not glossy,their stool was yellow and the frequency increased;Compared with the model group,the weight of the mice in the treatment group had no statistical significance at the 1st and 2nd week(P>0.05),but increased at the 3rd to 6th week(P< 0.001).The dietary intake,mental state,hair luster and defecation of the mice improved with the treatment week.2.Liver pathologicalHE staining showed that the hepatocytes of mice in the model group were disorganized,the hepatocyte cords were indistinct,and a large number of fat vacuoles appeared,accompanied by significant cytoplasmic loosening,ballooning and inflammatory cell infiltration.The results of Sudan IV suggestion that a host of red lipid droplets aggregation in the hepatocytes of NASH mice,and the boundary of hepatocytes was not clear,while TⅡA significantly reduced red lipid droplets.3.Serum biochemicalCompared with the normal group,ALT,AST,TBIL content of model group increased,TC and TG decreased,and the difference was statistically significant(P< 0.05);compared with the model group,the contents of ALT,AST and TBIL in the serum of the TⅡA mice were decreased,the contents of TC in the serum were increased(P< 0.05),and the contents of TG in the serum were slightly increased,with no statistical significance.4.Anti inflammatory effect of TⅡA on NASH miceTⅡA can reduce the expression levels of TNF-α m RNA,IL-6 m RNA and TGF-βm RNA in the liver of NASH mice.Compared with the control group,the relative expression of TNF-α,IL-6 and TGF-β m RNA in the liver of mice in the model group was increased(P< 0.05).Compared with the model group,TNF-α,IL-6 and TGF-β m RNA in the liver of TⅡA administered mice showed a decreasing trend(P< 0.05).5.Effect of TⅡA on MPO and Cit H3 expression during the formation of NETs in liver tissueTⅡA could reduce MPO and Cit H3 expression levels in the liver of NASH mice.Compared with the control group,increased positive expression of MPO was observed in the hepatocytes of mice in the model group(P< 0.05).Compared with the model group,the positive expression of MPO by TⅡA was significantly reduced(P < 0.05).Compared with the normal group,mice in the model group had increased hepatic Cit H3 protein expression(P< 0.05).Compared with the model group,the expression of Cit H3 protein in the liver of TⅡA mice was significantly reduced(P< 0.05).6.TⅡA inhibits hepatocyte apoptosisTⅡA reduced the expression levels of Caspase-3 and Bax proteins in the liver of NASH mice.Compared with the control group,Caspase-3 and Bax protein expression was increased in the liver of mice in the model group(P< 0.05).Compared with the model group,the expression of Caspase-3 and Bax protein in the liver of TⅡA treated mice was significantly reduced(P < 0.05).Conclusion1.Tanshinone ⅡA can reduce hepatic steatosis and inflammation,regulate liver function and blood lipid,and significantly improve the symptoms of NASH in mice.2.Tanshinone ⅡA may prevent and treat NASH by inhibiting the expression of MPO and Cit H3 and the formation of Neutrophil extracellular traps.3.Tanshinone ⅡA can also prevent and treat NASH by inhibiting hepatocyte apoptosis. |
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