| Objective:To explore the protective mechanism of Sagittaria sagittifolin polysaccharide(SSP)of liver injury model induced by isoniazid and rifampicin(INH/RFP)and non-alcoholic fatty liver model,thus provide experimental basis for the development of SSP as liver protection products.Method:1.Preventive protective effect and metabolomic study of Sagittaria sagittifolin polysaccharide on liver injury in mice treated with isoniazid and rifampicin1.1 Protective effect of Sagittaria sagittifolin polysaccharide on liver injury in mice treated with isoniazid and rifampicinTwenty-four BALB/C mice were randomly divided into normal control group(6 mice),model group(10 mice)and treatment group(8 mice).INH/RFP was used to establish the model,and SSP was administered to the treatment group at the same time.All mice were given saline(0.2g/kg)twice a day for the first time,and INH(0.1g/kg)+RFP(0.1g/kg)for the model group and treatment group.The second time wad in 4 hours later,the normal control group and model group were given saline(0.8g/kg),and the treatment group was given SSP(0.8g/kg).After 30 days,all mice were fasting for 12 hours,weighed and executed.Blood and liver samples were then collected.Blood was collected from the eyeball and centrifuged to obtain serum.The serum levels of ALT,AST and HE staining were detected by automatic biochemical analyzer,respectively,to evaluate the preventive effect of SSP on liver inj ury induced by INH/RFP.1.2 Serum metabolomic study of Sagittaria sagittifolin polysaccharide on isoniazid and rifampicin-induced liver inj ury in miceGrouping and administration were the same as 1.1.UPLC-HRMS was used to analyze the serum samples of mice with liver injury induced by isoniazid and rifampicin.The serum metabolites spectrum was obtained.PCA combined with PLS-DA was used to identify the possible specific biomarkers of SSP intervention on liver injury,explore the protective mechanism of SSP,and detect Nrf2 and heme HO-1 protein expression levels,thereby verifying the tricarboxylic acid cycle pathway in metabolomics research results.2.Preventive protective effect of Sagittaria sagittifolin polysaccharide on liver injury in non-alcoholic fatty liver mice and its metabolomic study2.1 Protective effect of Sagittaria sagittifolin polysaccharide on liver injury in mice with nonalcoholic fatty liver disease2.1.1 Evaluate the preventive effect of Sagittaria sagittifolin polysaccharide on glycolipid metabolism disorder and inflammation in non-alcoholic fatty liver disease mice.Twenty-four male ICR mice were randomly divided into three groups(8 mice in each group):normal control group was given normal feed+normal saline(0.8g/kg),model group was given methionine-choline deficiency feed(MCD)+normal saline(0.8g/kg)and treatment group got MCD+SSP(0.8g/kg).After 12weeks of feeding,all mice fasting for 12 hours,after anesthesia eyeball blood,centrifugal blood for serum,and take out liver tissue.The levels of serum TG,TC,FBQ FFA were detected by automatic biochemical analyzer,and the expressions of TNF-α,IL-6 and PGC-1α mRNA were detected by fluorescence quantitative polymerase chain reaction(RT-QPCR).HE staining and oil red O staining were used for liver histopathological examination.2.1.2 Evaluate the protective effect of Sagittaria sagittifolin polysaccharide on hepatocyte apoptosis in non-alcoholic fatty liver disease miceGrouping and administration were the same as 2.1.1.The levels of ALT,AST,HDLC and LDLC in serum were detected by automatic biochemical analyzer,and the levels of Bax,Bcl-2,Sirtl,Nrf2 and HO-1 mRNA in liver tissue were detected by RT-QPCR.Liver histopathology was examined by microscopy and Nrf2 protein was detected by Western-blot.2.2 Serum metabolomic study of Sagittaria sagittifolin polysaccharide on nonalcoholic fatty liver disease miceGrouping and administration were the same as 2.1.1.UPLC-HRMS was used to analyze the serum samples of non-alcoholic fatty liver disease mice treated with SSP and obtain the serum metabolites spectrum.PCA combined with OPLS-DA was used to identify the specific biomarkers of the possible intervention of SSP on liver injury,to find the marker metabolites,to explore the protective mechanism of SSP,and to detect the Nrf2 and HO-1 protein expression by immunohistochemical method.The metabolic pathway of arachidonic acid in metabolomics was verified.Result:1 Preventive protective effect and metabolomic study of Sagittaria sagittifolin polysaccharide on liver injury in mice treated with isoniazid and rifampicin1.1 Protective effect of Sagittaria sagittifolin polysaccharide on liver injury in mice treated with isoniazid and rifampicinThe results of serum biochemical test showed that the contents of AST and ALT in the model group were higher than those in the control group(P<0.01),while the contents of AST and ALT in the polysaccharide group were lower than those in the model group(P<0.05).HE staining showed that compared with the control group,inflammatory infiltration and necrosis of liver tissue appeared in the model group;compared with the model group,inflammatory infiltration and necrosis of liver tissue improved in the polysaccharide group.1.2 Metabonomic study on the liver inj ury of mice induced by isoniazid and rifampicinUPLC-HRMS showed that SSP could induce changes in 14 metabolites such as palmitic acid,ornithine,aspartic acid,succinic acid and fumaric acid,and regulate the metabolic pathways such as tricarboxylic acid cycle,ornithine cycle and amino acid cycle.The expression of Nrf2 and HO-1 was up-regulated by SSP.The results showed that SSP could up-regulate the expression of tricarboxylic acid cycle pathway.2 Preventive protective effect of Sagittaria sagittifolin polysaccharide on non-alcoholic fatty liver disease mice and its metabolomic study2.1 Protective effect of Sagittaria sagittifolin polysaccharide on non-alcoholic fatty liver disease mice2.1.1 Evaluate the preventive effect of Sagittaria sagittifolin polysaccharide on glycolipid metabolism disorder and inflammation in non-alcoholic fatty liver disease miceThe results of serum biochemical analysis showed that the levels of serum TG TC,FBG and FFA in model group were significantly higher than those in control group(P<0.01),while the levels of serum TG,TC,FBG and FFA in polysaccharide group were significantly lower than those in model group(P<0.05 or P<0.01).HE staining showed moderate steatosis of liver tissue in model group,accompanied by inflammation and necrosis,and steatosis in polysaccharide group was improved.Oil red O staining showed that hepatic steatosis was obvious in the model group and improved in the polysaccharide group.RT-QPCR showed that compared with the normal control group,the mRNA expression of TNF-α,IL-6 and PGC-1αin liver tissue of mice in the model group increased(P<0.05),while the mRNA expression of PGC-1α decreased(P<0.01);compared with the model group,the mRNA expression of TNF-a and IL-6 in liver tissue of mice in the polysaccharide group decreased significantly(P<0.05 or P<0.01),and the mRNA expression of PGC-la increased(P<0.01).2.1.2 Evaluate the protective effect of Sagittaria sagittifolin polysaccharide on hepatocyte apoptosis in non-alcoholic fatty liver disease miceThe results of serum biochemical analysis showed that the levels of ALT,AST and LDLC in the model group were significantly higher than those in the control group(P<0.01),while the levels of HDLC were significantly lower(P<0.05).Compared with the model group,the levels of serum ALT,AST and LDLC in the polysaccharide group decreased significantly(P<0.05 or P<0.01),while the levels of HDLC increased significantly(P<0.05).Mitochondria in liver tissue were observed by transmission electron microscopy.The swelling,deformation and mitochondrial ridge disappearance of liver mitochondria in model group were observed.The swelling,deformation and mitochondrial ridge disappearance of liver mitochondria in polysaccharide group were improved.RT-Q PCR showed that compared with the normal control group,the mRNA expression of Bax in the liver tissue of the model group increased(P<0.01),while the mRNA expression of Bcl-2,Sirtl,Nrf2 and HO-1 decreased(P<0.05);compared with the model group,the mRNA expression of Bax in the liver tissue of the polysaccharide group decreased(P<0.01),while the mRNA expression of Bcl-2,Sirtl,Nrf2 and HO-1 increased(P<0.05).Western-blot analysis showed that the expression of Nrf2 protein decreased in the model group(P<0.05),but increased in the polysaccharide group(P<0.05).2.2 Metabonomic study of Sagittaria sagittifolin polysaccharide on nonalcoholic fatty liver disease miceSSP can change 33 biomarkers,mainly the metabolic pathway of arachidonic acid.UPLC-HRMS showed that SSP could induce 33 metabolites such as palmitic acid,arachidonic acid,leukotriene A4 and leukotriene B4,and regulate the metabolic pathways of arachidonic acid.Immunohistochemical analysis showed that SSP could induce the up-regulation of Nrf2 and HO-1 protein expression,which indicated that SSP could regulate arachidonic acid metabolism pathway with high reliability.Conclusion:SSP can protect mice from INH/RFP-induced liver injury by regulating the pathways of tricarboxylic acid cycle,taurine metabolism,branched-chain amino acid metabolism and fatty acid metabolism.The protective effect of SSP on NAFLD is mainly related to improving mitochondrial damage,inhibiting inflammation,apoptosis and regulating arachidonic acid metabolism. |
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