| Objective: This experiment aims to screen proteins that are differentially expressed in mice liver before and after processing of fructus meliae toosendan by proteomics,and the physiological functions of differential proteins will be further analyzed to investigate the hepatotoxicity and attenuation mechanism of mice induced by fructus meliae toosendan.Methods: 1.Study on hepatotoxicity and hepatic oxidative damage in mice induced by continuous administration of different prepared products of fructus meliae toosendanThe Kunming mice were randomly divided into the normal group and the different prepared products of fructus meliae toosendan group.Each group used 2/3 MTD as the dose,and they were continuously administered for 7days,14 days and 28 days.Blood was taken 2 hours after the last administration to detect the activity of ALT,AST and ALP in the serum,and the liver,spleen,kidney,heart and lung were weighed and the organ coefficient was calculated.The activities of SOD,GSH-Px,CAT,XOD and the contents of MDA and GSH in liver tissues were measured,and the degree of peroxidation damage between the processed products was compared.2.Proteomics study on hepatotoxicity in mice induced by different prepared products of fructus meliae toosendanKunming mice were randomly divided into the normal group,the toosendan crude sample group,the vinegar-broiled sample group,the alcohol-broiled sample group,the fried sample group and the salt-broiled sample group.Each group used 2/3MTD as the dose,and they were continuously administered for7 days.The liver was taken two hours after the last administration,and each group of 10 liver samples was mixed in equal amounts and labeled with i TRAQ reagent after the protease cleavage;The liver protein was isolated by LC-MS/MS and protein data was retrieved using proteinpilot software 5.0(AB Sciex)to obtain a reliable protein with quantitative information.On the basis of the false discovery rate FDR<0.05,the expression ratio Ration(B/A)>1.3 or Ration(B/A)<0.77 between the two groups was set as the threshold for up or down for screening differentially expressed proteins.The selected differential proteins were used to GO classification and KEGG Pathway analysis,and the target proteins related to fatty liver formation were screened based on the above analysis results and the literature.1.Results: 1.Study on hepatotoxicity and hepatic oxidative damage in mice induced by continuous administration of different prepared products of f Fructus meliae toosendan1.1 The results of ALT,AST and ALP activities in the serum of mice showed that,compared with the normal group,the ALT activity was significantly increased(P<0.05 or P<0.01)after continuous administration of the toosendan crude sample group for 7 days,14 days and 28 days,and the ALT activity was significantly increased(P<0.05)after 14 days of continuous administration of the salt-broiled sample group.No significant changes in the other groups.After continuous administration for 28 days,the AST activity of the toosendan crude sample group and the fried sample group was significantly increased(P<0.01),and there was no significant difference in AST activity in the other groups.After 7 days of continuous administration,the ALP activity of the toosendan crude sample group,the alcohol-broiled sample group and the salt-broiled sample group was significantly increased(P<0.05 or P<0.01).After 14 days of continuous administration,the ALP activity of the alcohol-broiled sample group was significantly increased(P<0.05),and there was no significant change in the other groups.1.2 The results of the organ coefficient of the mice showed that compared with the normal group,the liver coefficient of the toosendan crude sample group,the vinegar-broiled sample group and the alcohol-broiled sample group was significantly increased(P<0.05 or P<0.01)after 7 days of continuous administration;after 14 days of continuous administration,the liver coefficient of the toosendan crude sample group was significantly increased(P<0.05),and the liver coefficient of the salt-broiled sample group was significantly reduced(P<0.01).After 28 days of continuous administration,the liver coefficient of the vinegar-broiled sample group and the alcohol-broiled sample group was significantly increased(P<0.05 or P<0.01).Compared with the normal group,the spleen coefficient of the vinegar-broiled sample group,the alcohol-broiled sample group,the fried sample group and the salt-broiled sample group was significantly reduced(P<0.01)after 7 days of continuous administration;after 14 days of continuous administration,the spleen coefficient of the vinegar-broiled sample group and the alcohol-broiled sample group was significantly reduced(P<0.01);after continuous administration for 28 days,there was no significant change in the spleen coefficient of each group.There were no significant differences in the results of heart,kidney,and lung organ coefficients in mice.1.3 The results of oxidative damage of liver tissue in mice showed that compared with the normal group,when the toosendan crude sample group was continuously administered for 7 days and 14 days,the alcohol-broiled sample group was continuously administered for 28 days,and the salt-broiled sample group was continuously administered for 7 days and 28 days,the content of MDA was significantly increased(P<0.05 or P<0.01);there was no significant difference between the vinegar-broiled sample group and the fried sample group.Compared with the normal group,when the vinegar-broiled sample group was continuously administered for 7 days,the toosendan crude sample group and the other groups were continuously administered for 14 days,the vinegar-broiled sample group,the alcohol-broiled sample group and the salt-broiled sample group were continuously administered for 28 days,the activity of SOD was significantly improved(P<0.05 or P<0.01);when the toosendan crude sample group,the alcohol-broiled sample group,the fried sample group,the salt-broiled sample group were continuously administered for 7 days,and the fried sample group were continuously administered for 28 days,the activity of SOD was not significantly improved.Compared with the normal group,the continuous administration of the toosendan crude sample group for 7 days,the vinegar-broiled sample group and the fried sample group were continuously administered for 28 days,the GSH-Px activity was significantly decreased(P<0.05 or P<0.01);when the salt-broiled sample group was continuously administered for 14 days,the activity of GSH-Px was significantly increased(P<0.01).Compared with the normal group,the content of GSH was significantly decreased(P<0.01)when the toosendan crude sample group,the fried sample group and the salt-broiled sample group were continuously administered for 7 days.When the salt-broiled sample group was continuously administered for 14 days and 28 days,the content of GSH was significantly increased(P<0.05).Compared with the normal group,when the vinegar-broiled sample group and the fried sample group were continuously administered for 14 days,when the vinegar-broiled sample group,the alcohol-broiled sample group,and the fried sample group were continuously administered for 28 days,the activity of CAT was significantly improved(P<0.05 或 P<0.01).Compared with the normal group,the activity of XOD was significantly decreased(P<0.01)when the alcohol-broiled sample group,the fried sample group and the salt-broiled sample group were continuously administered for 7 days;the activity of XOD was significantly increased(P<0.05)when the toosendan crude sample group was continuously administered for 28 days.2.Proteomics study on hepatotoxicity in mice induced by different prepared products of fructus meliae toosendanA total of 4359 proteins were identified.Compared with the normal group,the differential protein of the toosendan crude sample group was 96,the differential protein of the vinegar-broiled sample groupp was 78,the differential protein of the alcohol-broiled sample group was 139,the differential protein of the fried sample group was 127 and the difference protein of the salt-broiled sample group was 145.The expression of 67 differentially expressed proteins in the toosendan crude sample group returned to normal levels in each of the processed sample group.Through cluster analysis,GO enrichment and KEGG pathway analysis of 67 differential proteins,these proteins are involved in the interaction between ribosomal subtype enzymes,glutathione S-transferase metabolism,CYP450 enzyme metabolism and PPAR signaling pathway.The focus was on RPL3,RPL4,RPL26,RPL29,RPL10,RPL15,RPL37,RPS6,RPS2,gst-p,ACBP,FABPL,APOA2,GSTA1,GSTM2,GSTM3,GSTP1,CYP4A14,which was abnormally expressed in the toosendan crude sample group,but its expression returned to normal in each of the processed sample groups.Conclusion: Different prepared products of fructus meliae toosendan can alleviate the damage process of liver tissue.The attenuating mechanism of prepared products may be to reduce the abnormal expression of RPL3,,RPL4,RPL26,RPL29,RPL10,RPL15,RPL37,RPS6,RPS2,gst-p,ACBP,FABPL,APOA2,GSTA1,GSTM2,GSTM3,GSTP1,CYP4A14,thereby reducing the resulting decrease in protein synthesis,DNA damage,abnormal lipid metabolism,and oxidative damage. |
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