Protective effect of Lycium barbarum polysaccharides on Hcy-induced mouse hepatocyte injury and its mechanism
Objective To investigate the effect and mechanism of lycium barbarum polysaccharide(LBP)on hepatocyte injury induced by homocysteine(Hcy).Methods Normal C3H/An mouse hepatocytes(NCTC 1469)were cultured in vitro and treated with different concentrations of Hcy(0,50,100,200,500 μmol/L).The optimal concentrations of Hcy-treated NCTC 1469 cells were detected by MTT assay.When the cells reached the logarithmic growth stage,the conditions were set up as follows:(1)control group(cultured with DMEM medium supplemented with 10%horse serum)and Hcy group(treated with 100 μmol/L Hcy solution for 48 h),and the cells were collected.Cell viability staining was used to detect apoptosis,aspartate aminotransferase(AST)/alanine aminotransferase(ALT)activity detection kit was used to detect AST and ALT activities,RT-qPCR was used to detect the expression levels of YAP1,DNMT1,DNMT3a and DNMT3b mRAN,and Western blotting was used to detect the expression of YAP1 protein,nested methylation specific PCR(nMS-PCR)was used to detect DNA methylation rates in the YAP1 promoter region.(2)Control group,LBP group,Hcy group and Hcy+LBP group.LBP group was treated with 4 mg/ml LBP solution for 2 h,Hcy group and Hcy+LBP group were treated with 100 μmol/L Hcy solution for 48 h,and Hcy+LBP group was treated with 4 mg/ml LBP solution at 46 h,and the cells were collected.The expression levels of YAP1,DNMT1,DNMT3a and DNMT3b mRAN were detected by RT-qPCR;the expression of YAP1,Bax and Bcl-2 proteins was detected by Western blotting;AST/ALT activity detection kit was used to detect AST and ALT activities.Prediction of DNA methylation CpG islands in YAP1 promoter region by bioinformatics.Results NCTC 1469 cells were treated with 100 μmol/L Hcy according to the results of MTT assay.Compared with control group,the apoptosis rate of Hcy group increased(P<0.01),the activities of ALT and AST increased(P<0.001),the mRAN and protein expression levels of YAP1 decreased(P<0.001),and the methylation rate of YAP1 promoter region increased(P<0.01),the mRNA expression levels of DNMT1,DNMT3a and DNMT3b increased(P<0.01 or P<0.001).Compared with Hcy group,the mRNA expression levels of DNMT1,DNMT3a and DNMT3b in the Hcy+LBP group decreased(P<0.001),the mRAN and protein expression levels of YAP1 significantly increased(P<0.01 or P<0.001).In addition,in the Hcy+LBP group,cells showed significantly elevated of Bcl-2 protein(P<0.001),but decreased Bax protein(P<0.001),and decreased activities of ALT and AST(P<0.001).Conclusions The decrease of YAP1 expression may be the key process of Hcy induced injury of NCTC 1469 cells,and the methylation of the YAP1 promoter region may be the molecular mechanism of Hcy induced YAP1 expression change.LBP may improve NCTC 1469 cell damage induced by Hcy by positively regulating YAP1 expression.
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