枸杞多糖对Hcy诱导的小鼠肝细胞损伤的保护作用及其机制  

Protective effect of Lycium barbarum polysaccharides on Hcy-induced mouse hepatocyte injury and its mechanism

作　　者：王佩佩[1] 岳云 曹丽翠[1] 李宏伟 刘丽[1] 李航鹰 王晓丽[1] Wang Pei-Pei;Yue Yun;Cao Li-Cui;Li Hong-Wei;Liu Li;Li Hang-Ying;Wang Xiao-Li(Department of Traditional Chinese Medicine,Yinchuan First People's Hospital,Yinchuan,Ningxia 750004,China;Acupuncture Department of Zhongwei Hospital of Traditional Chinese Medicine,Zhongwei,Ningxia 755000,China;Department of Traditional Chinese Medicine,Ningxia Hui Autonomous Region People's Hospital,Yinchuan,Ningxia 750004,China;College of Pharmacy,Ningxia Medical University,Yinchuan,Ningxia 750004,China)

机构地区：[1]银川市第一人民医院中医科,宁夏银川750004 [2]中卫市中医院针灸科,宁夏中卫755000 [3]宁夏回族自治区人民医院中医科,宁夏银川750004 [4]宁夏医科大学药学院,宁夏银川750004

出　　处：《解放军医学杂志》2024年第5期542-549,共8页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金(222607017)。

摘　　要：目的探讨枸杞多糖(LBP)对同型半胱氨酸(Hcy)诱导的小鼠肝细胞损伤的作用及其机制。方法体外培养正常C3H/An小鼠肝细胞(NCTC 1469),采用不同浓度的Hcy(0、50、100、200、500μmol/L)处理NCTC 1469细胞,使用MTT法检测不同浓度Hcy对细胞活性的影响。取对数生长期细胞,设置:(1)对照组(采用10%马血清的DMEM培养基培养)与Hcy组(采用100μmol/L Hcy溶液处理48 h),收集细胞。采用细胞活力染色检测细胞凋亡情况,谷草转氨酶(AST)/谷丙转氨酶(ALT)活性检测试剂盒检测AST、ALT活性,RT-qPCR检测YAP1(Yes相关蛋白1)、DNMT1、DNMT3a、DNMT3b mRAN的表达,Western blotting检测YAP1蛋白的表达,巢式甲基化特异性PCR(nMS-PCR)检测YAP1启动子区DNA甲基化率。(2)对照组、LBP组、Hcy组与Hcy+LBP组,LBP组采用4 mg/ml LBP溶液处理2 h,Hcy组、Hcy+LBP采用100μmol/L Hcy溶液处理48 h,46 h时Hcy+LBP组加入4 mg/ml LBP溶液处理,收集细胞。采用RT-qPCR检测YAP1、DNMT1、DNMT3a、DNMT3b mRAN的表达,Western blotting检测YAP1、Bax、Bcl-2蛋白的表达,AST/ALT活性检测试剂盒检测AST、ALT活性。采用生物信息学方法预测YAP1启动子区DNA甲基化CpG岛。结果根据MTT法检测结果,选择100μmol/L Hcy处理NCTC 1469细胞进行后续实验。与对照组比较,Hcy组细胞凋亡率增高(P<0.01),ALT、AST活性增高(P<0.001),YAP1 mRAN和蛋白相对表达水平降低(P<0.001),YAP1启动子区域甲基化率增高(P<0.01),DNA甲基化酶DNMT1、DNMT3a及DNMT3b mRNA相对表达水平增高(P<0.01或P<0.001)。与Hcy组比较,Hcy+LBP组DNA甲基化酶DNMT1、DNMT3a及DNMT3b mRNA相对表达水平降低(P<0.001),YAP1 mRAN和蛋白相对表达水平明显增高(P<0.01或P<0.001),Bcl-2蛋白相对表达水平明显升高(P<0.001),Bax蛋白相对表达水平明显降低(P<0.001),ALT、AST活性降低(P<0.001)。结论YAP1表达降低可能是Hcy诱导的小鼠NCTC 1469细胞损伤的关键过程,YAP1启动子区域甲基化的改变可能是Hcy引起YAP1表达变化�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(0,50,100,200,500μmol/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 100μmol/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 100μmol/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 100μmol/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 expressi

关 键 词：枸杞多糖 同型半胱氨酸 肝细胞损伤 DNA甲基化 

分 类 号：R36[医药卫生—病理学]