芥子碱对H2O2诱导BMSCs成脂分化的影响

摘要

目的:探讨中药有效单体芥子碱对过氧化氢(H2O2)诱导骨髓间充质干细胞(bone marrow mesen-chymal stem cells,BMSCs)成脂分化的影响.方法:先通过全骨髓培养法和流式细胞术鉴定并筛选未分化的大鼠BMSCs;一方面利用H2O2诱导BMSCs成脂分化建立模型,一方面采用CCK-8实验检测芥子碱对BMSCs的毒性作用;模型建立成功与芥子碱药用浓度确定后进行油红O半定量实验检测细胞中脂滴含量,筛选出最佳的药物浓度;随后采用油红O染色拍片直接观察药物干预24 h后对BMSCs成脂分化的影响,并且采用qPCR和Western blot 实验检测BMSCs中成脂分化相关蛋白——脂肪细胞蛋白2(aP2)、过氧化物酶体增殖物激活受体γ(PPARγ)和葡萄糖转运蛋白4(Glut4)的变化情况.结果:浓度为200 μmol/L的H2O2作用1 h可诱导BMSCs成脂分化;CCK-8实验结果显示在芥子碱浓度<40 μmol/L的条件下对BMSCs的活力没有显著影响,同时结合油红O半定量实验结果筛选出芥子碱抑制其成脂分化的最佳浓度为15 μmol/L;油红O染色实验观察到芥子碱组(15 μmol/L)组脂滴较模型组数量明显减少,qPCR和Western blot实验结果亦显示正常对照组和芥子碱组aP2、PPARγ和Glut4表达均低于模型组(P<0.01).结论:芥子碱能够抑制H2O2诱导BMSCs向脂肪细胞分化,其机制可能与PPARγ/AMPK信号通路有关.%AIM:To investigate the effects of sinapine,an effective monomer of Chinese medicine,on hydro-gen peroxide(H2O2)-induced adipogenic differentiation of bone marrow mesenchymal stem cells(BMSCs).METHODS:The undifferentiated rat BMSCs were identified and screened by flow cytometry.The adipogenic differentiation of BMSCs was induced by H2O2,and the toxicity of sinapine on BMSCs was tested by CCK-8 assay.After the modeling method and the concentration range of sinapine were determined,the lipid droplets in the cells were detected by Oil Red O semi-quanti-tative assay,and the optimal drug concentration was selected.Finally,Oil Red O assay was observed 24 h after drug inter-vention,and the expression of adipogenic differentiation-related proteins,adipocyte protein 2(aP2),peroxisome prolifera-tor-activated receptor γ(PPARγ)and glucose transporter 4(Glut4),at mRNA and protein levels in the BMSCs was deter-mined by qPCR and Western blot.RESULTS:Treatment with H2O2at 200 μmol/L for 1 h induced BMSCs to differentiate into adipocytes.Below the concentration of 40 μmol/L,sinapine had no toxicity to BMSCs.The best inhibitory concentra-tion of sinapine on adipogenic differentiation was at 15 μmol/L.The number of lipid droplets in sinapine(15 μmol/L) group was significantly lower than that in model group.In sinapine group,the expression of aP2,PPARγand Glut4 at mR-NA and protein levels was lower than that in model group(P<0.01).CONCLUSION: Sinapine inhibits H2O2-induced adipogenic differentiation of rat BMSCs.The mechanism may be related to the PPARγ/AMPK signaling pathway.