芥子碱对H_2O_2诱导BMSCs成脂分化的影响

Effect of sinapine on H_2O_2-induced adipogenic differentiation of bone marrow mesenchymal stem cells

目的:探讨中药有效单体芥子碱对过氧化氢(H_2O_2)诱导骨髓间充质干细胞(bone marrow mesenchymal 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H_2O_2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200μmol/L的H_2O_2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<40μmol/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15μmol/L;油红O染色实验观察到芥子碱组(15μmol/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H_2O_2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 hydrogen 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-quantitative assay,and the optimal drug concentration was selected. Finally,Oil Red O assay was observed 24 h after drug intervention,and the expression of adipogenic differentiation-related proteins,adipocyte protein 2（ aP2）,peroxisome proliferator-activated receptor γ（ PPARγ） and glucose transporter 4（ Glut4）,at mRNA and protein levels in the BMSCs was determined by q PCR and Western blot. RESULTS： Treatment with H2O2 at 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 concentration 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,PPARγ and Glut4 at mRNA 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.