豆茶决明冲剂含药血清对3T3-L1前脂肪细胞分化的抑制作用

Inhibitory effect of serums containing Cassia nomame granule on the differentiation of 3T3-L1 preadipocytes

目的:探讨豆茶决明冲剂含药血清对前脂肪细胞增殖和分化的影响及其机制。方法:制备豆茶决明冲剂含药血清,培养3T3-L1前脂肪细胞,采用噻唑蓝(MTT)比色法检测豆茶决明冲剂含药血清(5%、10%、20%)对3T3-L1前脂肪细胞增殖过程的影响;采用油红O脂肪染色观察及评价细胞分化情况;异丙醇萃取法、甘油三酯(TG)试剂盒检测分析不同浓度豆茶决明冲剂含药血清对3T3-L1前脂肪细胞分化过程中脂质合成的影响;采用逆转录聚合酶链式反应(RT-PCR)法检测关键转录因子CCAAT增强子结合蛋白(C/EBPβ、C/EBPα)、过氧化物酶体增殖物活化受体γ(PPARγ)mRNA表达水平;采用Western blot法检测关键转录因子C/EBPβ、C/EBPα、PPARγ蛋白表达水平。结果:与对照组(Control)相比,0.9g/kg豆茶决明冲剂含药血清(5%、10%、20%)对3T3-L1前脂肪细胞增殖无显著性影响;采用"鸡尾酒法"成功诱导前脂肪细胞成脂分化为成熟脂肪细胞,豆茶决明冲剂含药血清(5%、10%、20%)干预前脂肪细胞,分化后胞浆中脂滴数量显著性降低。脂滴中TG含量显著性下降,C/EBPβ、C/EBPα、PPARγmRNA和蛋白表达显著性降低。结论:豆茶决明冲剂含药血清对3T3-L1前脂肪细胞分化有抑制作用,其分子机制可能与下调PPARγ、C/EBPα和C/EBPβmRNA和蛋白表达相关。

Objective： To study effects and mechanisms of serum containing cassia nomame granule on the proliferation and differentiation of preadipocytes. Methods： Rat serums containing drug were prepared for cell culture. 3T3-L1 preadipocytes were cultured in the presence of serum containing cassia nomame granule（5%,10%,20%）. MTT assay was applied to evaluate the cell proliferation. The accumulation of lipid synthesis in the cytoplasm of differentiated preadipocytes was observed by oil red O staining and the content of triglyceride（TG） were measured,mRNA and protein expressions of CCAAT enhancer binding protein（C/EBPα and C/EBPβ） and Peroxisome proliferator-activated receptor γ（PPARγ） were detected by reverse transcription polymerase chain reaction（RT-PCR） and western blotting. Results： Compared with the control group,there was no significant effect of serum containing cassia nomame granule on the proliferation of 3T3-L1 preadipocytes（P〈0. 05）. 3T3-L1 preadipocytes were successfully induced into mature adipicytes by hormonal cocktail. It showed that the lipid droplets in the cytoplasm and TG content in the preadipocytes treated with serum containing cassia nomame granule（5%,10%,20%） were significantly decreased compared with the control group（P〈0. 05）. mRNA and protein expressions of C/EBPβ、C/EBPα and PPARγ significantly decreased（P〈0. 05）. Conclusions： Inhibitory effect of serum containing cassia nomame granule on the differentiation of 3T3-L1 preadipocytes is found,which may be related to the decrease of mRNA and protein expressions of C/EBPβ、C/EBPα and PPARγ.