白藜芦醇对猪前体脂肪细胞凋亡的作用及机理

Effect and underlying mechanism of resveratol on porcine primary preadipocyte apoptosis

摘要:

旨在研究白藜芦醇对猪前体脂肪细胞凋亡的作用，探讨其分子机制。以50 μmol/L、100 μmol/L、200 μmol/L、400 μmol/L白藜芦醇处理猪前体脂肪细胞，采用Hoechst 33258染色剂染色，光学和荧光显微镜分别观察细胞的形态学变化。semi-qRT-PCR和Western blotting方法检测凋亡相关基因sirt1、caspase-3、bcl-2、bax、p53、NF-κB的mRNA和蛋白的表达变化。结果表明，白藜芦醇处理后，前体脂肪细胞出现明显的细胞凋亡，伴随细胞体积缩小，染色质凝集，核质固缩等特征显现，与对照组相比200 μmol/L处理组细胞的凋亡率显著升高 (P＜0.05)。凋亡相关基因sirt1、caspase-3和bax的mRNA和蛋白表达水平显著上调 (P＜0.05)，而bcl-2、p53、NF-κB等基因的表达水平明显下调 (P＜0.05)。进一步证实白藜芦醇特异性地增加sirt1的表达活性，而sirt1的上调影响caspase-3和bcl-2家族因子的活性，同时参与调控p53和NF-κB的转录表达。因此，推测sirt1调控凋亡相关因子表达是白藜芦醇诱导前体脂肪细胞凋亡的关键原因。

Abstract:

We demonstrated the effect of resveratrol on porcine primary preadipocytes apoptosis, to study the intracellular molecular mechanism. Porcine primary preadipocyte was treated with different concentration of resveratrol (0 μmol/L, 50 μmol/L, 100 μmol/L, 200 μmol/L, 400 μmol/L). We used optical microscope and fluorescence microscope to observe morphological changes during apoptosis after Hoechst 33258 Fluorescent dyes staining; and RT-PCR and Western blotting to measure the expression of apoptosis-associated gene sirt1, caspase-3, bcl-2, bax, p53, NF-κB. Primary preadipocyte apoptosis was apparent, accompanied by reduced cell volume, chromatin condensation, and nuclear shrinkage. Compared to the control and low concentration group, high dose group (200 μmol/L) significantly increased the ratio of primary preadipocyte apoptosis. The expression of sirt1, caspase-3, and bax was up-regulated markedly in response to resveratrol; in contrast, apoptotic inhibitor bcl-2, p53, NF-κB down-regulated. We further proved fact that resveratrol can specifically promote the activity of sirt1; moreover, activated sirt1 modulates the activity of caspase-3 and bcl-2 family, involving in transcriptional regulation of p53 and NF-κB through antagonizing factor-induced acetylation. Taken together, our data established resveratrol as new regulator in porcine primary preadipocyte apoptosis via activating the expression of sirt1, modulating activity of apoptotic-associated factor.