白藜芦醇介导SIRT1干预地塞米松诱导成骨细胞线粒体自噬的研究

Resveratrol inhibits dexamethasone-induced mitophagy in osteoblasts via SIRT1

目的研究白藜芦醇(RES)参与糖皮质激素诱导的成骨细胞线粒体自噬的作用机制。方法体外培养人成骨细胞hFob1.19,地塞米松(DEX)诱导细胞线粒体自噬,使用不同浓度的RES(10^(-8)~10^(-6)mol/L)处理成骨细胞5~120 min,CCK-8法检测成骨细胞的增殖,透射电镜检测成骨细胞内的自噬小体,酶联免疫吸附实验、实时荧光定量PCR和Western blotting分别检测成骨细胞内碱性磷酸酶(ALP)、caspase 3、转录沉默信息调节因子1(SIRT1)、细胞自噬标志蛋白(LC3和Beclin1)和细胞线粒体自噬标志蛋白(TOM20和Hsp60)的活性表达。结果DEX和RES作用成骨细胞2 h后,RES可明显减弱DEX对细胞增殖的抑制作用(P<0.05);透射电镜显示,DEX+RES组自噬小体明显少于DEX组(P<0.05);DEX可明显抑制成骨细胞内ALP活性,而RES明显改善了DEX对ALP蛋白活性的抑制作用(P<0.05);RES可导致成骨细胞内caspase 3的蛋白活性明显降低(P<0.05);RES以剂量依赖性和时间依赖性的方式增强成骨细胞中SIRT1的活性表达;RES单独作用成骨细胞,可显著抑制LC3和Beclin1 mRNA表达(P<0.05),并降低细胞内TOM20(线粒体自噬标记蛋白)和Hsp60(线粒体基质自噬标记蛋白)的蛋白活性;RES可部分抑制DEX对成骨细胞线粒体自噬的激活效应。结论RES通过上调SIRT1而增强成骨细胞的增殖活性,并抑制DEX诱导的成骨细胞线粒体自噬的发生。

Objective To study the mechanism of action of resveratrol(RES)in glucocorticoid-induced mitophagy in osteoblasts.Methods Human osteoblast cells(hFob1.19)were cultured in vitro and treated with dexamethasone(DEX)to induce mitophagy.The cells were then treated with different concentrations of RES(10^(-8)to 10^(-6)mol/L)for 5 to 120 min.The proliferation of osteoblasts was determined using the CCK-8 assay.Autophagosomes within the osteoblasts were detected by transmission electron microscopy.The expression of alkaline phosphatase(ALP),caspase 3,sirtuin 1(SIRT1),cellular autophagy marker 1(LC3 and Beclin1),and cellular mitophagy marker 11(TOM20 and Hsp60)in osteoblasts was detected by enzyme-linked immunosorbent assay,real-time PCR,and Western blotting,respectively.Results Following treatment of osteoblasts with DEX and RES for 2 h,RES significantly attenuated the inhibitory effect of DEX on cell proliferation(P<0.05).Transmission electron microscopy revealed significantly fewer autophagosomes in the DEX+RES group compared to those in the DEX group.DEX significantly inhibited ALP activity,whereas RES clearly ameliorated the inhibitory effect of DEX on ALP protein activity in the osteoblasts(P<0.05).RES significantly reduced caspase 3 activity,whereas it enhanced the expression of SIRT1 in a dose-and time-dependent manner in osteoblasts.RES alone significantly inhibited the mRNA expression of LC3 and Beclin1 and reduced the protein activity of TOM20(mitophagy marker)and Hsp60(mitochondrial matrix autophagy marker)in the cells.Furthermore,RES partially inhibited DEX-mediated activation of mitophagy in osteoblasts.Conclusion Our results suggest that RES enhances the proliferative activity of osteoblasts by upregulating SIRT1 and suppresses DEX-induced mitophagy in osteoblasts.