杨梅素对人卵巢癌SKOV3细胞自噬的诱导作用及线粒体分裂的促进作用

Induction effect of myricetin on autophagy in SKOV3 cells and promoting effect on mitochondrial fission

目的:观察杨梅素作用于人卵巢癌SKOV3细胞后细胞自噬的发生和线粒体分裂程度,探讨杨梅素对其线粒体分裂的影响及自噬的诱导作用。方法:体外培养SKOV3细胞,杨梅素给药剂量按0、20、40和60g·L^(-1)分为对照组和低、中、高剂量杨梅素组,均作用12h。流式细胞术检测各组细胞线粒体膜电位变化,MitoTracker Red荧光探针特异性标记线粒体观察其形态,Western blotting法检测动力相关蛋白1(Drp1)、线粒体分裂蛋白1(Fis1)和自噬相关蛋白LC3-Ⅰ及LC3-Ⅱ蛋白表达水平,采用间接免疫荧光法检测自噬相关蛋白LC3表达强度。结果:与对照组比较,各剂量杨梅素组细胞线粒体膜电位下降比例明显升高(t=3.27,t=6.85,t=5.49,P<0.05)。与对照组比较,各剂量杨梅素组细胞中线粒体数目增加,且线粒体砂砾感增强。与对照组比较,各剂量杨梅素组细胞中Drp1(t=4.35,t=3.28,t=6.17,P<0.05)和Fis1(t=8.32,t=6.74,t=9.27,P<0.05)蛋白表达水平明显升高,中和高剂量杨梅素组细胞中LC3-Ⅱ/LC3-Ⅰ比值明显升高(t=3.28,t=4.21,P<0.05)。间接免疫荧光检测,与对照组比较,随着杨梅素给药剂量增加,LC3表达强度逐渐增强。结论:杨梅素能够诱导人卵巢癌SKOV3细胞发生自噬,并对线粒体动分裂有明显促进作用。

Objective.- To observe the effects of myricetin on autophagy and mitochondrial fission in the human ovarian cancer SKOV3 cells, and to explore its induction effect on autophagy and promoting effect on mitochondrial fission. Methods： The SKOV3 cells were cultured in vitro. 0, 20, 40, and 60 g·L^-1 myricetin were used in control group and low, middle, high doses of myricetin groups for 12 h. The changes of mitochondrial membrane potential were detected by flow cytometry; the morphology of mitochondria was observed by MitoTracker Red; the expression levels of dynamin related protein1 （Drpl） and fission 1 （Fisl） in various groups were detected by Western blotting method, and the expression levels of autophagy related protein LC3 were detected by both Western blotting method and immunofluorescence method. Results.. Compared with control group, the ratios of decreased mitochondria in different doses of myricetin groups were significantly increased （t= 3.27, t=6.85, t= 5.49, P〈 0.05）. Compared with control group, the numbers of mitochondria in different doses of myricetin groups were increased, and the mitochondria looked more like gravel. Compared with control group, the expression levels of Drpl in different doses of myricetin groups were significantly increased （t= 4.35, t= 3.28, t = 6.17, P%0. 05）, and the expression levels of Fisl were increased also （t = 8.32, t = 6.74, t = 9.27） . The immunofluorescence results showed that the expression levels of LC3 in different doses of myricetin groups were significantly increased with the increase of myricetin dose compared with control group. The Western blotting results showed that the ratios of LC3- Ⅱ/LC3-Ⅰ in middle and high doses of myricetin groups were significantly increased compared with control group （t = 3.28, t = 4.21, P〈0.05）. Conclusion： Myricetin can induce the autophagy of SKOV3 cells, and it can also promote the mitochondrial fission.