基于SIRT1/FoxO1通路探究小檗碱抑制卵巢颗粒细胞凋亡与自噬的调节机制

Regulatory Mechanism of Berberine in Inhibiting Apoptosis and Autophagy in Ovarian Granulosa Cells Based on SIRT1/FoxO1 Pathway

目的:通过观察小檗碱(BBR)对卵巢颗粒细胞衰老的影响,探究其保护作用及调节机制。方法:应用H_(2)O_(2)诱导建立人卵巢颗粒样肿瘤(KGN)细胞衰老模型;设置空白组、模型组、BBR高剂量(1μmol·L^(-1))组和BBR低剂量(0.5μmol·L^(-1))组,模型组与BBR组加入浓度为10μmol·L^(-1)H_(2)O_(2),孵育40 min。通过细胞增殖与活性检测(CCK-8)分析检测BBR对KGN细胞增殖的影响;通过β-半乳糖苷酶染色检测BBR对KGN细胞衰老状态的影响;应用流式细胞术检测BBR对KGN细胞凋亡和ROS含量的影响;实时荧光定量聚合酶链式反应(Real-time PCR)检测BBR对KGN细胞抗凋亡蛋白B细胞淋巴瘤-2(Bcl-2)/促凋亡蛋白Bcl-2相关X蛋白(Bax)比值、胱天蛋白酶-3(Caspase-3)、叉头框转录因子O1(FoxO1)及过氧化氢酶(CAT)mRNA表达的影响;蛋白免疫印迹法(Western blot)检测BBR对KGN细胞沉默信息调节因子1(SIRT1)、超氧化物歧化酶2(SOD2)、c-Jun氨基末端激酶(JNK)、FoxO1、自噬相关蛋白微管相关蛋白轻链3Ⅱ(LC3BⅡ)、自噬关键分子酵母Atg6(Beclin-1)及泛素结合蛋白p62蛋白表达的影响。结果:H_(2)O_(2)诱导40 min后,与空白组比较,模型组细胞增殖率显著下降(P<0.01);与模型组比较,BBR干预组细胞增殖率明显上升(P<0.05);β-半乳糖苷酶染色结果显示,与空白组比较,模型组细胞呈现明显的衰老状态(P<0.01),BBR干预组细胞衰老情况较模型组显著降低(P<0.01);流式细胞术检测显示,与空白组比较,模型组细胞凋亡率显著上升(P<0.01),BBR干预组细胞凋亡率较模型组明显降低(P<0.05);同时,与空白组比较,模型组ROS含量显著增加(P<0.01);与模型组比较,BBR干预组细胞ROS含量显著降低(P<0.01);Real-time PCR结果显示,与空白组比较,模型组KGN细胞CAT、Bcl-2/Bax mRNA表达明显降低,Caspase-3与FoxO1 mRNA表达明显增加(P<0.05);与模型组比较,BBR干预后KGN细胞CAT与Bcl-2/Bax mRNA表达明显增加(P<0.05),Caspase-3与FoxO1 mRNA表达较模型组明显降低(P<0.05)。Western blot结果显示,与空白组比较,模型组SIRT1、SOD2及p62蛋白水平显著降低(P<0.01),JNK、FoxO1、LC3BⅡ与Beclin-1蛋白水平明显升高(P<0.05);BBR干预后,SIRT1、SOD2及p62蛋白水平较模型组显著增加(P<0.01),JNK、FoxO1、LC3BⅡ与Beclin-1蛋白水平较模型组明显降低(P<0.05)。结论:BBR具有抑制卵巢颗粒细胞衰老效应,其机制与通过SIRT1/FoxO1通路介导抑制细胞凋亡与自噬有关。

Objective:To investigate the protective effect and regulatory mechanism of berberine(BBR)against the senescence of ovarian granulosa cells.Method:A cell senescence model in the human ovarian granulosa-like tumor(KGN)cell line was induced by H_(2)O_(2).A control group,a model group,and highdose(1μmol·L^(-1))and low-dose(0.5μmol·L^(-1))BBR groups were set up.The cells in the model group and the BBR groups were incubated with 10μmol·L^(-1)H_(2)O_(2)for 40 min.The effect of BBR on KGN cell proliferation was detected by cell counting kit-8(CCK-8)assay.The effect of BBR on the senescence of KGN cells was detected byβ-galactosidase staining.The effects of BBR on the apoptosis and ROS content of KGN cells were detected by flow cytometry.The effects of BBR on the mRNA expression of B-cell lymphoma-2(Bcl-2)/Bcl-2-associated X protein(Bax),cysteinyl aspartate-specific protease-3(Caspase-3),forkhead transcription factor O1(FoxO1),and catalase(CAT)was detected by Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR).Western blot was used to detect the effects of BBR on protein expression of silent information regulator1(SIRT1),superoxide dismutase 2(SOD2),c-Jun N-terminal kinase(JNK),FoxO1,autophagy-associated protein microtubule-associated protein light chain 3Ⅱ(LC3BⅡ),mammalian ortholog of yeast Atg6(Beclin-1),and ubiquitin-binding protein p62.Result:After H_(2)O_(2)induction for 40 min,the cell proliferation rate of the model group decreased compared with that of the control group(P<0.01),and the cell proliferation rates of the BBR groups increased compared with that of the model group(P<0.05).The results ofβ-galactosidase staining showed that the cells of the model group showed significant senescence compared with those of the control group(P<0.01),and the cellular senescence in the BBR groups was reduced compared with that of the model group(P<0.01).As revealed by flow cytometry,compared with the control group,the model group showed increased apoptosis rate(P<0.01),and compared with the model group,BBR groups showed decreased apoptosis rates(P<0.05).Meanwhile,the ROS content in the model group increased compared with that in the control group(P<0.01),and compared with the model group,the BBR groups showed reduced cellular ROS content(P<0.01).The Real-time PCR results showed that compared with the control group,the model group showed decreased mRNA expression of CAT and Bcl-2/Bax in KGN cells and increased mRNA expression of Caspase-3and FoxO1(P<0.05),and compared with the model group,the BBR groups showed increased mRNA expression of CAT and Bcl-2/Bax(P<0.05)and reduced mRNA expression of Caspase-3 and FoxO1 in KGN cells(P<0.05).As revealed by Western blot results,SIRT1,SOD2,and p62 protein levels decreased in the model group compared with those in the control group(P<0.01),and JNK FoxO1,LC3BⅡ,and Beclin-1 protein levels increased(P<0.05).After BBR intervention,SIRT1,SOD2,and p62 protein levels increased(P<0.01),and JNK,FoxO1,LC3BⅡ,and Beclin-1 protein levels decreased compared with those in the model group(P<0.05).Conclusion:BBR has an inhibitory effect on ovarian granulosa cell senescence,and the mechanism is related to the inhibition of apoptosis and autophagy mediated by the SIRT1/FoxO1 pathway.