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Regulatory Mechanism of Berberine in Inhibiting Apoptosis and Autophagy in Ovarian Granulosa Cells Based on SIRT1/FoxO1 Pathway
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 29, Issue 6, Pages: 79-87(2023)
- 作者机构：
北京中医药大学 生命科学学院，北京 102488
- 作者简介：
- 基金信息：
DOI：10.13422/j.cnki.syfjx.20230104
CLC： R2-0;R22;R285.5;R289;R33Published：20 March 2023，
Published Online：29 November 2022，
Received：29 August 2022，
- 稿件说明：
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刘姣,杨阳,何悦双等.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[J].中国实验方剂学杂志,2023,29(06):79-87.
LIU Jiao,YANG Yang,HE Yueshuang,et al.Regulatory Mechanism of Berberine in Inhibiting Apoptosis and Autophagy in Ovarian Granulosa Cells Based on SIRT1/FoxO1 Pathway[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(06):79-87.
刘姣,杨阳,何悦双等.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[J].中国实验方剂学杂志,2023,29(06):79-87. DOI： 10.13422/j.cnki.syfjx.20230104.
LIU Jiao,YANG Yang,HE Yueshuang,et al.Regulatory Mechanism of Berberine in Inhibiting Apoptosis and Autophagy in Ovarian Granulosa Cells Based on SIRT1/FoxO1 Pathway[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(06):79-87. DOI： 10.13422/j.cnki.syfjx.20230104.
摘要
目的
2
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
2
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（1 μmol·L
-1
）组和BBR低剂量（0.5 μmol·L
-1
）组，模型组与BBR组加入浓度为10 μmol·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
2
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
2
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。
Abstract
Objective
2
To investigate the protective effect and regulatory mechanism of berberine （BBR） against the senescence of ovarian granulosa cells.
Method
2
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 high-dose （1 μmol·L
-1
） and low-dose （0.5 μmol·L
-1
） BBR groups were set up. The cells in the model group and the BBR groups were incubated with 10 μmol·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 by
β
-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
2
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 of
β
-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-3 and 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
2
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.
关键词
小檗碱卵巢颗粒细胞氧化损伤凋亡自噬细胞沉默信息调节因子1（SIRT1）/叉头框转录因子O1（FoxO1）信号通路
Keywords
berberineovarian granulosa cellsoxidative damageapoptosisautophagysilent information regulator1 （SIRT1）/forkhead transcription factor O1 （FoxO1） signaling pathway
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