银杏内酯B通过PI3K/AKT信号通路抑制MH7A人成纤维样滑膜细胞增殖及其促细胞凋亡

刘璘琛; 徐晓龑; 孙春萌; 俞济荣; 施青; 孙君君; 逄丹丹; 卫斐然; 刘兴

doi:10.11665/j.issn.1000-5048.2024091403

银杏内酯B通过PI3K/AKT信号通路抑制MH7A人成纤维样滑膜细胞增殖及其促细胞凋亡

1.
东南大学附属中大医院风湿科, 南京210009
2.
中国药科大学药学院药剂系, 南京210009
3.
东南大学医学院, 南京210009

基金项目: 国家自然科学基金项目（No. 22278442）

详细信息

通讯作者:
刘兴: Tel：18066068585　E-mail：llc2430782663@163.com

中图分类号: R965
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出版历程
- 收稿日期: 2024-09-13
- 刊出日期: 2025-04-24

Ginkgolide B inhibits cell proliferation and promotes cell apoptosis of MH7A human fibroblast-like synoviocytes through PI3K/AKT pathway

1.
Department of Rheumatology, Southeast University, Zhongda Hospital, Nanjing 210009
2.
Department of Pharmaceutics, School of Pharmacy, China Pharmaceutical University, Nanjing 210009
3.
School of Medicine, Southeast University, Nanjing 210009, China

Funds: This study was supported by the National Natural Science Foundation of China (No. 22278442)

摘要

摘要:
探讨银杏内酯B（ginkgolide B，GB）对MH7A人成纤维样滑膜细胞（fibroblast-like synoviocytes，FLS）的增殖抑制作用及其潜在机制。采用20 μg/L肿瘤坏死因子-α（tumor necrosis factor-a，TNF-α）刺激MH7A构建关节炎细胞模型。经不同浓度GB作用于MH7A细胞后，CCK-8法检测细胞活力；Transwell实验检测细胞侵袭力；流式细胞术检测细胞凋亡率和细胞周期；实时荧光定量PCR（Real-time quantitative PCR，RT-qPCR）和蛋白免疫印迹分别检测基因转录和蛋白表达量。与对照组相比，GB对细胞活力的抑制作用呈现出一定的浓度和时间依赖性；GB显著抑制细胞侵袭力、增加细胞凋亡率和G₀/G₁期比例；GB显著上调细胞Bcl-2相关X蛋白（Bcl-2-associated X protein，Bax）和p21 mRNA和下降Bcl-2、髓系白血病1（myeloid cell leukemia 1，Mcl-1）、蛋白激酶B（protein kinase B，PKB；又称AKT）、磷脂酰肌醇-3激酶（phosphatidylinositol 3-kinase，PI3K）、Cyclin D1和细胞周期调节蛋白激酶4（cyclin-dependent kinase 4，CDK4）mRNA转录水平；同时，GB显著上调Bax、p21和Cleaved-caspase 3蛋白和下调Bcl-2、Mcl-1、p-AKT、p-PI3K、Cyclin D1和CDK4蛋白表达量，且伴有p-PI3K/PI3K、p-AKT/AKT和Bcl-2/Bax比值的降低。综上，GB通过抑制PI3K/AKT信号通路，阻滞MH7A细胞G₁期向S期转化、抑制细胞活力和侵袭力，并诱导MH7A人成纤维样滑膜细胞凋亡。
- 银杏内酯B /
- 磷脂酰肌醇3-激酶/蛋白激酶B /
- 类风湿性关节炎 /
- MH7A细胞 /
- 凋亡
Abstract:
To explore the inhibitory effect of ginkgolide B (GB) on MH7A human fibroblast-like synoviocytes (FLS) and its potential mechanism. Firstly, 20 μg/L tumor necrosis factor-α (TNF-α) was pretreated with MH7A to establish a cell model of arthritis. After incubation of MH7A cells with various concentrations of GB, CCK-8 assay, Transwell assay, and flow cytometry (FCM) were separately used to detect cell viability, cell invasion, and cell apoptosis rate and cell cycle; Real-time quantitative PCR and Western blot assay were performed to detect the apoptosis- and cycle-related gene transcriptions and protein expressions, respectively. The results showed that compared with the control group, GB dose- and time-dependently suppressed cell viability to a greater extent; GB significantly reduced cell invasive ability and increased cell apoptosis rate and proportion of G₀/G₁ phase in MH7A cells, along with increased transcription levels of Bcl-2-associated X protein (Bax) and p21 mRNA and decreased transcription levels of Bcl-2, myeloid cell leukemia 1(Mcl-1), protein kinase B (PKB; AKT), IP3K, Cyclin D1 and cyclin-dependent kinase 4 (CDK4) mRNA; GB remarkably increased expression levels of Bax, p21, and cleaved-Caspase 3 protein and decreased expression levels of Bcl-2, Mcl-1, p-AKT, p-PI3K, Cyclin D1, and CDK4 protein, with decreased ratios of p-PI3K/PI3K, p-AKT/AKT, and Bcl-2/Bax. In conclusion, GB blocks the G₁-to-S cell cycle transition, suppresses cell viability and cell invasion and induces cell apoptosis of MH7A human RA-FLS via suppressing the PI3K/AKT signaling pathway.
- ginkgolide B /
- PI3K/AKT /
- rheumatoid arthritis /
- MH7A cell /
- cell apoptosis

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Figure 1. Effects of ginkgolide B (GB) on MH7A cell viability and cell invasion ($ \bar{x } \pm s $，n=6)

A: Cell viability determined by CCK-8 assay after incubation of MH7A cells with 0 (control group),10,20,40, and 80 μg/mL GB for 24,48 and 72 h; B: Representative image of cell invasion detected by Transwell assay after incubation of MH7A cells with 0 (control group),10,20 and 40 μg/mL GB for 24 h; C: Bar graph of cell invasion ability in MH7A cells ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs control group; ^#P<0.05, ^##P < 0.01 vs 10 μg/mL GB group

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Figure 2. GB on cell apoptosis ($\bar{x } \pm s $, n=3)

A: Representative flow cytometry analysis of cell apoptosis after incubation of MH7A cells with 0 (control group),10,20, and 40 μg/mL GB for 48 h; B: Bar graph of cell apoptosis in MH7A cells ^*P < 0.05,^***P < 0.001 vs control group;^#P < 0.05,^##P < 0.01 vs 10 μg/mL GB group

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Figure 3. Effect of GB on cell cycle ($ \bar{x } \pm s $, n=3)

A: Representative FCM analysis of cell cycle after incubation of MH7A cells with 0 (control group),10,20 and 40 μg/mL GB for 48 h; B: Bar graph of cell cycle distribution in MH7A cells ^*P < 0.05,^**P < 0.01,^***P < 0.001 vs control group;^#P < 0.05,^##P < 0.01 vs 10 μg/mL GB group

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Figure 4. Effect of GB on the transcription levels of mRNA ($ \bar{x } \pm s $, n=3)

qRT-PCR analysis of levels of transcription mRNA after incubation of MH7A cells with 0 (control group),10,20, and 40 μg/mL GB for 48 h ^*P < 0.05, ^**P < 0.01,^*** P < 0.001 vs control group;^# P < 0.05,^##P < 0.01 vs 10 μg/mL GB group;^&P < 0.05 vs 20 μg/mL GB group

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Figure 5. Effect of GB on the expression levels of apoptosis-related protein ($ \bar{x } \pm s $, n=3)

A: Representative image of Western blot assay after incubation of MH7A cells with 0 (control group),10,20, and 40 μg/mL GB for 48 h; B: Relative expression levels of apoptosis-related proteins; C: Ratio of phosphorylated to non-phosphorylated proteins; D: Ratio of Bcl-2 to Bax protein ^*P < 0.05,^**P < 0.01,^*** P < 0.001 vs control group;^#P<0.05,^##P<0.01 vs 10 μg/mL GB group;^&P<0.05 vs 20 μg/mL GB group

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Figure 6. Effect of GB on the expression levels of cell cycle-related protein ($ \bar{x } \pm s $, n=3)

MH7A cells were collected and the expression levels of protein were quantitatively determined by Western blot assay after incubation with 0 (control group),10,20, and 40 μg/mL GB for 48 h ^*P < 0.05,^**P < 0.01,^***P < 0.001 vs control group;^#P < 0.05,^##P < 0.01 vs 10 μg/mL GB group;^&P<0.05 vs 20 μg/mL GB group

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