天麻素通过蛋白激酶B/p38丝裂原活化蛋白激酶信号通路抑制H9c2心肌细胞凋亡

doi:10.16098/j.issn.0529-1356.2019.01.008

解剖学报 ›› 2019, Vol. 50 ›› Issue (1): 40-48.doi: 10.16098/j.issn.0529-1356.2019.01.008

天麻素通过蛋白激酶B/p38丝裂原活化蛋白激酶信号通路抑制H9c2心肌细胞凋亡

张玲¹杨萍² 姜永良³普俞维³谢立秋³孙林^3* 陆地^1*

1. 昆明医科大学生物医学工程研究中心，昆明 650500; 2. 昆明医科大学基础医学院人体解剖学与组织学胚胎学系，昆明 650500; 3.昆明医科大学第二附属医院心血管内科，昆明 650101

收稿日期:2018-03-14 修回日期:2018-04-23 出版日期:2019-02-06 发布日期:2019-04-18
通讯作者: 孙林;陆地 E-mail:ludi20040609@126.com
基金资助:
天麻素在心肌缺血再灌注损伤治疗中的作用及其信号机制研究;天麻素在动脉粥样硬化治疗中的信号调控机制;mTORC1 信号在天麻素治疗心肌缺血再灌注损伤中的作用及其调节机制;mTORC1 在天麻素抗心肌缺血再灌注损伤中的作用及其信号机制;人参皂苷 Rb1 在心肌缺血再灌注损伤治疗中的信号调控机制;mTOR信号通路在天麻素抗心肌缺血再灌注损伤中的调控机制

Gastrodin inhibits apoptosis of H9c2 cardiomyocytes through protein kinase B/p38mitogen actived protein kinase signaling pathway

ZHANG Ling¹YANG Ping² JIANG Yong-liang³PU Yu-wei³ XIE Li-qiu³SUN Lin ^3* LU Di ^1*

1.Biomedical Engineering Research Center, Kunming Medical University, Kunming 650500, China; 2.Department of Human Anatomy and Histology，Kunming Medical University College of Basic Medicine, Kunming 650500, China; 3.Department of Cardiology，the Second Affiliated Hospital of Kunming Medical University，Kunming 650101，China

Received:2018-03-14 Revised:2018-04-23 Online:2019-02-06 Published:2019-04-18
Contact: SUN Lin;LU Di E-mail:ludi20040609@126.com
Supported by:
The role of gastrodine in the treatment of myocardial ischemia reperfusion injury and its signal mechanism.

摘要/Abstract

摘要：

目的采用血清剥夺/复灌的大鼠H9c2心肌细胞模拟心肌缺血/再灌注损伤（I/R)，从细胞层面探讨天麻素(gastrodin)抗H9c2心肌细胞凋亡的作用及其机制。方法体外培养H9c2心肌细胞随机分为对照组、血清剥夺（0.5~6 h）处理组、血清剥夺/复灌(2/4 h)处理组、天麻素(10、20和40 μmol/L)处理组、天麻素(20 μmol/L) +渥曼青霉素(wortmannin) (1 μmol/L)处理组、wortmannin (1 μmol/L)处理组。采用免疫印迹法检测p38丝裂原活化蛋白激酶（p38MAPK）、磷酸化p38MAPK（p-p38MAPK）、蛋白激酶B（Akt）、p-Akt以及凋亡相关蛋白cleaved Caspase-3、Bcl-2 和Bax的表达变化；采用流式细胞术检测细胞凋亡；通过实时定量聚合酶链反应（Real-time PCR）检测Caspase-3、Bcl-2和Bax的mRNA表达变化；采用免疫荧光双标记法检测细胞内p-Akt蛋白水平表达的变化。结果血清剥夺(0.5~6 h)和血清剥夺/复灌(2/4 h)处理诱导细胞凋亡水平增加；加入不同浓度天麻素处理，与血清剥夺(0.5~6 h)和血清剥夺/复灌(2/4 h)处理组比较，凋亡相关蛋白cleaved Caspase-3表达降低，Bcl-2/Bax蛋白表达的比值上调(P<0.05)，基因转录水平检测的Caspase-3和Bax 表达量降低，Bcl-2表达量上调（P<0.05)，流式细胞术凋亡检测结果也同样显示，天麻素能抑制血清剥夺/复灌(2/4 h)诱导的细胞凋亡(P<0.05)；同时，血清剥夺(0.5~6 h)和血清剥夺/复灌(2/4 h)处理能抑制Akt/p38MAPK信号通路；加入不同浓度的天麻素(10、20和40 μmol/L)处理后，p-Akt的表达水平增多，p-p38MAPK蛋白表达减少(P<0.05)。加入Akt的特异性抑制剂wortmannin(1μmol/L)处理后，天麻素对上述因子的调控作用被反转(P<0.05)。结论在H9c2心肌细胞中，天麻素通过激活Akt/p38MAPK信号通路，抑制血清剥夺/复灌诱导的细胞凋亡，从而发挥抗心肌I/R损伤的保护作用。

关键词: 天麻素, 心肌缺血/再灌注损伤, p38丝裂原活化蛋白激酶, 免疫印迹法, 免疫荧光双标记法, 流式细胞术, 大鼠

Abstract:

Objective To investigate the effect and precise molecular mechanisms for explaining how gastrodin suppresses the apoptosis in the serum deprivation/reperfusion-induced H9c2 cardiomyocytes. Methods Rat H9c2 cardiomyocytes were cultured and treated differently in vitroand randomly divided into following groups, including the control group, the serum deprivation-stimulated (0.5-6 hours) groups, the serum deprivation/reperfusion(2/4 hours) group, gastrodin (10, 20 and 40 μmol/L) treatment groups, wortmannin (1 μmol/L) and gastrodin(20 μmol/L) co-treatment group, wortmannin (1 μmol/L) treatment group. The variation of protein expression levels of p-p38 mitogen actived protein kinase(MAPK), p-Akt and apoptosis related cleaved caspase-3, Bcl-2 and Bax were analyzed by Western blotting.H9c2 cardiomyocytes were analyzed by flow cytometry.The level of RNA expression levels of apoptosis related Caspase-3,Bcl-2 and Bax were analyzed by Real-time PCR assay. The expression level of p-Akt was assayed by double immunofluorescence labeling. Results The results showed that serum deprivation (0.5-6 hours) and serum deprivation/reperfusion (2/4 hours) induced apoptosis increasing; Using Western blotting and Real-time PCR assay, we found that treatment with gastrodin significantly alleviated cleaved Caspase-3 activation and increased the ratio of Bcl-2/Bax in a concentration-dependent manner after serum deprivation and serum deprivation/reperfusion exposure. The same result has been showed by flow cytometer. Serum deprivation and serum deprivation/reperfusion inhibited the Akt/p38MAPK signaling pathway; After treatment with gastrodin in a concentration-dependent manner induced the expression of p-Akt and decreased the expression of p-p38MAPK. The regulation effects of gastrodin were reversed by the Akt specific inhibitor wortmannin. Conclusion In conclusion , the present study demonstrated that gastrodin exerted protective effects against serum deprivation/reperfusion-induced apoptosis in H9c2 cardiomyocytes, at least partly via the Akt/p38MAPK pathway.

Key words: Gastrodin, Myocardial ischemia/reperfusion injury, p38 mitogen actived protein kinase, Western blotting, Double immunofluorescence labeling, Flow cytometry, Rat

张玲杨萍姜永良普俞维谢立秋孙林陆地. 天麻素通过蛋白激酶B/p38丝裂原活化蛋白激酶信号通路抑制H9c2心肌细胞凋亡[J]. 解剖学报, 2019, 50(1): 40-48.

ZHANG Ling YANG Ping JIANG Yong-liang PU Yu-wei XIE Li-qiu SUN Lin LU Di. Gastrodin inhibits apoptosis of H9c2 cardiomyocytes through protein kinase B/p38mitogen actived protein kinase signaling pathway[J]. Acta Anatomica Sinica, 2019, 50(1): 40-48.

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天麻素通过蛋白激酶B/p38丝裂原活化蛋白激酶信号通路抑制H9c2心肌细胞凋亡

Gastrodin inhibits apoptosis of H9c2 cardiomyocytes through protein kinase B/p38mitogen actived protein kinase signaling pathway

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