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Protective mechanisms of hypaconitine and glycyrrhetinic acid compatibility in oxygen and glucose deprivation injury

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Abstract

This study investigated the protective effect of the compatibility of hypaconitine (HA) and glycyrrhetinic acid (GA) on H9c2 cells under oxygen and glucose deprivation (OGD)-induced injury, and the possible mechanisms. We found that HA+GA significantly improved pathology and morphology of the nucleus and ultrastructure of H9c2 cells under OGD as determined by Hoechst 33342 staining and transmission electron microscopy (TEM) tests. It also reduced the releases of lactate dehydrogenase (LDH), creatine kinase-myocardial band isoenzyme (CK-MB), and aspartate transaminase (AST) from the cultured supernatant of H9c2 cells, which were tested by enzyme-linked immune sorbent assay (ELISA) kits. In addition, it lessened the apoptotic rate as determined by a fluorescein isothiocyanate-annexin V/propidium iodide (FITC-AV/PI) double staining assay. It was also found that HA+GA might regulate the protein expression associated with the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Overall, the study demonstrated that HA+GA protected H9c2 cells against OGD-induced injury, and the signaling mechanism might be related to the PI3K/Akt signaling pathway.

中文概要

题目

次乌头碱配伍甘草次酸对缺氧缺糖损伤H9c2 心肌细胞的保护作用及其分子机制

目的

探讨次乌头碱(HA)配伍甘草次酸(GA)对缺 氧缺糖(OGD)损伤H9c2 心肌细胞的保护作用 及其作用机制。

创新点

首次在OGD 模型中证明HA+GA 对H9c2 心肌细 胞有明显的保护作用。此作用与减少细胞坏死和 凋亡有关系,且其作用机制与磷脂酰肌醇-3-激酶/ 蛋白质丝氨酸-苏氨酸激酶(PI3K/Akt)信号通路 有关。

方法

采用H9c2 心肌细胞为研究对象,将其分为七组: 正常组、OGD 模型组、OGD+HA 组、OGD+GA 组、OGD+HA+GA 组、OGD+LY294002 组、 OGD+HA+GA+LY294002 组。采用Hoechst 33342 染色荧光显微镜及透射电镜观察前五组的H9c2 心肌细胞的形态学改变;采用酶联免疫吸附测定 法(ELISA)检测前五组细胞上清液中乳酸脱氢 酶(LDH)、肌酸激酶同工酶(CK-MB)以及天 门冬氨酸氨基转移酶(AST)的释放量的改变; 采用异硫氰酸荧光素-磷脂结合蛋白V/碘化丙啶 (FITC-AV/PI)双染色法检测前五组细胞凋亡率 的情况;采用蛋白质免疫印迹法(Western blot) 检测加入抑制剂LY294002 前后丝苏氨酸蛋白激 酶(Akt)、磷酸化丝苏氨酸蛋白激酶(p-Akt)、 B 细胞淋巴瘤/白血病-2 相关x 蛋白(Bax)、B 细胞淋巴瘤/白血病-2(Bcl-2)及半胱氨酸天冬氨 酸蛋白酶-9(caspase-9)等细胞作用信号通路 PI3K/Akt 相关蛋白的情况。

结论

( 1 ) Hoechst 33342 染色荧光显微镜显示 OGD+HA+GA 组抗凋亡作用最明显;(2)透射 电镜观察OGD+HA+GA 组凋亡现象改善最多; (3)LDH、CK-MB 及AST 的含量变化显示 OGD+HA+GA 组心肌细胞损伤指标降低最多 (P<0.05);(4)Western blot 法检测结果显示 HA+GA可以减少OGD对H9c2心肌细胞的损伤, 其作用机制与PI3K/Akt 信号通路有关。

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  1. The two authors contributed equally to this work

Authors and Affiliations

  1. Cardio-Cerebro Vascular Research Institute, Zhejiang Chinese Medical University, Hangzhou, 310053, China

    Li-qin Wang, Yu He, Hao-fang Wan, Hui-fen Zhou, Jie-hong Yang & Hai-tong Wan

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  1. Li-qin Wang
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  2. Yu He
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  3. Hao-fang Wan
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Correspondence to Jie-hong Yang.

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Project supported by the National Natural Science Foundation of China (Nos. 81473412, 81573868, and 81630105), the Zhejiang Provincial Natural Science Foundation of China (No. LZ17H270001), and the Zhejiang Provincial Program for the Cultivation of High-Level Innovative Health Talents, China

ORCID: Li-qin WANG, http://orcid.org/0000-0003-1181-116X

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Wang, Lq., He, Y., Wan, Hf. et al. Protective mechanisms of hypaconitine and glycyrrhetinic acid compatibility in oxygen and glucose deprivation injury. J. Zhejiang Univ. Sci. B 18, 586–596 (2017). https://doi.org/10.1631/jzus.B1600270

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