夏枯草提取物对缺氧/复氧诱导心肌细胞氧化应激损伤保护作用及抗凋亡的机制
| 作者: |
1李锦绣,
1焦宪法,
1王红宇,
1郭宇红,
1赵云峰
1 郑州人民医院重症医学科，郑州 450003 |
| 通讯: |
李锦绣
Email: wwsxu54@163.com |
| DOI: | 10.3978/j.issn.2095-6959.2021.04.001 |
摘要
目的：观察不同浓度的夏枯草提取物(Prunellae Spica extract，PSE)对心肌细胞H9c2缺氧/复氧(hypoxia-reoxygenation，H/R)损伤的保护作用，探讨其可能的作用机制。方法：体外培养心肌细胞H9c2，构建心肌细胞H/R模型。实验分为对照组、H/R组、H/R+PSE 20 μg/mL组、H/R+PSE 40 μg/mL组和H/R+PSE 80 μg/mL组。采用CCK-8法检测H9c2心肌细胞存活率。检测天冬氨酸氨基转移酶(aspartate aminotransferase，AST)、磷酸肌酸激酶(creatine phosphate kinase，CPK)、乳酸脱氢酶(lactate dehydrogenase，LDH)、超氧化物歧化酶(superoxide dismutase，SOD)、丙二醛(malondialdehyde，MDA)、活性氧(reactive oxygen species，ROS)水平的变化。采用流式细胞术检测细胞凋亡情况，蛋白质印迹法检测细胞凋亡相关蛋白和PI3K/AKT信号通路相关蛋白的表达。结果：与对照组相比，H/R组H9c2细胞的存活率显著降低(P<0.05)，凋亡率显著增加，AST、CPK、LDH、MDA和ROS水平显著增加，SOD水平显著降低(均P<0.05)。与H/R组相比，PSE能够显著促进细胞存活，抑制细胞凋亡，降低AST、CPK、LDH、MDA和ROS水平，提高SOD水平(均P<0.05)，并呈浓度依赖性。蛋白质印迹法检测显示：与对照组相比，H/R组H9c2细胞Bax和cleaved-caspase-3蛋白的表达显著增加(均P<0.05)，Bcl-2、p-PI3K和p-AKT蛋白的表达显著降低(均P<0.05)；与H/R组相比，PSE能够显著抑制Bax和cleaved-caspase-3蛋白的表达(均P<0.05)，促进Bcl-2、p-PI3K和p-AKT蛋白的表达(均P<0.05)。结论：PSE对H9c2心肌细胞的H/R损伤具有保护作用，其机制可能与激活PI3K/AKT信号通路有关。
关键词：
夏枯草提取物；H9c2细胞；缺氧/复氧；氧化应激；PI3K/AKT信号通路
Protection of Prunellae Spica extract on oxidative stress injury of cardiomyocytes induced by hypoxia/reoxygenation and its anti-apoptosis effect on cardiomyocytes
CorrespondingAuthor: LI Jinxiu Email: wwsxu54@163.com
DOI: 10.3978/j.issn.2095-6959.2021.04.001
Abstract
Objective: To observe the protective effects of different concentrations of Prunellae Spica extract (PSE) on H9c2 hhypoxia/reoxygenation (H/R) injury, and to explore its underlying mechanism. Methods: Cardiomyocyte H9c2 was cultured in vitro to construct an anoxic/reoxygenation model of cardiomyocytes. The experiment was divided into a control group, an H/R group, an H/R+PSE 20 μg/mL group, an H/R+PSE 40 μg/mL group, and an H/R+PSE 80 μg/mL group. The CCK-8 assay was used to detect the survival rate of cardiomyocyte H9c2. The changes of aspartate aminotransferase (AST), creatine phosphate kinase (CPK), lactate dehydrogenase (LDH), superoxide dismutase (SOD), malondialdehyde (MDA), and reactive oxygen species (ROS) were detected. Flow cytometry was used to detect the apoptosis of cardiomyocytes. The western blot was used to detect the expression of apoptosis-related proteins and PI3K/AKT signaling pathway-related proteins. Results: Compared with the control group, observation in the H/R group include: the survival rate of H9c2 cells was significantly decreased (P<0.05), the apoptosis rate was significantly increased, the levels of AST, CPK, LDH, MDA and ROS were significantly increased and the level of SOD declined significantly (all P<0.05). Compared with the H/R group, PSE could significantly promote cell survival, inhibit cell apoptosis, reduce the levels of AST, CPK, LDH, MDA and ROS, and increase the level of SOD in a concentration-dependent manner (all P<0.05). The Western blotting showed that compared with the control group, the expression of Bax and cleaved-caspase-3 protein in H9c2 cells was significantly increased, and the expressions of Bcl-2, p-PI3K and p-AKT protein was significantly increased in H/R group(all P<0.05); compared with the H/R group, PSE could significantly inhibited the expressions of Bax and cleaved-caspase-3 proteins and promoted the expressions of bcl-2, p-PI3K and p-AKT proteins (all P<0.05). Conclusion: PSE shows protective effects on hypoxia-reoxygenation injury of H9c2 cardiomyocytes, which may be related to the activation of PI3K/AKT signaling pathway.
Keywords：
Prunellae Spica extract; H9c2 cells; hypoxia/reoxygenation; oxidative stress; PI3K/AKT signaling pathway