1.西藏民族大学医学院藏药检测技术教育部工程研究中心,陕西 咸阳 712082;2.西藏民族大学医学院西藏自治区高原疾病分子遗传机制与干预研究重点实验室,陕西 咸阳 712082
R-33
LI Hongyan
1. Engineering Research Center of Tibetan Medicine Detection Technology, Ministry of Education, School of Medicine, Xizang Minzu University, Xianyang 712082, China. 2. Key Laboratory for Molecular Genetic Mechanisms and Intervention Research on High Altitude Disease of Tibet Autonomous Region, School of Medicine, Xizang Minzu University, Xianyang 712082SUN Fangyun
1. Engineering Research Center of Tibetan Medicine Detection Technology, Ministry of Education, School of Medicine, Xizang Minzu University, Xianyang 712082, China. 2. Key Laboratory for Molecular Genetic Mechanisms and Intervention Research on High Altitude Disease of Tibet Autonomous Region, School of Medicine, Xizang Minzu University, Xianyang 7120821. Engineering Research Center of Tibetan Medicine Detection Technology, Ministry of Education, School of Medicine, Xizang Minzu University, Xianyang 712082, China. 2. Key Laboratory for Molecular Genetic Mechanisms and Intervention Research on High Altitude Disease of Tibet Autonomous Region, School of Medicine, Xizang Minzu University, Xianyang 712082
目的 探讨甘松新酮(nardosinone, Nar)通过调控PI3K/ Akt/ mTOR 通路对低氧诱导H9c2 心肌细胞凋亡的影响及机制。 方法 采用氯化钴(CoCl2 ) 制备心肌细胞低氧损伤模型,CCK-8 试剂盒检测细胞活性;Hoechst 33342 进行细胞染色,激光共聚焦显微镜下观察细胞凋亡现象;网络药理学预测Nar 治疗IHD 相关信号通路;Western blot 检测凋亡、自噬和PI3K/ Akt/ mTOR 通路相关蛋白表达。 结果 PI3K/ Akt/ mTOR 通路是Nar 治疗IHD 关键信号通路之一;与对照组比,CoCl2(400 μmol/ L)组细胞活性降低,细胞核碎片增多,凋亡蛋白Cleaved-Caspase-3 表达升高、Bcl-2/ Bax 比降低,PI3K、Akt 和mTOR 磷酸化水平降低;P62 表达降低,LC3II/ LC3I 比和Beclin-1 表达升高(P<0. 05)。与CoCl2 组比,Nar(50 μmol/ L)预处理显著增加了细胞活性、减少细胞核凋亡碎片,降低Cleaved-Caspase-3 表达,升高Bcl-2/ Bax 比值,增加了PI3K、Akt 和mTOR 磷酸化水平及P62 表达,降低LC3II/ LC3I比和Beclin-1 表达(P<0. 05)。PI3K 特异性抑制剂LY294002 预作用后抵消了Nar 对CoCl2 引起的以上凋亡、PI3K/Akt/ mTOR 通路相关蛋白和P62 蛋白的影响,但没有抵消对LC3II/ LC3I 比和Beclin-1 表达的影响。 结论 Nar 激活PI3K/ Akt/ mTOR 通路抑制了CoCl2 诱导的H9c2 心肌细胞低氧凋亡,但可能通过其他途径缓解了过度自噬对细胞造成的损伤。
Objective To investigate the effect and mechanism of Nardosinone (Nar) in hypoxia-induced apoptosis of H9c2 cardiomyocytes meditated through the PI3K/ Akt/ mTOR pathway. Methods Cobalt chloride (CoCl2 ) was used to establish a hypoxic injury model in H9c2 cardiomyocytes. Cell proliferation was assessed by CCK-8 assays. Hoechst 33342 was applied to counterstain cells, and laser scanning confocal microscopy was used to observe apoptosis. The potential signaling pathway of Nar in treating IHD was predicted by network pharmacology. Western blot was used to detect expression of apoptosis-, autophagy-, and PI3K/ Akt/ mTOR pathway-related proteins. Results The PI3K/ Akt/mTOR pathway is major signaling pathway in Nar treatment of IHD. Compared with the control group, CoCl2 (400 μmol/ L)decreased cell proliferation, the Bcl-2/ Bax ratio, PI3K, Akt and mTOR protein phosphorylation levels, and P62 protein expression, while increasing nuclear apoptotic fragmentation, the LC3II/ LC3I ratio, and Cleaved-Caspase-3 and Beclin-1 protein expression (P<0. 05). Compared with the CoCl2 group, Nar (50 μmol/ L) pretreatment significantly increased cell proliferation, alleviated nuclear apoptotic fragmentation, decreased Cleaved-Caspase-3 protein expression, increased the Bcl-2/ Bax ratio, and increased PI3K, Akt and mTOR phosphorylation levels and P62 protein expression. The LC3II/ LC3I ratio and the Beclin-1 protein expression were also decreased (P< 0. 05). Pretreatment with PI3K specific inhibitor LY294002 offset the effects of Nar on CoCl2-induced apoptosis, PI3K/ Akt/ mTOR pathway-related proteins, and P62 protein expression, but had no effect on the LC3II/ LC3I ratio or Beclin-1 expression. Conclusions Nar inhibited CoCl2-induced H9c2 cardiomyocyte apoptosis by activating the PI3K/ Akt/ mTOR pathway, but probably alleviated the damage caused by excessive autophagy in cells through other pathways.
李红艳,梅显运,杨慧雅,李艺康,冯 佩,孙芳云.基于PI3K/ Akt/ mTOR 通路探讨甘松新酮对H9c2 心肌细胞低氧损伤的保护作用[J].中国比较医学杂志,2023,33(7):41~47.
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