高糖诱导的大鼠原代心肌细胞损伤对程序性坏死的影响及其机制*

doi:10.12047/j.cjap.5710.2019.035

摘要/Abstract

摘要： 目的: 探讨程序性坏死在高糖诱导的大鼠原代心肌细胞损伤中的变化及可能机制。方法: 原代大鼠心肌细胞随机分为4组(n=9):正常对照组(Control,5.5 mmol/L葡萄糖培养心肌细胞48 h)、高糖组(HG,30 mmol/L葡萄糖培养心肌细胞48 h)、HG+Nec-1(30 mmol/L葡萄糖+100 μmol/L程序性坏死关键蛋白RIP1抑制剂Nec-1共同培养心肌细胞48 h)组、高渗组(HPG,5.5 mmol/L葡萄糖+24.5 mmol/L甘露醇共同培养心肌细胞48 h)。MTT法检测各组心肌细胞活力,DHE荧光染色检测细胞氧化应激水平,ELISA法检测心肌细胞TNF-α、IL-6及IL-1β水平,Real-time PCR和Western blot分别检测各组程序性坏死关键蛋白RIP1、RIP3、MLKL mRNA和蛋白水平的表达情况。结果: 与Control组相比,HG组心肌细胞活力明显降低(P＜0.01),氧化应激水平明显增高(P＜0.01),TNF-α、IL-6及IL-1β水平升高明显(P＜0.01),RIP1、RIP3、MLKL mRNA及蛋白水平表达均明显升高(P＜0.05);与HG组相比,HG+Nec-1组心肌细胞活力明显升高(P＜0.01),氧化应激水平明显下降(P＜0.01),TNF-α、IL-6及 IL-1β水平明显降低(P＜0.01), RIP1、RIP3、MLKL mRNA及蛋白水平表达均下降(P＜0.05)。结论: 高糖诱导的原代大鼠心肌细胞损伤可引起程序性坏死的发生;抑制程序性坏死可减轻细胞损伤的机制,可能与抑制氧化应激、减轻炎症反应有关。

关键词: 原代心肌细胞, 大鼠, 高糖, 受体相互作用蛋白激酶1, 受体相互作用蛋白激酶3, 混合激酶结构域样蛋白

Abstract: Objective: To observe whether necroptosis was happened in high glucose (HG) - induced primary cardiomyocytes injury and to investigate the likely mechanism. Methods: The primary cultured cardiomyocytes were divided into 4 groups (n=9): control group (the cardiomyocytes were incubated with 5.5 mmol/L glucose for 48 h), HG group (the cardiomyocytes were incubated with 30 mmol/L glucose for 48 h), HG + necrostatin-1 (Nec-1) group (the cardiomyocytes was co-incubated with necroptosis inhibitor Nec-1 at 100 μmol/L and HG for 48 h) and hypertonic pressure group (HPG, the cardiomyocytes was co-incubated with 5.5 mmol/L glucose and 24.5 mmol/L mannitol for 48 h). Cell viability was measured by MTT method, reactive oxygen species (ROS) generation was measured by DHE staining. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) were tested by ELISA method. The mRNA and protein expressions of necroptosis related genes receptor interacting serine/threonine protein kinase 1 (RIP1), RIP3, mixed lineage kinase domain-like protein (MLKL) were tested by quantitative real-time PCR and Western blot. Results: The results showed HG intervention decreased cardiomyocytes viability, increased ROS generation, up-regulated the levels of TNF-α, IL-6 and IL-1β, increased RIP1, RIP3, MLKL expressions at mRNA and protein levels. Nec-1 treatment attenuated HG-induced increased cardiomyocytes viability, reduced ROS generation, down-regulated the levels of TNF-α, IL-6 and IL-1β, decreased RIP1, RIP3, MLKL expressions at mRNA and protein levels. Conclusion: Necroptosis was happened in high glucose-induced primary cardiomyocytes injury. Inhibition of necroptosis can reduce high glucose-induced cardiomyocytes damage, may be related to inhibition of oxidative stress and depression of inflammative factors releasing.

Key words: primary cardiomyocytes, rat, high glucose, RIP1, RIP3, MLKL

方婷婷, 曹瑞平, 叶红伟, 马善峰, 高琴. 高糖诱导的大鼠原代心肌细胞损伤对程序性坏死的影响及其机制^*[J]. 中国应用生理学杂志, 2019, 35(2): 160-164.

FANG Ting-ting, CAO Rui-ping, YE Hong-wei, MA Shan-feng, GAO Qin. Effects of high glucose induced primary cardiomyocytes injury on necroptosis and the related mechanism[J]. CJAP, 2019, 35(2): 160-164.

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高糖诱导的大鼠原代心肌细胞损伤对程序性坏死的影响及其机制^*

Effects of high glucose induced primary cardiomyocytes injury on necroptosis and the related mechanism

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