南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (1): 36-44.doi: 10.12122/j.issn.1673-4254.2022.01.04

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棕榈酸通过cGAS-STING-IRF3通路降低心肌细胞的自噬功能

余蕙麟，刘 谦，郭永正，夏 勇，罗素新   

  1. 重庆医科大学附属第一医院心血管内科，重庆 400016
  • 出版日期:2022-01-20 发布日期:2022-03-02

Palmitic acid suppresses autophagy in neonatal rat cardiomyocytes via the cGAS-STING-IRF3 pathway

YU Huilin, LIU Qian, GUO Yongzheng, XIA Yong, LUO Suxin   

  1. Department of Cardiology, First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
  • Online:2022-01-20 Published:2022-03-02

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摘要： 目的 探究棕榈酸（PA）对心肌细胞自噬功能的影响及潜在机制。方法 提取大鼠乳鼠心肌细胞（NRCMs）并体外培养24 h，分别加入 10%BSA 以及不同浓度的 PA（0、0.1、0.3、0.5、0.7 mmol/L）共培养 24 h。Western blotting 检测 NRCMs 中自噬指标（PINK1、Parkin、p62、LC3Ⅱ/LC3Ⅰ）以及cGAS、STING、p-IRF3/IRF3的蛋白表达水平。CCK8法检测细胞活性，筛选0.7 mmol/LPA用于后续实验。进一步将cGAS siRNA转染入NRCMs中以敲降cGAS的表达，将NRCMs细胞分为：空白对照组（Control组，无特殊处理）、阴性对照组（NC组，转染NC序列）、cGAS siRNA组（转染cGAS-siRNA敲降cGAS）、PA组（0.7 mmol/L PA处理24 h）、cGAS siRNA+PA组（转染cGAS-siRNA后予以0.7 mmol/L PA处理24 h）。Western blot检测NRCMs自噬相关蛋白指标表达变化，CCK8法检测细胞活性，免疫荧光检测p62和LC3阳性着色情况。结果 经不同浓度PA作用24 h后，NRCMs中PINK1、Parkin，LC3Ⅱ/LC3Ⅰ和LC3Ⅱ/LC3Ⅰ+Ⅱ蛋白表达量明显降低（P<0.05），p62蛋白表达量明显增高（P<0.05），且心肌细胞活性明显下降（P<0.05）。将cGAS敲降后，可明显逆转PA诱导的NRCMs自噬功能降低，并改善心肌细胞活性（P<0.05）。结论 PA通过介导cGAS-STING-IRF3通路激活，抑制心肌细胞的自噬功能，导致细胞活性降低。

关键词: 棕榈酸；cGAS-STING-IRF3；心肌细胞；自噬

Abstract: Objective To investigate the effect of palmitic acid (PA) on autophagy in neonatal rat cardiomyocytes (NRCMs) and explore the underlying mechanism. Methods NRCMs were isolated and cultured for 24 h before exposure to 10% BSA and 0.1, 0.3, 0.5, or 0.7 mmol/L PA for 24 h. After the treatments, the expressions of Parkin, PINK1, p62, LC3Ⅱ/ LC3Ⅰ, cGAS, STING and p-IRF3/IRF3 were detected using Western blotting and the cell viability was assessed with CCK8 assay, based on which 0.7 mmol/L was selected as the optimal concentration in subsequent experiments. The effects of cGAS knockdown mediated by cGAS siRNA in the presence of PA on autophagy-related proteins in the NRCMs were determined using Western blotting, and the expressions of P62 and LC3 in the treated cells were examined using immunofluorescence assay. Results PA at different concentrations significantly lowered the expressions of Parkin, PINK1, LC3 II/LC3 I and LC3 II/LC3 I+II (P<0.05), increased the expression of p62 (P<0.05), and inhibited the viability of NRCMs (P<0.05). Knockdown of cGAS obviously blocked the autophagy-suppressing effect of PA and improved the viability of NRCMs (P<0.05). Conclusion PA inhibits autophagy by activating the cGAS-STING-IRF3 pathway to reduce the viability of NRCMs.

Key words: palmitic acid; cGAS-STING-IRF3; cardiomyocytes; autophagy