cGAS-STING通路介导酸及去氧胆酸诱导人正常食管上皮细胞炎症的机制研究
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南京医科大学第一附属医院

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国家自然科学基金项目(面上项目,重点项目,重大项目)


Mechanism of cGAS-STING pathway mediating acid and deoxycholic acid inducing inflammation of human esophageal epithelial cell
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the First Affiliated hospital of Nanjing Medical University

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The National Nature Science Foundation of China(General Program,Key Program,Major Research Plan)

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    摘要:

    【摘要】目的:探讨cGAS-STING通路在酸及去氧胆酸(deoxycholic acid,DCA)诱导人正常食管上皮细胞(HEEC)炎症中的作用及潜在机制。方法:体外培养HEEC,CCK8法检测酸及DCA处理后细胞的存活率;光学显微镜观察药物对细胞形态学的影响;荧光显微镜及流式细胞术检测酸及DCA处理后细胞活性氧(ROS)、线粒体活性氧(mtROS)及线粒体膜电位(MMP)的变化;试剂盒检测ATP含量变化;透射电镜观察线粒体超微结构变化;免疫荧光及蛋白质印迹法检测γH2AX表达水平;免疫荧光检测线粒体及DNA的共定位;qPCR检测线粒体DNA(mtDNA)拷贝数变化;蛋白质印迹法检测cGAS、STING、p-NF-κB p65及NF-κB p65的蛋白表达水平;qPCR检测炎症因子IL-6及IL-1β的mRNA表达水平。结果:酸及DCA处理后细胞存活率呈剂量-时间依赖性抑制;药物处理后细胞内ROS、mtROS产生增多,同时MMP降低,ATP含量减少;与阴性对照组相比,酸及DCA刺激后γH2AX的表达水平升高;mtDNA释放入胞质,mtDNA拷贝数减少;cGAS、STING和p-NF-κB p65蛋白表达升高;炎症因子IL-6及IL-1β水平升高;cGAS抑制剂RU.521处理后抑制cGAS、STING的表达水平及部分抑制p-NF-κB p65的表达水平,炎症因子IL-6及IL-1β水平降低。结论:酸及DCA可诱导HEEC线粒体功能障碍及mtDNA释放,介导HEEC炎症,其机制可能与cGAS-STING通路的激活有关。

    Abstract:

    【Abstract】Objective:This study aims to investigate the function and mechanism of cGAS-STING pathway in acid and deoxycholic acid induced human esophageal epithelial cell inflammation. Methods:HEECs were cultured in vitro. The viability of cells treated with acid and DCA was measured by CCK8 assay. The effects of drugs on cell morphology were observed by optical microscope. Changes of cellular reactive oxygen species, mitochondrial reactive oxygen species,mitochondrial membrane potential were detected by fluorescence microscope and flow cytometry. ATP was detected by the kit. The ultrastructure of mitochondria was observed by transmission electron microscope. The expression of γH2AX was detected by immunofluorescence and Western blotting. The colocalization of mitochondria and DNA was detected by immunofluorescence. The mtDNA copy number was evaluated by qPCR. The expressions of cGAS、STING、p-NF-κB p65 and NF-κB p65 were detected by Western blotting. The mRNA expressions of inflammatory cytokines IL-6 and IL-1β were detected by qPCR. Results: CCK8 assay showed that acid and DCA inhibited the viability of HEECs in a concentration- and time- dependent manner. Intracellular ROS and mtROS were increased after drug treatment, along with reduced MMP and ATP. The expression of γH2AX was elevated. Compared with the negative control group,the expression of γH2AX was increased after acid and DCA stimulation, mtDNA released into the cytoplasm, mtDNA copy number was reduced, the expressions of cGAS, STING and p-NF-κB p65 were increased, and the expressions of inflammatory cytokines IL-6 and IL-1β were elevated. After treatment with cGAS inhibitor RU.521, the expression levels of cGAS and STING were inhibited and the expression of p-NF-κB p65 was partially inhibited, and the levels of inflammatory cytokines IL-6 and IL-1β were decreased. Conclusion: Acid and DCA can induce mitochondrial dysfunction, mtDNA release and mediate the inflammation of HEEC, which may be associated with the activation of cGAS-STING pathway.

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  • 收稿日期:2023-01-19
  • 最后修改日期:2023-04-14
  • 录用日期:2023-07-13
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