cGAS-STING通路调控NCOA4介导的铁自噬在HT22细胞缺氧复氧损伤中的作用

The role of cGAS STING pathway regulation of NCOA4 mediated iron autophagy in hypoxia reoxygenation injury of HT22 cells

目的探讨环鸟苷酸—腺苷酸合成酶(cGAS)-干扰素基因刺激蛋白(STING)通路调控核受体共激活因子4(NCOA4)介导的铁自噬在小鼠海马神经细胞(HT22)缺氧复氧损伤中的作用。方法2020年9月—2022年12月于武汉大学人民医院中心实验室进行实验。将HT22细胞系随机分为4组,对照组(Ctrl组)、缺氧复氧组(HR组)、缺氧复氧+cGAS抑制剂(RU.521)组(HR+RU组)、缺氧复氧+RU.521+NCOA4过表达组(HR+RU+NCOA4组)。HR组于无糖缺氧条件下培养6 h再复糖复氧培养24 h构建缺氧复氧模型,HR+RU组及HR+RU+NCOA4组提前给予3.0μmol/L的RU.521处理24 h后构建缺氧复氧模型,HR+RU+NCOA4组于缺氧复氧前5 d给予NCOA4慢病毒转染,对照组常规培养。免疫荧光检测各组ROS,ELISA检测CCK8、LDH、SOD、MDA及亚铁离子,Western blot检测cGAS、STING、NCOA4、LC3B和铁蛋白。结果与Ctrl组比较,HR组细胞活力CCK8、SOD降低,LDH、ROS、MDA、cGAS、STING、NCOA4、LC3B、铁蛋白、亚铁离子升高(P<0.05);与HR组比较,HR+RU组细胞活力CCK8、SOD、铁蛋白升高,LDH、ROS、MDA、cGAS、STING、NCOA4、LC3B、亚铁离子降低(P<0.05);与HR+RU组比较,HR+RU+NCOA4组细胞活力CCK8、SOD、铁蛋白降低(P<0.05),LDH、ROS、MDA、NCOA4、LC3B、亚铁离子升高(P<0.05),cGAS、STING差异无统计学意义(P>0.05)。结论缺氧复氧后HT22细胞cGAS-STING通路上调,NCOA4介导的铁自噬过度激活进而加重细胞损伤。

Objective To investigate the role of cGAS interferon gene stimulating protein(STING)pathway regulation of nuclear receptor co activator 4(NCOA4)mediated iron autophagy in hypoxia reoxygenation injury of mouse hippocampal neurons(HT22).Methods From September 2020 to December 2022,experiments were conducted in the Central Laboratory of People's Hospital of Wuhan University.HT22 cell lines were randomly divided into four groups:control group(Ctrl group),hypoxia reoxygenation group(HR group),hypoxia reoxygenation+cGAS inhibitor(RU.521)group(HR+RU group),and hypoxia reoxygenation+RU.521+NCOA4 overexpression group(HR+RU+NCOA4 group).The HR group was cultured for 6 hours under sugar free hypoxia conditions and then re cultured for 24 hours to construct a hypoxia reoxygenation model.The HR+RU group and HR+RU+NCOA4 group were given 3.0 in advanceμAfter 24 hours of treatment with RU.521 at a concentration of mol/L,a hypoxia reoxygenation model was constructed.The HR+RU+NCOA4 group was transfected with NCOA4 lentivirus 5 days before hypoxia reoxygenation,while the control group was cultured routinely.Immunofluorescence was used to detect ROS in each group,ELISA was used to detect CCK8,LDH,SOD,MDA,and ferrous ions,Western blot was used to detect cGAS,STING,NCOA4,LC3B,and ferritin.Results Compared with the Ctrl group,the cell viability CCK8 and SOD in the HR group decreased,while LDH,ROS,MDA,cGAS,STING,NCOA4,LC3B,ferritin,and ferrous ions increased(P<0.05);Compared with the HR group,the HR+RU group showed an increase in cell viability CCK8,SOD,and ferritin,while LDH,ROS,MDA,cGAS,STING,NCOA4,LC3B,and ferrous ions decreased(P<0.05);Compared with the HR+RU group,the cell viability CCK8,SOD,and ferritin in the HR+RU+NCOA4 group decreased(P<0.05),while LDH,ROS,MDA,NCOA4,LC3B,and ferrous ions increased.There was no statistically significant difference in cGAS and STING(P>0.05).Conclusion After hypoxia and reoxygenation,the cGAS STING pathway is upregulated,and NCOA4 mediated excessive activation of iron autophagy exacerbates cell damage.