褪黑素调控自噬对高糖环境下HT22细胞缺氧复氧损伤的影响  

Influence of melatonin on hypoxia reoxygenation injury by regulating autophagy in high-glucose cultured HT22 cells

作　　者：李冰玉 刘恋[1] 侯家保[1] 吴洋[1] 赵博[1] 夏中元[1] LI Bing-yu;LIU Lian;HOU Jia-bao;WU Yang;ZHAO Bo;XIA Zhong-yuan(Department of Anesthesiology,Renmin Hospital of Wuhan University,Wuhan,Hubei 430060,China)

机构地区：[1]武汉大学人民医院麻醉科,湖北武汉430060

出　　处：《中华实用诊断与治疗杂志》2022年第9期884-888,共5页Journal of Chinese Practical Diagnosis and Therapy

基　　金：国家自然科学基金(81970722,81901994,82001119)。

摘　　要：目的 构建高糖环境下缺氧复氧损伤小鼠海马神经元HT22细胞模型,探讨褪黑素(melatonin, Mel)调控自噬对高糖环境下HT22细胞缺氧复氧损伤的影响。方法 小鼠海马神经元HT22细胞分为高糖组、高糖缺氧复氧组、高糖缺氧复氧+Mel组、高糖缺氧复氧+Mel+3-甲基腺嘌呤(3-methyladenine, 3-MA)组。高糖组于高糖培养基、常氧环境中培养,建立高糖模型;高糖缺氧复氧组在建立高糖模型后,进行细胞周期同步化处理,无糖无血清培养基、低氧环境中培养6 h,高糖培养基、常氧环境中培养24 h,建立缺氧复氧模型;高糖缺氧复氧+Mel组和高糖缺氧复氧+Mel+3-MA组在建立高糖模型、细胞周期同步化处理后,分别加入100μmol/L Mel、100μmol/L Mel+5 mmol/L 3-MA,于高糖培养基、常氧环境中预培养4 h,再建立缺氧复氧模型。造模后取4组细胞,采用CCK-8法检测细胞活力,流式细胞术检测细胞凋亡率,DCFH-DA荧光探针检测活性氧水平,TBA法检测丙二醛(malondialdehyde, MDA)水平,WST-1法检测超氧化物歧化酶(superoxide dismutase, SOD)水平,Western blot法检测微管相关蛋白1轻链3(microtubule associated protein 1 light chain 3, LC3)Ⅰ、LC3Ⅱ、Beclin1、自噬相关蛋白14(autophagy related 14, Atg14)蛋白相对表达量并计算LC3Ⅱ/Ⅰ。结果 高糖缺氧复氧组细胞活力(0.37±0.14)、SOD水平[(51.88±11.76)u/mg]、LC3Ⅱ/Ⅰ(0.35±0.16)及Beclin1(0.31±0.15)、Atg14(0.32±0.16)蛋白相对表达量均低于高糖组[1.00±0.09、(151.54±24.25)u/mg、1.00±0.14、1.00±0.13、1.00±0.11](P<0.05),细胞凋亡率[(28.26±5.37)%]、活性氧(5.38±0.85)、MDA [(20.77±3.58)nmol/mg]水平均高于高糖组[(7.83±2.35)%、1.01±0.69、(6.13±1.40)nmol/mg](P<0.05);高糖缺氧复氧+Mel组细胞活力(0.79±0.13)、SOD水平[(112.60±17.69)u/mg]、LC3Ⅱ/Ⅰ(0.69±0.16)及Beclin1(0.75±0.12)、Atg14(0.74±0.14)蛋白相对表达量均高于高糖缺氧复氧组(P<0.05),细胞凋亡率[(15.16±3.23)%]、活性氧(2.Objective To prepare the high-glucose(HG) cultured hippocampal neuron HT22 cells models in hypoxia reoxygenation injury mice, and to investigate the influence of melatonin(Mel) on hypoxia reoxygenation injury by regulating autophagy. Methods The HT22 cells were randomly divided into HG group, HG hypoxic reoxygenation(HHR) group, HHR+Mel group, and HHR+Mel+3-methyladenine(3-MA) group. HG group was cultured in normoxia HG medium to establish a HG model. In HHR group, after establishment of HG model, the cells were synchronized and cultured in serum-free and glucose-free hypoxia medium for 6 h, followed by 24 h culture in normoxia HG medium to establish hypoxic reoxygenation model. In HHR+Mel group and HHR+MEL+3-MA group, after HG modeling and synchronized treatment, the hyperglycemic cells were administered 100 μmol/L Mel and 100 μmol/L Mel+5mmol/L 3-MA,followed by preculture in normoxia HG medium for 4hto prepare hypoxia reoxygenation model.The cell viability was detected by CCK-8,the apoptosis rate was detected by flow cytometry,the reactive oxygen species(ROS)level was monitored by DCFH-DA probe,the malondialdehyde(MDA)content was determined by TBA test,the superoxide dismutase(SOD)activity was determined by WST-1test,the relative expressions of microtubule associated protein 1light chain 3(LC3)Ⅱ/Ⅰ,Beclin1and autophagy related 14(Atg14)were detected by Western blot,and the ratio of LC3Ⅱ/Ⅰ was calculated.Results The cell viability,SOD,LC3Ⅱ/Ⅰ,Beclin1and Atg14were lower in HHR group[0.37±0.14,(51.88±11.76)u/mg,0.35±0.16,0.31±0.15,0.32±0.16]than those in HG group[1.00±0.09,(151.54±24.25)u/mg,1.00±0.14,1.00±0.13,1.00±0.11](P<0.05),and the apoptosis rate,ROS and MDA were higher in HHR group[(28.26±5.37)%,5.38±0.85,(20.77±3.58)nmol/mg]than those in HG group[(7.83±2.35)%,1.01±0.69,(6.13±1.40)nmol/mg](P<0.05).The cell viability,SOD,LC3Ⅱ/Ⅰ,Beclin1and Atg14were higher in HHR+Mel group[0.79±0.13,(112.60±17.69)u/mg,0.69±0.16,0.75±0.12,0.74±0.14]than those in HHR group(P<0.05),and the ap

关 键 词：缺氧复氧损伤 褪黑素 自噬 高糖 HT22细胞 小鼠 

分 类 号：R743.3[医药卫生—神经病学与精神病学]