减少氧糖剥夺/复氧后脊髓星形胶质细胞凋亡:抑制高迁移率族蛋白B1/核转录因子κB通路的作用  

Inhibition of high mobility group box 1/nuclear factor-kappa B pathway reduces apoptosis in spinal cord astrocytes after oxygen-glucose deprivation/reoxygenation

作　　者：吕聪 孙麟 冯皓宇 马迅 贺亚军 李季声 LüCong;Sun Lin;Feng Haoyu;Ma Xun;He Yajun;Li Jisheng(Department of Orthopedics,Shanxi Dayi Hospital Affiliated to Shanxi Medical University,Taiyuan 030032,Shanxi Province,China)

机构地区：[1]山西医科大学附属大医院骨科

出　　处：《中国组织工程研究》2019年第33期5353-5359,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金(81870976),项目负责人:孙麟~~

摘　　要：背景:脊髓损伤后,星形胶质细胞核内高迁移率族蛋白B1被释放到细胞外,通过与细胞膜表面受体结合激活核转录因子κB引起脊髓水肿或者炎症等一系列病理反应,但有关高迁移率族蛋白B1/核转录因子κB通路调节氧糖剥夺/复氧损伤后脊髓星形胶质细胞凋亡的研究尚少。目的:分析高迁移率族蛋白B1/核转录因子κB通路对氧糖剥夺/复氧后脊髓星形胶质细胞凋亡的调节作用。方法:体外培养新生一二天SD大鼠(山西医科大学动物中心提供)脊髓星形胶质细胞,建立氧糖剥夺细胞损伤模型,并分别复氧培养6,12,24 h,ELISA法检测培养基内高迁移率族蛋白B1质量浓度,Western blot法检测细胞内高迁移率族蛋B1、Bcl-2、BAX蛋白表达量,MTT法检测细胞存活率,以此筛选最佳复氧时间进行以下实验。取第4代脊髓星形胶质细胞,分4组培养:正常组、氧糖剥夺/复氧组、氧糖剥夺/复氧+丙酮酸乙酯组、氧糖剥夺/复氧+核转录因子κB抑制剂组,复氧培养24h后,ELISA法检测培养基内高迁移率族蛋白B1质量浓度,Westernblot法检测细胞内高迁移率族蛋B1、核转录因子κB、Bcl-2、BAX蛋白表达量,MTT法检测细胞存活率,流式细胞仪检测细胞凋亡。结果与结论:(1)各检测结果显示复氧培养24 h为最佳复氧时间,用于后续实验;(2)与正常组比较,氧糖剥夺6 h/复氧24 h组高迁移率族蛋B1、核转录因子κB蛋白表达及细胞凋亡率升高(P<0.05),细胞存活率、Bcl-2/BAX比值降低(P<0.05);与氧糖剥夺6 h/复氧24 h组比较,氧糖剥夺6 h/复氧24 h+丙酮酸乙酯组、氧糖剥夺6 h/复氧24 h+核转录因子κB抑制剂组细胞存活率、Bcl-2/BAX比值升高(P<0.05),核转录因子κB蛋白表达、细胞凋亡率降低(P<0.05);(3)结果表明,高迁移率族蛋白B1/核转录因子κB通路参与调节氧糖剥夺/复氧后脊髓星形胶质细胞的凋亡。BACKGROUND: Spinal cord injuries triggers the release of high mobility group box 1 (HMGB1) from nerve cells to activate the nuclearfactor-kappa B (NF-κB) by binding to cell membrane surface receptor, thereby inducing a series of pathological reactions, such as spinal cordedema or inflammation. However, little is reported on whether inhibition of HMGB1/NF-κB pathway could attenuate spinal cord astrocytesapoptosis after oxygen-glucose deprivation/reoxygenation (OGD/R).OBJECTIVE: To investigate the effect of HMGB1/NF-κB pathway on spinal cord astrocytes apoptosis after OGD/R.METHODS: Spinal cord astrocytes of newborn Sprague-Dawley rats (provided by the Laboratory Animal Center of Shanxi Medical University,China) were cultured in vitro and an OGD/R model was established. Spinal cord astrocytes were subjected to reoxygenation 6, 12, and 24hours. Release of HMGB1 in the culture medium was detected by ELISA. The expression of HMGB1, Bcl-2 and Bax were detected by westernblot and the cell survival rate was determined by MTT. The optimal reoxygenation time was then selected for the following experiments. Spinalcord astrocytes at passage 4 were divided into normal group, OGD6h/R24h group, OGD6h/R24h+ethyl pyruvate group, OGD6h/R24h+Bay11-7082 group. After 24 hours of reoxygenation, the release of HMGB1 was detected by ELISA, the expression of HMGB1, NF-κB, Bcl-2 andBAX was analyzed by western blot, the survival rate of astrocytes was determined by MTT, and the apoptosis of astrocytes was measured byflow cytometry.RESULTS AND CONCLUSION: (1) The results showed that the best reoxygenation time was 24 hours, which was used for subsequentexperiments. (2) Compared with the normal group, the release and expression of HMGB1 and NF-κB as well as the apoptotic rate ofastrocytes were obviously increased in the OGD6h/R24h group (P < 0.05), while the survival rate of astrocytes and the ratio of Bcl-2/Bax wereobviously declined in the OGD6h/R24h group (P < 0.05). Compared with the OGD6h/R24h group, the astrocyte survival rate and

关 键 词：脊髓损伤 星形胶质细胞 高迁移率族蛋白B1 核转录因子ΚB 细胞凋亡 氧糖剥夺/复氧 Bcl-2 BAX 

分 类 号：R459.9[医药卫生—治疗学] R365[医药卫生—临床医学]