HMGB1基因沉默对氧糖剥夺/复氧所致星形胶质细胞损伤的保护作用

Protective effect of HMGBl gene silence on astrocyte injury caused by oxygen-glucose deprivation/reoxygenation

目的探讨高迁移率族蛋白1(HMGB1)基因沉默对氧糖剥夺/复氧导致的星形胶质细胞损伤的保护作用。方法将星形胶质细胞分为对照组、模型组、HMGB1 siRNA组、非特异性siRNA组。模型组星形胶质细胞在无糖DMEM培养基、1%O2的培养条件下行2、4、6h的氧糖剥夺后复氧至24h。HMGB1 siRNA组星形胶质细胞在氧糖剥夺/复氧前转染载有HMGB1干扰序列的慢病毒,然后氧糖剥夺6h复氧至24h。采用RT-PCR和Western blotting检测各组星形胶质细胞HMGB1 mRNA及蛋白的表达状况,ELISA法测定星形胶质细胞培养上清液中HMGB1蛋白的浓度变化,并通过乳酸脱氢酶(LDH)漏出率、MTT法检测星形胶质细胞的损伤情况及存活率。结果 RNA干扰可明显抑制星形胶质细胞HMGB1蛋白的表达(P<0.01),RT-PCR及ELISA法检测显示,与对照组相比,模型组星形胶质细胞HMGB1 mRNA表达量和细胞培养上清液中HMGB1的浓度均有所增加(P<0.01),且随着氧糖剥夺时间的延长呈上升趋势(P<0.01)。与对照组相比,模型组星形胶质细胞明显损伤,随着氧糖剥夺时间的延长,星形胶质细胞损伤加重,LDH漏出率逐渐增高(P<0.01),存活率逐渐下降(P<0.01);与模型组相比,HMGB1 siRNA组细胞损伤明显减轻,LDH漏出率水平明显下降(P<0.01),细胞存活率显著上升(P<0.01)。结论通过RNA干扰技术可抑制星形胶质细胞HMGB1的表达。HMGB1过表达可能是氧糖剥夺/复氧后引起细胞损伤的重要因素之一。

Objective To investigate the protective effect of high mobility group box 1 （HMGB1） gene silence on astrocyte injury caused by oxygen-glucose deprivation/reoxygenation （OGD/R） in vitro. Methods Astrocytes were divided into control group, OGD/R group and HMGB1 siRNA group. Astrocytes in OGD/R group were cultured with glucose free DMEM under 1% 02 condition, then they were treated with OGD for 2, 4 and 6h respectively, and then reoxygenated for 24h. In HMGB1 siRNA group, astrocytes that transfected with HMGB1 small interference RNA （siRNA） lentivirus-vector were treated with OGD 6h, and then reoxygenated for 24h. After 24h reoxygenation, the mRNA and protein expression of HMGB 1 in the astrocytes were determined by RT-PCR and Western blotting. HMGB1 protein in culture supernatant of astrocytes was determined by ELISA. The cell injury and survival rate were assessed by LDH activity （LDH%） and MTT assay. Results Compared with the astrocytes without transfection of HMGB 1 siRNA lentivirus-vector, the protein expression of HMGB 1 was suppressed by siRNA. Compared with the control group, with prolongation of the OGD time, the mRNA and protein expression of HMGB 1 increased gradually （P〈0.0S） after OGD/R, and it further increased with elapse of time. OGD resulted in significant injuries with time extention, and the LDH% increased （P〈0.05） with marked lowering of survival rate （P〈0.05）. Compared with the OGD/R group, cell injury in HMGB1 siRNA group alleviated remarkably, and survival rate was elevated si!~nificantly （P〈O.O1）. Conclusion The expression of HMGB1 in astrocvtes can be inhibited by siRNA, and over-expression of HMGB 1 might be an important factor in 0 GD/R-induced cell iniury.