抑制高迁移率族蛋白1减轻大鼠脊髓星形胶质细胞氧糖剥夺/复氧后的损伤

Inhibition of high mobility group protein attenuates spinal astrocyte injury in rats after oxygen glucose deprivation/reoxygenation

背景:既往对于抑制大鼠体内高迁移率族蛋白1(high mobility group protein,HMGB1)的释放改善脊髓功能的恢复较多,但对于体外培养脊髓星形胶质细胞,抑制HMGB1改善细胞氧糖剥夺/复氧的损伤研究尚少。目的:探讨抑制大鼠脊髓星形胶质细胞HMGB1后减轻细胞氧糖剥夺/复氧的损伤作用。方法:星形胶质细胞从新生SD大鼠脊髓提取,分离,培养鉴定。实验分两部分:第一部分实验,体外培养大鼠脊髓原代星形胶质细胞,对细胞行氧糖剥夺/复氧处理,实验分组:对照组,正常培养;氧糖剥夺6 h/复氧6,12,24 h组。第二部分实验,星形胶质细胞分组:对照组,正常培养;氧糖剥夺6 h/复氧24 h组;特异性RNA干扰组;非特异性RNA干扰组;丙酮酸乙酯组。利用Western blot和ELISA法检测各部分实验各组星形胶质细胞HMGB1的表达和释放量,利用乳酸脱氢酶的漏出率和MTT法检测细胞的损伤和存活率,光镜下观察细胞损伤后细胞形态结构的变化。结果与结论:(1)脊髓星形胶质细胞氧糖剥夺/复氧后,HMGB1蛋白表达及分泌升高(P<0.01),且复氧至24 h时达到最高值(P<0.01);特异性RNA干扰组及丙酮酸乙酯组HMGB1的表达量明显降低,提示特异的HMGB1shRNA和丙酮酸乙酯能有效抑制HMGB1表达;(2)细胞氧糖剥夺/复氧后,乳酸脱氢酶漏出率均明显增加,细胞存活率降低(P<0.01),复氧至24 h时损伤最为严重(P<0.01)。与氧糖剥夺6 h/复氧24 h组相比,特异性RNA干扰组及丙酮酸乙酯组细胞的乳酸脱氢酶漏出率明显下降;(3)光镜下观察细胞在氧糖剥夺/复氧后,细胞出现肿胀,分解等损伤变化,而特异RNA干扰和丙酮酸乙酯组细胞损伤明显减轻;(4)结果提示,HMGB1在脊髓星形胶质细胞损伤发生与发展中起到关键性的作用,且抑制HMGB1后能减轻氧糖剥夺/复氧对大鼠脊髓星形胶质细胞的损伤作用。

BACKGROUND： Many investigations focus on the inhibition of high mobility group protein（HMGB1） for improving the functional recovery of spinal cord in mice, but how to culture spinal cord astrocytes in vitro and inhibit the expression of HMGB1 for attenuating oxygen glucose deprivation/reoxygenation（OGD/R） injury is rarely reported. OBJECTIVE： To investigate the effect of HMGB1 inhibition on spinal cord astrocytes after OGD/R in vitro. METHODS： Astrocytes were isolated from rat spinal cord, and were then cultured and identified.（1） The rat primary spinal cord astrocytes were subjected to 6-hour OGD, followed by 6-, 12-, and 24-hour reoxygenation, respectively. The rat primary spinal cord astrocytes cultured in the normal medium were used as controls.（2） The rat spinal cord astrocytes were divided into five groups： control group（normal medium）; 6-hour OGD/24-hour R group; 6-hour OGD/24-hour R plus HMGB1 sh RNA group; 6-hour OGD/24-hour R plus non-targeting sh RNA group; 6-hour OGD/24-hour R plus ethyl pyruvate group. The expression and release of HMGB1 in astrocytes were determined by western blot assay and ELISA. The cell injury and survival rate were assessed by lactate dehydrogenase and MTT assay. The morphological changes of the cells were observed under light microscope. RESULTS AND CONCLUSION： The expression and release of HMGB1 protein was increased after OGD/R（P〈0.01）, and reached the highest value at reoxygenation for 24 hours（P〈0.01）. The expression level of HMGB1 in the HMGB1 sh RNA and ethyl pyruvate groups was signficnatly decreased, suggesting that specific RNA interference and ethyl pyruvate could effectively inhibit the expression of HMGB1. The leakage rate of lactate dehydrogenase was increased and cell survival rate was decreased after OGD/R, and cell injury was the most serious at reoxygenation for 24 hours（P〈0.01）. Compared with the 6-hour OGD/24-hour R group, the leakage rate of lactate dehydrogenase in the HMGB1 sh RNA and ethyl pyruvate groups was significantly reduced. There were cell swelling, decomposition and other damage changes after OGD/R, while HMGB1 sh RNA and ethyl pyruvate RNA could significantly reduce cell injury. Our findings imply that HMGB1 plays an important role in the occurrence and development of spinal cord astrocyte injury, and inhibition of HMGB1 can alleviate spinal cord astrocyte injury in rats after OGD/R.