摘要目的 探讨受体相互作用蛋白激酶(RIP)1和RIP3通路是否介导谷氨酸诱导的HT-22海马神经细胞死亡. 方法 (1)体外培养小鼠海马神经细胞株HT-22,分为空白对照组、zVAD组、Nec-1组、谷氨酸组、谷氨酸+zVAD组、谷氨酸+zVAD+Nec-1组、谷氨酸+Nec-1组,zVAD、Nec-1、谷氨酸的终浓度分别为20 μmol/L、30 μmol/L、3 mmol/L,作用24 h后用Cell titer glo发光法细胞活力检测试剂盒(CTG)检测细胞存活率;碘化丙啶(PD及Hoechst细胞染色检测细胞的坏死;(2)将HT-22细胞分为空白对照组、谷氨酸组、谷氨酸+Nee-1组,浓度和作用时间同上,应用MitoSox red线粒体超氧化物指示剂检测细胞线粒体内活性氧自由基(ROS)水平;(3)将HT-22细胞分为空白对照组、谷氨酸组、谷氨酸+叔丁基茴香醚(BHA)组,BHA的终浓度为100 μmol/L,作用24 h后用PI及Hoechst细胞染色检测细胞的坏死;(4)将HT-22细胞分为RIP3 siRNA组、对照组,分别转染RIP3 siRNA、siRNA对照序列,72 h后Western blotting检测2组细胞RIP3蛋白的表达;(5)将HT-22细胞分为对照组、R1P3 siRNA组、谷氨酸组、RIP3 siRNA+谷氨酸组,分别各自转染siRNA对照序列、RIP3 siRNA,48 h后加入3 mmol/L谷氨酸,24 h后用PI及Hoechst细胞染色检测细胞的坏死,CTG检测细胞存活率. 结果 (1)与空白对照组比较,谷氨酸组、谷氨酸+zVAD组PI阳性细胞百分比升高,细胞存活率降低,差异有统计学意义(P＜0.05).与谷氨酸组比较,谷氨酸+Nec-1组PI阳性细胞百分比明显减少,细胞存活率增加,差异有统计学意义(P＜0.05);(2)谷氨酸组HT-22细胞内的ROS水平明显高于空白对照组,谷氨酸+Nec-1组细胞内的ROS水平明显低于谷氨酸组,差异有统计学意义(P＜0.05);(3)谷氨酸组PI阳性细胞百分比高于空白对照组,谷氨酸+BHA组PI阳性细胞百分比低于谷氨酸组,差异有统计学意义(P＜0.05);(4)与对照组比较,RIP3siRNA组细胞内RIP3蛋白水平明显下调;(5)与对照组比较,谷氨酸组PI阳性细胞百分比升高、细胞存活率降低,与谷氨酸组比较,RIP3 siRNA+谷氨酸组阳性细胞百分比降低、细胞存活率升高,差异均有统计学意义(P＜0.05). 结论 RIP1、RIP3通路及ROS介导谷氨酸诱导的HT-22海马神经细胞死亡.

abstractsObjective To explore whether receptor interacting protein (RIP)1/RIP3 pathways participate in glutamate induced cell death in HT-22 neuronal cells and investigate the potential neuroprotection ofnecrostatin-1 in glutamate induced cell death in HT-22.Methods (1) In vitro cultured mouse hippocampal neuronal HT-22 cells were divided into control group,zVAD group,necrostatin-1 (Nec-1) group,glutamate group,glutamate+zVAD group,glutamate+zVAD+Nec-1 group and glutamate+Nec-1 group;they were treated with zVAD,Nec-1 and glutamate at the final concentrations of 20 μmol/L,30 μmol/L and 3 mmol/L for 24 h.Cell viability was detected using a luminescence-based commercial kit Cell Titer-Glo (CTG).Necrotic cell death was measured by propidium iodide (PI) and HE stainings.(2) HT-22 cells were divided into control group Ⅰ,glutamate group Ⅰ and glutamate+Nec-1 group Ⅰ;MitoSox Red was used to detect mitochondrial reactive oxygen species (ROS) level.(3) HT-22 cells were divided into control group Ⅱ,glutamate group Ⅱ and glutamate+tertiary butyl-hydroxyanisole (BHA) group;the final concentration of BHA was 100 μmol/L;necrotic cell death was measured by PI and HE stainings after 24 h of treatment.(4) HT-22 cells were divided into RIP3 siRNA and control group Ⅲ,and then,they were transfected with RIP3 siRNA or negative siRNA,respectively;the RIP3 protein expression was determined by Westem blotting after 72 h of treatment.(5) HT-22 cells were divided into negative siRNA+Control,RIP3 siRNA,negative siRNA+glutamate and RIP3 siRNA+glutamate groups;the cells were transfected with RIP3 siRNA or Negative siNRA,respectively;48 h later,the glutamate groups were treated with 3 mmol/L glutamate;PI positive cells and cell viability were measured by PI and HE stainings and CTG at 24 h after glutamate treatment.Results (1) As compared with control group,percentage of PI positive cells was greatly increased and cell viability was decreased in glutamate group and glutamate+zVAD group,with statistically significant differences (P＜0.05);as compared with those in the glutamate group,percentage of percentage of PI positive cells was was significantly decreased and cell viability was statistically increased in glutamate+Nec-1 group (P＜0.05).(2) ROS level in HT-22 cells of the glutamate group was significantly increased than that in the control group Ⅰ (P＜0.05);however,ROS level in HT-22 cells of glutamate+Nec-1 group Ⅰ was significantly decreased than that in glutamate group Ⅰ (P＜0.05).(3)Percentage of PI positive cells in the glutamate group Ⅱ was significantly higher than that in the control group Ⅱ (P＜0.05),and that in the glutamate+BHA group was statistically lower than that in the glutamate group Ⅱ (P＜0.05).(4) The RIP3 protein expression in the RIP3 siRNA group was obviously down-regulated as compared with that in the control group Ⅲ.(5) As compared with those in the negative siRNA group,percentage of PI positive cells was statistically increased and cell viabilities were statistically decreased in glutamate group (P＜0.05);however,percentage of PI positive cells was significantly decreased and cell viability was significantly increased in RIP3 siNRA+glutamate group as compared with those in the glutamate group (P＜0.05).Conclusion RIP1/RIP3 pathway and ROS might mediate glutamate induced cell death in HT-22 cells.