Methamphetamine is one of the most prevalent drugs abused in the world.Methamphetamine abusers usually present with hyperpyrexia (39℃),hallucination and other psychiatric symptoms.However,the detailed mechanism under...Methamphetamine is one of the most prevalent drugs abused in the world.Methamphetamine abusers usually present with hyperpyrexia (39℃),hallucination and other psychiatric symptoms.However,the detailed mechanism underlying its neurotoxic action remains elusive.This study investigated the effects of methamphetamine + 39℃ on primary cortical neurons from the cortex of embryonic Sprague-Dawley rats.Primary cortex neurons were exposed to 1 mM methamphetamine + 39℃.Propidium iodide staining and lactate dehydrogenase release detection showed that methamphetamine + 39℃ triggered obvious necrosis-like death in cultured primary cortical neurons,which could be partially inhibited by receptor-interacting protein-1 (RIP1) inhibitor Necrostatin-1 partially.Western blot assay results showed that there were increases in the expressions of receptor-interacting protein-3 (RIP3) and mixed lineage kinase domain-like protein (MLKL) in the primary cortical neurons treated with 1 mM methamphetamine + 39℃ for 3 hours.After pre-treatment with RIP3 inhibitor GSK’872,propidium iodide staining and lactate dehydrogenase release detection showed that neuronal necrosis rate was significantly decreased;RIP3 and MLKL protein expression significantly decreased.Immunohistochemistry staining results also showed that the expressions of RIP3 and MLKL were up-regulated in brain specimens from humans who had died of methamphetamine abuse.Taken together,the above results suggest that methamphetamine + 39℃ can induce RIP3/MLKL regulated necroptosis,thereby resulting in neurotoxicity.The study protocol was approved by the Medical Ethics Committee of the Third Xiangya Hospital of Central South University,China (approval numbers: 2017-S026 and 2017-S033) on March 7,2017.展开更多
目的 考察坏死性凋亡抑制剂及分子量为4 000的聚乙二醇(PEG-4000)对小鼠肾小管上皮细胞TCMK-1表面一水草酸钙(COM)晶体黏附沉积的影响。方法 分别用400、800μg/m L COM作用于TCMK-1细胞,或先用受体相互作用的丝氨酸/苏氨酸蛋白激酶(RIP...目的 考察坏死性凋亡抑制剂及分子量为4 000的聚乙二醇(PEG-4000)对小鼠肾小管上皮细胞TCMK-1表面一水草酸钙(COM)晶体黏附沉积的影响。方法 分别用400、800μg/m L COM作用于TCMK-1细胞,或先用受体相互作用的丝氨酸/苏氨酸蛋白激酶(RIPK) 3抑制剂GSK-872预处理后再加入400、800μg/m L COM处理TCMK-1细胞,37℃孵育12 h后在倒置相差显微镜下观察细胞表面晶体黏附情况,用CCK-8法检测细胞增殖活性,2’,7’-二氯二氢荧光素二乙酸酯(DCFH-DA)探针法检测细胞氧化应激水平,蛋白质印迹法检测坏死性凋亡相关蛋白RIPK1、RIPK3、磷酸化混合谱系激酶结构域样蛋白(p-MLKL)的表达,电感耦合等离子体发射光谱法(ICP)检测细胞表面晶体黏附量。将TCMK-1细胞分为3组,分别用800μg/m L COM、先用PEG-4000溶液再加入800μg/m L COM、先用800μg/m L COM再加入PEG-4000溶液处理细胞,37℃孵育12 h后在倒置相差显微镜下观察细胞表面晶体黏附情况,CCK-8法检测细胞增殖活性、DCFH-DA探针法检测细胞氧化应激水平,ICP检测细胞表面晶体黏附量。结果 400μg/m L COM作用时,与COM处理组相比,GSK-872预处理组中TCMK-1细胞晶体黏附量减少、细胞增殖活性增强(P<0.05)、氧化应激水平降低(P<0.05);800μg/m L COM作用时,与COM处理组相比,GSK-872预处理组中TCMK-1细胞晶体黏附量无明显变化、细胞增殖活性增强(P<0.05)、氧化应激水平降低(P<0.05)、RIPK3和p-MLKL表达减少(P<0.05)。与COM处理组相比,PEG-4000预处理组TCMK-1细胞晶体黏附量明显减少、细胞增殖活性增强、氧化应激水平降低(P均<0.05),而后加入PEG-4000组与COM处理组相比晶体黏附量、细胞增殖活性、氧化应激水平均无明显变化(P均>0.05)。结论 用GSK-872抑制坏死性凋亡可以一定程度减少COM在细胞表面黏附沉积,但在较高的晶体负荷下晶体可在细胞表面聚集形成不定型沉淀。在培养基中使用PEG-4000预处理能够使COM微晶粒在悬液中保持悬浮稳定,减少晶体聚集沉积及细胞黏附和细胞氧化应激损伤;但充分接触COM晶体后的TCMK-1细胞再加入PEG-4000不能逆转晶体细胞黏附聚集沉淀。展开更多
基金funded by the National Natural Science Foundation of China,No.81971891(to KX),81571939(to KX),81772134(to KX),81772024(to JY),and 81860781(to FXL)the Key Research and Development Program of Hunan Province of China,No.2018SK2091(to KX)+1 种基金the Natural Science Foundation of Hunan Province of China,No.2017JJ2339(to JY)the Wu Jie-Ping Medical Foundation of the Minister of Health of China,No.320.6750.14118(to KX)
文摘Methamphetamine is one of the most prevalent drugs abused in the world.Methamphetamine abusers usually present with hyperpyrexia (39℃),hallucination and other psychiatric symptoms.However,the detailed mechanism underlying its neurotoxic action remains elusive.This study investigated the effects of methamphetamine + 39℃ on primary cortical neurons from the cortex of embryonic Sprague-Dawley rats.Primary cortex neurons were exposed to 1 mM methamphetamine + 39℃.Propidium iodide staining and lactate dehydrogenase release detection showed that methamphetamine + 39℃ triggered obvious necrosis-like death in cultured primary cortical neurons,which could be partially inhibited by receptor-interacting protein-1 (RIP1) inhibitor Necrostatin-1 partially.Western blot assay results showed that there were increases in the expressions of receptor-interacting protein-3 (RIP3) and mixed lineage kinase domain-like protein (MLKL) in the primary cortical neurons treated with 1 mM methamphetamine + 39℃ for 3 hours.After pre-treatment with RIP3 inhibitor GSK’872,propidium iodide staining and lactate dehydrogenase release detection showed that neuronal necrosis rate was significantly decreased;RIP3 and MLKL protein expression significantly decreased.Immunohistochemistry staining results also showed that the expressions of RIP3 and MLKL were up-regulated in brain specimens from humans who had died of methamphetamine abuse.Taken together,the above results suggest that methamphetamine + 39℃ can induce RIP3/MLKL regulated necroptosis,thereby resulting in neurotoxicity.The study protocol was approved by the Medical Ethics Committee of the Third Xiangya Hospital of Central South University,China (approval numbers: 2017-S026 and 2017-S033) on March 7,2017.
文摘目的 考察坏死性凋亡抑制剂及分子量为4 000的聚乙二醇(PEG-4000)对小鼠肾小管上皮细胞TCMK-1表面一水草酸钙(COM)晶体黏附沉积的影响。方法 分别用400、800μg/m L COM作用于TCMK-1细胞,或先用受体相互作用的丝氨酸/苏氨酸蛋白激酶(RIPK) 3抑制剂GSK-872预处理后再加入400、800μg/m L COM处理TCMK-1细胞,37℃孵育12 h后在倒置相差显微镜下观察细胞表面晶体黏附情况,用CCK-8法检测细胞增殖活性,2’,7’-二氯二氢荧光素二乙酸酯(DCFH-DA)探针法检测细胞氧化应激水平,蛋白质印迹法检测坏死性凋亡相关蛋白RIPK1、RIPK3、磷酸化混合谱系激酶结构域样蛋白(p-MLKL)的表达,电感耦合等离子体发射光谱法(ICP)检测细胞表面晶体黏附量。将TCMK-1细胞分为3组,分别用800μg/m L COM、先用PEG-4000溶液再加入800μg/m L COM、先用800μg/m L COM再加入PEG-4000溶液处理细胞,37℃孵育12 h后在倒置相差显微镜下观察细胞表面晶体黏附情况,CCK-8法检测细胞增殖活性、DCFH-DA探针法检测细胞氧化应激水平,ICP检测细胞表面晶体黏附量。结果 400μg/m L COM作用时,与COM处理组相比,GSK-872预处理组中TCMK-1细胞晶体黏附量减少、细胞增殖活性增强(P<0.05)、氧化应激水平降低(P<0.05);800μg/m L COM作用时,与COM处理组相比,GSK-872预处理组中TCMK-1细胞晶体黏附量无明显变化、细胞增殖活性增强(P<0.05)、氧化应激水平降低(P<0.05)、RIPK3和p-MLKL表达减少(P<0.05)。与COM处理组相比,PEG-4000预处理组TCMK-1细胞晶体黏附量明显减少、细胞增殖活性增强、氧化应激水平降低(P均<0.05),而后加入PEG-4000组与COM处理组相比晶体黏附量、细胞增殖活性、氧化应激水平均无明显变化(P均>0.05)。结论 用GSK-872抑制坏死性凋亡可以一定程度减少COM在细胞表面黏附沉积,但在较高的晶体负荷下晶体可在细胞表面聚集形成不定型沉淀。在培养基中使用PEG-4000预处理能够使COM微晶粒在悬液中保持悬浮稳定,减少晶体聚集沉积及细胞黏附和细胞氧化应激损伤;但充分接触COM晶体后的TCMK-1细胞再加入PEG-4000不能逆转晶体细胞黏附聚集沉淀。
