芍药苷抑制醋酸铅诱导大鼠海马神经元凋亡的机制  

Mechanism of paeoniflorin inhibiting apoptosis of hippocampal neurons of rats induced by lead acetate

作　　者：严伟伟[1] 李国辉[1] 赵佳骏[2] 贾仰民[1] 娄懿[1] 干晓瑜 Yan Weiwei;Li Guohui;Zhao Jiajun;Jia Yangmin;Lou Yi;Gan Xiaoyu(Department of Occupational Medicine,Hangzhou Red Cross Hospital,Hangzhou 310003,China;Department of Radiology,Hangzhou Hospital for the Prevention of Occupational Disease,Hangzhou 310003,China)

机构地区：[1]杭州市红十字会医院职业病科,杭州310003 [2]杭州市职业病防治院放射科,杭州310003

出　　处：《中华劳动卫生职业病杂志》2022年第3期170-176,共7页Chinese Journal of Industrial Hygiene and Occupational Diseases

基　　金：浙江省中医药科技计划项目(2020ZA082)。

摘　　要：目的探讨芍药苷对醋酸铅诱导海马神经元凋亡的影响及潜在机制。方法于2020年9月,从胎鼠中分离培养原代海马神经元细胞,并利用细胞免疫荧光法进行鉴定。MTT法测定细胞活力确定醋酸铅诱导海马神经元凋亡浓度及时间,芍药苷细胞毒性研究评价芍药苷浓度及其对醋酸铅诱导海马神经元凋亡的影响。依据结果选取不同浓度芍药苷干预海马神经元细胞,作用24 h后,加醋酸铅染毒,同时设空白组和模型组,测定海马神经元细胞中活性氧(ROS)、超氧化物歧化酶(SOD)、乳酸脱氢酶(LDH)、丙二醛(MDA)及半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)含量;蛋白免疫印迹(Western blotting)检测海马神经元细胞中细胞外信号调节激酶(ERK)、磷酸化ERK(p-ERK)、p38丝裂原活化蛋白激酶(MAPK)、磷酸化p38 MAPK(p-p38MAPK)、c-Jun氨基端激酶(JNK)、磷酸化JNK(p-JNK)蛋白表达。结果分离培养的海马神经元细胞经免疫荧光化学染色鉴定后,给予醋酸铅处理,MTT结果显示醋酸铅浓度25μmol/L,处理24 h毒性效果最佳。80μmol/L以下浓度芍药苷对海马神经元细胞均无细胞毒性作用;与模型组比较,20、40、80μmol/L芍药苷干预组海马神经元细胞活力均明显升高(P<0.05)。与空白组比较,模型组海马神经元细胞中ROS活力、LDH释放水平、MDA和Caspase-3含量均明显升高(P<0.01),SOD活力明显降低(P<0.01);与模型组比较,40、80μmol/L芍药苷干预组海马神经元细胞中ROS活力、LDH释放水平、MDA和Caspase-3含量明显降低(P<0.05),SOD活力明显升高(P<0.05)。与模型组比较,40、80μmol/L芍药苷干预组海马神经元细胞中p-ERK/ERK比值明显升高(P<0.01),p-p38MAPK/p38MAPK和p-JNK/JNK比值明显降低(P<0.05)。结论芍药苷可能通过MAPK信号通路,下调p-p38MAPK、p-JNK蛋白表达,上调p-ERK蛋白表达,抑制醋酸铅诱导的大鼠海马神经元凋亡。Objective To investigate the effect and underlying mechanism of paeoniflorin on hippocampal neuron apoptosis induced by lead acetate.Methods In September 2020,primary hippocampal neuronal cells were isolated and cultured from fetal rats,and identified using cellular immunofluorescent.MTT assay was used to measure the cell viability to determine the concentration and time of lead acetate-induced hippocampal neuron apoptosis.MTT was also used to evaluate the effect of paeoniflorin concentration on the apoptosis of hippocampal neurons induced by lead acetate.According to the results,different concentrations of paeoniflorin were selected to intervene hippocampal neuron cells,after 24 h,lead acetate was added to the cells,meanwhile,blank and model groups were set up,the content of reactive oxygen species(ROS),superoxide dismutase(SOD),lactate dehydrogenase(LDH),malondialdehyde(MDA)and Caspase-3 were measured.Extracellular signal regulated kinase(ERK),phosphorylated ERK(p-ERK),p38 mitogen-activated protein kinases(p38MAPK),phosphorylated p38MAPK(p-p38MAPK),c-Jun N-terminal kinase(JNK)and phosphorylated JNK(p-JNK)protein expression in hippocampal neuronal cells were determined by Western blotting.Results The isolated and cultured hippocampal neurons were identified by immunofluorescence chemical staining and then treated with lead acetate,MTT results showed that lead acetate had the best toxicity effect when treated for 24 h at a concentration of 25μmol/L.Paeoniflorin showed no cytotoxic effect on hippocampal neuronal cells when the concentrations below 80μmol/L.Compared with the model group,the activity of hippocampal neuronal cells was significantly increased after treating with 20,40 or 80μmol/L paeoniflorin(P<0.05).Compared with the blank group,the ROS activity,LDH release level,MDA content and caspase-3 content were significantly increased(P<0.01),and the SOD activity was significantly decreased(P<0.01)in the hippocampal neuronal cells of the model group.Compared with the model group,the ROS activity,LDH release

关 键 词：海马 神经元 细胞凋亡 芍药苷 醋酸铅 丝裂原活化蛋白激酶 大鼠 

分 类 号：R285.5[医药卫生—中药学]