通窍活血汤含药脑脊液调控ASK1/MKK4/JNK信号通路对OGD/R损伤HT22细胞的保护作用  

Protective effect of Tongqiao Huoxue Decoction containing cerebrospinal fluid on OGD/R-damaged HT22 cells via regulation of ASK1/MKK4/JNK signaling pathway

作　　者：袁美玲 张云 汪光云 吴倩[1,2,3] 王艳 汪宁[1,2,3] YUAN Mei-ling;ZHANG Yun;WANG Guang-yun;WU Qian;WANG Yan;WANG Ning(Anhui University of Chinese Medicine,Hefei 230012,China;Laboratory of Research and Development of Chinese Medicine of Anhui Province,Hefei 230012,China;Institute for Pharmacodynamics and Safety Evaluation of Chinese Medicine,Anhui Academy of Chinese Medicine,Hefei 230012,China)

机构地区：[1]安徽中医药大学,安徽合肥230012 [2]安徽省中药研究与开发重点实验室,安徽合肥23001 [3]安徽省中医科学院中药药效与安全性评价研究所,安徽合肥230012

出　　处：《中国中药杂志》2022年第19期5274-5283,共10页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金面上项目(81773933);安徽省高校自然科学研究重点项目(KJ2019A0434);安徽省第十三批115产业创新团队“针药结合防治脑病创新团队”项目(皖人才办[2020]4号)。

摘　　要：该文探讨通窍活血汤含药脑脊液(TQHXD-CSF)对氧糖剥夺/复糖复氧(OGD/R)损伤小鼠海马神经元(HT22)细胞的保护作用及其作用机制是否与调控ASK1/MKK4/JNK信号通路有关.将HT22细胞随机分为5组,分别为空白脑脊液组(control组)、OGD/R组、TQHXD-CSF组、凋亡抑制剂组(Z-VAD-FMK,20μmol·L^(-1))、TQHXD-CSF+凋亡抑制剂组.除control组外,其余各组细胞在氧糖剥夺6 h后迅速复糖复氧12 h进行OGD/R造模并分组给药.CCK8法检测HT22细胞活力,LDH试剂盒检测乳酸脱氢酶释放量,倒置显微镜观察HT22细胞形态.Western blot检测Bax、Bcl-2和caspase-3的蛋白表达情况,实时荧光定量PCR(qRT-PCR)检测Bax、Bcl-2和caspase-3的mRNA水平.再将HT22细胞随机分为6组,分别为control组、OGD/R组、si-NC组、si-ASK1组、TQHXD-CSF组、TQHXD-CSF+si-ASK1组.CCK8法检测细胞活力,细胞动态分析仪动态监测细胞增殖,Hoechst染色和流式细胞术检测HT22细胞凋亡情况,Western blot检测MKK4、p-MKK4、JNK、p-JNK、c-Jun、p-c-Jun、Cyt C、Bax、Bcl-2和caspase-3的蛋白表达情况.结果显示,与OGD/R组相比,TQHXD-CSF可以显著提高细胞存活率,改善细胞形态,降低Bax/Bcl-2、caspase-3蛋白和mRNA的表达量.当沉默ASK1后,与OGD/R组相比,TQHXD-CSF组细胞存活率显著提升,降低细胞荧光强度,降低细胞凋亡率,p-MKK4、p-JNK、p-c-Jun、Cyt C、Bax/Bcl-2和caspase-3的蛋白表达量减少,但其效果不及TQHXD-CSF+si-ASK1组.综上,TQHXD-CSF可抑制OGD/R损伤的HT22细胞ASK1/MKK4/JNK通路介导的细胞凋亡,对缺血再灌注神经元细胞损伤具有保护作用.To investigate the protective effect of Tongqiao Huoxue Decoction containing cerebrospinal fluid(TQHXD-CSF)on HT22 cells damaged by oxygen-glucose deprivation/reoxygenation(OGD/R)and whether the mechanism is related to the regulation of ASK1/MKK4/JNK signaling pathway.HT22 cells were subjected to OGD/R to simulate cerebral ischemia-reperfusion injury(CIRI).Then the cells were randomly divided into five groups:blank cerebrospinal fluid(control group),OGD/R group,TQHXD-CSF group,Z-VAD-FMK group(20μmol·L^(-1)) and TQHXD-CSF+Z-VAD-FMK group.Except the control group,cells in the other groups were reoxygenated for 12 h after 6 h of oxygen and glucose deprivation for modeling OGD/R,and group administration was performed.Cell viability and cytotoxicity were detected by CCK8 and LDH assay kit,respectively and the morphology of HT22 cells was observed by inverted microscope.Western blot and qRT-PCR were employed to detect the protein and mRNA expression levels of Bax,Bcl-2 and caspase-3,respectively.Then HT22 cells were assigned into the control group,OGD/R group,si-NC group,si-ASK1 group,TQHXD-CSF group and TQHXD-CSF+si-ASK1 group.Cell viability,proliferation and apoptosis were determined by CCK8,electric cell-substrate impedance sensing(ECIS),and Hoechst staining and flow cytometry,respectively.The protein expression of MKK4,p-MKK4,JNK,p-JNK,c-Jun,p-c-Jun,Cyt C,Bax,Bcl-2 and caspase-3 was tested by Western blot.The results showed that compared with OGD/R group,TQHXD-CSF significantly enhanced cell viability,improved cell morphology and reduced the protein and mRNA expression levels of Bax,Bcl-2 and caspase-3.In addition,when ASK1 was silenced,compared with OGD/R group,TQHXD-CSF remarkably improved cell viability,and decreased apoptosis rate and the protein expression levels of p-MKK4,p-JNK,p-c-Jun,Cyt C,Bax/Bcl-2 and caspase-3,but the effect was not as good as that of TQHXD-CSF+si-ASK1 group.In conclusion,TQHXD-CSF can inhibit apoptosis mediated by ASK1/MKK4/JNK signaling pathway in OGD/R-damaged HT22 cells,and has pro

关 键 词：通窍活血汤 脑脊液 氧糖剥夺/复糖复氧 HT22细胞 细胞凋亡 

分 类 号：R285[医药卫生—中药学]