补阳还五汤通过miR-148a-3p抑制大鼠星形胶质细胞凋亡而减轻脑缺血再灌注损伤  

Buyang-Huanwu decoction alleviates cerebral ischemia-reperfusion injury by inhibiting rat astrocyte apoptosis through miR-148a-3p

作　　者：田甜 蔡国英 叶佳蓓 单玉栋 周晓红 郭书翰 高维娟 TIAN Tian;CAI Guoying;YE Jiabei;SHAN Yudong;ZHOU Xiaohong;GUO Shu-han;GAO Weijuan(Hebei University of Chinese Medicine,Heibei Key Laboratory of Chinese Medicine Research on Cardio-cerebrovascular Disease,Shijiazhuang 050091,China;Department of Pathophysiology,Chengde Medical College,Chengde 067000,China;Department of Acupuncture and Moxibustion,the First Affiliated Hospital of Hebei College of Traditional Chinese Medicine,Shijiazhuang 050013,China;Hebei Cangzhou Hospital of Integrated Traditional Chinese and Western Medicine Affiliated to Hebei College of Traditional Chinese Medicine,Cangzhou 061012,China;Hebei University of Chinese Medicine,Shijiazhuang 050200,China)

机构地区：[1]河北中医药大学,河北省心脑血管病中医药防治研究重点实验室,河北石家庄050091 [2]承德医学院病理生理学教研室,河北承德067000 [3]河北中医药大学第一附属医院针灸科,河北石家庄050013 [4]河北中医药大学附属河北省沧州中西医结合医院,河北沧州061012 [5]河北中医药大学,河北石家庄050200

出　　处：《中国病理生理杂志》2023年第8期1431-1439,共9页Chinese Journal of Pathophysiology

基　　金：中央引导地方科技发展资金(No.206Z7706G);河北省科技研发平台与新型研发机构建设专项(No.20567626H);河北省属高校基本科研业务费基础研究专项(No.JCYJ2022009)。

摘　　要：目的:探究补阳还五汤是否通过激活微小RNA-148a-3p(miR-148a-3p)靶向调控胶质细胞原纤维酸性蛋白(GFAP)而抑制大鼠氧糖剥夺/复氧复糖(OGD/R)星形胶质细胞凋亡。方法:取对数生长期的大鼠星形胶质细胞系CTX TNA2,随机分为5组:空白对照组(CON组)、模型组(OGD/R组)、补阳还五汤含药血清(BYHWDS)组、miR-148a-3p抑制物组(inhibitor组)和BYHWDS+inhibitor组,每组每种检测重复3次。除CON组外,其余各组氧糖剥夺6 h后复氧复糖24 h,并于复氧复糖的同时给予相应药物处理(但转染于造模前操作)。采用倒置相差显微镜观察细胞形态变化;MTS法检测各组细胞活力变化;qRT-PCR检测miR-148a-3p、GFAP mRNA和caspase-3 mRNA的表达水平;Western blot检测GFAP、Bax、Bcl-2、caspase-3、cleaved caspase-3、caspase-9和cleaved caspase-9蛋白水平;annexin V-FITC/PI染色检测CTX TNA2细胞的凋亡情况;免疫荧光双染观察GFAP、caspase-3、Bcl-2和Bax的表达情况。结果:与CON组相比,OGD/R组和inhibitor组的细胞皱缩,突起严重萎缩、变形,细胞碎片增多,细胞活力显著降低(P<0.01),miR-148a-3p表达水平降低(P<0.01),GFAP和caspase-3的mRNA表达水平均显著升高(P<0.01),GFAP蛋白表达水平,cleaved caspase-3/caspase-3、Bax/Bcl-2和cleaved caspase-9/caspase-9比值,以及细胞凋亡水平均显著升高(P<0.05)。与OGD/R组和inhibitor组相比,BYHWDS组和BYHWDS+inhibitor组细胞形态较损伤后有所恢复,突起萎缩减轻,连接有所增加,细胞活力显著升高(P<0.01),miR-148a-3p表达水平升高(P<0.01),GFAP和caspase-3的mRNA表达水平均显著降低(P<0.01),GFAP蛋白表达水平,cleaved caspase-3/caspase-3、Bax/Bcl-2和cleaved caspase-9/caspase-9比值,以及细胞凋亡水平均显著降低(P<0.05)。结论:补阳还五汤能上调miR-148a-3p表达而调低靶基因GFAP,从而抑制OGD/R大鼠CTX TNA2细胞凋亡,减轻脑缺血再灌注损伤,发挥神经保护作用。AIM:To explore the mechanism of Buyang-Huanwu decoction in alleviating cerebral ischemia-reperfusion injury.METHODS:Rat astrocyte CTX TNA2 cells at the logarithmic growth stage were randomly divided into 5 groups:blank control group(CON group),model group[(oxygen-glucose deprivation/reoxygenation(OGD/R)group],Buyang-Huanwu decoction-medicated serum(BYHWDS)group,miR-148a-3p inhibitor group(inhibitor group),and BYHWDS+inhibitor group.All experiments were conducted three times with three replicates.Apart from the cells in CON group,the cells in the other groups were subjected to OGD for 6 h and reoxygenation for 24 h,and were then treated with the drugs at the same time of reoxygenation(transfection was performed before modeling).Cell morphology was observed by inverted phase-contrast microscopy and viability was measured by MTS method.The expression of microRNA-148a-3p(miR-148a-3p),glial fibrillary acidic protein(GFAP)mRNA and caspase-3 mRNA was measured by qRT-PCR,and the protein levels of GFAP,Bcl-2,Bax,caspase-3,cleaved caspase-3,caspase-9 and cleaved caspase-9 were assessed by Western blot.Apoptosis was measured using an annexin V-FITC/PI kit.The expression of GFAP,caspase-3,Bcl-2 and Bax was also evaluated by immunofluorescence double-staining.RESULTS:Compared with CON group,the cells in OGD/R and inhibitor groups were observed to be shrunken and deformed,with increased cell debris and significantly reduced viability(P<0.01).The expression of miR-148a-3p was significantly decreased(P<0.01),while the mRNA levels of GFAP and caspase-3 were significantly increased(P<0.01).The protein expression of GFAP,the cleaved caspase-3/caspase-3,Bax/Bcl-2 and cleaved caspase-9/caspase-9 ratios,and the apoptosis level were significantly increased(P<0.05).Compared with OGD/R and inhibitor groups,the cell morphology of BYHWDS and BYHWDS+inhibitor groups recovered after injury,with reduced atrophy,increased connections,and significantly increased cell viability(P<0.01).The expression of miR-148a-3p was significantly increased(P<

关 键 词：补阳还五汤 微小RNA-148a-3p 胶质细胞原纤维酸性蛋白 星形胶质细胞 细胞凋亡 脑缺血再灌注损伤 

分 类 号：R743.3[医药卫生—神经病学与精神病学] R364.1[医药卫生—临床医学] R285.5