毛蕊异黄酮介导GP130/JAK/STAT信号通路对脊髓星形胶质细胞氧化损伤的影响  

Effect of Calycosin-mediated GP130/JAK/STAT Signaling Pathway on Oxidative Injury of Astrocytes in Spinal Cord

作　　者：郭德华[1] 刘小舟 吴成林 许洋 张国福[2] GUO Dehua;LIU Xiaozhou;WU Chenglin;XU Yang;ZHANG Guofu(Jiangxi University of Chinese Medicine,Nanchang 330004,China;The Affiliated Hospital of Jiangxi University of Chinese Medicine,Nanchang 330006,China)

机构地区：[1]江西中医药大学,南昌330004 [2]江西中医药大学附属医院,南昌330006

出　　处：《中国实验方剂学杂志》2022年第18期54-61,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81860856);江西省科学技术厅省临床研究中心项目(20212BCG74004);江西中医药大学校级研究生创新专项资金项目(JZYC21S23);江西中医药大学校级研究生创新专项资金项目(JZYC20S17);江西省研究生创新专项资金项目(YC2021-S524)。

摘　　要：目的探究毛蕊异黄酮介导糖蛋白130(GP130)/Janus酪氨酸蛋白激酶(JAK)/信号转导及转录激活因子(STAT)信号通路对脊髓星形胶质细胞氧化损伤的影响。方法原代分离大鼠脊髓星形胶质细胞,并利用免疫荧光检测胶质纤维酸性蛋白(GFAP)进行鉴定。分别加入5、10、20μmol·L^(-1)毛蕊异黄酮预处理脊髓星形胶质细胞12 h,然后加入100μmol·L^(-1)过氧化氢(H_(2)O_(2))处理24 h造成氧化损伤模型,细胞增殖与活性检测(CCK-8)法检测各组细胞增殖情况选择合适的毛蕊异黄酮处理浓度。实验分为空白组、模型组(H_(2)O_(2)100μmol·L^(-1))、毛蕊异黄酮组(毛蕊异黄酮20μmol·L^(-1))、毛蕊异黄酮+LY294002组(毛蕊异黄酮20μmol·L^(-1)+LY29400210μmol·L^(-1))、毛蕊异黄酮+Stattic组(毛蕊异黄酮20μmol·L^(-1)+Stattic 3μmol·L^(-1))。CCK-8法、免疫荧光检测各组细胞增殖情况,流式细胞术检测各组细胞凋亡和周期情况;蛋白免疫印迹法(Western blot)检测各组细胞中磷酸化(p)-JAK2、p-STAT3、p-蛋白激酶B(Akt)、GP130、白细胞介素-6(IL-6)的蛋白表达水平。结果与空白组比较,模型组细胞的增殖活力明显降低,细胞凋亡率明显升高(P<0.05);与模型组比较,毛蕊异黄酮(20μmol·L^(-1))组细胞的增殖活力明显增加,细胞凋亡率明显降低(P<0.05);与毛蕊异黄酮(20μmol·L^(-1))组比较,PI3K抑制剂LY294002和STAT3抑制剂Stattic均能明显降低细胞的增殖活力,增加细胞凋亡率(P<0.05)。与空白组比较,模型组细胞中p-JAK2、p-STAT3、p-Akt、GP130、IL-6的蛋白表达水平明显增加(P<0.05);与模型组比较,各用药组p-JAK2、p-STAT3、p-Akt、GP130、IL-6的蛋白表达水平均明显降低(P<0.05)。结论毛蕊异黄酮能够促进氧化损伤的星形胶质细胞增殖并抑制其凋亡,并能通过抑制磷脂酰肌醇3-激酶(PI3K)/Akt通路磷酸化、JAK2/STAT3通路磷酸化起作用。Objective To investigate the effect of calycosin-mediated glucoprotein130/Janus kinase/signal transducer and activator of transcription factor(GP130/JAK/STAT)signaling pathway on oxidative injury of astrocytes in spinal cord.Method Astrocytes in rat spinal cord were isolated and identified by immunofluorescence detection of glial fibrillary acidic protein(GFAP).The cells were respectively pre-treated with 5,10,20μmol·L^(-1) calycosin for 12 h,and then 100μmol·L^(-1) H_(2)O_(2)(24 h)was added to induce oxidative injury.Cell counting kit-8(CCK-8)assay was employed to detect cell proliferation and select the optimal concentration of calycosin.The following experimental groups were designed:control group,model group(100μmol·L^(-1) H_(2)O_(2)),calycosin group(20μmol·L^(-1) calycosin),calycosin+LY294002 group(20μmol·L^(-1) calycosin+10μmol·L^(-1) LY294002),and calycosin+Stattic group(20μmol·L^(-1) calycosin+3μmol·L^(-1) Stattic).CCK-8 assay and immunofluorescence method were used to detect the proliferation of cells and flow cytometry was applied to detect cell apoptosis and cycle.The protein expression of phosphorylated(p)-JAK2,p-STAT3,p-protein kinase B(Akt),GP130,and interleukin-6(IL-6)was detected by Western blotting.Result Compared with the control group,the model group showed low proliferation activity and high apoptosis rate of cells(P<0.05).Compared with the model group,calycosin(20μmol·L^(-1))group displayed high proliferation activity and low apoptosis rate of cells(P<0.05).Compared with calycosin(20μmol·L^(-1))group,both phosphatidylinosirtol-3-kinases(PI3K)inhibitor LY294002 and STAT3 inhibitor Stattic significantly reduced the proliferation activity and increased the apoptosis rate of cells(P<0.05).The protein expression of p-JAK2,p-STAT3,p-Akt,GP130,and IL-6 in the model group was higher than that in the control group(P<0.05),and the expression of the above indicators was lower in each treatment group than in the model group(P<0.05).Conclusion Calycosin can promote the proliferation an

关 键 词：脊髓星形胶质细胞 毛蕊异黄酮 氧化损伤 毛蕊异黄酮介导糖蛋白130(GP130)/Janus酪氨酸蛋白激酶(JAK)/信号转导及转录激活因子(STAT)信号通路 脊髓损伤 补阳还五汤 

分 类 号：R285.5[医药卫生—中药学] R966[医药卫生—中医学] R744