黄芩苷通过调控巨噬细胞M2极化对脊髓损伤大鼠炎症反应的抑制作用  

Inhibitory effect of baicalin on inflammation in rats with spinal cord injury by regulating macrophage M2 polarization

作　　者：许大勇[1] 李云朋[1] 魏景梅[1] 刘汝银[1] XU Dayong;LI Yunpeng;WEI Jingmei;LIU Ruyin(Department of Spine Surgery,Henan Provincial Hospital of Traditional Chinese Medicine,Zhengzhou 450002,China)

机构地区：[1]河南省中医院脊柱科,河南郑州450002

出　　处：《吉林大学学报（医学版）》2021年第1期158-167,共10页Journal of Jilin University：Medicine Edition

基　　金：河南省中医药拔尖人才培养项目(2019ZYBJ18)。

摘　　要：目的:探讨黄芩苷对大鼠脊髓损伤(SCI)的保护作用,阐明其作用机制。方法:体内实验应用改良Allen氏打击法建立大鼠SCI模型,50只健康SD大鼠随机分为假手术组(给予生理盐水)、模型组(给予生理盐水,建立SCI模型)、黄芩苷组(SCI大鼠给予200 mg·kg^-1黄芩苷)、黄芩苷+DMSO组(SCI大鼠给予200 mg·kg^-1黄芩苷和0.2%DMSO)和黄芩苷+AS1517499(STAT6通路抑制剂)组(SCI大鼠给予200 mg·kg^-1黄芩苷和10 mg·kg^-1 AS1517499),每组10只。造模成功后用贝索-比蒂-布雷斯纳汉(BBB)评分评价大鼠脊髓运动功能。体外实验用2.5μg·L^-1脂多糖(LPS)和0.125μg·L^-1干扰素γ(IFN-γ)诱导巨噬细胞(RAW264.7)M1型极化,10μg·L^-1白细胞介素4(IL-4)诱导RAW264.7细胞M2型极化。RAW264.7细胞分为对照组、M1型极化组、M2型极化组、M1型极化+黄芩苷组、M2型极化+黄芩苷组、M1型极化+黄芩苷+DMSO组和M1型极化+黄芩苷+AS1517499组。酶联免疫吸附试验(ELISA)检测大鼠脊髓组织和RAW264.7细胞中炎症因子[白细胞介素12(IL-12)、TNF-α、IL-4和白细胞介素10(IL-10)]水平,Western blotting法检测大鼠脊髓组织和RAW264.7细胞中M1型巨噬细胞标记蛋白[诱导型一氧化氮合酶(iNOS)和趋化因子5(CCL-5)]及M2型巨噬细胞标记蛋白[精氨酸酶1(Arg1)、甘露糖受体C1(MRC1)和Janus激酶1/信号转导及转录激活蛋白6(JAK1/STAT6)通路相关蛋白]表达水平。结果:体内实验,与模型组比较,黄芩苷组大鼠BBB评分明显升高(P<0.05),脊髓组织中IL-12和TNF-α水平及iNOS、CCL-5和磷酸化信号转导及转录激活蛋白1(p-STAT1)蛋白表达水平明显降低(P<0.05),IL-4和IL-10水平及Arg1、MRC1、磷酸化JAK1(p-JAK1)和磷酸化STAT6(p-STAT6)蛋白表达水平明显升高(P<0.05或P<0.01);与黄芩苷+DMSO组比较,黄芩苷+AS1517499组大鼠脊髓组织中IL-12和TNF-α水平及iNOS、CCL-5和p-STAT1蛋白表达水平明显升高(P<0.01),IL-4和IL-10水平及Arg1、MRC1和p-STAT6蛋白表达水平明Objective:To explore the protective effect of baicalin on the spinal cord injury(SCI)in the rats,and to clarify its mechanism.Methods:The SCI models of rats were established by modified Allen’s method in vivo.Fifty healthy SD rats were randomly divided into sham operation group(given normal saline),model group(modeling,given normal saline),baicalin group(modeling,given 200 mg·kg^-1 baicalin),baicalin+DMSO group(modeling,given 200 mg·kg^-1 baicalin and 0.2%DMSO)and baicalin+AS1517499(STAT6 pathway inhibitor)group(modeling,given 200 mg·kg^-1 baicalin and 10 mg·kg^-1 AS1517499)(n=10).After the models were successfully established,the spinal cord motor function of rats was evaluated by using the Basso-Beattie-Bresnahan(BBB)method.In vitro,2.5μg·L^-1 lipopolysaccharide(LPS)and 0.125μg·L^-1 interferon-γ(IFN-γ)were used to induce M1 polarization of macrophages(RAW264.7),and 10μg·L^-1 interleukin-4(IL-4)was used to induce M2 polarization of macrophages.The RAW264.7 cells were divided into control group,M1 type polarization group,M2 type polarization group,M1 type polarization+baicalin group,M2 type polarization+baicalin group,M1 type polarization+baicalin+AS1517499 group,and M1 type polarization+baicalin+DMSO group.ELISA assay was used to detect the levels of inflammatory factors interleukin-12(IL-12),tumor necrosis factor-α(TNF-α),interleukin-4(IL-4)and interleukin-10(IL-10)in the spinal cord tissue and RAW264.7 cells of the rats;Western blotting method was used to detect the expression levels of M1 type macrophage marker proteins inducible nitric oxide synthase(iNOS)and chemokine-5(CCL-5),M2 type macrophage marker proteins arginase 1(Arg1)and mannose receptor C1(MRC1)and Janus kinase 1/signal transducer and activator of transcription 6(JAK1/STAT6)pathway related proteins in spinal cord tissue and RAW264.7 cells of the rats.Results:In vivo experiments,compared with model group,the BBB score of the rats in baicalin group was significantly increased(P<0.05),the levels of IL-12 and TNF-αand the expression

关 键 词：脊髓损伤 黄芩苷 炎症反应 Janus激酶1 信号转导及转录激活蛋白6 信号通路 

分 类 号：R285.5[医药卫生—中药学] R651.2[医药卫生—中医学]