基于JAK2/STAT3信号通路苦参素对EAE小鼠中枢小胶质细胞向M1型极化的抑制作用及机制研究

Inhibitory Effect and Mechanism of Matrine on M1 Type Polarization of Central Microglia in EAE Mice Based on JAK2/STAT3 Signaling Pathway

目的探讨苦参素通过酪氨酸激酶2/信号转导子和转录激活子3(JAK2/STAT3)信号通路对实验性自身免疫性脑脊髓炎(EAE)小鼠中枢小胶质细胞向M1型极化的抑制作用及影响机制。方法50只小鼠分为正常对照组、EAE组、EAE+苦参素200组、EAE+苦参素400组及EAE+醋酸泼尼松组,各10只。除正常对照组外均建立EAE模型,于发病日开始给药,EAE+苦参素200组、EAE+苦参素400组腹腔注射200、400 mg·kg^(-1)苦参素注射液,EAE组和正常对照组腹腔注射生理盐水,EAE+醋酸泼尼松组灌胃7 mg·kg^(-1)醋酸泼尼松溶液,1次·d^(-1),连续14 d。给药期间进行神经功能评分,采用苏木精-伊红染色及劳克坚牢蓝染色检测腰髓炎性细胞浸润、髓鞘脱失情况,流式细胞术分析M1型、M2型小胶质细胞占比,实时荧光定量聚合酶链反应(RT-qPCR)测定诱导型一氧化氮合酶(iNOS)、γ-干扰素(IFN-γ)、肿瘤坏死因子-α(TNF-α)、精氨酸酶1(Arg-1)、白细胞介素(IL)-4、IL-10 mRNA表达量,蛋白质印迹法测JAK2、p-STAT3蛋白表达量。结果与EAE组比较,EAE+苦参素200组给药5~14 d、EAE+苦参素400组及EAE+醋酸泼尼松组给药3~14 d神经功能评分均较低(P<0.05);与EAE+苦参素200组比较,EAE+苦参素400组及EAE+醋酸泼尼松组给药5~14 d神经功能评分均较低(P<0.05)。与EAE组比较,EAE+苦参素200组、400组,EAE+醋酸泼尼松组炎症浸润评分、髓鞘脱失评分、M1型小胶质细胞占比、iNOS、IFN-γ、TNF-αmRNA相对表达量及JAK2、p-STAT3蛋白相对表达量均降低,但仍高于正常对照组(P<0.05),且EAE+苦参素400组、EAE+醋酸泼尼松组低于EAE+苦参素200组(P<0.05);与正常对照组和EAE组比较,EAE+苦参素200组、400组,EAE+醋酸泼尼松组M2型小胶质细胞占比及Arg-1、IL-4、IL-10 mRNA相对表达量均升高(P<0.05),且EAE+苦参素400组和EAE+醋酸泼尼松组高于EAE+苦参素200组(P<0.05)。结论苦参素可有效改善EAE小鼠脊髓炎症及髓鞘脱失情况,抑制中枢小胶质细胞向M1型极化,且与给药剂量相关,其作用机制可能与抑制JAK2/STAT3信号通路有关。

OBJECTIVE To investigate the inhibitory effect and mechanism of matrine on the M1 polarization of central microglia in experimental autoimmune encephalomyelitis(EAE)mice through the signal pathway of tyrosine kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3).METHODS Fifty mice were divided into control group,model group,model+matrine 200 group,model+matrine 400 group and model+prednisone acetate group,10 mice in each group.EAE models were successfully established except the control group.The drug was administered on the day of onset.The model+matrine 200 group and the model+matrine 400 group were injected intraperitoneally with 200 and 400 mg·kg^(-1)matrine injection.The model group and the control group were injected intraperitoneally with normal saline.The model+prednisone acetate group was given intragastrically with 7 mg·kg^(-1)body weight prednisone acetate solution once a day for 14 d.Neurological scores were determined during administration.Hematoxylin eosin staining and Luxol Fast Blue staining were used to detect inflammatory cell infiltration and demyelination.The proportion of M1 and M2 microglia in central nervous system was analyzed by flow cytometry.The expressions of iNOS,IFN-γ,TNF-α,Arg-1,IL-4,IL-10 mRNA were tested by RT-qPCR and the expressions of JAK2 and p-STAT3 protein were tested by Western blot.RESULTS Compared with the model group,the neurological function scores of the model+matrine 200 group administered for 5 to 14 d,the model+matrine 400 group,and the model+prednisone acetate group administered for 3 to 14 d were lower(P<0.05).Compared with the model+matrine 200 group,the model+matrine 400 group and model+prednisone acetate group had lower neurological scores at 5 to 14 d after administration(P<0.05).Compared with the model group,the scores of inflammatory infiltration and demyelination,the proportions of M1 microglia,iNOS,IFN-γ,TNF-αmRNA expressions and the relative expressions of JAK2 and p-STAT3 protein in the model+matrine 200 group,model+matrine 400 group and model+prednisone acetate group were lower,but still higher than those in the control group(P<0.05),and the model+matrine 400 group and model+prednisone acetate group were lower than those in the model+matrine 200 group(P<0.05).Compared with the control group and model group,the proportions of M2 microglia and Arg-1,IL-4,IL-10 mRNA expressions in the model+matrine 200 group,model+matrine 400 group and model+prednisone acetate group increased(P<0.05),and the model+matrine 400 group and model+prednisone acetate group were higher than that of the model+matrine 200 group(P<0.05).CONCLUSION Matrine can effectively improve the spinal cord inflammation and demyelination of EAE mice and inhibit the central microglia polarization to M1 type in a dose-dependent manner,for which the mechanism of action may be inhibiting the JAK2/STAT3 signaling pathway.