运脾解毒通络祛湿方靶向IRE1α/XBP1/PDK1抑制CD4^(+)T细胞中枢迁移改善CIA小鼠中枢敏化的机制研究

Mechanism study of Yunpi Jiedu Tongluo Qushi Decoction on central sensitization of collagen-induced arthritis mice via suppressing CD4~+T cell migration by targeting IRE1α/XBP1/PDK1

目的:探究运脾解毒通络祛湿方通过靶向肌醇需要酶1α(IRE1α)/X-box结合蛋白1(XBP1)/丙酮酸脱氢酶激酶1(PDK1)抑制CD4^(+)T细胞中枢迁移改善胶原诱导关节炎(CIA)小鼠中枢敏化的疗效机制。方法:32只雄性DBA/1小鼠经适应性喂养后随机分为4组:空白组、CIA组、中药组、西药组,每组8只。向小鼠尾部2次注射胶原佐剂以构建CIA模型。初次免疫后,各药物组连续42 d接受相应药物治疗。自初次免疫后28 d起每周观察并记录各组小鼠痛阈及关节炎症评分情况。干预结束后分离与收集外周血、脊髓组织。此外,利用磁珠分选术收集空白组、CIA组及中药组脾脏CD4^(+)T细胞,并且将空白组随机分为:空白组与IRE1α激动剂组,其中IRE1α激动剂组予以毒胡萝卜素(Thapsigargin)1μmol/L处理24 h。采用ELISA法检测各组小鼠血清白细胞介素(IL)1β、IL-6的含量;免疫荧光检测各组小鼠脊髓背角CD4、cFos、IBA1及IRE1α/XBP1/PDK1通路相关蛋白荧光强度;乳酸试剂盒检测各组脊髓背角组织乳酸含量;此外,应用免疫荧光检测各组CD4^(+)T细胞IRE1α/XBP1/PDK1通路相关蛋白荧光强度,乳酸试剂盒检测各组CD4^(+)T细胞乳酸含量,Transwell实验观察各组CD4^(+)T细胞迁移能力。结果:与CIA组比较,中药组小鼠机械痛阈与热痛阈均显著升高(P<0.05),关节炎症评分显著减低(P<0.01),血清IL-1β、IL-6水平显著减低(P<0.05),中药组脊髓背角CD4、cFos及IBA1的荧光强度显著减低(P<0.05),IRE1α、XBP1、PDK1荧光强度显著减低(P<0.05),乳酸浓度显著减少(P<0.05)。体外实验表明:与空白组比较,CIA组、IRE1α激动剂组CD4^(+)T细胞IRE1α、XBP1、PDK1表达水平显著增加(P<0.01,P<0.05),乳酸浓度显著升高(P<0.01,P<0.05),CD4^(+)T细胞迁移能力显著增强(P<0.05)。与CIA组比较,中药组IRE1α、XBP1、PDK1表达水平显著下降(P<0.01,P<0.05),细胞乳酸浓度显著下降(P<0.05),CD4^(+)T细胞迁移能力显著减弱(P<0.05)。结论:运脾解毒通络祛湿方可通过靶向IRE1α/XBP1/PDK1,抑制CD4^(+)T细胞向糖酵解重编程,减少CD4^(+)T细胞中枢迁移,从而改善CIA小鼠中枢敏化。

Objective:To investigate the mechanism of Yunpi Jiedu Tongluo Qushi Decoction on central sensitization of collagen-induced arthritis(CIA)mice via suppressing CD4^(+)T cell migration by targeting IRE1α/XBP1/PDK1.Methods:Thirty-two male DBA/1 mice were randomly divided into 4 groups after adaptive feeding,including blank group,CIA group,traditional Chinese medicine(TCM)group and western medicine group,with 8 mice in each group.The CIA model was constructed by collagen injection.After the initial immunization,each drug group received the corresponding drug treatment for 42 consecutive days.Pain threshold and arthritis scores in each group were assessed weekly from day 28.After the intervention,peripheral blood and spinal cord tissues in each group were separated and collected.In addition,spleen CD4^(+)T cells in blank group,CIA group and Chinese medicine group were collected by magnetic activated cell sorting,and the blank group was randomly divided into blank group and IRE1αagonist group.The agonist group was treated with Thapsigargin 1μmol/L for 24h.The serum levels of interleukin(IL)-1βand IL-6 were detected by ELISA.The fluorescence intensity of CD4,c Fos,IBA1 and IRE1α/XBP1/PDK1 were detected by immunofluorescence.Lactic acid kit was used to detect the content of lactic acid in spinal dorsal horn.The fluorescence intensity of IRE1α/XBP1/PDK1 pathway related proteins was detected by immunofluorescence,the lactic acid content of cells in each group was detected by lactic acid kit,and the migration ability of CD4^(+)T cells in each group was observed by Transwell assay.Results:Compared with CIA group,the paw withdrawal threshold and paw withdrawal latency were significantly increased(P<0.05),the arthritis score(P<0.01)and serum IL-1βand IL-6 levels in TCM group were significantly decreased(P<0.05),the fluorescence intensity of CD4,c Fos and IBA1 in spinal dorsal horn of TCM group was significantly decreased(P<0.05),the fluorescence intensity of IRE1α,XBP1 and PDK1were significantly decreased(P<0.05),the lactate concentration was decreased significantly(P<0.05).In addition,in vitro experiments showed that compared with the blank group,the expression levels of IRE1α,XBP1 and PDK1 in CD4^(+)T cells were significantly increased(P<0.01,P<0.05),the lactate concentration was significantly increased(P<0.01,P<0.05),the migration ability of CD4^(+)T cell was significantly enhanced(P<0.05)in the CIA group and IRE1αagonist group.Compared with the CIA group,the expression levels of IRE1α,XBP1 and PDK1 were significantly decreased(P<0.01,P<0.05),the cellular lactate concentration decreased significantly(P<0.05),and the migration ability of CD4^(+)T cell was significantly decreased in the TCM group(P<0.05).Conclusion:Yunpi Jiedu Tongluo Qushi Decoction can inhibit the reprogramming of CD4^(+)T cells to glycolysis and the central migration of CD4^(+)T cells by targeting IRE1α/XBP1/PDK1,thereby improving the central sensitization of CIA mice.