鳖甲调控生发中心B细胞免疫应答在MRL/lpr狼疮小鼠中的疗效作用研究

Study on the therapeutic effect of turtle shell regulating the immune response of germinal center B cells in MRL/lpr lupus mice

目的:基于生发中心(GC)B细胞免疫应答探讨鳖甲治疗系统性红斑狼疮(SLE)的疗效及其作用研究。方法:21只6周龄雌性MRL/lpr小鼠按体质量随机分为模型组、羟氯喹组和鳖甲组,每组7只,7只MRL/MpJ小鼠作为正常组,共4组。第7周起分别以0.9%氯化钠溶液、硫酸羟氯喹片、鳖甲干预7周,计算脾脏及淋巴结指数,检测尿蛋白、肾脏病理切片进行HE染色、免疫组化C3、免疫荧光IgG分析;ELISA法检测小鼠血清中抗ds-DNA抗体、IgM和IgG水平;流式细胞术检测小鼠脾脏、淋巴结及外周血中GC B细胞、IgM和IgG表达情况。结果:与模型组比较,羟氯喹及鳖甲治疗组脾脏、淋巴结肿大得到显著缓解(P<0.01),14周尿蛋白表达显著下降(P<0.01),肾小球肿胀增大、炎症细胞浸润及大量C3、IgG沉积等病理改变减轻;血清ELISA结果表明药物治疗后抗ds-DNA抗体表达显著降低(P<0.01),鳖甲组IgM表达显著降低(P<0.01),羟氯喹组IgG表达显著减少(P<0.05);流式细胞仪结果表明羟氯喹及鳖甲治疗后脾脏中GC B细胞表达稍减少,IgM、IgG表达无明显差异,淋巴结中GC B细胞、IgM、IgG表达均显著降低(P<0.01),外周血中GC B细胞表达显著下降(P<0.01),羟氯喹组IgG表达显著减少(P<0.05),鳖甲组中IgM、IgG表达显著降低(P<0.05)。结论:鳖甲能够通过抑制MRL/lpr小鼠淋巴结、外周血中GC B细胞表达,降低IgM、IgG等自身抗体的形成,进一步减轻SLE疾病活动度,缓解肾脏病理损害,从而发挥疗效。

Objective:To explore the therapeutic effect and role of turtle shell in systemic lupus erythematosus(SLE)based on the B cell immune response of the germinal center(GC).Methods:Twenty one 6-week female MRL/lpr mice were randomly divided into model group,hydroxychloroquine group,and turtle shell group,7 mice in each group.Seven MRL/MpJ mice were used as normal control group.From the 7th week onwards,normal saline,hydroxychloroquine and turtle shell were used to intervene for 7 weeks.The spleen and lymph node indices were calculated,and urine protein and renal pathological sections were detected for HE staining,immunohistochemistry C3 and IgG analysis;ELISA method was used to detect the levels of anti ds-DNA antibodies,IgM,and IgG in mouse serum;Flow cytometry was used to detect the expression of GC B cells,IgM,and IgG in the spleen,lymph nodes,and peripheral blood of mice.Results:Compared with the model group,the hydroxychloroquine and turtle shell significantly alleviated splenic and lymph node swelling(P<0.01),significantly decreased urinary protein expression at 14 weeks(P<0.01),and reduced pathological changes such as increased glomerular swelling,infiltration of inflammatory cells,and deposition of large amounts of C3 and IgG;The serum ELISA results showed a significant decrease in anti ds-DNA antibodies(P<0.01)and IgM expression after turtle shell treatment(P<0.01),and a decrease in IgG expression in the hydroxychloroquine group(P<0.05);The results of flow cytometry showed that the expression of GC B cells in the spleen was slightly reduced after treatment with hydroxychloroquine and turtle shell,while there was no significant difference in the expression of IgM and IgG.The expression of GC B cells,IgM,and IgG in lymph nodes were significantly reduced(P<0.01).The expression of GC B cell in peripheral blood significantly decreased(P<0.01).The expression of IgG in the hydroxychloroquine group was reduced(P<0.05),while the expression of IgM,IgG in the turtle shell group was reduced(P<0.05).Conclusion:Turtle shell can inhibit the expression of GC B cells in lymph nodes and peripheral blood of MRL/lpr mice,reduce the formation of autoantibodies such as IgM and IgG,further reduce the activity of SLE disease,alleviate renal pathological damage,and exert therapeutic effects.