龙胆泻肝汤对实验性自身免疫性葡萄膜炎大鼠补体C4、MBL2表达的影响

Effect of Longdan Xiegan decoction on expression levels of C4 and MBL2 in experimental autoimmune uveitis rats

目的探讨龙胆泻肝汤(Longdan Xiegan decoction,LXD)对实验性自身免疫性葡萄膜炎(experimental autoimmune uveitis,EAU)大鼠的治疗作用及对血清中C4、MBL2蛋白表达水平的影响。方法54只Lewis大鼠用随机数字表法分为正常对照组、EAU组和LXD组,其中EAU组、LXD组大鼠制备EAU模型,LXD组造模后使用LXD每天灌胃处理,EAU组给予等量生理盐水灌胃。免疫后12 d使用激光扫描检眼镜(scanning laser ophthalmoscope,SLO)观察三组大鼠眼底炎症,并取三组大鼠同侧眼球进行病理切片,观察视网膜组织病理学变化;分离三组大鼠的脾脏和淋巴结,收集T淋巴细胞,流式细胞仪检测CD4^+/CD8^+细胞比例的变化;酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测血清中C4、MBL2蛋白的表达水平。结果SLO检查结果显示,与正常对照组相比,EAU组大鼠屈光间质不清,无法观察眼底视网膜及血管情况;LXD组大鼠眼底血管迂曲扩张,屈光间质混浊,视盘边界模糊不清,但较EAU组大鼠症状轻。组织病理学检查结果显示,与正常对照组相比,EAU组大鼠眼组织结构紊乱,视网膜全层破坏,视网膜内可见大量炎性细胞浸润;LXD组大鼠视网膜仅表现为轻、中度炎性细胞浸润。流式细胞仪检测结果发现,EAU组大鼠脾脏、淋巴结中CD4^+/CD8^+比值均高于正常对照组;LXD组大鼠的CD4^+T细胞表达水平下降,CD8^+T细胞表达水平升高,两者比例趋于平衡。ELISA检测结果发现,与正常对照组相比,EAU组大鼠免疫后12 d、16 d、20 d血清C4蛋白水平均显著升高,与EAU组相比,LXD组免疫后12 d、16 d、20 d血清C4蛋白水平均明显降低,差异均有统计学意义(均为P<0.05);同时,EAU组各时间点血清MBL2蛋白水平明显降低,而LXD组较EAU组各时间点血清MBL2蛋白水平均显著升高,差异均有统计学意义(均为P<0.01)。结论LXD可有效缓解EAU大鼠眼内炎症,改善脾脏、淋巴结中CD4^+/CD8^+细胞比例失衡,同时降低血清中补体C4蛋白表达水平,上调MBL2蛋白表达水平,促进补体系统恢复平衡,加快葡萄膜炎的炎症消退,从而达到治疗EAU的作用。

Objective To investigate the therapeutic effect of Longdan Xiegan decoction(LXD)on rats with experimental autoimmune uveitis(EAU)and its effect on serum levels of C4 and MBL2 proteins.Methods Totally 54 Lewis rats were randomly divided into normal control group,EAU group and LXD group by random number table.Rats in both EAU group and LXD group were used to establish EAU models.After modeling,rats in LXD group received intragastric administration of LXD every day,and those in EAU group received same volume of normal saline by gavage.After immune treatments for 12 days,the symptom of ocular fundus inflammation was observed under scanning laser ophthalmoscope(SLO)and then the pathological sections of eyeballs at the same side were extracted to observe the pathological changes of retina.Meanwhile,the spleen and lymph nodes were isolated to collect T lymphocytes for detecting the levels of CD4^+/CD8^+cells by flow cytometry.The enzyme-linked immunosorbent assay(ELISA)was used to detect the expressions levels of C4 and MBL2 proteins in serum.Results Compared with control group,SLO results showed that rats in EAU group had unclear refractive stroma,fundus retina and vessels could not be observed.Rats in LXD group had tortuous and distended fundus vessels,turbid refractive stroma and unclear boundary of optic disk,but the symptoms were milder than those in EAU group.Compared with normal control group,histopathological examination results demonstrated that structure of ocular tissues were in disorder and the full-thickness retina was damaged for rats in EAU group,in addition,the retina was infiltrated by a large number of inflammatory cells.However,the retina of the rats in LXD group showed mild to moderate infiltration of inflammatory cells.Flow cytometry results showed that the levels of CD4^+/CD8^+in spleen and lymph nodes for rats in EAU group were higher than those in normal control group.LXD group had lower levels of CD4^+T cells,whereas the levels of CD8^+cells increased,and the level of CD4^+/CD8^+tended to balance.ELISA test showed that serum C4 levels in EAU group were apparently higher than those in normal control group on day 12,16 and 20 after immune treatment,while the C4 levels in LXD group was apparently lower than those in EAU group on day 12,16 and 20,and statistical differences were observed(all P<0.05).In the meantime,serum MBL2 levels in EAU group apparently decreased at different time points,and were lower than those in LXD group(all P<0.01).Conclusion LXD can effectively alleviate the intraocular inflammation in EAU rats,improve the imbalance of CD4^+/CD8^+ratio in spleen and lymph nodes,decrease the expression level of C4 protein and up-regulate the expression level of MBL2 protein,promote the recovery of complement system balance,and accelerate the regression of inflammation of uveitis.LXD can play a role in treating EAU.