雷公藤甲素配伍菟丝子黄酮通过miR-125a调控Tfh-Tfr-Treg平衡治疗SLE的机制研究

Systemic lupus erythematosus (SLE) is one of the most typical autoimmune diseases. Follicular regulatory T cells (Tfr) are newly discovered subset of regulatory T cells (Treg), share phenotypic characteristics with follicular helper T cells (Tfh) and Treg, and play an important role in SLE. Clinical application of Tripterygium wilfordii is effective in the treatment of SLE, but the exact mechanism is not clear, and the side effects limit its widespread use in clinical. Our previous study found that miR-125a was downregulated in lupus, and triptolide could up-regulated miR-125a in vitro, and further regulated Treg and Tfh. Therefore, this project intends to further use the miR-125a antagonist technology in the tail vein of mice and investigate the expression of miR-125a and its target gene STAT3, which affects the balance of Tfh-Tfr-Treg. We also detect the effect of cuscuta favonoids on the toxicity of triptolide-induced lupus mice. This study explores the new mechanism of SLE in vivo and in vitro, and reflects the unique advantages of "balance" of traditional Chinese medicine and provide a theoretical basis for clinical treatment.

系统性红斑狼疮（SLE）是最典型的自身免疫性疾病。滤泡调节性T细胞(Tfr)是新发现的调节性T细胞（Treg）亚群，具有滤泡辅助性T细胞（Tfh）和Treg的双重特性，是目前免疫学研究的前沿热点。临床应用雷公藤治疗SLE有效，其确切机制尚不明确，且副作用限制其在临床广泛应用。申请人前期研究发现：miR-125a在狼疮中表达下降，雷公藤甲素体外上调miR-125a，进一步调控Treg和Tfh。因此本项目拟分别在体内外水平，进一步利用小鼠尾静脉注射miR-125a拮抗剂等技术，探讨雷公藤甲素及其配伍菟丝子黄酮通过调节miR-125a及其靶基因STAT3的表达，影响Tfh-Tfr-Treg的平衡；并检测菟丝子黄酮对雷公藤甲素所致狼疮鼠的毒性影响。本研究在体内、外水平分别探讨SLE发病新机制，体现中医药调节机体功能“平衡”的特色优势，为临床治疗提供理论依据和新思路。

系统性红斑狼疮（SLE）是最典型的自身免疫性疾病。滤泡调节性T细胞(Tfr)是新发现的调节性T细胞（Treg）亚群，具有滤泡辅助性T细胞（Tfh）和Treg的双重特性，是目前免疫学研究的前沿热点。临床应用雷公藤治疗SLE有效，其确切机制尚不明确，且副作用限制其在临床广泛应用。miR-125a在狼疮中表达下降，雷公藤甲素体外上调miR-125a，进一步调控Treg和Tfh细胞。本项目在体内外水平探讨雷公藤甲素及其配伍菟丝子黄酮通过调节miR-125a的表达，影响Tfh-Tfr-Treg的平衡。体外实验结果显示：与溶剂对照组相比，用雷公藤甲素和雷公藤甲素+菟丝子黄酮处理组治疗后miR-125a显著升高。雷公藤甲素和雷公藤甲素+菟丝子黄酮处理组能显着升高STAT3和pSTAT3水平。利用 miR-125a inhibitor下调 miR-125a 水平，miR-125a-inhibitor转染后能够抑制这一作用。体内实验结果显示：与野生型对照小鼠（C3H小鼠）相比，MRL/lpr小鼠脾脏中Foxp3+CXCR5+CD4+Tfr细胞的比例显著降低，而MRL/lpr小鼠脾脏中的Foxp3-CXCR5+CD4+Tfh细胞的比例显著增高。MRL/lpr小鼠脾脏中Tfr与Tfh的比率显著低于对照小鼠。与溶剂处理对照组比较，雷公藤甲素组及雷公藤甲素+菟丝子黄酮组的脾脏指数、抗dsDNA抗体定量、尿蛋白定量均降低。雷公藤甲素+菟丝子黄酮处理组与雷公藤甲素组比较能提高小鼠孕酮水平。雷公藤甲素和雷公藤甲素+菟丝子黄酮处理的小鼠肾脏损害得到缓解，肾小球炎症细胞浸润、肾小球硬化、肾间质和血管周围病变显着减少。与溶剂处理对照组比较，雷公藤甲素、菟丝子黄酮治疗组STAT3 mRNA表达降低，FoxP3、miR-125a mRNA表达增加。雷公藤甲素+菟丝子黄酮联用处理组效果较单药处理组更明显。与溶剂处理对照组相比，MRL/lpr小鼠药物处理组的Foxp3蛋白表达增加。雷公藤甲素处理的MRL/lpr小鼠脾脏中Tfr和Tfh的比率显著高于溶剂处理MRL/lpr小鼠，且雷公藤甲素可以抑制MRL/lpr小鼠的GC反应。本研究在体内、外水平分别探讨SLE发病新机制，体现中医药调节机体功能“平衡”的特色优势，为临床治疗提供理论依据和新思路。