基于“清热利湿”治法探讨积雪草酸调控microRNA-146a靶向TRAF6/NF-KB干预肠肾互动及肠道菌群对抗IgA肾病进展的炎性机制

Intestinal flora is closely related to chronic kidney disease (CKD) and end-stage renal disease (ESRD), and has become a new target for the treatment of chronic kidney disease. microRNA (microRNA) as a transcription regulator has a regulatory effect on intestinal flora, renal inflammation and renal fibrosis. microRNA-146a plays an important regulatory role in the immune response of the body, but The mechanism of regulates intestinal microflora ,renal fibrosis and anti- inflammation is still unclear. On the basis of previous studies, through IgA nephropathy animal model and flora interference model, in vitro cytological transfection of mimics and inhibitors of microRNA146 and in vivo adoptive transfusion, high-throughput 16S rDNA sequencing technology was used to detect intestinal microflora, flow cytometry was used to detect macrophage polarization, inflammatory factor liquid chip detection, Luciferase Report detection of target genes and other experimental methods, to clarify the pharmacodynamic effect of Asiatica Acid and its inflammatory mechanism against the progress of IgA nephropathy, and to explore the biological basis and scientific connotation of "clearing away heat and dampness" therapy, and to provide an effective target for delaying the progress of IgA nephropathy.

肠道菌群与慢性肾脏病（CKD）及终末期肾病（ESRD）密切相关，成为慢性肾脏病治疗的新靶点；微RNA（microRNA，miRNA）作为转录调节因子，对肠道菌群及肾脏炎症、肾纤维化过程均具有调控作用；miRNA146a对机体免疫应答具有重要调节功能，但其如何调控肠道菌群及对抗肾脏炎症的机制仍不明确。在前期研究基础上，通过IgA肾病动物模型及菌群干扰模型、miRNA146a模拟物及抑制剂体外细胞学转染及体内过继输注，采用高通量16S rDNA 测序技术、流式细胞术、炎症因子液相芯片检测、荧光素酶报告检测靶基因等实验方法，探讨miRNA146a对炎症信号通路TRAF6/NF-KB的调控作用，对肠道菌群及肠-肾免疫的影响，阐明积雪草酸的药效学作用及对抗IgA肾病进展的炎性机制；探讨“清热利湿”治法的生物学基础及科学内涵，为延缓IgA肾病进展提供有效靶点。

研究证实，炎症是慢性肾脏病进展至肾纤维化及终末期肾脏病的重要驱动因素；进行有效抗炎治疗，靶向防控疾病进展，可能为慢性肾脏病在内的多种疾病的精准治疗开辟新的途径。肠道微生物、微RNA、巨噬细胞与慢性肾脏病进展密切相关，但他们之间如何相互作用，参与IgA肾病进程尚不清楚。前期研究发现清热利湿中草药积雪草对慢性肾炎蛋白尿具有良好的治疗作用，推测其主要成分积雪草酸可能通过对抗肾脏炎症发挥作用，肠道微生物、miRNA-146a、巨噬细胞、炎性因子可能是其作用的重要靶点。通过IgA肾病动物模型及菌群干扰模型、miRNA146a模拟物及抑制剂体外细胞学转染及体内过继输注，采用高通量16S rDNA 测序技术、荧光素酶报告检测靶基因、双标免疫荧光、组织化学、qRT-PCR及westernblot 检测等实验方法，探讨miRNA146a对炎症信号通路TRAF6/NF-KB的调控作用，积雪草酸对肠道菌群、肠-肾免疫的影响，阐明积雪草酸的药效学作用及对抗IgA肾病进展的炎性机制。体外研究结果显示：对LPS诱导的HK2细胞炎症，积雪草酸可通过上调miRNA 146a-5p，抑制TRAF6、NF-KBp65mRNA及蛋白的表达，减少炎性细胞因子TNF-α、IL-6、IL-1β释放，发挥抗炎作用；动物实验研究结果显示：积雪草酸可减少蛋白尿、减轻肾小球内IgA沉积，减轻肾组织病理损伤，促进肾脏巨噬细胞向M2表型极化，促进肾脏修复；积雪草酸通过上调肾组织miRNA 146a-5p抑制 TRAF/NF-KB通路激活，降低肾脏炎性因子MCP-1、TNF-α、IL-6、IL-1β释放，发挥抗炎作用；同时AA可减轻结肠病理损伤，抑制肠粘膜sIgA分泌，上调结肠组织关键性蛋白Occludin、Calduin1 、ZO-1表达，调节肠道菌群，从而发挥抗炎作用，对抗IgA肾病进展。