盐酸青藤碱通过靶向巨噬细胞防治IgA肾病的机理研究

IgA nephropathy is the most prevalent primary chronic glomerular disease worldwide. Sinomenine is an alkaloid extracted from the stem of the Chinese medicinal plant of Sinomenium acutum which has been used to treat various rheumatic and arthritic diseases for over a thousand years. Recently, sinomenine hydrochloride has been used to protect and prevent IgA nephropathy in clinic, however the exact mechanism remains unclear. Numerous studies and our previous works suggest that macrophage plays an important role in the pathogenesis of IgA nephropathy; sinomenine hydrochloride shows potent inhibitory effects on proliferation, activation and migration of macrophages. Thus, sinomenine hydrochloride might protect IgA nephropathy by targeting macrophages. In order to prove this hypothesis, Near-Infrared fluorescence probes labeling technique, flow cytometry and immunofluorescence will be used to investigate the effect of sinomenine hydrochloride on proliferation, activation and migration of macrophages in the rat model of IgA nephropathy; macrophages clearance and supplement in the rat model of IgA nephropathy will be carried to prove the mechanism of sinomenine hydrochloride protected IgA nephropathy by targeting macrophages; Immunoblotting, electrophoretic mobility shift assay and gene transfection technique will be employed to explore the mechanism of inhibitory effects of sinomenine hydrochloride on macrophage migration. This project will provide key fundamentals for the clinical application of sinomenine hydrochloride for IgA nephropathy treatment, and pave a new way for drug discovery from the Traditional Chinese medicine.

IgA肾病为最常见的原发性肾小球肾炎。青藤碱是从祖国传统抗风湿中药青风藤中提取的生物碱单体。近年来，盐酸青藤碱在临床上被用于预防和治疗IgA肾病，然其作用机制未明。大量文献和我们先前的研究表明：巨噬细胞在IgA肾病的发生和发展过程中起着重要的病理作用；盐酸青藤碱在体内外实验中对巨噬细胞增殖、活化和迁移具有很强的抑制作用。因此，盐酸青藤碱防治IgA肾病可能通过靶向巨噬细胞。本实验采用近红外荧光探针标记技术、流式细胞仪检测技术、免疫荧光法等来探讨盐酸青藤碱对IgA肾病大鼠体内巨噬细胞增值、活化和迁移的影响；利用巨噬细胞清除和增补方法明确盐酸青藤碱通过靶向巨噬细胞来防治IgA肾病；通过免疫印迹法、凝胶迁移实验和基因转染技术来寻找盐酸青藤碱抑制巨噬细胞迁移机制；以此阐明盐酸青藤碱防治IgA肾病的机制。该研究可为盐酸青藤碱临床上防治IgA肾病奠定实验基础，同时为中医药的研究开发提供一种新的研究思路。

IgA肾病为临床最常见的原发性肾小球肾炎。青藤碱是从祖国传统抗风湿中药青风藤中提取的生物碱单体。近年来，盐酸青藤碱在临床上被用于预防和治疗IgA肾病，但其防治IgA肾病的作用机制未明。大量的文献和我们先前的研究表明：巨噬细胞在IgA肾病的发生和发展过程中起着重要的病理作用；盐酸青藤碱在体内外实验中对巨噬细胞增殖、活化和迁移具有很强的抑制作用。提示：盐酸青藤碱防治IgA肾病可能通过靶向巨噬细胞。本研究采用异种蛋白诱导建立实验性IgA肾病大鼠模型，从预防性和治疗性给药两个方面系统评价了盐酸青藤碱对IgA肾病的防治作用；采用近红外荧光探针标记技术、流式细胞仪检测技术、transwell小室法等探讨了盐酸青藤碱对IgA肾病大鼠体内巨噬细胞增值和迁移的影响；利用巨噬细胞增补方法明确盐酸青藤碱通过靶向巨噬细胞来防治IgA肾病；通过免疫印迹法和基因转染技术来寻找盐酸青藤碱抑制巨噬细胞迁移机制。研究发现，盐酸青藤碱预防性和治疗性干预均显著降低了IgA肾病大鼠24h尿蛋白水平和尿红细胞数、减少肾小球IgA和C3的沉积、减轻肾小球系膜细胞的增生，表现出较好的IgA肾病保护作用。机制研究发现，盐酸青藤碱通过下调mPGES-1的表达和Src/FAK/P130Cas 轴的激活从而抑制巨噬细胞的迁移、减少肾组织中巨噬细胞的浸润，保护IgA肾病。巨噬细胞补充实验进一步证实，巨噬细胞是盐酸青藤碱发挥IgA肾病保护作用的重要靶细胞。该研究为盐酸青藤碱临床上防治IgA肾病奠定实验基础，同时为中医药的研究开发提供了一种新的研究思路。