复方金钱草活性成分通过CD44/TGF-βR通路延缓草酸钙结晶诱导肾小管EMT的机制研究

Calcium oxalate crystal can induce EMT in tubular epithelium and initiate the process of chronic kidney disease. In this study, we found that Chinese medicine Fu-Fang-Jin-Qian-Chao herbal granules can improve crystal-induced renal EMT by regulating TGF-βR/Smad pathway. Previous studies have shown that CD44 plays an important role in crystal-induced EMT. CD44 and TGFβRs were reported to be co-localized and interacted under certain stimulation. It is speculated that CD44 may play a role in EMT by acting on TGFβRs. Previously, components with high affinity to CD44 could be screened from Fu-Fang-Jin-Qian-Chao herbal granules by cell membrane chromatography, which implied that the effect of Fu-Fang-Jin-Qian-Chao granules are mediated by CD44. This project intends to further study the protective mechanism of Fu-Fang-Jin-Qian-Chao’s active ingredients on crystal-induced renal tubular EMT through CD44/TGFβR pathway using methods of Co-Immunoprecipitation, GST-pull down and cell membrane chromatography. The compounds screened by cell membrane chromatography will be further validated as potential active ingredients. This study will provide theoretical support for the treatment of crystal-induced renal fibrosis by active ingredients of traditional Chinese medicine.

结石对肾脏的损害远早于梗阻的形成，草酸钙结晶可诱导小管上皮发生EMT，启动慢性肾脏病进程。本课题发现中医药制剂复方金钱草颗粒可通过调控TGF-βR/Smad通路改善结晶诱导肾脏EMT。前期研究表明CD44在结晶诱导EMT中有重要作用。有文献报道在一定刺激下CD44和TGF-βRs存在共定位，可发生相互作用，由此推测CD44可通过作用于TGF-βRs在EMT中发挥作用。前期通过细胞膜色谱法可从复方金钱草颗粒中筛选出与CD44高亲和性的成分化合物，佐证复方金钱草的作用可能是通过CD44介导的。本项目拟通过免疫共沉淀、GST-pull down、细胞膜色谱等分子生物学手段，进一步研究复方金钱草活性成分通过CD44/TGF-βR通路延缓结晶诱导肾小管EMT的分子机制。并将细胞膜色谱法所筛选出的成分化合物作为潜在活性成分进行验证，为中药有效成分治疗结晶诱导肾脏纤维化提供理论支持。

泌尿系结石是慢性肾脏病的重要病因，结石对肾脏的损害远早于梗阻的形成。结石的主要成分是草酸盐结晶,结晶导致的肾小管损伤是慢性肾脏病的重要病理基础。中医药制剂复方金钱草颗粒是祖国医学中防治泌尿系结石的经典药材,但其活性成分及具体的肾保护作用尚不完全清楚。本研究利用血、尿 生化,组织病理等检测手段,明确了复方金钱草对草酸盐结晶肾损伤小鼠模型的保护性效应。利用全二维HK-2和HK-2／CIKI（结晶肾损伤）细胞膜色谱比较系统分析发现，芒果苷成分在HK-2／CIKI细胞膜色谱柱上的亲和性高于HK-2细胞膜色谱柱。从而推测芒果苷是复方金钱草治疗结晶肾损伤的潜在活性成分。我们继续利用草酸结晶肾小管损伤细胞及小鼠模型成功验证了芒果苷延缓草酸结晶肾损伤的有效性。证实芒果苷能够下调CD44、OPN及MCP1被结晶诱导的表达，有效降低小鼠肾组织内草酸钙结晶的沉积，改善草酸钙结晶所致的小鼠肾组织病理损伤。为了探寻更多潜在的复方金钱草活性成分与分子机制，项目组利用小鼠尿液样品进行代谢分析，鉴别出肌酐、泛酸等15种代谢物为结晶肾损伤小鼠尿液的差异代谢物，主要参与氨基酸和脂肪酸代谢通路。其中13种代谢物在广金钱草干预后水平出现逆转趋势，从而说明广金钱草可通过改善代谢紊乱延缓结晶肾损伤。此外，我们利用网络药理学分析了复方金钱草治疗结晶肾损伤的多成分-多靶点-多代谢物的机制特点，成功寻找到雌激素受体2、雄激素受体等新的分子机制。综上所述，芒果苷是复方金钱草延缓草酸钙结晶肾损伤的潜在活性成分。复方金钱草因其多靶点的效应,可能通过综合调节作用延缓了结晶肾损伤的进展。上述研究极大地丰富了复方金钱草的药理特性，也发现了结晶肾损伤的新的潜在干预靶点。