益肾活血法通过调控外泌体及特定非编码RNA改善肾纤维化的机制研究

肾纤维化(RIF)的原因有肾小管上皮细胞老化与上皮间质转化（EMT），中医主要辨证肾虚血瘀，以益肾活血法制剂肾康灵治疗，临床疗效佳。申请人前期研究发现：肾康灵通过影响Notch1信号通路改善了RIF，机制有待深化；外泌体及其携带的Notch1通路相关的长链非编码RNA HOTAIR，既受肾康灵影响，又参与调控细胞老化、EMT及RIF。由此提出科学假说：益肾活血法中药肾康灵可以调控受损肾脏释放外泌体，并通过这些外泌体携带的非编码RNA参与的HOTAIR/miR-124/Notch1表观遗传通路，进而改善肾纤维化。拟以肾康灵干预UUO大鼠RIF模型，细胞老化与EMT肾小管上皮细胞模型，及肾小管上皮细胞与肾脏纤维母细胞Transwell共培养，在体内与体外检测外泌体及上述非编码RNA通路相关指标。以验证假说，阐明益肾活血法治疗RIF的机制，为肾康灵及其联合外泌体治疗RIF的临床应用提供实验依据。

Renal fibrosis (RIF) is attributed to the senescence of renal tubular epithelial cells and epithelial-mesenchymal transition (EMT), and is also caused by kidney deficiency and blood stasis in the perspective of traditional Chinese medicine, Shenkangling decoction, whose function is reinforcing kidney and activating blood circulation has clinical effect for treating patients with chronic kidney disease and renal fibrosis. Our previous study demonstrated that Shenkangling decoction can alleviate the progression of RIF through modulating Notch1 signaling pathway. Moreover, our results indicated that Notch1 signaling pathway of the exosomal non-coding RNA HOTAIR was involved in the regulation of cellular senescence, EMT and RIF, and the processes were affected by Shenkangling decoction. However, the mechanism needs further clarification. We hypothesize that Shenkangling decoction could effectively treat patients with RIF through modulating the secretion of exosomes by damaged kidney cells, and sequentially releasing non-coding RNAs, which is involved the epigenetic pathway of HOTAIR / miR-124 / Notch 1 axis. In this proposal, we design to detect the levels of the exosome, the non-coding RNAs and the molecular markers of the associated signal pathway of the non-coding RNAs using unilateral ureteral obstruction (UUO) RIF rat model in vivo, cellular senescence and EMT model of the renal tubular epithelial cells, and the simulated renal tissue model with transwell co-culture of renal tubular epithelial and renal fibroblast cells. The aim of this proposal is to prove our hypothesis and clarify the mechanism of Shenkangling decoction for RIF treatment, and provides experimental evidence for the clinical application of Shenkangling decoction alone and combined Shenkangling decoction and the exosomes for the treatment of RIF.