EMP3/TGF-β/Smad3恶性反馈环路在肾间质纤维化中的作用及活血消癥方的调控机制研究

肾纤维化(RF)是慢性疾病(CKD)进展为终末期肾病的关键环节,目前缺乏有效的药物治疗。项目组前期依据肾内“微癥积”理论以活血消癥法的肾纤康颗粒能显著延缓CKD进展，但作用机制尚不明确。RNA-seq测序显示肾纤康能特异性下调CKD小鼠上皮膜蛋白3(EMP3)的表达，进一步实验及生物信息学预测提示在RF发生过程中可能存在EMP3上调→活化的EMP3与TGFβ受体2(TGFBR2)互作激活TGF-β/Smad3信号通路→Smad3转录上调EMP3表达这一恶性正反馈回路。基于此发现，本研究拟借助体内外实验，采用蛋白免疫共沉淀、超声微泡、RfxCas13d介导的RNA静默等技术进一步分析EMP3介导的TGF-β/Smad3失衡与RF的相关性，探讨EMP3在RF中的作用及肾纤康分子机制，进一步深化CKD内在病理机制的认识及肾纤康治疗CKD生物学内涵的解释。

Renal fibrosis (RF) is the key part in the progression of chronic kidney disease (CKD) to end-stage renal disease (ESRD). Currently, there is a lack of effective drugs in clinical practice. Shenqiankang granule (SQK) developed by our research team, which is characterized by "huoxue xiaozheng" according to the traditional Chinese medicine viewpoint of “micro-zhengji” in the kidney, can significantly delay the progress of CKD, whereas the underlying mechanism remains to be elucidated. Our previous RNA-seq sequencing studies have first revealed that SQK could specifically down-regulate the expression of epithelial membrane protein 3 (EMP3) in CKD mice. Further experiments validation and bioinformatics prediction suggest that there may be a positive feedback loop in the process of RF in which increasing EMP3 expression→TGF-β receptor type 2 (TGFBR2) activated by interacting with EMP3 and leading to TGF-β/Smad3 signaling activation →increasing EMP3 expression transcribed by Smad3. Based on these findings, we aim to investigate the pathogenic role of EMP3 mediated by TGF-β/Smad3 in RF and to elucidate the intervention mechanism of SQK by utilizing coimmunoprecipitation, ultrasound-microbubble based gene delivery, RfxCas13d mediated RNA knocking down techniques, combined with animal and cell experiments. Collectively, the present study will provide important significance in identification of prevention and treatment target of CKD, exploring the role of EMP3 in RF and the molecular mechanism of SQK, and elucidating the inherent pathological mechanism of CKD and the biological connotation of SQK in the treatment of CKD.