基于Nrf2调控PINK1介导线粒体自噬的金水缓纤方治疗肺纤维化机制

Pulmonary fibrosis (PF) is a serious threat to human health and the lack of effective therapeutic agents. Myofibroblast Differentiation is the main pathological link, Transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) reduction and mitophagy deficiency play important roles in this process. Based on the pathogenesis of Zhengxu Luobi Jisun, we developed the Jinshui Huanxian formula (JHF) and it's effect is definite in clinical. Based on the regulation of Nrf2/PINK1 mediated mitophagy inhibition of myofibroblast activation may be the potential pathway of the JHF. The following researches will be carried out: ①based on bleomycin induced PF rat model, immunofluorescence, qPCR, Western blot and other techniques were used to observe the efficacy of JHF and its effect on Nrf2 and mitophagy. ②Based on the TGF-β1-induced cell model in MRC-5 cell, serum pharmacology, cell culture and other techniques were used to observe the effects of the JHF on the activation of myofibroblast and mitophagy. The effect of Nrf2/PINK1 on the regulation of mitophagy was investigated by Nrf2siRNA and PINK1 siRNA. The aim of this study is to explore the mechanism of the treatment of PF by JHF via activating Nrf2 to mediate the activation of mitophagy inhibiting myofibroblast differentiation.

肺纤维化严重危害人类健康，治疗措施有限。肌成纤维细胞活化是其主要病理环节，Nrf2降低和线粒体自噬不足起重要作用。前期基于“正虚络痹积损”病机而拟定的以补益肺肾为主，佐以活血化痰的金水缓纤方疗效确切。调控Nrf2促进PINK1介导线粒体自噬，以抑制肌成纤维细胞活化可能是金水缓纤方潜在作用途径。拟开展：①采用博来霉素诱导肺纤维化大鼠模型，应用免疫荧光、qPCR、Western blot等技术，观察金水缓纤方疗效及对Nrf2、线粒体形态与能量和自噬的影响。②基于TGF-β1诱导MRC-5细胞模型，采用血清药理学、细胞分子生物等技术，观察金水缓纤方对肌成纤维细胞活化和线粒体自噬的影响；采用RNA干扰Nrf2和PINK1表达，考察Nrf2调控PINK1介导线粒体自噬在金水缓纤方抑制肌成纤维细胞活化中的作用。从激活Nrf2介导线粒体自噬以抑制肌成纤维细胞活化角度揭示金水缓纤方治疗肺纤维化的机制。

成纤维细胞活化是肺纤维化发生发展的主要病理环节，氧化/抗氧化失调和线粒体自噬不足在其中起关键作用。本项目基于临床疗效确证的金水缓纤方及其组分方，从激活Nrf2促进线粒体自噬抑制成纤维细胞活化角度揭示金水缓纤方及其组分方治疗肺纤维化的机制。研究发现：①金水缓纤方及金水缓纤组分方显著缓解肺纤维化大鼠的纤维化程度，抑制氧化应激，促进线粒体自噬。金水缓纤方及金水缓纤组分方均能够显著降低博来霉素（BLM）诱导肺纤维化大鼠的炎细胞浸润，减轻肺泡结构破坏程度，降低肺组织HYP含量及Col-Ⅰ、Col-Ⅲ、α-SMA的表达；增强肺组织中Nrf2、HO-1、T-SOD水平，降低MDA含量。金水缓纤组分方能够促进肺纤维化大鼠线粒体自噬水平，有效改善肺纤维化大鼠肺组织成纤维细胞中的线粒体肿胀、嵴断裂及自噬缺陷等情况，并能够提高线粒体自噬相关蛋白PINK1、Park2、LC3B的表达，降低P62的表达。②金水缓纤组分方抑制成纤维细胞活化，上调Nrf2和PINK1/Parkin通路。建立了TGF-β1诱导HFL的成纤维细胞活化模型，金水缓纤组分方呈浓度依赖性抑制HFL中ACTA2、FN1和COL1A1 mRNA表达。30.63 μg/mL 和61.25μg/mL的金水缓纤组分方均能有效抑制α-SMA、FN和COL1蛋白表达，增强Nrf2、NQO1和HO-1的蛋白表达，显著增加TOM20与LC3B共定位点，提升PINK1、Parkin2蛋白表达，61.25 μg/mL金水缓纤组分方组能够显著增加LC3B Ⅱ蛋白表达。③金水缓纤方组分方激活Nrf2增强PINK1/Park2介导的线粒体自噬进而抑制肺纤维化。HFL转染 Nrf2siRNA后，30.63 μg/mL和61.25 μg/mL金水缓纤组分方组Nrf2、NQO1表达未见升高；α-SMA、FN表达未见显著降低，线粒体相关蛋白PINK1、Park2表达未见显著升高。. 综上，本研究证实了金水缓纤方及其组分方的疗效，金水缓纤组分方可通过激活Nrf2增强PINK1/Park2介导的线粒体自噬进而抑制肺纤维化。该研究为金水缓纤方的临床应用及新药研发提供依据。