MAPK/FMOD通路在氧化应激介导慢性胰腺炎纤维化中的机制研究

Chronic pancreatitis (CP) is a chronic irreversible disease, the most typical histopathological features of CP is pancreatic fibrosis, which is closely related to the activation of pancreatic stellate cells (PSCs). Oxidative stress (OS) is playing a key role in the activation of PSCs and pancreatic fibrosis, MAPK is one of the main signaling pathways of OS, but the downstream molecular mechanism of OS-activated MAPK is unclear. Fibromodulin (FMOD) is an OS-sensitive proteoglycan, and has an important functional role in tissue repair and fibrogenesis, however, little is known about its potential role in the development of pancreatic fibrosis. Our preliminary study confirmed that the expression levels of FMOD increased in both the serum and pancreatic tissue of CP, and bioinformatics analysis showed that FMOD promoter contained AP-1 binding sites, which is the common transcription factor of three well-characterized MAPK signaling pathways. In our study, we use cell transfection, cell behavior detection, RT-RCR, Western blotting and immunohistochemistry techniques to confirm the molecular mechanism of MAPK/FMOD pathway in OS-mediated activation of PSCs induced fibrosis of CP in cellular, animal and organizations levels. This study may further elucidate the pathogenesis of OS-activated PSCs and pancreatic fibrosis, and provide a novel approach to treat CP.

慢性胰腺炎（CP）是一种慢性不可逆性疾病，主要病理特征为胰腺纤维化，其与胰腺星形细胞（PSCs）活化密切相关。氧化应激（OS）在各种原因引起的PSCs活化及胰腺纤维化中均扮演重要角色，MAPK是OS主要的信号通路之一，然而OS激活MAPK信号通路后是如何介导胰腺纤维化的，其下游机制尚不明确。纤调蛋白（FMOD）是一种OS敏感分子，其参与了组织修复及纤维化过程，目前尚无FMOD与胰腺纤维化的研究。前期预实验发现CP患者血清及组织中FMOD的表达较正常人明显上调，生物信息学分析显示FMOD的启动子含有MAPK信号通路下游转录因子AP-1的结合位点。在此基础之上，本课题拟采用细胞转染、小分子干扰、体内外实验等技术深入探讨MAPK/FMOD通路在OS介导PSCs活化，进而促进胰腺纤维化发生中的作用机制。本课题的开展有助于进一步阐明OS活化PSCs及促胰腺纤维化的分子机制，并为治疗CP提供新的靶点。

慢性胰腺炎（CP）是一种不可逆性疾病，其与胰腺星形细胞（PSCs）活化密切相关。纤调蛋白（FMOD）是一种氧化应激敏感分子，其参与了组织修复与纤维化过程，然而FMOD与胰腺纤维化的关系尚不清。通过本课题研究首先采用DBTC单次尾静脉注射成功构建了慢性胰腺炎模型，CP组MDA含量明显高于对照组（2.63±1.42 vs. 1.50±0.64, P=0.046）证实氧化应激参与成模过程，通过免疫组化及Western Blot 实验发现FMOD、α-SMA、col I及MAPK信号通路磷酸化蛋白表达在CP组明显高于对照组，说明FMOD及MAPK信号通路与胰腺纤维化有关。接着在体外实验证实OS能促进PSCs活化，经MND处理PSCs后FMOD、α-SMA 及col Іa1 mRNA相对表达分别为3.39±1.17（P=0.036）、2.20±0.52（P=0.023）、2.53±0.38（P=0.017）；并且OS促进PSCs活化依赖于FMOD表达，下调FMOD表达后，MND促α-SMA 及col Іa1 表达作用明显下降，α-SMA 及col Іa1相对mRNA值分别为（2.07±0.11 vs. 3.64±0.78, P=0.026; 1.59±0.38 vs. 2.48±0.11, P=0.018），WB显示α-SMA 及col Іa1蛋白水平也明显降低，免疫荧光结果也发现MND+sh-FMOD组相比MND组α-SMA荧光值也明显降低（1.75±0.32 vs. 0.87±0.59, P=0.010）；通过小分子干扰实验发现MND+SP600125组FMOD、α-SMA及col Іa1表达较MND组明显降低，荧光素酶报告基因实验显示AP-1表达质粒组相对空载荧光强度明显增加（6.25±1.52 vs. 1.00±0.23, P<0.001），CHIP实验结果显示不转染AP-1慢病毒的对照组没有扩增出FMOD启动子区域的片段；而转染了AP-1慢病毒的实验组，检测到FMOD启动子区域片段，且富集度较阴性对照组显著升高（34.51±2.26 vs. 1.06±0.11, P<0.001）。最终证实JNK/FMOD信号通路参与了MND促PSCs活化作用，进而促进了胰腺纤维化。通过本课题阐明了OS介导PSCs活化及促进胰腺纤维化的分子机制，并为治疗CP提供了新的思路。