晚期氧化蛋白产物通过TNF/TNFR信号通路激活NF-κB促进滑膜细胞异常增殖的实验研究

Rheumatoid arthritis is an important disease which has an effect on the health of worldwide people. The excess proliferation of fibroblast-like synoviocytes (FLS) plays an important role in the genesis and development of RA. Advanced oxidation protein products (AOPP), which are formed by oxidative stress, are prevalent in RA patients. Previous studies have already demonstrated that the accumulation of AOPP in the body can raise the level of oxidation stress which can be a cause of many diseases. Our previous study had demonstrated AOPP take part in inflammatory reaction in RA by making TNF-α secretion via activating the signaling pathway of NF-κB. While TNF-α mediated Tumor necrosis factor/ Tumor necrosis factor receptor (TNF/TNFR) can active NF-κB to make abnormal proliferation of FLS happen. The preliminary experiments have demonstrated that AOPP could induce the abnormal proliferation proliferation of FLS but the specific molecular mechanism remained unclear. Therefore, we speculate that AOPP can induce cell proliferation of FLS through activating the signaling pathway of NF-κB which could be the new pathogenesis of RA. First, this project will study the mechanism of abnormal proliferation of FLS by CCK8. After that, co-immunoprecipitation will be used to explore whether AOPP can take part in abnormal proliferation of FLS via TNF/TNFR signaling pathway. Finally, we will use antagonist and the technology of siRNA to interdict TNF/TNFR signaling pathway in order to observe the AOPP’s effect on FLS. We expect this study can give us some new sights of AOPP on inducing abnormal proliferation of FLS and set up new methods for treatment of RA patients.

滑膜细胞（FLS）异常增值是类风湿性关节炎（RA）发生的重要环节。RA患者体内存在晚期氧化蛋白产物（AOPP）的潴留。研究证实AOPP可激活FLS内的NF-κB促进TNF-α分泌参与RA炎症反应的发生。而TNF-α介导的肿瘤坏死因子受体家族（TNF/TNFR）可激活NF-κB，在FLS异常增殖中扮演重要角色。预实验显示AOPP可促进FLS异常增殖，但具体分子机制尚不清楚。我们推测AOPP可以通过TNF-α介导的TNF/TNFR激活NF-κB促进FLS异常增殖，这可能是RA发病的新机制。本项目首先采用CCK8等方法观察AOPP对FLS异常增殖的作用，接着通过免疫共沉淀等手段验证AOPP是否可通过TNF/TNFR信号通路参与FLS异常增殖，最后利用拮抗剂及siRNA干扰技术不同层面阻断TNF/TNFR通路观察AOPP对FLS的影响，这将为揭示AOPP在FLS异常增殖的分子机制提供实验研究基础。

滑膜细胞（FLS）异常增殖是导致RA滑膜增生的重要原因之一。RA患者体内存在晚期氧化蛋白产物（AOPP）的潴留。我们采用SD大鼠FLS为模型，利用AOPP进行体外干预，我们发现伴随着AOPP浓度的升高，FLS出现异常增殖的现象。接着，我们利用免疫荧光法观察在AOPP干预下TNFR1在FLS的表达情况。我们发现，伴随着AOPP的浓度升高，TNFR1在FLS中表达越来越强，而且我们还检测到TNF-α与TNFR1结合也越来越强。这说明AOPP可以通过促进FLS分泌TNF-α激活TNF/TNFR信号通路。接着，我们分别用TNF-α拮抗剂英夫利昔单抗（Infliximab）、依那西普（Etanercept）以及TNFR1封闭肽预先处理FLS，再用AOPP进行刺激，我们发现TNFR1在FLS的表达减弱。同样地利用免疫共沉淀法也发现TNF-α与TNFR1相互作用情况较未封闭、阻断前弱，这进一步确定了AOPP激活了TNF/TNFR信号通路。.接着我们发现在AOPP的干预下，TNF/TNFR信号通路的下游分子TRADD、RIP、TRAF2在RNA水平和蛋白水平表达均增加。同样地，Infliximab、Etanercept以及TNFR1封闭肽均能抑制这些蛋白和mRNA的表达，这证实了AOPP可以激活TNF/TNFR信号通路的下游关键信号。以上实验结果均可被TRADD siRNA、RIP siRNA、TRAF2 siRNA、IKKβsiRNA、NF-κB特异抑制剂（SN50）所抑制，以上实验研究结果可以最终确定，AOPP是通过激活TNF/TNFR信号通路，进而激活NF-kB，促进滑膜细胞的异常增值的。本项目为AOPP在FLS异常增殖的分子机制提供了理论研究依据，为RA发病机制提供新理论，为RA的防治探索了新途径。.此外，类风湿关节炎的患者存在骨质疏松症，而类风湿模型大鼠体内存在AOPP升高的现象，那么AOPP会不会导致大鼠骨质疏松的发生呢？我们利用AOPP作用于SD大鼠，检测大鼠胫骨、股骨的骨强度、骨小梁结构以及骨代谢指标，我们发现AOPP能够加速大鼠骨量丢失，但这个过程不能被抗氧化酶SOD所抑制。以上结论提示AOPP可能参与了RA大鼠骨质疏松的发生发展，这为我们后续的研究-类风湿性关节炎导致的骨质疏松提供了研究基础，为我们今后行抗骨质疏松提供新靶点。