TLR4/NF-κB信号通路对肾型传支病毒致痛风鸡肾脏炎症的调控作用及机制

Avian gout, also known as urate deposition, is the most common disease in poultry. The nephrotrophic strains of infectious bronchitis virus infection is on of the main causes of avian gout. However, the pathogenesis of chicken gout induced by nephrotrophic strains of infectious bronchitis virus is still unclear. It is known that TLR4/NF-κB signaling pathway is an important pathway for the regulation of inflammatory responses. Our research group have found that the kidney with chicken gout induced by nephrotrophic strains of infectious bronchitis virus showed nephritis nephropathy. The results of high-throughput transcriptome sequencing analysis of gout chicken kidney caused by nephrotrophic strains of infectious bronchitis virus showed that TLR4 and NF-κB gene expression was significantly up-regulated in Toll-like receptor signaling pathway. Therefore, this study uses RNA interference, Real-time PCR, Western blotting, immunohistochemistry, computer image analysis techniques and clinical pathology detection technology to study the role of TLR4/NF-κB signaling pathway, in vivo and in vitro, and to reveal the the regulation and mechanism of TLR4/NF-κB signaling pathway on kidney inflammation of chicken gout induced by infectious bronchitis virus in nephrotrophic strains, and partially elucidate the potential mechanism, provide a theoretical basis for the precention and drug targets of chicken gout. Furthermore, this study will solve the industrial problem and contribute knowledge to the poultry science.

禽痛风又称尿酸盐沉积症，是家禽常见多发病。肾型传支病毒感染是目前生产上鸡痛风发生的常见病因之一。但肾型传支病毒致鸡痛风的发病机制并不清楚。TLR4/NF-κB信号通路是机体重要的调节炎症反应的通路。本课题组前期研究表明，肾型传支病毒致痛风鸡肾脏表现为肾炎肾病性变化，痛风鸡肾脏高通量转录组测序数据分析结果显示Toll样受体信号通路中TLR4和NF-κB基因表达显著上调。因此，本研究利用RNA干扰、荧光定量PCR、Western blotting、免疫组化、计算机图像分析技术和临床病理学等检测技术，体内和体外试验相结合，研究TLR4/NF-κB信号通路与肾型传支病毒所致痛风鸡肾脏炎症的相关性，从而揭示TLR4/NF-κB信号通路对肾型传支病毒致痛风鸡肾脏炎症的调控作用及机制，进而部分阐明肾型传支病毒致鸡痛风的机理，并为其有效防治提供新思路和药物作用的靶点，将对养鸡业发展具有重要学术和生产意义。

痛风又称为尿酸盐沉积症，是一种尿酸代谢性疾病，引起尿酸盐沉积症的原因多达 20 余种，其中肾型传染性支气管炎病毒（NIBV）感染是我国目前生产上鸡发生痛风的常见原因之一。NIBV致痛风鸡肾脏表现为肾炎肾病性变化，主要损害肾小管上皮细胞，但其发病机制并不清楚。TLR4/NF-KB 信号通路在炎症信号的传递中发挥着重要作用。本项目系统研究了人工复制NIBV致痛风鸡的临床病理学变化；NIBV致痛风对鸡肾脏TLR4/NF-κB 信号通路及其相关基因表达的影响；NIBV感染致痛风鸡多组学的研究；NIBV感染对原代鸡肾小管上皮细胞的影响；NIBV感染对鸡原代肾小管上皮细胞对模式识别受体的先天免疫调控及细胞焦亡的影响；TRAF6 抑制剂 C25-140 对病毒在肾小管上皮细胞的复制、MDA5/NF-κB和细胞焦亡信号通路相关因子的影响；NIBV感染诱发鸡原代肾小管上皮细胞发生细胞程序性坏死及凋亡；RIPK3的抑制剂GSK872对病毒在肾小管上皮细胞的复制、程序性坏死和细胞凋亡的影响。结果表明，NIBV感染成功诱发了鸡痛风，发病过程中，造成肾脏结构和功能受损， NIBV可在鸡的多个组织中进行复制，肾脏病毒复制最为活跃；NIBV感染激活了TLR4/NF-κB 信号通路，促进了炎症因子的活化；多组学研究发现NIBV感染显著下调过氧化物酶体的作用，并显著激活模式识别受体信号通路；嘌呤代谢发生改变并显示出尿酸合成增加的趋势；能够引起的盲肠菌群结构的改变并降低微生物的多样性；抑制TRAF6 的活性可以减少NIBV的复制，显著改善NIBV感染激活的鸡原代肾小管上皮细胞的模式识别受体所调控的先天免疫和细胞焦亡；抑制 RIPK3 的活性可以减少NIBV的复制、缓解细胞程序性坏死并抑制细胞凋亡。说明，NIBV感染诱发鸡痛风过程中，激活肾脏TLR4模式识别受体信号通路，从而诱导NF-κB介导的免疫应答反应，导致炎症因子活化，促进肾脏炎症及损伤，最终造成肾脏尿酸排泄障碍，这可能是NIBV致痛风鸡肾脏损伤的发病机制之一。总之，通过体内外研究，部分阐明了NIBV致痛风鸡肾脏损伤的发病机制，为禽痛风的防治提供了一定的理论基础。