NS1在HMGB1介导RSV感染后期气道炎症中的作用及机制

During the later stage of RSV infection, HMGB1 was significantly increased wihch could incude persistent airway inflammation and AHR by promoting Th2 cytokines. In the nucleus, HMGB1 as an architectural factor, sustains chromosome structure and stability, and its release was related with the stability of nucleosome. NS1 was the first expressed RSV protein, with the fuction of ubiquitination may ubiquitinated Histone, led to relaxation of nucleosome. NS1 may induced HMGB1 release, thus take part in airway inflammation and AHR, but the mechanism are not clear. So we plan to confirm that NS1 take part in airway inflammation and AHR by plasmid induced siRNA knockdown in vivo. By using plasmid induced overexpression, immunefluorescence and Western blot we want to investigate that if the NS1 was critical in HMGB1 release. Then we use Immunoprecipitation to revel that NS1 binded to H1 and ubiquitinated H1, thus led to HMGB1 translocate extracellular induce Th2 cytokine. By carrying out this funds will confirm the pathogenic mechanism of RSV-induced airway inflammation, provide a solid theoretical basis for seek out a new method to treating RSV induced airway inflammation.

呼吸道合胞病毒（RSV）感染后期内源性损伤相关分子HMGB1升高，可促进Th2类细胞因子产生，引起持续的气道炎症及AHR。HMGB1是染色质的结构因子，可与组蛋白H1结合，其从胞核解离与核小体稳定性密切相关。非结构蛋白1（NS1）是RSV感染后最先转录的蛋白成分，具有泛素酶特性，可泛素化组蛋白致核小体松弛，但NS1能否通过泛素化组蛋白H1促进HMGB1的释放尚不清楚。本项目拟采用siRNA基因沉默、NS1过表达、IP、泛素化等方法进一步探明RSV感染后期NS1是否通过泛素化组蛋白H1，促进HMGB1自胞核释放至胞外，致Th2类细胞因子升高，最终介导RSV感染后期气道炎症及AHR。研究结果将进一步揭示RSV感染后期气道炎症及AHR的形成机制，为防治RSV感染后远期相关气道疾病提供新的可能的干预靶点。

RSV感染后期内源性损伤因子HMGB1升高，介导RSV感染后期气道炎症及气道高反应。阻断HMGB1后RSV感染后期气道炎症及气道高反应降低。但RSV感染后HMGB1的释放机制并不清楚。HMGB1在胞核内与核小体中组蛋白H1结合，其从胞核内释放与核小体的稳定性密切相关。非结构蛋白NS1是RSV感染后最先表达且持续增高的蛋白，具有泛素酶特性，可能通过影响H1而促进HMGB1的释放，进而影响RSV感染后期的气道炎症。因此本研究主要从体内外探索NS1对HMGB1释放的影响及其对RSV感染后期气道炎症的影响。体内实验发现沉默NS1后RSV感染后期气道炎症降低，HMGB1水平下降，同时未发现Th2类因子改变。体外实验发现RSV感染后HMGB1表达水平增高，细胞上清中HMGB1分泌增加。过表达NS1后HMGB1与组蛋白H1表达水平均增加，胞浆胞核HMGB1表达均增加。沉默NS1后HMGB1表达水平降低，胞浆及胞外释放的HMGB1降低。通过IP发现HMGB1与H1有直接结合作用，RSV感染后HMGB1与H1结合降低。NS1可能通过促进HMGB1表达，降低HMGB1与H1的结合，促进其释放而发挥抗炎作用。此外，NS1过表达之后可抑制激素的抗炎作用，进一步发现NS1与宿主细胞内组蛋白去乙酰化酶SIRT1有直接结合作用。抑制SIRT1后NS1蛋白水平降低，提示SIRT1可调控NS1蛋白降解。研究结果首次发现RSV非结构蛋白NS1能够促进HMGB1表达和释放，且能影响RSV感染后期气道炎症。而目前RSV感染引起的气道炎症及气道高反应尚无特异性的治疗方案及疫苗。NS1作为RSV非结构蛋白，近年来越来越受到重视，深入对NS1的研究有助于深入了解RSV感染后的病理生理以及对RSV感染后的防治提供新的思路及干预靶点。