SENP6介导的NLRP3炎症小体的SUMO化修饰在机械通气肺纤维化中的调控作用与机制研究

Mechanical ventilation is an indispensible supportive therapy for patients with acute respiratory failure. However, lung overdistension induced by mechanical ventilation may also cause or aggravate pulmonary fibrosis, which seems to cause high-mortality observed in patients requiring prolonged mechanical ventilation. In our preliminary study, we found that mechanical ventilation upregulated NLRP3 expression in mouse lung tissues in a SUMOylation-dependent manner, as well as lung tissues harvested from mechanically ventilated patients. In addition, we found that gene knockdown of protease of SUMOylation SENP6 significantly aggravated mechanical ventilation-induced pulmonary fibrosis. By analyzing studies of ventilated mouse models and in vitro cyclic stretch-treated pulmonary vascular endothelial cells, it was found that SUMOylation were significantly reduced in the lung tissues of mice subjected to mechanical ventilation. Taken together, these findings suggest that SENP6 modulates the TLR inflammatory pathway to aggravate mechanical stretch-induced lung fibrosis. On the basis of these results, we plan to explore the mechanism of SUMOylation of NLRP3 in mechanical stretch-induced pulmonary fibrosis via double-immunofluorescence, western blot, RNA interference from four aspects: 1) The influence of SUMOylation in lung tissues on the outcome of patients with mechanical stretch-induced pulmonary fibrosis; 2) Verifying the protective effect of SUMOylation of NLRP3 in mechanical stretch-induced pulmonary fibrosis mouse model; 3) Screening of binding sites for NLRP3 inflammasome SUMOylation; 4) detecting major molecular pathways and key proteins that interact with NLRP3. Our findings will shed light on the mechanisms involved in the pathogenesis of mechanical ventilation-induced pulmonary fibrosis, which may provide one of the future treatment options of mechanical ventilation-induced pulmonary fibrosis.

机械通气在提供呼吸支持治疗同时也可能导致肺部纤维化病变，增加危重患者的死亡率。通过对临床样本和动物模型实验结果分析,我们发现机械通气可以激活肺组织NLRP3炎性体的表达，同时伴有NLRP3-SUMO化水平降低，基因敲低SENP6可以减轻机械通气诱导的肺部纤维化病变。通过进一步实验，我们发现SENP6可能通过调节TLR炎性信号通路，加重机械牵拉诱导的肺纤维化病变。据此，本课题组拟利用免疫荧光染色、免疫蛋白印迹、RNA干扰等技术手段，从4个层面开展研究：(1)探索NLRP3-SUMO化水平对机械通气相关肺纤维化患者疾病转归的影响；(2)验证NLRP3-SUMO化水平对机械通气肺纤维化模型小鼠的保护作用；(3)筛选NLRP3-SUMO化的结合位点；(4)探索与NLRP3相互作用的转录因子和关键蛋白。本课题的实施将有助于阐明机械通气相关肺纤维化的发生机制，有望为肺部纤维化病变的临床防治提供新思路。

机械通气（MV）在为全身麻醉和危重患者提供呼吸支持治疗的同时，还可能导致或进一步加重肺损伤。大量临床和实验室研究已证实机械通气诱导的肺损伤（VILI）可增加危重患者的死亡率，因此，VILI的早期诊断对VILI的预防和治疗具有重要意义。通过对临床样本和动物模型进行免疫组化、免疫蛋白印迹、RNA干扰、实时定量PCR等技术分析，我们发现大潮气量机械通气可引起肺部炎症，激活肺组织NLRP3炎性体的表达，同时，模型中的小鼠肺组织和血浆的代谢组学分析结果提示MV可导致小鼠γ-氨基丁酸（GABA）系统和尿素循环失调，柠檬酸循环（CAC）紊乱和氧化还原失衡，可能作为特定的代谢生物标志物来帮助识别VILI的表现和进展，并作为VILI潜在的治疗靶点，为临床实践中机械肺通气潮气量的设定提供及时预警和参考。