骨髓间充质干细胞外泌体抑制NLRP3和AIM2炎症小体激活改善体外循环急性肺损伤

Exosome from BMMSC Attenuates Cardiopulmonary Bypass-Induced Acute Lung Injury via Inhibiting NLRP3 and AIM2 Inflammasome Activation

摘要: 目的：探究骨髓间充质干细胞外泌体对体外循环所致急性肺损伤的作用及机制。方法：大鼠分为假手术组(Sham组)，体外循环1小时建立急性肺损伤模型(ALI组)及骨髓间充质干细胞外泌体治疗组(BMMSC-Exo组)，HE染色评估肺组织病理改变；ELISA检测肺泡灌洗液中炎症因子TNF-α、IL-6的表达情况。体外培养AM NR8383肺泡巨噬细胞系，按处理方式不同分为对照组(Control组)、LPS模拟肺损伤组(LPS组)以及外泌体治疗组(BMMSC-Exo组)，激动剂组，激动剂 + 外泌体治疗组，通过Western blot检测炎症小体蛋白、Cleaved caspase 1的表达情况，通过ELISA检测IL-1β和IL-18的释放情况。结果：与Sham组相比，ALI组肺组织炎症浸润增加，肺组织损伤评分明显升高(P < 0.05)，肺泡灌洗液中TNF-α、IL-6的表达增加(P < 0.05)；BMMSC-Exo组则可见肺部炎症缓解，肺组织损伤评分则较损伤组明显降低(P < 0.05)，肺泡灌洗液中TNF-α、IL-6的表达减少(P < 0.05)。体外研究提示：LPS组肺泡巨噬细胞中炎症小体NLRP3和AIM2的蛋白表达均增加；而BMMSC-Exo组中NLRP3和AIM2的蛋白表达均明显降低；炎症小体激动剂组增加Cleaved caspase 1、IL-18及IL-1β的表达，而给予外泌体处理后，上述蛋白的表达均被明显抑制(P < 0.05)。结论：体外循环所致急性肺损伤中炎症小体NLRP3和AIM2的表达增加，骨髓间充质干细胞外泌体可通过阻断炎症小体的激活，从而减少其下游蛋白及炎症因子的释放，从而改善体外循环所致急性肺损伤炎症反应。

Abstract: Objective: To investigate the effect and mechanism of exosomes derived from bone marrow mesenchymal stem cells on acute lung injury induced by cardiopulmonary bypass (CPB). Methods: The rats were divided into Sham operation group, acute lung injury (ALI) model established by cardiopulmonary bypass (CPB) for 1 hour (ALI group) and bone marrow mesenchymal stem cell exosome treatment group (BMMSCS-Exo group). The pathological changes of lung tissue were evaluated by HE staining. ELISA was used to detect the expression of TNF-α and IL-6 in bronchoalveolar lavage fluid. AM NR8383 alveolar macrophage cell line was cultured in vitro. According to different treatment methods, the cells were divided into Control group (Control group), LPS simulated lung injury group (LPS group) and exosome treatment group (BMMSCS-Exo group), agonist group, agonist + exosome treatment group. Western blot was used to detect the expression of inflammasome proteins and Cleaved caspase 1, and ELISA was used to detect the release of IL-1β and IL-18. Results: Compared with the Sham group, the ALI group had significantly increased inflammatory infiltration in lung tissue, significantly increased lung injury score (P < 0.05), and significantly increased expression of TNF-α and IL-6 in alveolar lavage fluid (P < 0.05). Compared with the injury group, the BMSC-Exo group had significantly reduced lung inflammation, significantly reduced lung tissue injury scores (P < 0.05), and significantly reduced expression of TNF-α and IL-6 in alveolar lavage fluid (P < 0.05). In vitro study showed that the protein expression of NLRP3 and AIM2 in alveolar macrophages increased in the LPS group. However, the protein expression of NLRP3 and AIM2 in BMMSCS Exo group was significantly decreased. The inflammasome agonist group increased the expression of Cleaved caspase 1, IL18, and IL-1β, while exosome treatment significantly inhibited the expression of these proteins (P < 0.05). Conclusion: The expression of inflammasome NLRP3 and AIM2 is increased in cardiopulmonary bypass (CPB)-induced acute lung injury. Exosomes derived from bone marrow mesenchymal stem cells can reduce the release of downstream proteins and inflammatory factors by blocking the activation of inflammasome, thereby improving the inflammatory response of CPB induced acute lung injury.