Role Of NLRP3 Inflammasome On Post-hemorrhagic Shock Mesenteric Lymph Mediating Acuted Lung Injury

The lung is the most easily affected and manifested organ during the episodes of severe shock. The massive systemic inflammatory response is a key link of the development of acute lung injury(ALI) in this paradigm.A large amount of studies indicate that return to systemic circulation of mesenteric lymph produced in the development of hemorrhagic shock is one of the main contributors of ALI and its mechanism is closely related to inflammation assaults. Inflammasome, one of protein complexes consists of a variety of cytoplasmic proteins, is the center of the inflammatory response. NOD-like receptor family pyrin domaincontaining protein(NLRP) 3 inflammasome can be activated by various molecules, bacterial and viral antigens, and then exerts diverse effects in the development of inflammation conditions, metabolic diseases and so on.NLRP3 is the most extensively studied type of inflammasome and plays a pivotal role in the pathogenesis of ALI induced by various reasons. Recent studies have shown that the activation of NLRP3 is one of the important mechanisms of ALI occured during the process of severe hemorrhagic shock. However, whether the process of shock mesenteric lymph mediating ALI is involved in the the activation of NLRP3, the process that blocking the return of shock mesenteric lymph alleviating the severity of ALI is associated with the activition of NLRP3 is remain to be further investigated.In this study, based on previous results that shock mesenteric lymph drainage blunted ALI, a mouse model of hemorrhagic shock plus fluid resuscitation is introduced and used to observe the influence of shockmesenteric lymph drainage on NLRP3 and its relating signal molecules and the changes of NLRP3 and inflammatory mediators in shock mesenteric lymph. Moreover, to further verify the role of NLRP3 playing in shock mesenteric lymph mediating ALI, the expression of NLRP3 in lung of normal mouse is determined after infusion shock mesenteric lymph venously.In first, eighteen male C57BL/6J mice were randomized to sham shock, hemorrhagic shock(HS), hemorrhagic shock plus mesenteric lymph drainage(shock+drainage) group. All of the animals were general anesthetized and underwent thigh and abdominal surgery. After a 30-min stabilization period, the animals were subjected to sham shock, shock and shock plus mesenteric lymph drainage. In the shock, shock+drainage groups, the arterial blood pressure(MAP) was reduced to 40±2 mmHg within 10 min and maintained at this level for 90 min. After hypotension,the shed blood and an equal volume of Ringer’s solution were perfused within 30 min through right femoral artery. The MAP was observed for 3h after the end of infusion. In shock+drainage group, after infusion completed, the mesenteric lymph duct was cannulated and mesenteric lymph was drained up to 3 h after shock. In the sham shock group, the mice were cannulated and operated as described above, but no blood was withdrawn or infused. At 3 h after infusion or at corresponding time point in each group, lungs from a fixed location were harvested and used for the observation of pulmonary histomorphology and the determination of NLRP3, TLR2, TLR4, MyD88, interleukin(IL)-1β and IL-18. The observations of histomorphology changes showed that there was normal structure in the pulmonary tissue of mice in sham group, the broadening alveolar septum with inflammatory cell infiltration appeared in the lung in the shock group, less pulmonary tissue damage in the shock+drainage group. The results from Western blotting indicated that the expression of NLRP3, TLR2, TLR4 and MyD88 protein in shock group weresignificantly increased when compared with those of sham group. The mesenteric lymph drainage decreased the expression of NLRP3, TLR2 and MyD88 in shock lungs significantly. Results from ELISA indicated that L-1β and IL-18 in shock group were significantly increased when compared with those of sham group. The mesenteric lymph drainage decreased the levels of L-1β and IL-18 in shock lungs significantly.Results from qRT-PCR indicated that the mRNA expression of NLRP3,TLR2, TLR4, and MyD88 in shock lungs increased significantly. The mesenteric lymph drainage decreased the expression of those indicators.Thereafter, normal wild-type mice were conducted anesthesia. Part of these animals was performed sham shock and drainage of mesenteric lymph for 1h. The remainder animals were performed shock and drainage of mesenteric lymph from completed fluid resuscitation up to 3h. The NLRP3 expression in in lymph was detected using western blotting method; the levels of IL-1? and IL-18 in lymph were measured using ELISA method. Results indicated that there were no statistical differences in the expression of NLRP3 and the levels of IL-1? and IL-18 between sham and shock mesenteric lymph.The present study indicates that the drainage of shock mesenteric lymph blunts the expression of TLR2, TLR4 and MyD88 in lung both in protein and mRNA levels, thereby, down-regulates the expression of NLRP3 and decreases the levels of proinflammatory mediators of IL-1βand IL-18. However, no significant differences are found in the expression of NLRP3 and the levels of IL-1? and IL-18 in shock mesenteric lymph compared those in sham lymph. It is thus clear that shock mesenteric lymph inducing activation of NLRP3 and the subsequent inflammatory response mediated by NLRP3 is one of the important mechanisms of ALI occured during the process of severe hemorrhagic shock. These results may provide an experimental evidence for the prevention and treatment of ALI caused by severe shock by targeting mesenteric lymph.