The Role Of TRPM2 Channel/NLRP3 Inflammasome In Lung Injury Following Hemorrhagic Shock

Lung is the first affected and most susceptible organs during the episode of hemorrhagic shock.It has been reported that there has been an80%incidence of acute lung injury（ALI）following hemorrhagic shock and the mortality would reach 40%approximately if ALI developed into acute respiratory distress syndrome（ARDS）.Therefore,it is important to illustrate the pathogenesis of ALI for the treatment of severe hemorrhagic shock.Recent studies have shown that nucleotide-binding oligomerization domain-like receptor protein 3（NLRP3）is one of the important mechanisms of ALI during the process of severe hemorrhagic shock.Activation of transient receptor potential melastatin 2（TRPM2）channel mediates Ca²⁺influx and participates in the regulation of many signaling pathways,and activation of TRPM2 channel is involved in the activation of NLRP3 inflammasome,secretion of inflammatory cytokines and the subsequence of inflammatory response induced by various factors.However,the role of TRPM2 channel and TRPM2 channel mediating NLRP3 activation in the pathogenesis of ALI after hemorrhagic shock remain to be clarified.To do it,the present study has observed the changes of lung tissue structure,water contents and the expression of related protein after hemorrhagic shock in TRPM2^-/-and NLRP3^-/-mice.Firstly,the genotypes of wild-type（WT）,NLRP3^-/-and TRPM2^-/-mice were verified.The results showed that the WT mice had specific amplification bands of NLRP3 and TRPM2 genes,while the NLRP3^-/-mice had no specific amplification bands of NLRP3 gene and the TRPM2^-/-mice had no specific amplification bands of TRPM2 gene.At the same time,the NLRP3^-/-mice had no NLRP3 protein expression,and the TRPM2^-/-mice had no TRPM2 protein expression.Then,the model of hemorrhagic shock was prepared according to routing method in our lab（hypotension of 40mm Hg for 90 min,resuscitation performed with whole blood plus equal Ringer’s fluid）in WT,TRPM2^-/-and NLRP3^-/-mice.The survival time between different groups was observed.Subsequently,WT,NLRP3^-/-and TRPM2^-/-mice were randomly divided into sham and shock groups.The lung was harvested at 3h after the end of hemorrhage plus resuscitation or at the corresponding control time point for the nest assay of morphology,water content and myeloperoxidase（MPO）expression.Besides,the levels of reactive oxygen species（ROS）and cytokines IL-1βand IL-18 were detected by ELISA assay.The expression of TRPM2 and NLRP3 in WT mice,and NLRP3 in TRPM2^-/-mice and TRPM2 in NLRP3^-/-mice were detected by the method of Western Blotting.The results indicated that the survival time of NLRP3^-/-and TRPM2^-/-mice after hemorrhagic shock were significantly higher than that of WT mice.Histo-morphological results showed that the lung tissue structure of the sham mice was basically normal.The lung tissue of the shock mice showed thickened pulmonary septum,inflammatory cell infiltration,alveolar edema or congestion as well as alveolar hemorrhage,and increased water content of the lung tissue.Compared with WT mice,TRPM2^-/-and NLRP3^-/-mice after shock had less lung tissue damage and lower lung tissue water content,indicating that knock out of TRPM2 and NLRP3genes alleviated ALI caused by hemorrhagic shock.The results also indicated that the expressions of MPO,NLRP3 and TRPM2 and the levels of ROS,IL-1βand IL-18 in lung tissue of WT sham group mice were obviously lower than those in the WT shock group.In addition,the expression of MPO and the levels of ROS,IL-1βand IL-18 in lung tissues of TRPM2^-/-and NLRP3^-/-shock mice were significantly lower than that of WT shock mice,and the expression of MPO in lung tissues of NLRP3^-/-shock mice was lower than that of TRPM2^-/-shock mice.The expression of NLRP3 in lung tissues of TRPM2^-/-mice was significantly lower than that of WT shock mice,and the expression of NLRP3^-/-shock mice was significantly lower than that of WT shock mice.Finally,in order to further reveal the molecular mechanisms of the interaction of TRPM2 channel and NLRP3 inflammasome,the current study screened protein interactions,common mi RNAs and transcription factors（TFs）focusing on the TRPM2 and NLRP3,using bioinformatics research techniques.Furthermore,the relationship among TRPM2 and NLRP3 gene,SMAD3,and mi RNA were analyzed and the eight-seven related mi RNAs were identified.In conclusion,the TRPM2 channel and NLRP3 inflammasome and their crosstalk are involved in the mechanism of lung injury caused by hemorrhagic shock.In addition,Bioinformatic analyses suggest that the interaction between TRPM2 channel and NLRP3 inflammasome might be related to TFs and mi RNAs,which could provide a foundation for further studies.