| Objective : To investigate whether the activation of NLRP3 inflammasome pathway is associated with the upstream regulation mechanism of mitophagy in acute lung injury（ALI）induced by intestinal ischemia-reperfusion（II/R）.To clarify whether dexmedetomidine（DEX）has lung protective effect,whether it can effectively reduce the inflammatory response of lung tissue in rats with ALI induced by II / R,and whether the lung protective mechanism of DEX is related to enhancing the level of mitophagy,improving mitochondrial function and inhibiting the activation of NLRP3 inflammasome in lung tissue of rats with ALI induced by II/R;it was further clarified whether the inhibitory effect of DEX on NLRP3 inflammasome pathway depended on mitophagy mediated by PINK1/Parkin signaling pathway.Methods : SD rats were randomly divided into 4 groups（n=7）.Sham group : only superior mesenteric artery（SMA）was exposed;model group（II/R）: blocking SMA for1 hour and reperfusion for 2 hours;DEX pretreatment model group（II/R+DEX）:ischemia-reperfusion was performed after DEX intervention;DEX and3-methyladenine pretreatment model group（II/R+DEX+3-MA）: DEX and 3-MA intervention after ischemia-reperfusion.The model of intestinal ischemia reperfusion injury was established by clamping SMA for 1 hour and reperfusion for 2 hours.The myeloperoxidase（MPO）activity,malondialdehyde（MDA）content,mitochondrial reactive oxygen species（mt ROS）content and wet/dry weight ratio（W/D）in lung tissues of rats in each group were detected.HE staining was used to evaluate the pathological changes of lung tissue and lung injury score.The expressions of tumor necrosis factor-α（TNF-α）,interleukin-18（IL-18）and IL-1β in lung tissue and serum were detected by enzyme-linked immunosorbent assay（ELISA）.Western Blot was used to detect the expressions of NLRP3,apoptosis-related spotted protein（ASC）,cysteine protein hydrolase-1（Caspase-1）and mitophagy-related protein PTEN-induced hypothetical kinase 1（PINK1）,E3 ubiquitin ligase（Parkin）,Beclin-1 and microtubule-related protein light chain 3-II（LC3-II）/LC3-I in lung tissue.Results :1.Changes of lung injury degree,mitophagy and NLRP3 inflammasome pathway in II/R model rats.1.1 Degree of lung injury : Compared with the Sham group,the alveolar structure in the lung tissue of rats in the II/R group was significantly damaged,alveolar rupture,bleeding,and a large number of inflammatory cell infiltration were observed.The pulmonary interstitial was significantly thickened,and the alveolar cavity was fused.The W/D value,lung injury score and MPO activity were significantly increased（P<0.05）.In addition,the expression of TNF-α in lung tissue and serum of rats was significantly increased（P<0.05）.1.2 Mitochondrial autophagy : Compared with Sham group,the expression of LC3-II/I and Beclin-1 protein in lung tissue of II/R group was significantly increased（P<0.05）.PINK1 and Parkin protein expression were also significantly increased（P<0.05）.1.3 NLRP3 inflammasome pathway : Compared with the Sham group,the expressions of NLRP3,ASC and Caspase-1 proteins in the lung tissue of rats in the II/R group were significantly increased（P<0.05）.In addition,the expressions of IL-1β and IL-18 in lung tissue and serum of rats were significantly increased（P<0.05）.2.Effect of DEX on lung injury in II/R model rats.Compared with the II/R group,the lung tissue of rats in the II/R+DEX group showed relatively slight lung injury.The alveolar exudation,inflammatory cell infiltration and alveolar septum in the II/R+DEX group were lighter than those in the II/R group,and the W/D value,lung injury score and MPO activity were significantly decreased（P<0.05）.In addition,the expression of TNF-α in lung tissue and serum of rats decreased significantly（P<0.05）.3.Regulation of DEX on mitochondrial autophagy in II/R model rats.Compared with the II/R group,the expression levels of LC3-II/I and Beclin-1 in the lung tissue of rats in the II/R+DEX group were further increased（P<0.05）.The expression of PINK1 and Parkin protein was further increased（P<0.05）.4.Effect of DEX on mt ROS and MDA in lung tissue of II/R model rats.Compared with Sham group,mt ROS and MDA levels in lung tissue of II/R group were significantly increased（P<0.05）.Compared with II/R group,mt ROS and MDA levels in lung tissue of II/R+DEX group decreased significantly（P<0.05）.5.Effect of DEX on NLRP3 inflammasome pathway in II/R model rats.Compared with the II/R group,the expressions of NLRP3,ASC and Caspase-1 in the lung tissue of rats in the II/R+DEX group were significantly decreased（P<0.05）.In addition,the expression levels of IL-1β and IL-18 in lung tissue and serum of rats were significantly decreased（P<0.05）.6.Effects of DEX and mitophagy inhibitor on mitophagy in II/R model rats.Compared with II / R + DEX group,the expression of LC3-II / I and Beclin-1 protein in lung tissue of II/R+DEX+3-MA group was significantly decreased（P<0.05）,and the expression of PINK1 and Parkin protein was also significantly decreased（P<0.05）.7.Effect of DEX combined with mitophagy inhibitor on NLRP3 inflammasome pathway in II/R model rats.Compared with II/R+DEX group,the expression of NLRP3,ASC and Caspase-1 protein in lung tissue of II/R+DEX+3-MA group was significantly increased（P<0.05）,and the expression of IL-1β and IL-18 in lung tissue and serum of rats was significantly increased（P<0.05）.Conclusion :1.In II/R injury model,the level of mitophagy in rat lung tissue is relatively low,and the NLRP3 inflammasome pathway is overactivated,which may be one of the important mechanisms of ALI induced by II/R.2.DEX can effectively reduce the inflammatory response of lung tissue in ALI rats induced by II/R,and has certain lung protection effect.3.The pharmacological mechanism of DEX lung protection may be related to up-regulating PINK1/Parkin signaling pathway-mediated mitophagy in lung tissue of II/R model rats,reducing mt ROS production,improving mitochondrial function,inhibiting NLRP3 inflammasome activation and reducing the release of inflammatory factors.4.The inhibitory effect of DEX on NLRP3 inflammasome pathway depends on its regulation of mitophagy mediated by PINK1/Parkin signaling pathway. |
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