棕榈酰化修饰的NOD_(2)在失血性休克动物模型中的作用

The role of palmitoylated NOD_(2)in animal model of hemorrhagic shock

目的探讨核苷酸结合寡聚化结构域2(NOD_(2))在失血性休克及缺血再灌注时对肺损伤的作用及机制。方法10只NOD_(2)基因敲除SD大鼠,经失血性休克模型制备后设为对照组(复苏初期予以生理盐水)。30只普通SD大鼠经失血性休克动物模型制备后,按随机数字表法分为3组:空白组(复苏初期予以生理盐水)、棕榈酰化修饰酶(PAT)抗体组(anti-PAT组,复苏初期予以PAT抗体)及NOD_(2)抗体组(anti-NOD_(2)组,复苏初期予以NOD_(2)抗体),每组10只。经复苏24 h采集血液标本后将其处死并采集肺组织。在实验开始、休克初期及再灌注后2 h分别进行血气分析。检测肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β、IL-6及肺组织丙二醛(MDA)、髓过氧化物酶(MPO)水平。免疫印迹法检测肺组织胞核高迁移率族蛋白B(HMGB1)、棕榈酰化转移蛋白(ZDHHC5)及NOD_(2)表达水平。荧光显微镜及光镜下观察肺组织病理切片。结果240 min时空白组氧分压[p(O2)]明显低于其他3组(P<0.05);30 min及240 min时空白组乳酸水平高于其他3组(P<0.05)。空白组TNF-α、IL-1β、IL-6、MDA及MPO水平最高(P<0.05),且对照组低于anti-PAT组和anti-NOD_(2)组。空白组HMGB1、NOD_(2)和ZDHHC5表达量最高(P<0.05),且对照组HMGB1和NOD_(2)表达量低于anti-PAT组和anti-NOD_(2)组(P<0.05)。空白组棕榈酰化修饰的NOD_(2)的免疫荧光表达量明显高于对照组、anti-PAT组及anti-NOD_(2)组。空白组较对照组、anti-PAT组及anti-NOD_(2)组肺泡内皮及肺泡组织破坏明显,炎性细胞浸润明显。结论在失血性休克及缺血再灌注动物模型中,NOD_(2)在ZDHHC5的调控下产生棕榈酰化修饰的NOD_(2),从而促使TNF-α、IL-1β、IL-6、MDA及MPO的释放,损伤肺组织。

Objective To investigate the effect and mechanism of nucleotide binding oligomerization domain 2(NOD_(2))on lung injury during hemorrhagic shock and ischemia-reperfusion.Methods Ten NOD_(2)knockout SD rats were set as the control group after the preparation of hemorrhagic shock model(normal saline was given at the initial stage of resuscitation).Thirty ordinary SD rats were randomly divided into three groups:the blank group(normal saline was given at the initial stage of resuscitation),the palmitoyl modification enzyme(PAT)antibody group(PAT antibody was given at the beginning of resuscitation)and the NOD_(2)antibody group(NOD_(2)antibody was given at the beginning of resuscitation),with 10 rats in each group.After 24 hours of resuscitation,blood samples were collected,and lung tissues were collected.Blood gas analysis was performed at the beginning of the experiment,at the beginning of shock and 2 hours after reperfusion.Plasma tumor necrosis factor(TNF)-α,interleukin(IL)-1β,IL-6,malondialdehyde(MDA)and myeloperoxidase(MPO)in lung tissue were detected.High mobility group B(HMGB1),palmitoylated transfer protein(ZDHHC5)and NOD_(2)were detected by Western blot assay.The pathological sections of lung tissue were observed under fluorescence microscope and light microscope.Results The oxygen partial pressure[p(O2)]at 240 min was significantly lower in the blank group than that in the other three groups(P<0.05).The lactate levels at 30 min and 240 min were significantly higher in the blank group than those in the other three groups(P<0.05).TNFα,IL-1β,IL-6,MDA and MPO levels were the highest in the blank group(P<0.05),and which was lower in the control group than that of the anti-PAT group and the anti-NOD_(2)group.The expression levels of HMGB1,NOD_(2)and ZDHHC5 were the highest in the blank group(P<0.05),and the expression levels of HMGB1 and NOD_(2)were lower in the control group than those in the anti-PAT group and anti-NOD_(2)group(P<0.05).The immunofluorescence expression of palmitoylated NOD_(2)was significantly higher in the blank group than that in the control group,anti-PTA group and anti-NOD_(2)group.Compared with the control group,the alveolar endothelium and alveolar tissue were significantly damaged and the inflammatory cell infiltration was obvious in the blank group,anti-PTA group and anti NOD_(2)group.Conclusion In hemorrhagic shock and ischemia-reperfusion animal models,NOD_(2)produces palmitoylated NOD_(2)under the regulation of ZDHHC5,thereby promoting the release of TNF-a,IL-1b,IL-6,MDA and MPO,resulting in lung injury.