Mechanisms Of Alveolar Epithelial Barrier Dysfunction Induced By Cardiopulmonary Bypass In Rat And The The Protective Effects Of KOR

IntroductionCardiopulmonary bypass(CPB)is an important means of open heart surgery.With the continuous improvement and improvement of CPB technique,cardioprotec-tion and cardiac surgical techniques,the incidence of complications and death of open heart surgery The rate of decline gradually.However,lung injury remains one of the major complications after CPB.Nature magazine for the first time in the early 2000 s referred to the vagal nerve and its main neurotransmitter acetylcholine(Ach)constitutes a neuro-immune regulatory pathway,namely-cholinergic anti-inflammatory pathway(CholinergicAnti-infammatory Pathway CAP).CAP is an endogenous mechanism of neural feedback regulation.When the body is noxious stimuli trigger inflammation,the vagus nerve release Ach to peripheral tissues,Ach and macrophages and other immune cells on the surface of the corresponding receptors,through the regulation of a series of physiological and pathological reactions within the body,and then play an anti-inflammatory effect.In this process,Ach mainly acts on muscarinic acetylcholine receptor(mAChR)and nicotinic acetylcholine receptor(nAChR),which are mainly distributed on the surface of vagal effector cells,Respectively,regulated by the G-protein family of receptor families and the family of ligand-gated ion channel receptorsKappa opioid receptor(KOR)consists of 380 amino acids and is a subtype of opioid receptors belonging to the G protein-coupled receptor family,mainly distributed in the central nervous system,by in situ hybridization The results showed that K opioid receptor mRNA was found in dentate gyrus,hypothalamus,some thalamic nuclei and cerebral cortex,caudate nucleus,olfactory bulb,nucleus accumbens and spinal cord in rat hippocampus.They not only have good analgesic activity,but also have no morphine-like(mu opioid receptor)side effects such as respiratory depression,constipation,addiction,and tolerance,and are also able to combat the drug dependence induced by mu opioid receptor agonism.It has been demonstrated that ?-agonists improve the memory impairment induced by ?-agonists,which act not only in opposition to ?-agonists but also in the regulation of ?-systems such as antinociception,effects on body temperature,The impact of dopaminergic release in the nucleus accumbens,and the release of methionine-enkephalin-like substance in the spinal cord.In addition to exploring the mechanism of functional impairment of pulmonary epithelial barrier by CPB at the cellular,molecular and genetic level,this study will further explore the feasibility of ?-opioid receptor protection on the protective pulmonary epithelial barrier after CPB.Aiming to provide experimental basis for its clinical application and also to provide effective prevention and treatment measures for reducing the incidence of acute lung injury after CPB surgery clinically.Materials and Methods Materials1.Animals: Adult male Wietar rats weighing 400 to 500 g were used in the present study.2.Kit and drugs: kits of LPS,TNF-?,IL-6,IL-10;MDA,MPO,SOD;MMP-9.3. Equipment: small animal ventilator,blood gasanalyzer,specially designed me-mbrane oxygenator,roller pump;Space monitor;Phillips CM-10 transmission electron microscope;MetaMorphimage analysis system;Olympus 1000microscropic camera.Methods1.The establishment of rat CPBMale Wistar rats were anesthetized by intraperitoneal injection of 10% chloral hydrate 350 mg · kg-1 and assisted by small-animal ventilator.Through the miniaturized CPB loop equipment,the right thoracic cavities were drained and left carotid artery perfusion was performed to establish the thoracotomy CPB model in rats.2.experimental groupingFifty-six SD rats were randomly divided into five groups: SH,CPB,CPB + Ach,CPB + U50488,CPB + U50488 SH + THAG2,and 24 rats in CPB group.SH group: Sham-operated group(SH).Rats were intubated only in the corresponding part but not CPB.CPB group: aortic open group,the end of CPB60 min and stop CPB after 2h three points.CPB + Ach,CPB + U50488,CPB + U50488 SH + THAG2 group: The rats in each group were given corresponding preconditioning before the operation.3.observe the indicators and test methods(1)Determination of extravascular lung water content in rats;(2)The changes of MDA,MPO and SOD in lung tissue;(3)The changes of plasma IL-6,IL-10,TNF-?,LPS and MMP-9;(4)The morphological changes of lung tissue were observed by HE and TEM.(5)Lung tissue ?7-nACh R protein and mRNA expression.(6)Statistical analysis was performed by using software SPSS11.0.Results1.The changes of lung epithelial barrier function in each groupThe levels of EVLW,plasma LPS and MMP9 in CPB group were significantly higher than those in CPB group(P <0.05).The difference was statistically significant.It suggested that the inflammatory response of rats was increased and the lung epithelial function was impaired after CPB.2.The changes of Lung tissue MDA,MPO and SOD levelsCompared with SH group,the concentration of MDA and MPO in lung tissue of CPB group increased significantly and the concentration of SOD decreased significantly(P <0.05),while the level of oxidative stress in UBF group Ease,given anticholinergic pathway agonists and inhibitors,U50488 this effect is enhanced and inhibited.3.The changes of plasma TNF-a,IL-6 and IL-10 levelsCompared with SH group,the levels of TNF-a and IL-6 in plasma of CPB group were significantly increased(P <0.05),and the concentrations of IL-10 and IL-10 in CPB group were significantly increased Reaction state eased,given anticholinergic pathway agonists and inhibitors,U50488 this effect is enhanced and inhibited.4.The changes of the lung tissue morphology of rats in each group(1)HE staining light microscopyThe lung tissue of rats in SH group was intact with clear alveoli and no alveolar septa.The alveolar wall in CPB2 h group was thicker,hyperemia / hemorrhage in alveolar cavity and infiltration of a large number of inflammatory cells.After administration of opioid receptor agonist U50488,the inflammation reaction State has eased,giving anticholinergic pathway agonists and inhibitors,U50488 this effect is enhanced and inhibited.(2)transmission electron microscope observationThe ultrastructure of lung tissue in SH group was normal and the morphology of vascular endothelial cells was normal.The ultrastructure of lung tissue in CPB2 h group was obviously damaged,the structure of blood-gas barrier was blurred,the edema was thick,part of alveolar epithelial cells were damaged,the rupture of vascular endothelial basement membrane,Destruction of mitochondrial structure was vacuolar changes,lamina small body emptying.The opiate receptor agonist U50488,the inflammatory response has eased,given anticholinergic pathway agonists and inhibitors,U50488 this effect is enhanced and inhibited.4.The protein ?7nAChR expression of lung tissue and pulmonary epithelial barrier functionThe expression of ?7nAChR protein in lung tissue was negatively correlated with EVLW,plasma LPS concentration,MMP9 concentration,MDA,MPO concentration in plasma and positively correlated with plasma SOD concentration5.Given ?7-nAChR agonist,the changes of inflammatory pathways in each groupAfter administration of anticholinergic pathway agonist Ach,the levels of plasma TNF-a and IL-6 in CPB + Ach group were significantly lower than those in CPB group,and the content of NF-?B was also significantly lower than that in CPB group Rats EVLW was significantly lower than the CPB group,the difference was statistically significant.Conclusions1.Rats after CPB EVLW,plasma LPS,MMP-9 levels were significantly increased lung epithelial barrier function is an important mechanism.2.The decrease of a7 nAchR protein and mRNA in the lung epithelium of CPB may be one of the mechanisms of CPB-induced pulmonary epithelial barrier dysfunction.3.systemic inflammatory response after CPB is caused by pulmonary epithelial barrier dysfunction,an important factor,and plays an important role in systemic damage.The correlation between a7 nAchR and lung injury,inflammatory reaction and NF-?B pathway may be one of the mechanisms by which a7 nAchR is involved in the regulation of lung injury after CPB.4.?-opioid receptor preconditioning can reduce the degree of ischemia-reperfusion injury and systemic inflammatory response after CPB,reduce the permeability of alveolar epithelial epithelial barrier play a protective effect,there is a dose of this effect Dependency.5.Pretreatment with ?-opioid receptor protects the lung epithelial barrier function may be through activating CAP-NF-?B signaling pathway,initiating endogenous protective mechanisms,anti-oxidative damage,and regulating inflammatory responses.