Role And Mechanisms Of AT1 Receptor In LPS-induced Acute Lung Injury

Role and mechanisms of AT1 receptor in LPS-induced acute lung injury1.IntroductionSepsis is the commonest complication in surgery and critical illness.Lung is one of the easiest impaired organs in sepsis.Acute lung injury(ALI) is a critical illness,which is characterized by the development of hypoxemia,alveolar-capillary barrier damage, permeability pulmonary edema,pulmonary inflammation and respiratory failure.The proinflammatory cytokines,such as tumor necrosis factor(TNF-α) and interleukin (IL)-1β,are the core of the cytokine-network and play a critical role in the development of ALI.AngiotensinⅡ,the main peptide of the renin-angiotensin system(RAS),exerts vasoconstrictive,growth,and remodeling effects.In mammalian cells,two pharmacologically distinct subtypes of angiotensinⅡreceptors have been characterized, named angiotensinⅡtype 1(AT1) receptor and angiotensinⅡtype 2(AT2) receptor. The evidence from pervious studies suggested that angiotensinⅡmediated most of its biological functions through its signals of AT1 receptor.AT1 receptor antagonists are widely used to treat patients with hypertension,cardiovascular and renal diseases. Recent studies have showed the other important function as an inflammatory mediator. AngiotensinⅡcan enhance vascular permeability and contribute to the recruitment of inflammatory cells by inducing the production of chemokines and adhesion molecules. Recent studies have demonstrated that AT1 receptor antagonist could suppress various cytokines and adhesion molecules,such as TNF-α,IL-6,IL-8,monocyte chemotactic protein-1(MCP-1),vascular cell adhesion molecule-1(VCAM-1),and E-selectin,and prevent atherosclerosis,rheumatoid arthritis and kidney damage.However,there has been little information regarding the role of AT1 receptor in acute lung injury.The objective of the present study was two-fold:1) to investigate the role of AT1 receptor in LPS-mediated ALI 2) and to elucidate the effect of antagonizing AT1 receptor on the release of proinflammatory cytokines and the activation of nuclear factors such as NF-κB and activated protein(AP)-1 in the lung.Here,this study is expected to investigate the mechanism of acute lung injury and provide a new way and approach for lung protection.2.Objectives(1) To study the effect of AT1 receptor antagonist ZD7155 on the proinflammatory cytokines and investigate AT1 receptor antagonism on the protection of lung after LPS induced ALI.(2) To investigate the effect of ZD 7155 on the AngⅡreceptors expression and nuclear factors activations and clarify the initial mechanism of AT1 receptor in LPS-induced ALI.3.Materials and methodsPartⅠRole of AT1 receptor in the lung after LPS-induced ALITwenty-four healthy adult male BLBA/C mice were divided into NS group,LPS group,and LPS plus ZD 7155 group randomly.Briefly,the mice were anesthetized by intraperitoneal injection of 1%pentobarbital sodium(60 mg/kg) before the surgical exposure of the trachea.Sterile NS alone as a normal control or E.coli LPS(2 mg/kg) was instilled directly into the lungs via a 20-gauge catheter.In another set of experiments,mice were pretreated with AT1 receptor antagonist ZD 7155(10 mg/kg) intraperitoneally 30 min prior to LPS exposure.At 24 h after LPS administration,the mice were anesthetized again and exsanguinated by aortic transaction.The lung was clamped and excised for histologic analysis,lung water content measurement,and the proinflammatory cytokines examination. PartⅡEffect of AT1 receptor antagonist ZD 7155 on activation of nuclear factors in mice lung after LPS-induced ALIEighty-eight male BLBA/C mice were divided into NS group(8 mice),LPS group (40 mice),and LPS plus ZD 7155 group(40 mice) randomly.The experiment process is same as the PartⅠ.One,3,6,12,and 24 h after LPS administration,the mice were anesthetized again and exsanguinated by aortic transaction.The lung tissue was harvested same as the PartⅠ.AT1 and AT2 receptor expression were measured by Western blot.EMSA analysis was for AP-1 and NF-κB activations and IHC detection was for AT1 receptor expression.4.ResultsPartⅠIn vivo,2 mg/kg LPS intratracheally administration resulted in elevated lung water content than normal sterile water group at 24 hour after post-injury(81.08±1.78%vs 75.83±1.27%,P＜0.01).Histopathology examination showed the worse histologic condition,and increased pulmonary TNF-αand IL-1βmRNA expressions(0.65±0.08 vs 0.10±0.02,P＜0.01;0.73±0.11 vs 0.09±0.02,P＜0.01 respectively).Administration of ZD7155,a special AT1 receptor antagonist,markedly decreased the mRNA expressions of TNF-αand IL-1βin the lung(0.29±0.05 vs 0.65±0.08 P＜0.05;0.31±0.07 vs 0.73±0.11,P＜0.05 respectively),reduced lung water content(79.06±2.36%vs 81408±1.78%,P＜0.05),and prevented LPS-mediated lung injury.PartⅡMinimal AT1 receptor activation appeared as brown in alveolar macrophages and endothelial cells in the saline alone group,but at 1 h and 3 h after LPS exposure,AT1 activation was markedly increased in alveolar wall cells as well as inflammatory cells (macrophages and polymorphonuclear neutrophils).And at 6 h after LPS exposure, there was a marked AT1 receptor expression throughout the lung,including all of the endothelial beds and interstitial macrophages.At 12 h and 24 h post-LPS exposure,AT1 receptor was also seen in above cells and up-regulation started to diminish back to baseline.Western blot analysis showed that expression of AT1 receptor was increased in lung tissue harvested from LPS stimulation at 6h and 24 h compared with those from NS group(3.44:0.72 and 2.37:1.26,P＜0.01 ratio toβ-actin respectively).Administration with ZD 7155 resulted in a marked decrease in AT1 receptor expression(1.90:3.44 and 1.53:2.37,P＜0.01 respectively vs LPS group).Otherwise,The AT2 receptor expression was significantly downregulated in the LPS group compared with NS group(0.76:1.31 and 0.64:1.80,P＜0.01 at 6h and 24h,respectively).This decrease was partly inhibited by pretreatment with ZD 7155(1.09:0.76 and 1.29:0.64,P＜0.01 respectively).EMSA showed that the increased NF-κB and AP-1 activity were maximal at 3 h and 6 h respectively after LPS exposure.The NF-κB and AP-1 DNA binding activities significantly increased in the LPS group in comparison to NS group at 3 and 6 hours (52.33±4.8 vs 5.47±0.5,42.78±4.1 vs 5.47±0.5 P＜0.05 and 61.14±5.7 vs 2.53±0.2, 73.25±7.1 vs 2.53±0.2 P＜0.05.respectively).Pretreatment with ZD 7155 significantly inhibited the DNA binding activities of NF-κB and AP-1 in the lung(20.53±2.4 vs 52.33±4.8,18.51±1.7 vs 42.78±4.1 P＜0.05 and 17.56±1.6 vs 61.14±5.7,21.54±2.2 vs 73.25±7.1 P＜0.05 respectively).5.Conclusions(1) AT1 receptor mediates the release of TNF-αand IL-1βand plays an important regulatory role in LPS-induced acute lung injury.(2) AngiotensinⅡmediates the activations of NF-κB and AP-1 and the release of proinflammatory cytokines through AT1 receptor and contributes to LPS-induced acute lung injury.