The Relative Relationship Beteen Bmi Index And The Severity Of Lung Injury In Rat Ali Model

Introduction:The basic nature of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is local manifestation of systemic inflammatory response syndrome in lung tissues.Although every kind of breath support technic and drug treatment targeted for ALI/ARDS have made great progress,there is no significant improvement of incidence of ARDS from1994to2009through a meta-regression analysis in2009and the mortality is up to40%in China.Some clinical studies have confirmed that the incidence and severity of lung injury are much higher in high BMI patients group than those in normal BMI patients group in the ICU department,but it is not reported whether the obesity related-metabolism plays a critical function to its pathogenesis so far.Angpt2(Angiopoietin-like protein2) is secreted by fat cells and has a silmilar domain and certain homology, the study clearly closely with obesity-related lipid-derived inflammatory factors..Recently,more and more research on Angptl2was studied,especially in the field of obesity and inflammation,and Angptl2was clearly proved to be lipid-derived inflammatory factor that is closely related to obesity.But whether Angptl2plays some function to the pathogenesis and progression of ALI in patients is not reported, especially in obese patients. So ALI model is established in obese mice fed by high fat diet. Based on the idea that obesity is a condition of chronic inflammation,we study the relationship between fat metabolism and the level of inflammatory mediators in the progress of ALI to find out potential methods that can alleviate the incidence and mortality of lung injury,which not only can promote the prognosis,but also can provide some new strategies to predict and cure ALI caused by many extrapulmonary causes.1ALI is a manifestation of systemic inflammatory response syndrome in lung tissues:the function of inflammatory mediators especially cytokines to pathogenesis of ALI is emphasized increasingly.TNF-α is one of the most important proinflammatory cytokine together with IL-1β to irritate the inflammatory reaction in lung resulting in ARDS,what is more,the activation of oxidative stress and protease is also involved in the pathogenesis of lung injury;NF-κB is commonly known as the cytokine that plays a cruel role in the pathogenesis and progression of ALI.2obesity is a condition of chronic inflammation John N and his colleague found that the level of TNF-α,IL-6,CRP,FABP-4,PGE2,IL-8,PAI-1,MCP-1,Leptin,HGF,LPL,ICA M-1et al. was elevated in obese patients, and all of them are secreted by fat cell and closely related to the pathogenesis of lung injury.Some clinical studies have confirmed that the incidence and severity of lung injury are much higher in high BMI patients group than those in normal BMI patients group in the ICU department.Here we conduct this project in rats ALI model induced by OA to observe the behavior of rats,to evaluate the injury degree with the use of histopathology analysis,to measure the rate of wet/dry weight (W/D),the amount of NF-κ Bp65and protein in lung tissues,to detect ratio of NF-kBp65nuclear translocation in situ by indirect immunofluorescence and level of TNF-α、IL-1β in peripheral blood.1Objective1.1To determine the difference of lung histopathology scores,level of TNF-β IL-1β in peripheral blood,NF-KB p65leve of lung tissue in rats with different severity of ALI.1.2To evaluate the severity of ALI by measuring lung histopathology scores,TNF-α,IL-1β,NF-κB p65together,and make the relevance analysis between BMI index and severity of lung injury.1.3By analyzing the BMI index group Angptl2mRNA and protein levels from Angptl2is obesity’s fat-derived inflammatory cytokines in the angle of departure to explore the correlation between BMI and ALI possible mechanism pathway.2Method2.136healthy male SD rats(168±5.31g) provided by the experimental animal center of Chongqing Medical University were randomly divided into four group(n=9),they were C group(Normal),OA group(Normal+OA),CH group(obesity control),OH group(obesity+OA). rats in C and CA group were fed with normal diet,rats in CH and OH group were fed with high fat diet(20%lard,10%milk powder,10%eggs,60%normal diet) to induce obese rats model,BMI and level of blood-fat of all rats were measured after8-week feeding.B. ALI model in OA and OH group was induced by injection of oleic acid(OA0.1ml/kg) through caudal vein,while rats in C and CH group were injected with an equal amount of physiological saline in the same way.The behavior、color of skin-mucous membrane and respiratory rate of all rats were observated to judge whether the ALI model was successful or not after4-hour injection of OA.Then all rats were fully anesthetized by3.5%chloral hydrate(10ml/kg),Jugular vein was separated out,vein blood was extracted and plasma was separated out for use; the hemorrhage and edema of lung tissues were observed after thoracotomy,the left lung tissues were excised for measurement of lung wet/dry weight(W/D)ratio and histopathology analysis;the right lung tissues were packed to store at-80℃refrigerator for use to detect the level of TNF-α、IL-1β and blood-fat、ratio of NF-κBp65nuclear translocation by immunofluorescence and NF-κB mRNA in lung tissue by PCR,Quantification of Angptl2mRNA by RT-PCR assay,Semi-quantification of Angptl2protein by Western blotting.3Result 3.1The behavior,color of skin-mucous membrane and respiratory rate of all rats.The rats in C group and C-H group,their normal condition and mental state were well, breath steadily and respiratory rate ((44±12)、(42±8),no cyanosis and no death.In OA group,respiratory rate increased up to (98±13)/min, body hairs bristled, cyanosis emerged,and no death after1-4hour injection of OA.In O-H group,respiratory rate significantly increased,body hairs erected,big wheezing,and cyanosis of lips was more obvious than that in OA group,one death in4hours.The level of serum lipid,weight and length of all rats were measured.There was no significant difference in the level of TG and BMI index in C group and OA group The level of TG and BMI index in CH group and OH group are more higher than those in both former groups and P<0.05.3.2The ratio of W/D and lung histomorphology observationIn C group,there was smooth,pale pink, no congestion and edema in lung tissues,In C-H group,lung tissue was much more hypertrophy than normal group,and seen some yellow fat tissue on the surface;In OA group,part of the lung tissues was dark red and wide rang of point or spotty hemorrhage, histopathology analysis showed that typical change of lung injury such as infiltration of inflammatory cells and exudation of edema fluid,hemorrhage were significantly seen;In OH group,lung tissue was significantly inflated to become dark red,Histomorphology showed much edema fluid exudated and inflammatory cells infiltrated;Result of histopathology scores in four groups according to the reference [8] showed that the scores among CH,OH,OA group was higher than that in C group (P<0.05),there was significant difference between OH and OA group (P<0.05).but there was no statistically significant difference between OH and OA group from the data of W/D analysis(P>0.05).3.3The level of TNF-α IL-1β in four groupThe level of TNF-α and IL-1β in supernatant after homogenate of lung tissue was used to evaluate the degree of inflammatory reaction in lung.The level of TNF-α and IL-1β was higher in OA and OH group than that in C group after4-hour injection of OA,and the level of TNF-α and IL-1β in OH group was elevated much more significantly.3.4Ratio of NF-κBp65nuclear translocation in situ by indirect immunofluorescenceThe circular green fluorescence represented NF-KBp65protein positive cells,and fluorescence was centralized in nucleus district.There was little positive cells in C group,higher in OA group,and highest in OH group.The optical density of pictures in each group was determined by Image J software,the result of OH group was higher than OA group,and the difference was statistically significant (P<0.05);After electrophoresis,the expression of NF-κB mRNA at348bp was significantly increased in OH group compared to OA group (P<0.05). 3.5Semi-quantification of NF-κB mRNA in lung tissue by PCRThe gray value of NF-κB mRNA was lowest in C group,higher in OA group,and the difference was statistically significant (P<0.05); The gray value was significant higher in OH group than that in CH group,the difference was statistically significant (P<0.05);In OH and OA group,the gray value was much higher in OH group (P<0.05).3.6Relevance analysis between BMI index and W/D,histopathology scores,TNF-α、IL-1βBIM index was positive correlation with them,and best correlation with histopathology scores and IL-1β,but indefinite correlation with W/D.No injection of OA group:BMI with W/D:0.62;histopathology scores:0.78;TNF-α:0.83;IL-1β:0.84;The ALI model group:BMI with W/D:0.29,histopathology scores:0.85,TNF-α:0.65,IL-1β:0.79.3.7Quantification of Angptl2mRNA by RT-PCR assay,Semi-quantification of Angptl2protein by Western blottingThe amounts of Angptl2protein were significantly increased in O-H group and OA group after injection of OA.The amounts of Angptl2protein were significantly,in addition,the leve higher in O-H group than OA group(P<0.05).4Discussion4.1Lung histopathology scores,level of TNF-α、IL-1β in peripheral blood,NF-κB p65leve of lung tissue in rats increased with increasing BMI index. 4.2BMI index,Lung histopathology scores,TNF-α,IL-1β,NF-κB p65together,have well correlation with the degree of lung injury.4.3The leve of Angptl2protein expression differences in different BMI groups,receptor mediated through the alveolar wall,lead to increased permeability of the alveolar wall edema fluid exudation and alveolar wall collapse,as a result,cause lung injury differences between groups.