The Expression Of SIRT1、TNF-α Protein And The Effect Of Resveratrol On Acute Hyperoxia-induced Lung Injury In Neonatal Rats

Objective:1.To study acute hyperoxia- induced lung injury in neonatal rats;2.To observe the expression of SIRT1 and TNF-α protein in the pathogenesis of acute hyperoxia-induced lung injury and whether resveratrol can change the expression of them. And to explore the protective effects of resveratrol on acute hyperoxia- induced lung injury.Methods: One hundred and eight neonatal rats were randomly assigned to three groups at 48 hours after the birth:Group Ⅰ:control group(air+DMSO,n=36);Group Ⅱ:hyperoxia group(hyperoxia+DMSO,n=36);Group Ⅲ: resveratrol group(hyperoxia+ resveratrol,n=36);The rats of Group Ⅰwere exposed to normoxia(room air) and the rats of group Ⅱ、Ⅲ were exposed to hyperoxia(90-95% oxygen)in a specially constructed boxes for 7 days.The rats in the group Ⅲ were given intraperitoneal injection of resveratrol solution(resveratrol 20 mg,100%DMSO 100 ul,0.9% normal saline 1.9ml)5ml/kg,once a day for 7 consecutive days from the beginning of hyperoxia exposure.The rats in the group Ⅰand group Ⅱ were given intraperitoneal injection of with the same volume of DMSO solution(100%DMSO 100 ul,0.9% normal saline 1.9ml), once a day for 7 consecutive days.Each group was divided into 3 subgroups,the 1-day,3-day and 7-day.Twelve rats from each subgroup were randomly selected,and killed to measure the following indices:(1) The total protein concentration in BALF;(2) Lung wet-to-dry ratio:The left lung were isolated and weighed,then the dried lungs were weighed,and calculated the lung wet/dry ratio;(3) Lung histology:Lung sections from the right lung were stained with HE,and examined for the histological changes;(4) Western blotting were used to detect the protein of SIRT1 and TNF-α;Results:1.The general condition of the rats in each group:Group Ⅰ, the growth of rats was in normal: shiny fur, normal diet, activity, good weight gains.Group Ⅱ,with oxygen time increases, poor mental reactions, shiny fur worse than before, eat less, weight slow growth.The general condition of rats in Group Ⅲ compared with Group Ⅱ was improved.2. The pathological morphology of lung tissue:Group Ⅰ, the lung becomes mature and alveolar size gradually converge, alveolar septa become thinner with time,there is no inflammation and bleeding. Group Ⅱ, at 1th day,the lung structure still intact, a small amount of red blood cells in the alveoli were seen; at 3th day,vascular congestion, interstitial edema and redbloodcell,inflammatory infiltrates were found.; at 7th day, severe hemorrhage, exudation, inflammatory infiltrates were seen. Lung parenchyma structure becomes disordered, alveolar structure is simple, the number of alveoli is significantly reduced.The exudation,cell infiltration in alveolar cavity were obviously decreased in Group Ⅲ compared with Group Ⅱ.3. The lung wet/dry ratio:(1)Comparison in each group: Group Ⅰand Group Ⅲ,there was no significant difference in the lung W/D rations of rats at each test point(P> 0.05); As oxygen time goes on, the lung W/D ration of rats is increased gradually in Group Ⅱ.(2)Comparison between different groups:At first experimental day,there was no significant difference in lung W/D ration among the three groups.After 3 and 7 days of hyperoxia,the lung W/D ration of rats was higher in Group Ⅱ compared with Group Ⅰ;and the lung W/D ration in Group Ⅲ was lower than that in Group Ⅱ(P<0.05).4. The total protein concentration in BALF:(1)Comparison in each group:Group Ⅰand Group Ⅲ,there was no significant difference in the TP of BALF at each test point(P> 0.05); As oxygen time goes on, the TP of BALF is increased gradually in Group Ⅱ.(2)Comparison between different groups:There was insignificant difference in the TP of BALF among the three groups at experimental day 1(P>0.05);At 3th and 7th day,the TP concentration in the BLAF of rats was higher in Group Ⅱ compared with Group Ⅰ(P<0.05),and the TP of BALF in the Group Ⅲ was lower than that in the Group Ⅱ(P<0.05).5. The expression of SIRT1 and TNF-α protein in lung tissue:(1)Comparison in each group:Group Ⅰ,there was no significant difference in the expression of SIRT1 and TNF-α at each test point(P> 0.05).As oxygen time goes on, the expression of SIRT1 is increased and the expression of TNF-α is decreased gradually,no matter in Group Ⅱor Group Ⅲ.(2)Comparison between different groups:There was no marked difference in the expression of SIRT1 and TNF-α among the three groups at 1th day(P>0.05). At 3th and 7th day, TNF-α protein expression in Group Ⅱwas significantly increased as compared with that in Group Ⅰ(P<0.05);SIRT1 protein expression in Group Ⅱ was significantly decreased as compared with that in Group Ⅰ(P<0.05);The level of SIRT1 protein expression in Group Ⅲwas higher than that in Group Ⅱbut lower than that in Group Ⅰ(P<0.05),and the level of TNF-α protein expression in Group Ⅲ was lower than that in Group Ⅱ with statistical significance but higher than that in Group Ⅰ(P<0.05),no matter at 3th or 7th day.Conclusion:1.Neonatal rats were exposed to a high-oxygen environment for a certain period of time developed acute lung injury.The main pathological changes of the lung tissues are local hyperemia, hemorrhage, inflammatory exudates, structure turbulence of the pulmonary tissue,with severe inflammatory cell aggregation.2.The expression of TNF-α protein in the lung of rats with acute hyperoxia-induced lung injury increased significantly,as time goes on, the expression level increased gradually.The expression of SIRT1 protein in the lung of rats with acute hyperoxia-induced lung injury increased significantly,and as time goes on, the expression level decreased gradually.These findings suggest that the SIRT1 and TNF-α may responsible for acute hyperoxia-induced lung injury.3.Resveratrol may provide protection for acute hyperoxia-induced lung injury via up-regulation of SIRT1 protein expression,and down-regulation of TNF-α protein expression.