The Effect Of Annexina1 Mimetic Peptide Ac2-26 On Pulmonary Injury And P38MAPK/NF-κB Induced By Cardiopulmonary Bypass In Rats

Objective:To observe the effects of Annexin A1 mimic peptide Ac2-26 on the expression of p38MAPK/NF-κB and explore the protective effect and mechanism of Ac2-26 on lung injury induced by cardiopulmonary bypass in rats.Methods:Eighteen adult healthy SD rats（male,350⁴50g）were randomly divided into three groups（n=6）:Sham operation group（Sham group）,left lung ischemia-reperfusion injury group（I/R group）,Annexin A1 mimic peptide Ac2-26 group（Ac2-26 group）.After anesthesia,the three groups of rats were placed with tail vein puncture,and supine position fixation.The right femoral artery,vein,left femoral vein,and right common carotid were punctured and placed.After the tracheal intubation was connected to the ventilator under laryngoscope,and mechanical ventilation.The right femoral artery connected with biological information collection and processing system.In the sham group,the left lung was only exposed by puncture and thoracotomy.In the I/R group and Ac2-26 group,the rats were connected to the extracorporeal circulation pipeline（the right common carotid artery was connected to the peristaltic pump,and the left and right femoral veins were connected to the membrane oxygenator）.After the connection,the extracorporeal circulation was started.After 10 minutes,the lung injury model was established by clamping the left lung hilus.In group Ac2-26,Ac2-26 was given before blocking the left pulmonary hilum,while in group I/R,the same volume of normal saline was given at the same time.Before cardiopulmonary bypass（T₁）,at the time of opening the left pulmonary hilum（T₂）and at the end of the experiment（T₃）,arterial blood gas analysis of right femoral artery was taken to calculate the oxygenation index（OI）and respiratory index（RI）,and the vital signs of the rats were recorded.At the end of the experiment（T₃）,the left lung bronchoalveolar lavage fluid（BALF）and left lung tissues were taken.The changes of lung tissue structure and pathological scores were observed under light microscopy.The levels of TNF-αand IL-10in lung tissue,and IL-6 and total protein in BALF were detected by ELISA respectively.Westernblotting was used to detect the expression of p38MAPK、p-p38MAPK、NF-κBp65、p-NF-κBp65 and AnxA1.Results:（1）Changes of heart rate（HR）of three groups.Heart rate（HR）at different time points in rats of three groups:HR at T₁,T₂ and T₃ time points in rats of group Sham had no significant difference（P>0.05）.HR at T₂ and T₃ in I/R and Ac2-26 groups was lower than that at T₁（P<0.05）.HR at T₃ was significantly lower in group I/R（P<0.05）and had no difference in group Ac2-26（P>0.05）than that at T₂.Simultaneous point comparison:At T₁,there was no significant difference in HR of three groups（P>0.05）.At T₂,HR in I/R and Ac2-26 groups was significantly lower than that in Sham group（P<0.05）,and in I/R group was same as that in Ac2-26 group（P>0.05）.At T3,HR in I/R and Ac2-26 groups was lower than that in Sham group（P<0.05）,and Ac2-26group increased significantly than that in I/R group（P<0.05）.（2）Changes of mean artery pressure（MAP）of three groups.Mean artery pressure（MAP）at different time points in rats of three groups:MAP at T₁,T₂and T₃ time points in rats of group Sham had no significant difference（P>0.05）.MAP at T₂and T₃ in I/R and Ac2-26 groups was significantly lower than that at T₁（P<0.05）.MAP at T₃ increased significantly in I/R and Ac2-26 groups than that at T₂（P<0.05）.Simultaneous points comparison:At T₁,there was no significant difference in MAP of three groups（P>0.05）.At T₂,MAP in I/R and Ac2-26 groups was significantly lower than that in Sham group（P<0.05）,and in I/R group was same as that in Ac2-26 group（P>0.05）.At T₃,MAP in I/R and Ac2-26 groups was lower than that in Sham group（P<0.05）,and in I/R group was same as that in Ac2-26 group（P>0.05）.（3）Changes of oxygen and index（OI）and respiratory index（RI）of three groups.Oxygen and index（OI）and respiratory index（RI）at different time points in rats of three groups:OI and RI at T₁,T₂ and T₃ time points in rats of group Sham had no significant difference（P>0.05）.Compared with T₁ time points,OI decreased and RI increased significantly in I/R and Ac2-26 groups at T₂ and T₃ time points（P<0.05）.Compared with T₂time points,OI decreased and RI increased significantly at T₃ time points in I/R and Ac2-26groups（P<0.05）.At the same time point OI and RI comparison:At T₁,there was no significant difference in OI and RI of the three groups（P>0.05）.At T₂,OI was lower and RI was higher significantly in I/R and Ac2-26 groups than those in Sham group（P<0.05）.At the same time,OI and RI in I/R group were same as those in Ac2-26 group（P>0.05）.At T₃,OI was lower and RI was higher significantly in I/R and Ac2-26 groups than those in Sham group（P<0.05）,and OI increased and RI decreased significantly in Ac2-26 group than those in I/R group（P<0.05）.（4）Changes of TNF-αand IL-10 contents in left lung tissue of three groups at the end of experiment（T₃）.The levels of TNF-αand IL-10 were higher in I/R and Ac2-26 groups than those in Sham group（P<0.05）.TNF-αwas lower and IL-10 was higher significantly in Ac2-26 group than those in I/R group（P<0.05）.（5）Changes of IL-6 and total protein content in the left lung bronchoalveolar lavage fluid（BALF）of three groups at the end of experiment（T₃）.Compared with Sham group,the contents of IL-6 and total protein in I/R and Ac2-26 groups increased significantly（P<0.05）.The contents of IL-6 and total protein in BALF of Ac2-26group were lower than those in I/R group（P<0.05）.（6）Light microscopic results and pathological damage scores of lung tissue of three groups at the end of experiment（T₃）.In the Sham group,the left lung tissue was vesicular with complete structure.In the I/R group,the left lung tissue structure was obviously disordered,the lung interstitium was obviously widened,there were plenty of red blood cells and inflammatory cells infiltration in the alveolar hemorrhage and edema,and the pathological score was significantly higher than that in the Sham group（P<0.05）.In the Ac2-26 group,the left lung tissue structure was relatively complete,the lung interstitium was widened,there were a small quantity of red blood cells and inflammatory cells infiltration in the alveolar hemorrhage and edema,and the pathological score of Ac2-26 group was higher than that of Sham group and lower than that of I/R group.（7）Changes of p38MAPK and p-p38MAPK in left lung tissue of three groups at the end of experiment（T₃）.There was no difference in p38MAPK of the three groups（P<0.05）.Compared with Sham group,the expression of p-p38MAPK and p-p38MAPK/p38MAPK in the left lung of I/R and Ac2-26 groups were significantly increased（P<0.05）.Compared with I/R group,the expression of p-p38MAPK and p-p38MAPK/p38MAPK in Ac2-26 group decreased significantly（P<0.05）.（8）Changes of NF-κBp65 and p-NF-κBp65 in left lung tissue of three groups at the end of experiment（T₃）.Compared with Sham group,the expression of NF-κBp65,p-NF-κBp65,and p-NF-κBp65/NF-κBp65 in the left lung of I/R and Ac2-26 groups were up-regulated（P<0.05）.Compared with I/R group,the expression of NF-κBp65,p-NF-κBp65,and p-NF-κBp65/NF-κBp65 in Ac2-26 group were down regulated（P<0.05）.（9）Changes of AnxA1 in left lung tissue of three groups at the end of experiment（T₃）.Compared with Sham group,the expression of AnxA1（37kD）and AnxA1（33kD）in lung tissue of I/R and Ac2-26 groups were up-regulated（P<0.05）.Compared with I/R group,the expression of AnxA1（37kD）and AnxA1（33kD）in Ac2-26 group were down regulated（P<0.05）.Conclusion:（1）Ac2-26 can ameliorate the lung function of rats suffering from CPB,alleviate pulmonary injury and inflammatory exudation,so as to reduce the pulmonary injury of rats after CPB.（2）The mechanism of Ac2-26 reducing pulmonary injury of rats suffering from CPB may be related to the inhibition of p38MAPK/NF-κB signaling pathway to reduce the content of TNF-α,IL-6 and increase the concentration of IL-10.