Study On The Relationship Between The Inflammatory Protection Of Metformin On Lung And AMPK Pathway In Septic Rats

Objective:The purpose of the study was to survey the pulmonary inflammatory protective effect of metformin(Met)on septic rats caused by cecal ligation and puncture(CLP),the protective effect could be inhibited by Compound C(CC),an inhibitor of adenosine monophosphate activated protein kinase(AMPK),to evaluate the effect of Met on AMPK in lung tissue of septic rats,to investigate the inflammatory protective effect of Met on lung in septic rats and its mechanism,to provide a new idea for relative research.Methods:100 adult male SD rats for clean grade were fed adaptively for one week,a total of 100 adult male SD rats were randomly selected by digital sampling,adopting digital random sampling method 100 adult male SD rats.They were randomly divided into 5 groups: control group(Con),sham group(Sham),sepsis group(CLP),Met group(CLP+Met),CC group(CLP+Met+CC),there were 20 rats in each group.Each group include 12 h group,24 h group and 10 rats in each subgroup.CLP+Met group and were given 200 mg/kg Met intragastrically once a day for 14 days.Con group,Sham group and CLP group were fed with the same amount of saline compared with Met solution for 14 days.CLP+Met+CC group was given injection of CC 20 mg/kg intraperitoneally one hour before the establishment of the model,while the other four groups were injected the same amount of physiological saline compared with CC at the same time.No special operation was performed in the Con group.Sham group was treated with sham operation,to open the abdominal cavity layer by layer after anesthesia.The abdominal cavity was sutured immediately layer by layer after gently turning the pericecal intestine of the rat.Septic rat models were made by CLP in CLP group,CLP+Met group and CLP+Met+CC group.The general state of CLP group,CLP+Met group and CLP+Met+CC group were observed at 12 h and 24 h after CLP.The intestinal changes were observed by laparotomy,and the lung tissue and serological samples of the corresponding groups were taken.The left lung tissue was fixed,embedded in paraffin and stained with hematoxylin-eosin(HE).The pathological changes were observed under pathological microscope,and the pathological score of lung injury was scored.At the same time the middle lobe of the right lung tissue was taken,the wet and dry weight were detected and get W/D index,it indicates the degree of pulmonary edema in rats.WB was used to detect the expression of AMPK and phosphorylated adenylate-activated protein kinase(p-AMPK)in the remaining lung tissue,and the degree of AMPK activation was expressed by p-AMPK/AMPK.Find the abdominal aorta of the rat,take rat arterial blood.Detection of serum interleukin-6 and the tumor necrosis factor-α expression by ELISA kit.The general state of rats in Sham group was observed 12 hours and 24 hours after sham operation,and the intestinal tract was observed by laparotomy.Lung samples and serological samples were taken to detect the above indexes in the corresponding group.The rats in the Con group were also observed after 12 hours and 24 hours,and the intestinal manifestations were observed by laparotomy.The lung and serological samples were taken in the corresponding group,and the method was the same as before.The statistical data analysis is used by SPSS20.0,the measurement data accord with the normal distribution,mean ±standard deviation is described the measurement data,the comparison between more than three groups is shown by single factor analysis of variance,and the comparison was conducted by T test.The measurement data do not meet to the normal distribute ion,the statistical description of measurement data is expressed by median(quartile interval).Nonparametric rank sum test was used in difference analysis.P<0.05,means obvious difference.Results:1.The rats in Sham group and Con group were in good general condition at 12 h and 24 h,the intestinal appearance was normal after laparotomy,the structure of lung tissue was normal under light microscope,there was no obvious injury and inflammation in lung tissue,and there was no significant difference in lung pathological injury score.The expression levels of IL-6 and TNF-α,the number of W/D in lung tissue and the expression level of AMPK,p-AMPK,pAMPK/AMPK in lung tissue were all in the normal range.There was no obvious difference(P>0.05).2.The general state of rats in CLP group was worse than the Sham group at 12 h and 24 h,with corresponding manifestations of sepsis,stench of laparotomy,intestinal adhesion necrosis,ascites formation and other intestinal changes.The lung tissue showed severe destruction of endothelial cells and epithelial cells,pulmonary hemorrhage,disappearance of normal morphology of lung tissue and infiltration of a large number of inflammatory cells.The score of lung pathological injury,the level of serum IL-6 and TNF-α and the number of W/D in lung tissue were increased significantly,and levels of AMPK,p AMPK and p-AMPK/AMPK were significantly higher than those of control group.There were significant difference in statistics(P<0.05).3.The inflammatory changes of lung tissue caused by CLP were significantly inhibited in CLP+Met group compared with CLP group at 12 h and 24 h.The general state of rats was improved and the intestinal reaction of laparotomy was alleviated.The lung pathological injury score,the expression level of IL-6,TNF-α and the value of W/D in lung tissue were decreased significantly,while the levels of AMPK,p-AMPK and p-AMPK/AMPK were significantly increased.There were significant difference in statistics(P<0.05).4.The general state of rats worse in CLP+Met+CC group was compared with that in CLP+Met group at 12 h and 24 h,stench after laparotomy,intestinal necrosis and other appearance aggravated,lung tissue inflammatory cell infiltration,alveolar hemorrhage and other pathological injuries were more serious.The lung pathological injury score,serum IL-6,TNF-αand lung tissue W/D increased,while the levels of AMPK,p-AMPK and p-AMPK/AMPK decreased significantly.There were significant difference in statistics(P<0.05).5.Compared with 12 h group,the rats in Con group and Sham group were in good condition,normal intestinal reaction,normal lung tissue stained by HE,no inflammatory infiltration and other pathological manifestations in 24 h group and 12 h group.There was no significant difference in lung pathological injury score,TNF-α,IL-6,W/D,AMPK,p-AMPK and pAMPK/AMPK expression.There has no obvious difference in statistics(P>0.05).6.Compared with the 12 h group,the general state of 24-hour rats in the CLP group,CLP+Met group and CLP+Met+CC group was worse than that in the 12-hour group,and the intestinal reaction was aggravated in the 24-hour group.The pathological injury score of lung tissue,IL-6,TNF-α and Whamp D increased significantly,while the levels of AMPK,p-AMPK and p-AMPK/AMPK decreased significantly(P<0.05).Conclusions:1.Met can protect the lungs of septic rats by inhibiting the inflammatory response of the lungs.2.The inhibitory effect of metformin on lung inflammation in septic rats is achieved by activating AMPK signal pathway and can be blocked by CC.