Effect Of Metfromin On Pathogenesis Of Abdominal Aortic Aneurysm Through AMPK/mTOR Signal Pathway

Objective:Abdominal aortic aneurysm(AAA)is a leading cause of mortality and morbidity in the elderly.Currently,there remain no effective drugs that could prevent the growth of aneurysms and delay aneurysm rupture in the clinical setting.The main risk factors are age older than 65 years,male sex,and smoking history.Other risk factors include a family history of abdominal aortic aneurysm,coronary artery disease,hypertension,peripheral artery disease,and previous myocardial infarction.Therefore,the prevention and early diagnosis of aneurysms are very important.Diabetes can lead to aortic atherosclerosis and vascular endothelial dysfunction,and it is also an important independent risk factor for cardiovascular disease.But epidemiological evidence has shown that diabetes could restrain the progress of the AAA,as a protective factor for AAA.Some scholars have investigated that hypoglycemic agent maybe the key role on limiting the progression of AAA disease.However,its molecular mechanism is unclear.Metformin as an hypoglycemic agent,its close relationship with AMPK/mTOR signal pathway caught our attention.The AMP-Activated Protein Kinase(AMPK)as an energy sensor that regulates cellular metabolism and glucose homeostasis,which is also the core of the study of diabetes and other metabolic diseases.AMPK has many other functions including regulation of autophagy,cell proliferation,and ECM synthesis and degradation.The inhibitions of expression and activity of MMP-2/9 by activation of AMPK have been shown in previous studies conducted on different types of cells.The high expression of MMPs can lead to the formation of AAA.According to records,the activation of AMPK can inhibit the expression of mTOR.As an inhibitor of mTOR,rapamycin can also inhibit the development of abdominal aortic aneurysm,which has been shown in previous study.A study showed that there were no significant difference in total AMPK protein levels between control aortic tissues and AAA tissues.But p-AMPK level was significantly lower in AAA tissues than in control aortic tissues,indicating the reduced activation of AMPK signal pathway in human AAA.Previous studies have revealed that mTOR signaling is upregulated in thoracic aneurysm and dissection and that rapamycin can protect against this phenotype by preserving contractile function of VSMC.At present,the research has shown that autophagy was closely related to the onset of AAA.Because autophagy can resist excessive inflammatory reaction in cells,it may suggest that autophagy also plays an important role in the development of AAA.Moreover,AMPK/mTOR signaling pathway is the core of autophagy mediation.Thus,we deduce that metformin activates AMPK,inhibiting mTOR at the same time,which could be the key link to prevent the development of AAA.This study provides theoretical basis for prevention and treatment of AAA with metformin.Methods: In this study,human AAA tissue was used as the experimental group(AAA),normal abdominal aorta tissue as a negative control group(NC),by Western blot detection of AMPK,p-mTOR protein expression,The expressions of p-AMPK and p-mTOR were detected by immunohistochemistry.Cultured human VSMC(HVSMC),identified and counted cells,respectively,with a gradient concentration of AMPK agonist AICAR and inhibitor Compound C role of cells,CCK8 assay HVSMC proliferation activity changes,to determine the optimal drug concentration,respectively,with AICAR and Compound C was used to intervene cells.Western blot was used to detect the expression of p-AMPK and p-mTOR after intervention.Compound C(C.C)was used as AMPK inhibitor.Metformin(MET)was administered to activate AMPK.AAA models were induced by intraluminal porcine pancreatic elastase(PPE)infusion.Fourty male SD rats weighing(200±20g)were divided into control group,MET+AAA group,AAA group and C.C+AAA group,(n=10).Immunohistochemistry analysis of the contents of macrophages,neovasculars and SMCs(smooth muscle cells)in these different groups.The expression of p-AMPK,MMP-2/9,p-mTOR,?-SMA,OPN,Beclin-1and LC3-II were analyzed by Western blot.An AMPK inhibitor(C.C)was used to confirm whether AMPK/mTOR was the key role of MET limiting aneurysm enlargement or disease progression in AAA.Results: 1.The expression of p-AMPK in NC group was significantly higher than that in AAA group(P <0.05),while the expression of p-mTOR in NC group was significantly lower than that in AAA group(P <0.05).2.p-AMPK and p-mTOR mainly expressed in vascular smooth muscle cells.3.In HVSMC cultured in vitro,the expression of p-mTOR can be directly or indirectly regulated by activating AMPK expression;inhibiting the expression of AMPK will increase the expression of p-mTOR.Compared with AAA model rats group,MET significantly reduces AAA diameter in MET+AAA group,but this effect was inhibited by C.C in C.C+AAA group.The contents of macrophages and neovasculars were significantly decreased in MET+AAA group compared to the AAA group and C.C+AAA group,whereas the contents of VSMC were increased in MET+AAA group compared with AAA group and C.C+AAA group.Using western blotting,we found that p-AMPK,?-SMA,Beclin-I and LC3-II levels were higher in MET+AAA group than AAA group and C.C+AAA group,but p-mTOR,OPN and MMP-2/9 levels were significantly lower in MET+AAA group.Conclusions:1.Expression levels of p-AMPK were significant higher in control group than that in AAA patients,but expression levels of p-mTOR were lower.2.Activating AMPK could inhibit the expression of p-mTOR.3.AMPK/mTOR signaling pathway may be a new target of AAA treatment.4.Metformin could reduce aneurysm enlargement or disease progression.By enhancing AMPK activity with MET,reducing inflammation reaction and enhancing the level of autophagy,MET decreased the expressions of MMP-2/9 in abdominal aortic aneurysm tissues.Further,MET increased the extracellular matrix,improving expression of vascular smooth muscle contractile proteins ?-SMA and inhibiting expression of vascular smooth muscle synthetic protein OPN at the same time.Eventually it inhibited the formation and development of abdominal aortic aneurysm.Thus,we deduce that metformin activates AMPK,inhibiting mTOR at the same time,which could be the key link to prevent the development of AAA.These results highlight the important role of the AMPK/mTOR signal pathway in aneurysm progression and the potential application of MET as a therapeutic candidate for AAA.