Molecular Mechanism Study Of The Anti-abdominal Aortic Aneurysm Effect Of Metformin

BackgroundAbdominal Aortic Aneurysm(AAA)is a partial permanent expansion of the abdominal aorta wall due to local lesions or injuries.It is usually happened in elderly men with about 5-10%incidence in men aged 65 to 79.Smoking,hypertension,hyperlipidemia,and atherosclerosis are the main causes for the formation of AAA.Once ruptures,the mortality of AAA can reach 90%,which is an important cause of death worldwide.Aneurysm resection and artificial vascular replacement and endoluminal stent repair are effective ways to prevent AAA rupture.Patients who fail to reach the surgical indication need long-term follow-up review,which brings huge economic burden and long-term psychological pressure to the patient.There is no clinically effective drug to control the progression of the abdominal aortic aneurysm or to reverse it.Many population-based studies have shown a strong negative correlation between the prevalence of diabetes and the prevalence of AAA,growth,and rupture.Metformin(Met)is the most commonly used hypoglycemic drug worldwide.It is ofter used as first-line drug treatment for type 2 diabetes.Experimental evidence shows that it can aiffect a variety of mechanisms in addition to hypoglycemic effects,including inhibition of inflammation and inhibition extracellular matrix remodeling and reduction of oxidative stress.Naoki Fujimura and others performed logistic regression analysis on cardiovascular risk factors and drug prescriptions of 58 patients with abdominal aortic aneurysm with diabetes,and the results showed that after correcting the effects of gender,age,smoking status and obesity,in the 11 different type of drug treatment,only the use of metformin is negatively correlated with the expansion of AAA,which suggests that metformin may have an inhibitory effect on the occurrence and development of abdominal aortic aneurysms,but the specific mechanism is not clear.Phosphoinositide 3-kinase(PI3K)/Protein kinase B(AKT)/Mammalian target of rapamycin(mTOR)signaling pathways was involved in the regulation of cell growth and proliferation,apoptosis,autophagy and other cell functions.At present,most opinions suggest that the formation,expansion,and rupture of AAA are related to the imbalance of extracellular matrix metabolism,tissue hypoxia,atherosclerosis,inflammation and immune response,apoptosis,blood pressure regulation and hemodynamic abnormalities,while PI3 K/AKT/mTOR signaling pathway is cross-linked and talks with the pathways involved in the aforementioned pathophysiological processes.Previous studies have shown that the PI3K/AKT/mTOR signaling pathway is activated in AAA,and inhibition of the PI3K/AKT/mTOR signaling pathway can inhibit the occurrence and development of AAA.In recent years,a large number of experiments have confirmed that Met can inactivate the PI3K/AKT/mTOR signaling pathway and inhibit the growth of many tumors.Whether the PI3K/AKT/mTOR signaling pathway is involved in Met’s anti-AAA has not been reported.Therefore,this study mainly analyzed the internal molecular mechanism of Met on inhibiting the growth of AAA,in order to provide experimental evidence for the use of Met to prevent and treat AAA.PurposesThe expression of PI3K/AKT/mTOR signaling pathway in AAA tissues was analyzed to further clarify its effect on the occurrence and development of AAA.Angiotensin Ⅱ(Ang-Ⅱ)was used to establish AAA model in mice.Met was given for some mice by drinking.The tumor formation rate,the changes of tumor tissue microstructure,the fracture of arterial wall elastic fiber,the changes of collagen fiber and muscle fiber expression,the apoptosis and autophagy of tissue cells and the expression of PI3K/AKT/mTOR pathway in tissue cells were analyzed to study the anti-tumor effect of Met on AAA.Rat thoracic aorta smooth muscle cells(VSMCs)were used as experimental cells in vitro.After Ang-Ⅱ and/or Met treatment,the proliferation,apoptosis,cell cycle arrest,autophagy,migration and PI3K/AKT/mTOR pathway were detected to further study the anti-AAA effects of Met.PI3K inhibitor(LY294002)and PI3K activator(740 Y-P)were added to analyze the role of the PI3K/AKT/mTOR pathway in this process.si-RNA targeting autophagy-related gene 7(si-Atg7)and Atg7 overexpression plasmid(OE-Atg7)were designed and transfected into VSMCs respectively to further study the possible molecular mechanism of Met on VSMCs autophagy.MethodChapter 1:Met inhibitd the occurrence and development of AAA in mice by inhibiting PI3K/AKT/mTOR pathway and autophagyPatients with AAA were recruited.The AAA tissues were collected and preserved during the procedure.Normal abdominal aorta tissue was utilized as control.Western blotting was used to detect the expression levels of PI3K,AKT,mTOR and their phosphorylated forms p-PI3K,p-AKT,and p-mTOR.A micro-osmotic pump was implanted in Apolipoprotein E gene knockout(ApoE-/-)mice and Ang-II was continuously given to establish AAA mouse model.Met was given for some mice by drinking.Mice was divided into Sham group(physiological saline was obtained by a microosmotic pump),Model group(AAA group,1.44 mg/kg of Ang-Ⅱ was obtained by microosmotic pump),and Met treatment group(AAA+Met group,1.44 mg/kg of Ang-Ⅱ by microosmotic pump and 100 mg/kg Met by drinking).After 28 days,the maximum diameter of the abdominal aorta was detected by Doppler ultrasound Abodominal aorta tissue was removed from mice after inhaled anesthesia with sodium pentobarbital.Hematoxylin-Eosin(HE)staining,EVG staining,Masson staining and Sirius Red staining were used to observe the microstructural changes of abdominal aortic tissue,the fracture of elastic fibers,the expression of collagen fibers and muscle fibers and the changes of collagen fibers.Tunel staining was used to observe the apoptosis of abdominal aortic cells.Immunohistochemistry was utilized to analyze the expressions of platelet endothelial cell adhension molecule-1(PECAM-1/CD31,CD31)and cluster of differentiation(CD68)to assess the neovascularization and macrophage infiltration in the aneurysm wall;Immunohistochemistry was also applied to analyze the expressions of matrix metalloproteinase 2(MMP-2)and matrix metalloproteinase 9(MMP-9)to assess the degradation of the extracellular matrix in the aneurysm wall;Immunohistochemistry was also applied to analyze PI3K,p-PI3K,AKT,p-AKT,mTOR,p-mTOR,microtubule-associated protein 1 light chain 3(LC3B)and Beclin 1 in abdominal aortic tissues.western blotting was utilized to detect the protein expression levels of PI3K,p-PI3K,AKT,p-AKT,mTOR,p-mTOR,LC3B and Beclin 1.qRT-PCR was used to measure the mRNA expression levels of PI3K,p-PI3K,AKT,p-AKT,mTOR,p-mTOR,LC3B and Beclin 1 in abdominal aortic tissues.Chapter 2:Exploring the effects of Met on the function of Ang-Ⅱ-induced VSMCs in vitroVSMCs were used as experimental cells.The experiment was divided into Control group(normally cultured VSMCs),Ang-Ⅱ group(1 μM Ang-Ⅱ treatment);Ang-Ⅱ+Met group(1 μM Ang-Ⅱ+10 mM Met treatment),Ang-Ⅱ+Met+LY294002 group(1 μM Ang-Ⅱ + 10 mM Met+10 μM LY294002 treatment),Ang-II+Met+740 Y-P group(1 μM Ang-Ⅱ+10 mM Met+25 mM 740Y-P treatment).EdU staining was used to detect VSMCs proliferation activity.Annexin V-FITC/PI double staining and flow cytometry were used to detect the apoptosis of VSMCs.PI single staining and flow cytometry were used to detect the VSMCs cycle distribution.Transmission electron microscopy was used to observe the changes of autophagosomes in VSMCs.Immunofluorescence experiments was utilized to analyze the expression of α-SMA and LC3B expression in VSMCs.Scratch and Transwell experiments were used to detect the migration ability of VSMCs.qRT-PCR was used to detect PI3K,AKT,mTOR,LC3B in VSMCs and Beclin 1 mRNA expression levels.Western blotting was utilized to measure the Bax,Bcl-2,PI3K,p-PI3K,AKT,p-AKT,mTOR,p-mTOR,LC3B and Beclin 1 protein expression levels in VSMCs.Chapter 3:Met relieved Ang-II-induced VSMCs autophagy via inhibiting Atg7 expressionVSMCs were used as experimental cells.Western blotting was utilized to detect the autophagy-related geng 4(Atg4)and autophagy-related geng 7(Atg7)expression in VSMCs after Ang-Ⅱ and Met treatment.siRNAs targeting Atg7(si-Atg7)and Atg7 overexpression plasmids(OE-Atg7)were synthezied and transfected into VSMCs.Atg7 expression level was detected by immunofluorescence assay.Western blotting were used to detect the protein expression levels of Atg7,LC3B and Beclin 1.ResultChapter 1:Met inhibits the occurrence and development of AAA in mice by inhibiting the PI3K/AKT/mTOR pathway and autophagyCompared to normal abdominal aortic tissues,the protein expression ratio of p-PI3K and PI3K(p-PI3K/PI3K),the protein expression ratio of p-AKT and AKT(p-AKT/AKT)and the proteion expression ratio of p-mTOR and mTOR(p-mTOR/mTOR)were significantly increased in AAA tissues(P<0.05 or P<0.01).Compared to the mice in the Sham group,the maximum diameter of abdominal aorta in the AAA group was significantly increased(P<0.01).Met treatment significantly inhibited the increase of the abdominal aorta diameter in the mice(P<0.01).The AAA formation rates of mice in Sham group was 0%,in the AAA group was 78%and in the AAA+Met group was 25%.HE staining revealed that compared to Sham group,the abdominal aorta wall of mice in the AAA group was significantly thickened,and the number of infiltrating cells in the medial and adventitia of the arteries was increased.Met treatment attenuated these phenomenon.EVG staining showed that compared to Sham group,the abdominal aortic wall in the AAA group had significantly fewer elastic fibers and rupture,which was accompanied with the loss of original bump shape.AAA+Met group had more elastic fibers and no rupture than the AAA group.The abdominal aortic wall still exerts bump shape.Masson staining revealed that collagen fibers in the abdominal aorta of mice in the AAA group become significantly thicker,cells were arranged disordered and unevenly distributed,and muscle fiber content increased.Met treatment attenuated these phenomenon.Sirius Red staining showed that the fibrosis degree of collagen fibers in abdominal aorta tissue of mice in AAA group were significantly increased.Met treatment also attenuated these phenomenon.Tunel staining showed that Met reduced the apoptosis of mouse abdominal aorta tissue caused by Ang-Ⅱ.Immunohistochemistry revealed that the expressions of CD31,CD68,MMP-2,p-PI3K,p-AKT,p-mTOR,LC3B,and Beclin 1 in the aorta tissue of mice in the AAA group increased(P<0.01).Met treatment significantly reduced the expressions of CD31,CD68,MMP--2,p-PI3K,p-AKT,p-mTOR,LC3B,and Beclin 1(P<0.01).qRT-PCR experiments showed that the mRNA expressions of LC3B and Beclin 1 in abdominal aorta tissue of mice in AAA group was significantly increased(P<0.01).Met treatment reduced the mRNA expressions of LC3B and Beclin 1(P<0.01).Western blotting experiments showed that the protein expression levels of p-PI3K/PI3K,p-AKT/AKT,p-mTOR/mTOR,the ratio of LC3B Ⅱ and Ⅰ(LC3B-Ⅱ/Ⅰ)and Beclin 1 in the aorta tissue of mice in the AAA group were significantly increased(P<0.01).Met significantly alleviated these phenomenon(P<0.05 or P<0.01).Chapter 2:Exploring the effects of Met on the function of Ang-Ⅱ-treated VSMCs in vitroAng-Ⅱ treatment significantly enhanced the proliferation activity of VSMCs(P<0.01)and increased the distribution of VSMCs in the S phase(P<0.01).Met incubation inhibitd the proliferation of VSMCs(P<0.05)and reduced the distribution of VSMCs in the S phase(P<0.01).LY294002 enhanced the effect of Met on VSMCs proliferation(P<0.05),while 740 Y-P reduced the effect of Met on VSMCs proliferation and S-phase distribution(P<0.05 or P<0.01).After Ang-Ⅱ treatment,the apoptosis rate of VSMCs was increased(P<0.01),which was accompanied with the increase of apoptotic promiting protein Bax expression level and the decrease of apoptotis inhibitory protein Bcl-2 expression level in VSMCs.Met treatment reduced the apoptosis rate of VSMCs(P<0.01),lowered the Bax expression,as well as enhanced theBcl-2 expression.LY294002 increased the effect of Met on the apoptosis of VSMCs(P<0.05),as well as the expressions of Bax and Bcl-2,while 740 Y-P had opposite effects(P<0.01).Transmission electron microscopy experiments observed that Ang-Ⅱ treatment increased the number of autophagosomes in VSMCs Immunofluorescence experiments found that Ang-Ⅱ treatment increasedthe expression of LC3B in VSMCs.Met incubation reduced the number of autophagosomes and reduced the expression of LC3B in VSMCs.After the addition of LY294002,the number of autophagosomes was further reduced,accompanied with the decrease of LC3B expression.However,after the addition of 740 Y-P,the number of autophagosomes was increased and the expression of LC3B was also increased.Scratch experiments and Transwell experiments found that Ang-II treatment significantly improved VSMCs migratory ability(P<0.01).Met inhibitd VSMCs migration(P<0.01).LY294002 enhanced the effect of Met on VSMCs migration(P<0.05),while 740 Y-P had opposite effect(P<0.01).qRT-PCR experiments showed that the mRNA expressions of LC3B and Beclin 1 in VSMCs treated with Ang-Ⅱwere significantly increased(P<0.01).Met treatment reduced the mRNA expressions of LC3B and Beclin 1(P<0.01).After adding LY294002,the mRNA expressions of LC3B and Beclin 1 was further inhibited(P<0.05 or P<0.01).After dding 740 Y-P,the mRNA expressions of LC3B and Beclin 1 was increased(P<0.01).Western blotting experiments showed that the protein expression levels of p-PI3K/PI3K,p-AKT/AKT,p-mTOR/mTOR,LC3B-Ⅱ/Ⅰ and Beclin 1 in VSMCs were significantly increased after Ang-II treatment(P<0.01).Met significantly decreased the protein expressions of p-PI3K/PI3K,p-AKT/AKT,p-mTOR/mTOR,LC3B-Ⅱ/Ⅰand Beclin 1 in VSMCs(P<0.01).LY294002 enhanced the effect of Met(P<0.05 or P<0.01),while 740 Y-P reduced the effect of Met(P<0.01)Chapter 3:Met relieved Ang-II-induced VSMCs autophagy via inhibiting Atg7 expressionAng-Ⅱ treatment significantly increased the Atg4 and Atg7 protein expression level in VSMCs,while Met incubation reduced the Atg4 and Atg7 expression level.After transfection with si-Atg7,the expression of Atg7 in VSMCs was decreased,while the expression of Atg7 was enhanced after transfection with OE-Atg7.Overexpression of Atg7 significantly enhanced the autophagy of VSMCs caused by Ang-Ⅱ,as well as increased the protein expression levels of LC3 Ⅱ/Ⅰ and Beclin 1.Inhibition of Atg7 expression had opposite effects.Met treatment significantly reduced the Atg7 expression in VSMCs,as well as inhibited the protein expression levels of LC3 Ⅱ/Ⅰ and Beclin 1.ConclusionThis study analyzed the expression of PI3K/AKT/mTOR signaling pathway in clinical samples of AAA.By establishing mouse model of AAA using Ang-II,the inhibitory effect of Met on AAA was discussed.Using VSMCs as experimental cells,the molecular mechanism of the anti-AAA effect of Met was further analyzed.We first explored the inhibitory effect of Met on AAA and its molecular mechanism through complete animal and cell experiments.In-depth study of changes in the PI3K/AKT/mTOR signaling pathway,autophagy,arterial wall microstructure,and smooth muscle cell function after Met treatment provides a theoretical basis for subsequent drug treatment.The main conclusions were as follows:1.PI3K/AKT/mTOR signaling pathway was activated in AAA tissues;2.Met significantly reduced the incidence of Ang-Ⅱ-induced AAA in ApoE-/-mice,attenuated abdominal aortic wall thickening,reduced the number of infiltration cells in the medial and adventitia of the arteries,enhanced elastic fibers in the arterial wall,protectd the breakage of the elastic fibers,as well as alleviated the changes of collagen fibersaortic wall.Moreover,Met attenuated the cell apoptosis and autophagy in abdominal aortic wall,as well as inhibited the activation of PI3K/AKT/mTOR pathway;3.Met relieved Ang-Ⅱ-induced VSMCs proliferation,S-phase arrest,apoptosis,autophagy,migration,and the activation of PI3K/AKT/mTOR signaling pathways in VSMCs.The inactivation of PI3K/AKT/mTOR pathway caused by Met played key roles in the anti-tumor effect of Met on AAA.4.Met alleviates the increase of Atg4 and Atg7 expression in VSMCs caused by Ang-Ⅱ.Overexpression of Atg7 enhanced the autophagy of VSMCs caused by Ang-II.Inhibition of Atg7 had opposite effect.Met relieved Ang-Ⅱ-induced autophagy of VSMCs by inhibiting Atg7 expression.