The Role Of Gallic Acid In The Development Of Angiotensin Ⅱ-induced Atrial Fibrillation And The Underlying Molecular Mechanism

Background:Atrial fibrillation(AF)is the most common clinical arrhythmia disease,and its prevalence increases with age.Hypertension is one of the most common complication among many risk factors causing AF.Epidemiological studies have shown that hypertension could activate renin-angiotensin-aldosterone system(RAAS)and increase angiotensin Ⅱ(Ang II),which plays a critical role in the regulation of inflammatory response,oxidative stress,and fibrosis,the common pathological alterations in AF.Increasing evidence shows that Ang II exerts these actions mainly through angiotensin type I receptor(ATIR)and activation of NF-kB signaling.The ubiquitin-proteasome system(UPS)plays an important role in controlling protein degradation in the cardiac hypertrophy process,and the 26S proteasome contains two structures:the 20S core particle and the 19S regulatory particle.The core particle consists of two pairs of rings,and the proteolytic β subunits reside in the inner two rings.Among them,three standard catalytic subunits,including β1,β2,and β5 are responsible for caspase-like,trypsin-like,and chymotrypsin-like activities,respectively.When cells are stimulated by inflammatory stimuli such as IFN-y,three of the standard catalytic subunits are replaced with three inducible subunits,including β1i,β2i and β5i.This modified proteasome is called the immunoproteasome.Our recent data revealed that the immunoproteasome catalytic subunits play important roel in the regulation of cardiac hypertrophic remodeling,heart failure,atrial fibrillation(AF),and atherosclerosis in mice after DOCAsalt treatment or Ang II infusion.Gallic acid(GA),also known as trimethylbenzoic acid,is a natural plant extract.It has been proved that it has multiple functions such as reduction of blood pressure,antiinflammatory,anti-fibrosis and free radical scavenging and so on.It has been reported that GA is able to reduce Ang Ⅱ-indcued hypertension and improve cardiac remodleing in mice.However,whether GA can regulate the activity of immune proteasomes thereby inhibiting Ang Ⅱ-induced AF has not been studied.Objective:To explore the role of GAin the regulation of AF and the underlying molecular mechanism.Method:1.AF model was induced by Ang Ⅱ(2000 ng/kg/min)infusion using an osmotic minipump for 1-3 weeks in Male 8-10 weeks old mice.Electrical stimulation was used to induce AF occurrence.2.The different doses of GA(5 mg and 20 mg/kg.BW)were administered once a day for total 21 days;3.Echocardiography was performed to measure left atrial volume by using a Vevo 2100 High-Resolution Imaging System;4.Hematoxylin&Eosin(H&E)and Masson’s trichrome staining were used to analyze inflammatory cell infiltration and the extent of atrial fibrosis in the atria;5.Dihydroethidium(DHE)staining was used to detect superoxide production.6.Real-time quantitative PCR was performed to detect the expression levels of inflammation and fibrosis related genes,including α-SMA,collagen I,collagen III,IL-1β,IL-6,TNF-α,MCP-1,NOX2,NOX4,β1 β2,β5,β1i,β2i and β5i;Western blot analysis was performed to detect the expression levels of inflammation and fibrosis related signaling mediators,including TGF-β1,p-Smad2/3,Smad2/3,p-p65,p65,Cx43,PTEN,p-AKT1,AKT1,β2i and β5i.7.The proteasome activities were measured using fluorogenic peptide substrates.8.Statistical analysis:All results are presented as mean ± SD.GraphPad Prism 5 was used for statistical analysis and graphing.P value<0.05 was considered statistically significant.Result:1.Compared with that of the Ang Ⅱ infusion group,GA treatment dose-dependently inhibited Ang II-induced elevation of systolic blood pressure,left atrial dilation,and incidence and duration of AF in mice.2.Compared with that of the Ang Ⅱ infusion group,GA treatment dose-dependently reduced Ang II-induced increase of inflammatory cell infiltration,atrial fibrosis as well as the mRNA expression levels of fibrosis markers(α-SMA,collagen Ⅰ and Ⅲ)and inflammatory cytokines(IL-1β,IL-6,TNF-α and MCP-1)in atrial tissues.GA treatment also significantly inhibited the activation of IKKα/β-IKBα-NF-kB signaling pathway in atrial tissues induced by Ang Ⅱ infusion.3.GA treatment dose-dependently attenuated Ang Ⅱ-induced enhancement of ROS level and the mRNA levels of NADPH oxidase subunits(NOX2,NOX4 and p22)in the atrial tissues compared with that of the Ang Ⅱ infusion group.4.Ang Ⅱ infusion markedly increased 26S proteasome activities(trypsin-like and chymotrypsin-like)and the mRNA levels of the immunoproteasome catalytic subunits(βi and β5i)in atrial tissues compared with saline-infused control.However,GA treatment dose-dependently inhibited these effects.5.Ang II infusion significantly decreased the protein levels of PTEN and increased the downstream protein levels of p-Akt1,TGF-β,and p-Smad2/3 compared with salineinfused control.In contrast,GA treatment dose-dependently reversed the levels of these proteins.Conclusion:GAsignificantly inhibited Ang Ⅱ-induced hypertension,atrial remodleing and AF inducibility in mice.These protective effects were associated with GA-medicated inhibition of the immunoproteasome subunit expression and activities,which blocks PTEN degradation,leading to attenautation of pro-fibrotic,pro-inflammatory and oxidative signaling pathways(AKT1,TGF-β/p-Smad2/3,IKK/NF-kB and NOXs).These results suggest that GA may be a new small molecular compound for the prevention and treatment of AF.