Protective Effects Of NADPH Oxidase Inhibitor Apocynin On Atrial Remodeling In Alloxan- Induced Diabetic Rabbits

Objective Atrial fibrillation（AF）is the most common sustained cardiac arrhythmia in clinical practice,associated with excess mortality.the prevalence of AF increases with aging.Diabetes mellitus（DM）is a strong AF risk factor and give reasons for10–25%of AF patients.However,the underlying mechanisms between DM and AF remains speculative.Previous experiments indicate that reactive oxidant species（ROS）may be involved not only in promoting but also in maintaining AF.We recently indicate that oxidative stress may play an important role for the trigger of AF in an alloxan-induced diabetic rabbit model.The primary sources of ROS include mitochondria,xanthine oxidases,uncoupled NO synthases（NOS）and the nicotinamide adenine dinucleotide phosphate（NADPH）oxidases,and NADPH oxidase（NOX）is thought to play a critical role in redox signaling in a wide range of cardiovascular diseases.Recent studies have found that ROS produced by activated NOX can reinforces atrial structural and electrical remodeling in patients with AF.As an extract from several plant sources,Apocynin is a potent inhibitor of NOX via inhibition of p47phox assembly to the core of NOX.The aim of this study was to evaluate the potential effects of apocynin in atrial electrical and structural remodeling and AF promotion in alloxan-induced diabetic rabbits.Methods For this study,90 Japanese rabbits were randomized to control group（Control,n=30）,alloxan-induced diabetic group（DM,n=30）and apocynin-treated diabetic group（APO,n=30）.Rabbits in the APO group were orally administered apocynin（15 mg/kg/day）for 8 weeks.After 8 weeks,Echocardiography was performed.Aortic diastolic and systolic blood pressure（DBP and SBP）and the LV end-diastolic pressure（LVEDP）were measured.Serum biochemical and malonaldehyde（MDA）,superoxide dismutase（SOD）levels and left atrial tissue NADPH oxidase（NOX）activities were measured.Isolated rabbit hearts were Langendorff perfused.Atrial refractory effective period（AERP）,atrial effective refractory period dispersion（AERPD）,interatrial conduction time（IACT）and vulnerability to AF were measured.I_Ca,L was measured by whole-cell voltage-clamp techniques in left atrial myocytes isolated from the rabbits of three groups.Atrial interstitial fibrosis was evaluated by masson’s trichrome staining.The protein expression of NF-κB,TGF-β,p38,P-p38,JNK,P-JNK,ERK and P-ERK were measured by Western-blotting analysis.Results⑴compared with the Control group,the Left atrial diameter（LAD）,Interventricular septal thickness（IVST）,Left ventricular posterior wall thickness（LVPWT）,Left ventricular end diastolic diameter（LVEDD）were significantly increased in the DM group（P<0.05）,IVST and LVPWT were suppressed by treatment with apocynin（P<0.05）.⑵the IACT was prolonged and AERPD was increased in the DM group（P<0.05）,while apocynin attenuated IACT and AERPD in diabetic rabbits（P<0.05）.Wenckbach cycle length of AV conduction（AVWCL）was decreased in the DM group（P<0.05）and partially suppressed by treatment with apocynin（P<0.05）.AF vulnerability in DM group was significantly higher than control group（46/450 vs.5/450,P<0.05）and was markedly reduced by apocynin（12/450 vs.46/450,P<0.05）.⑶DM group had increased atrium weight ratio,LV weight ratio,and heart weight ratio（P<0.05）.Treatment with apocynin decreased atrium weight ratio,LV weight ratio,and heart weight ratio,but there were no significant statistical differences among the 3 groups.⑷The Left atrium Collagen volume fraction（LACVF）was significantly increased in the DM group compared with control group（P<0.05）and was attenuated by apocynin treatment（P<0.05）.⑸Serum MDA level had apparently increased（P<0.05）.Apocynin significantly increased serum SOD activity（P<0.05）and decreased serum MDA level（P<0.05）in APO group.left atrial tissue NOX activities were apparently increased in DM group compared with Control group.Apocynin treatment reduced the increase of myocardial NOX activities in the diabetic rabbits.⑹The density of I_Ca,L current was apparently increased in diabetic myocytes than that in the control,apocynin significantly reduced it.⑺the expressions of TGF-β,P-p38,NF-κB,P-JNK,ERK and P-ERK apparently increased in the DM group（P<0.05）,treatment with apocynin in DM rabbits（APO group）significantly reduced their expressions（P<0.05）.⑻The expressions of rac1 increased in the DM group（P<0.05）,and apocynin reduced its expressions（P<0.05）.Conclusion Apocynin attenuates atrial remodeling and suppresses AF promotion in alloxan-induced diabetic rabbits by inhibiting the activation of NADPH oxidase.The inhibition of overexpression of NF-κB,TGF-β,P-p38,P-JNK,ERK and P-ERK may contribute to its protective effects of atrial remodeling.The potential benefits of apocynin on ionic channel remodeling in alloxan-induced diabetic rabbits need further investigation.