Effect Of Shensongyangxin Capsule On Cx40 Of Diabetic Rats

Objective: Diabetes is one of principal diseases which threaten human health.According to a statistic,the mortality of diabetic cardiovascular complications up to 75%,most of them die of coronary heart disease related to diabetic.Clinically,diabetic can be with all kinds of arrhythmias,but the pathogenesis is still not very clear.Meanwhile,the effective treatment is scarce in clinic,such as antiarrhythmic drugs,radiofrequency ablation(RFA),the Pacemaker in the treatment of bradyarrhythmia and ICD implantation for VT/VF.These treatments are positive and effective,but there are remain limitations.Recently people propose a new conception named upstream therapy of arrhythmia which focus on the pathophysiology of arrhythmia such as the myocardial ion current disorder,reentrant and ectopia triggered activity.Gap junction(GJ)is a special connection mode between two myocardial cells,which often refer to intercalated disc.Connexins(Cx)in GJ play an important role in cell coupling.There are three main subtypes in the heart:Cx40,Cx43 and Cx45.Research has proved that Cx40 locate in atrium and ventricle.Cx43 mainly locate in ventric and merely low degree express in atrium.Cx45 locate in conduction system of cardiac.Comparatively speaking on velocity of conduction Cx40 and Cx43 have rapid conduction feature.While conduction velocity of Cx45 is much slower,which may regulate the conduction of electrocardiogram.Arrhythmia of DM is related to disorder of ectopia triggered activity and conduction of electrocardiogram.Also the conduction is contingent on GJ.Research has proved in myocardium of DM over activation of RAAS,oxidation stress,fibrosis of myocardium and so on can lead disorder of Cx expression and distribution.The research is to discuss the effect of Shensongyangxin(SSYX)capsule and Benazepril on Cx40 through analyzing the disorder of Cx40 expression and distribution of diabetic rats myocardium.Methods: Male SD rats(n=50)purchaseed from Nanjing Qinglongshan animal breeding centre were randomly divided into a group of control(n=10)and a group of mode of DM(n=40).After one week adaptive breeding,group of mode were one-time injected Streptozotocin(STZ)65mg/kg from enterocoelia.Blood glucose(BG)were measured from caudal vein 3days later,BG?16.7mmol/l were defined as successful mode.The group of mode(n=37)ultimately were divided into group of DM(n=12),group of Benazepril(n=12)and group of SSYX(n=13).Group of Benazepril were given Benazepril 7.5mg/kg.d by gavage,group of SSYX were given SSYX capsule 1g/kg.d by gavage and group of control and group of DM were given same amount of distilled water by gavage.Each group were fed by sufficient food and water 8 weeks.After anesthesia using 25% Urethane 6ml/kg by intraperitoneal injection,each group were examed by electrocardiogram.After decollation,we exposed the heart,took out the heart and rinsed the heart in 4? saline.Then we separated the left atrium and left ventricle by incision along macroaxis and atrioventricular groove of the heart.Each sample divided into two parts,one section were immersed in Formaldehyde solution,and the other section preserved in-80? refrigerator.Then the level of expression of Cx40 of each group were analyzed by Western blotting.The structural modification were observed by HE stain.The distribution of Cx40 were investigated by immunofluorescence.Results: 1.Feeding 8 weeks later,diabetic rats appeared the so-called typical clinical polydipsia,polyuria,and more food and weight loss of “a little more than three”symptoms obviously.Various arrhythmias can be seen from DM group's ECG.Heart rate(HR)of DM group decrease significantly compared to other three groups(P<0.05).Compared to group of control and Benazepril,P wave width of group of DM are increased significantly(P<0.05),While compared to group of SSYX,there are no statistically significant.There are no statistical significance of QRS duration and QTc among 4 groups.2.The Cx40 expression level of group of DM myocardium(atrium and ventricle)are decreased significantly compared to group of control(P<0.05).Compared to group of DM,Cx40 in atrium and ventricle of group of Benazepril and atrium of SSXY have a rising trend(P<0.05),but there are no statistical significance of Cx40 in ventricle between group of DM and SSXY(P>0.05).3.Pathology observation from HE staining show that myocardium of group of control arrang orderliness,while group of DM are disorganized,and group of Benazepril and SSXY can see fibrous tissue and vascular inflammatory reaction though the myocardium arrang orderliness.4.Immunofluorescence show in group of control,Cx40 are expressed mainly in the intercalated discs between neighbouring myocytes just like ladder-like distribution.Occasionally Cx40 are located in lateral of myocytes.Cx40 of group of DM are mainly expressed in lateral of myocytes and the distribution are disorganized.Group of Benazepril Cx40 are expressed in not only intercalated discs but also the lateral of myocytes.Although WB show the level of Cx40 of group of SSYX atrium are increased significantly,Cx40 are located mainly in lateral of myocytes.Conclusion: 1.Various arrhythmia and HR decrease can be found in DM group.Benazepril and SSYX capsule can increase the HR of DM rats.Benazepril can modulate P wave width,but the effect is limited for QRS duration and QTc.The effect of SSYX is limitated for P wave width,QRS duration and QTc.2.Cx40 mainly expressed in atrium less expressed in ventricle.The level of Cx40 is decreased in DM rats myocardium and the distribution is mainly in lateral of myocytes.3.Benazepril can improve myocardial Cx40 level and distribution.SSYX can up-regulate the atrium Cx40 level,but have less impact for the distribution.There are no obvious effect on Cx40 expression and distribution of diabetic rats' ventricle treated by SSYX capsule.