Effects And Mechanisms Of Hyperoside Attenuates Apoptosis And Arrhythmia Caused By Myocardial Ischemia Reperfusion Injury

During the treatment for acute myocardial infarction and ischemia heart disease, there is myocardial ischemia reperfusion injury, which causes damage to the re-vascularized tissue. A lot research revealed the underlining mechanism of MIRI is complicated, including oxidative stress, the accumulation and release of inflammatory factors, and disturbance of intracellular calcium cycling et al. Different regulating factors interacts with each other, leading to myocyte apoptosis. Attenuating measurements includes pre-conditioning, post-conditioning and usage of protective medicine. Pretreatment with protective drugs is most important and applicable. Drugs with antioxidative effects shows relief for MIRI.Hyperoside is a kind of flavonol glycosides found in a variety of herb medicine. Lots research on it revealed anti-inflammatory, anti-lipid peroxidation and inhibiting calcium overload and other pharmacological activities. To investigate the protection effect of Hyperoside during MIRI, we established cellular model, isolated heart ex vivo, in vivo model of I/R, to testify the possible protection effect of Hyperoside for MIRI.Part I:The protective mechanisms of Hyperoside on primary neonatal rat cardiomyocytes during Hypoxia/Reoxygenation injuryObjective:To study the protective mechanisms of Hyperoside on primary neonatal rat cardiomyocytes during Hypoxia/Reoxygenation injury.Methods:Primary neonatal rat ventricular myocytes were prepared and cultured as experimental subjects, were randomly distributed into different groups as follows: control group (A); H/R group (B):cardiomyocytes were incubated with anaerobic solution for 4h hypoxia followed by 4h reoxygenation; H/R+HYPlowW group (C):cardiomyocytes are incubated with 10?mol/L hyperoside during 4h hypoxia/4h reoxygenation; H/R+HYPhighgroup(D):cardiomyocytes are incubated with 50?mol/L hyperoside during 4h hypoxia/4h reoxygenation. After H/R period, cells were observed under microscope, Cell Counting Kit-8 assays were used for the cell viability assay. SOD activity were measured, LDH?MDA level in culture solution were measured. Cell apoptosis was measured by flow cytometry.The activation of JNK?p38 and expression level of Caspase-3 were measured by western blotting.Results:(1) Primary neonatal rat ventricular myocytes were prepared and cultured successfully; (2)Compared with control group, cell viability was significantly decreased after H/R; compared with H/R and H/R+HYPlow group, cellular survival rate of H/R+HYPhigh was higher; (3) Compared with control group, cellular contain of SOD was significantly lower after H/R; compared with H/R and H/R+HYPlow group, cellular SOD of H/R+HYPhigh group was higher; (4) Compared with control group, contain of LDH and MDA in culture solution was significantly higher after H/R; compared with H/R and H/R+HYPlow group, contain of LDH and MDA in culture solution was lower;(5) Compared with control group, activation of JNK?p38 pathway was significantly higher after H/R; compared with H/R and H/R+HYPlow group, activation of JNK?p38 pathway of H/R+HYPhigh group was lower;(6) Compared with control group, protein level of Caspase-3 protein were higher after H/R; compared with H/R and H/R+HYPlow group, protein level of Caspase-3 in H/R+HYPhigh group was lower.Conclusion:Hyperoside reduces oxidative stress and cardiomyocyte apoptosis during H/R injury, the possible mechanism is to reduce activation of JNK?p38, reduce protein level of Caspase-3.Part ?:The protective mechanisms of Hyperoside during I/R injury on Langendorff perfused rat heart in vitroObjective:To investigate the protection effect of Hyperoside for I/R model of isolated rat heart during Langendorff perfusion, and its influence of JNK> p38 signal pathway.Methods:Rats orally received Hyperoside (200mg/kg/d) for 14 days, then myocardial I/R model was established by 30min/2h period on Langendorff perfusion, by stop/reperfusion. The level of SOD and MDA in heart tissue were measured. Tunnel staining of left ventricular heart tissue to evaluate apoptosis rate. The phosphorylation of JNK?p38 and protein level of Bax/Bcl-2?TNF-? and NF-?B were detected by western blot analysis.Results:Hyperoside attenuated decrease of SOD and increase of MDA in heart tissue during perfusion I/R, inhibited phosphorylation of JNK?p38; reduced the increase of Bax/Bcl-2 ratio post I/R; reduced myocardial apoptosis and TNF-?, NF-?B expression during perfusion I/R.Conclusion:Hyperoside attenuates the oxidation production and cell apoptosis, inhibited phosphorylation of JNK?p38, reduced myocardial apoptosis and TNF-?, NF-?B during I/R model of ex vivo rat heart on Langendorff perfusion,Part ?:Effect of hyperoside on ventricular electric-physiological characters of isolated rat heart after I/R perfusionObjective:To observe and testify the possible attenuation effect of Hyperoside on changes of ventricular electric-physiological characters of isolated rat heart after I/R perfusion.Methods:Rat were pretreated with hyperoside and perfused under Langendorff according to groups as Part.?. After I/R period, ADP?ERP?APDR of different site of left ventricle(LAB?LAP?LPB?LPP?LFW)?threshold of APD alternation inducibility of ventricular arrhythmias were measured by MAP recording and electrical programmed pacing.Results:(1)Compared with control group, APD and ERP of different sites of left ventricle in I/R group prolonged significantly; compared with I/R group, I/R+HYP attenuates the prolongation of these parameters.;(2) compared with control group, Smax of APDR curve at different sites of left ventricle in I/R group increased significantly; compared with I/R group, I/R+HYP attenuates the increase of Smax; (3) compared with control group, threshold of APD alteration in I/R group increased significantly; compared with I/R group, I/R+HYP attenuates the increase of threshold of APD alteration; (4) compared with control group, inducibility of ventricular arrhythmias in I/R group increased significantly; compared with I/R group, I/R+HYP attenuates the increase of inducibility of ventricular arrhythmias.Conclusion:Hyperoside attenuates abnormal changes of characters of ventricular electrical-physiological parameters induced by I/R, and decreased the inducibility of reperfusion ventricular arrhythmias.Part IV:Protection effect of hyperoside on disturbance of myocardial calcium cycling after ischemia reperfusionObjective:To study the protection effect of Hyperoside of MIRI in vivo, and observe its influence on myocyte calcium cycling.Methods:Rat were anaesthetized for left anterior descending coronary artery temporary ligation for 30min and 2h reperfusion. Before operation, rats received hyperoside according to group strategy as Part.II, Sham group receive all procedure without ligation of silk underpass coronary artery. After reperfusion period, ventricular hemodynamic parameters were measured by carotid artery intubation. CK, LDH level in serum were measured. Tissue surffered MIRI were sampled for HE staining, immunohistochemical staining. Single myocyte of I/R injured ventricle was isolated with enzyme under Langendorff perfusion. Calcium sparking and parameters of calcium transient in myocytes were measured under confocal laser scanning microscope.Results:In vivo model of MIRI by coronary artery ligation were established successfully. (1)Compared with Sham group, ventricular hemodynamic parameters were decreased after I/R, hyperoside improved myocardial function compared with I/R group; (2) Hyperoside attenuated the increase of CK, LDH in serum; (3) Hyperoside attenuated inflammation and apoptosis caused by MIRI; (4)Compared with Sham group, Calcium sparking incresed and calcium transient decresed in myocytes affected by I/R, Hyperoside attenuates the change of calcium cycling by MIRI.Conclusion:Hyperoside attenuates MIRI of rat model in vivo. Hyperoside attenuates disturbance of calcium cycling of ventricular myocytes induced by I/R.