Cardiac Protective Effects of Resveratrol Glucoside in Rat Models with Myocardial Ischemia Reperfusion

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1 Cardiac Protective Effects of Resveratrol Glucoside in Rat Models with Myocardial Ischemia Reperfusion Jihong Li 1, Haiyan Yu 2, Jingya Yang 3 1.Zhang Zhongjing School of Chinese Medicine, Nangyang Institute of Technology, Nangyang , China 2.Institute of Biology and Chemical Engineering, Nangyang Institute of Technology, Nangyang , China Abstract 3.College of food science and technology, Shanghai ocean university, Shanghai , China Myocardial ischemia is a common clinical symptom. It leads to decrease of cardiac oxygen supply due to heart hemoperfusion decline during disease onset. Therefore, the myocardial energy metabolism may appear disorders resulting in unable to support the heart working properly. It may severely threaten the patient s life when it becomes seriously. This study has analyzed the myocardial reperfusion in rat models in order to discuss the protective effects of resveratrol glucoside on myocardial ischemia reperfusion, as well as related protective mechanisms, thereby estimating those relevant influencing factors and providing references for clinical practice. Keywords: Resveratrol Glucoside, Myocardial Ischemia, Rat Models. 1. STUDY BACKGROUND 1.1 Literature Review Myocardial ischemia is commonly seen clinically. There are a lot of pathogenic factors of which the most important fundamental factor is a high demand on myocardial oxygen. However, the supply is unable to meet the demand (Dong et al., 2011). Early researches believe that the cardiac blood-supply ability (He et al., 2011) can be fast recovered if the reperfusion is performed in patients with myocardial ischemia. However, in later studies, during the reperfusion and thrombolysis, the clinical results achieved through decreasing the infarct size are likely limited (Zhang et al., 2013) by ischemia reperfusion injuries. Resveratrol glucoside is a sample with strong active capacity. In majority of studies, the intervention of resveratrol glucoside is expected to enhance the cardiac protection role thereby improving the clinical application results of myocardial ischemia reperfusion. 1.1 Study Objective Rat models are animal model samples generally used in medical clinical researches. They have a high reference value (Fan et al., 2013) with respect to experimental iatreusiology, experimental pathology and experimental physiology studies. Moreover, using these rat models to study the cardioprotection of resveratrol glucoside on myocardial ischemia reperfusion, it is also an experimental method (Wu et al., 2013) provided with strong evidence. This study has established a damage model with myocardial ischemia among rat models using coronary artery ligation and relaxing method in order to analyze the cardioprotection of resveratrol glucoside on myocardial damages thereby providing theoretical references for intervention of resveratrol glucoside in clinical studies. 1. Summary of Resveratrol Glucoside The ingredient of resveratrol glucoside is derived from the roots of polygonum cuspidatum - a plant of polygonaceae. It also can be extracted from roots of polygonum multiflorum, barks of piceaglehnii masters and eucalyptus of myrtaceae. The samples of resveratrol glucoside selected in this study are some samples extracted from polygonum cuspidatum and belong to the natural active ingredients (Zhao et al., 2010) of polydatin. Its 342

2 English name is known as Polydatin and the full name as resveratrol glucoside. Its chemical formula of physicochemical properties is shown as follows: SMILES: Oc1ccc(cc1)/C=C/c1cc(O)cc(c1) O Resveratrol glucoside is mainly produced in southern Gansu, southern Shaanxi, the east, middle and south China, as well as Sichuan and Guizhou areas. It is a perennial arbuscular herbal, up to 1 m or above. Its rhizome lies in the deep soil, with yellowish-brown nodular structure in the surface. While the stem is upright cylindrically. The surface is smooth and hairless, present with multiple red spots and showing hollow. It is externally featured with leaves alternate, simple, ovate nearly rounded, 12 cm the longest and 9 cm the widest. Its front end is short and sharp but round or wedge-shape in the stem base. The flowering phase is between July and September. The fruitful period for medicine is in October (Zhang et al., 2010). Its clinical pharmacological function is mainly including eliminating phlegm, relieving cough and asthma, antibiosis, regulating blood-lipids, scavenging free radicals and lowering cholesterol. With respect to antibacterial actions, it has a high inhibiting effect mainly specific to staphylococcus aureus, type A and B streptococcus, micrococcus ascoformans catarrhalis and escherichia coli. For antivirus activities, it has a strong inhibiting effect mainly to Asian influenza A (Beijing) 68-1 strain virus, herpes simplex virus and orphan virus. This study has mainly discussed the clinical application of resveratrol glucoside and observed its role (Zhao et al., 2010) of protective mechanisms in rat models with myocardial damages, thereby estimating the leading effect of resveratrol glucoside in cardiac function under myocardial ischemia reperfusion, including cardiac systolic function, myocardial infraction (MI) areas, perfusion-induced arrhythmia, myocardial superoxide dismutase activity, nitric oxide synthase activity and malondialdehyde levels. 1. EXPERIMENTAL DATA SELECTION METHODS AND STUDY METHODS OF RESVERATROL GLUCOSIDE 2.1 Experimental Animals and Biochemical Substances A total of 26 male adult rats were selected as study samples with the wight of a rat around g. The study protocol was edited by Research Committee for (National) Guide for the Care and Use of Laboratory Animals. Experimental samples of resveratrol glucoside were supplied by China ZONP Pharmaceutical Co., Ltd. 2.2 In Vivo Myocardial Ischemia Reperfusion Injury Samples The study materials of resveratrol glucoside were based on rat s heart models and the study adopted a random allocation method to investigate the pharmacological mechanisms. Pentobarbital sodium injection was given to those adult rats for anesthesia and the dose was calculate under a ratio of 30 mg/kg. The body temperature of those rat models was controlled at (36.8±1.0) ºC. The respiration rate of rat models was stabilized at approximately 1.5 m L/100 g. The arterial blood pressure values were recorded by ECG. These monitored data results were recorded and used to evaluate whether these physical indexes of rat models have met the study criteria. The conventional heart rate of rat models was measured and calculated by ECG intervals and monitored though the left femoral venous duct. An incision for left thoracotomy was decided in the space between the 3 rd and 4 th rib in order to expose the heart model of rats. Silk suture was applied starting from the descending branch of left coronary artery (Jian et al,2015). After keeping a stable cardiac function in the heart model of rats, the conditions of myocardial ischemia was recorded for 15 minutes. The stability of heart rate and blood pressure was studied via ligation and reperfusion. The signs of myocardial ischemia in rat models were distinguished based on arterial occlusion after the coronary artery was successfully managed, showing the ascending and descending trends in ECG after the ligation. The blood pressure was also monitored and assessed. 2.2 Measurement of Sample Indexes of Arrhythmia In this study, the arrhythmia was a corresponding indicator measured in the rat models of the treatment group after receiving the ischemia reperfusion for 60 minutes. Intravenous injection of resveratrol glucoside 100 g was given to the rats to measure the physical signs. While 0.9% normal saline was mixed in the resveratrol glucoside with same volume in the control group based on index parameters of resveratrol glucoside as the evaluation indicators. Rat s ventricular rhythm and premature beats were measured in the preliminary trial based on ventricular fibrillation and tachycardia as references. Arrhythmia evaluation indexes were used to estimate specific patterns of arrhythmia in the ischemia period based on Lambeth Conven - tions as references, and 343

3 decide the impacts of resveratrol glucoside via respectively recording VPC and VT indicators. 2.2 Measurement of Effective Values of Myocardial Infraction Areas The ischemia reperfusion therapy was performed to those rats for 30 minutes in the trial based on 60 minutes as the basic treatment time. The concentration of resveratrol glucoside was set as 0.1% and a dose of 0.2 ml per 100 g body weight was mixed with 0.9% normal saline for injection 10 minutes before femoral vein ischemia. A complete aorta ligation was carried out after injecting the resveratrol glucoside. It was injected into the heart via the left ventricular wall. Then the transfer and freeze staining was carried out for 15 minutes. If TTC-stained normal myocardium shows blue, it means the ischemic myocardium is red and swelling; if TTC showed pale, it means non-staining (Meng et al, 2014). Systematic analysis was conducted when related data were incoming Terminal Server. The scope (dangerous zone) of ischemic myocardium represents a value from ischemic percentage to a specific interval of left ventricle while the degree of myocardial infraction is a percentage of ischemic infarction size in the ventricle. 3. MATHEMATICAL MODELS OF STATISTIC ANALYSIS SPECIFIC TO STUDY RESULTS Relevant experimental data of resveratrol glucoside collected from the rat models with myocardial ischemia reperfusion were entered into SPSS 19.0 statistical software for mathematical analysis and statistics. Enumeration data is represented by ((X ± S) and tested by x 2. (P<0.05) means the difference is statistically significant. Meanwhile, the variance formula is used for mathematical result analysis. The smaller the variance yields are, the more stable of mathematical results in this study will be. They are close to the expected study direction. On the contrary, it represents that the study data are not stable and needs further studies by collecting samples again. Sample variance measurement formula of rat models: S 2 = n i 1 (X S) 2 n 1 (1) In this formula, S represents the sample of assessment data; n means sample quantity collected; ((X ± S) indicates the measurement value of data samples in this study group. The relevant categories of index changes such as infarct size, myocardial enzyme activities and lactic dehydrogenase are clearly displayed in the formula. The facticity of mathematical statistics of research results are evaluated in the meantime. The parent variance is used for data validation, thereby checking whether the data range of the results is close to the anticipated index or not. Calculation formula of parent variance: ς 2 = n i=1 (xi x ) n (2) 4. STUDY RESULTS OF RESVERATROL GLUCOSIDE IN RAT MODELS WITH MYOCARDIAL ISCHEMIA REPERFUSION 4.1 Impacts of Resveratrol Glucoside on Ecg In Rat Models with Myocardial Ischemia Reperfusion The impacts on ST-T segment changes in rats with myocardial ischemia reperfusion are shown in Table 1. No extremely significant dynamic changes were produced at ST-T segment during the experiment in the sham-operation group. However, the ST-T segment was significantly elevated in the rat models in the coronary artery ligation group at 30 minutes and 2 hours after reperfusion. It was relatively significant as compared to the sham-operation group. The difference was statistically significant (P <0.05). However, in the treatment group, resveratrol glucoside was given to rats in advance, these rats experienced the symptoms of anti-myocardial ischemia in varying degrees. It is clearly that the reperfusion is also a major factor (Wang, 2014) that may induce the ST-T elevating. When adding the dose of resveratrol glucoside, the drug action time was also increasing in rat models. The stability in the large-dose treatment group was higher than that of model control group. The difference of data comparison was significant among groups (P <0.05). Table 1 Comparison and analysis of the effects of myocardial ischemia reperfusion on ECG ST-T changes group Model group Small dose group Dose group control group normal 0.142± ± ± ±

4 Ligation 30min Reperfusio n 2h 0.164± ± ± ± ± ± ± ± Myocardial Infarction Category and Activity Index of Resveratrol Glucoside in Rats with Myocardial Ischemia Reperfusion Impacts of resveratrol glucoside on MI scope and serum CK and LDH activities in rats with myocardial ischemia reperfusion are shown in Table 2. Table 2 Impacts of resveratrol glucoside on MI scope and serum CK and LDH activities in rats with myocardial ischemia reperfusion group Infarction range Myocardial mildew Dehydrogenati on activity of lactic acid Model group 39.52± ± ± Small dose 35.48± ± ±1983. group 6 Dose group 32.67± ± ± High dose 29.12± ± ± group The control 22.05± ± ±1986. group Pathological Impacts of Resveratrol Glucoside on Myocardium in Rats with Myocardial Ischemia Reperfusion These experimental samples were executed to death when the trail was finished in order to take out the heart models. The contrast samples were collected from the left ventricle; soaked by formalin (5mol/L); and given relevant paraffin embedding and sectioning so as to examine the corresponding HE chromosome images and observe the results, as well as the alternate shape of myocardial model. The rat s left ventricular fibers were arranged regularly in the sham-operation group. It expresses a normal staining in the myocardial nucleus. Meanwhile the sarcoplasmic reticulum was longitudinally and clearly seen, with no related necrosis or pathological changes discovered. The lesions of rat s myocardial fibers showed the diffused distribution in the ischemia reperfusion group, mainly showing muscle fiber structure disorder with apparent basophilia and a small part of granular changes, as well as myolysis and focal liquefied necrosis (Fang et al, 2014) in some lesions. After giving the pretreatment with 10mg /kg of resveratrol glucoside, no obvious abnormalities were seen in the myocardium after myocardial ischemia reperfusion under microscope, only the focal or patchy distribution could be seen. Effects of resveratrol glucoside on MDA levels and SOD activities in rats with myocardial ischemia reperfusion are shown in Table 3. Table 3 Effects of resveratrol glucoside on MDA levels and SOD activities in rats with myocardial ischemia reperfusion group MDA(n mol/ml) SOD(nU/mL) Model group 6.85± ±21.7 Small dose group 4.92± ±25.6 Dose group 4.15± ±26.7 High dose group 3.99± ±16.5 The control group 3.85± ±

5 5. ANALYSIS ON CARDIAC PROTECTIVE EFFECTS OF RESVERATROL GLUCOSIDE IN RATS WITH MYOCARDIAL ISCHEMIA REPERFUSION At the present stage, there are many measures used in detection of myocardial infarction. This study has adopted the myocardial sample chemical staining assay and indirectly evaluating active enzyme indexes (Xu et al, 2014) via assessing MI scope. If these obtained results are consistent, it means resveratrol glucoside plays a remarkable role of cardiac protection in rat models with myocardial ischemia reperfusion. Meanwhile, the drug dependence of resveratrol glucoside was lower for MI scope in rats with myocardial ischemia reperfusion. Moreover, its protective actions can be learned from cardiac cells releasing LDH and CK when performing the reperfusion. With respect to the progression of acute myocardial infarction (AMI), it can be observed after the revascularization. While the myocardial ischemia reperfusion is an important factor that affects the ventricular fibrillation. The sudden death rate is rather high among patients with coronary heart disease. However, those selected rat models are close to the study results. When the transient myocardial ischemia is happened in those animals, the arrhythmia can be effectively controlled if blood supply is restored. In this study, when the heart rate of rat models went fast, the dynamic changes of ST segment and T wave were mutually overlapped and then no significant changes were discovered in ST segment. It only investigated the ST-T combination variation reaction interval (Xiong and Wei, 2014). However, it also found that the potential difference (PD) was rather large between myocardial ischemic region and normal tissue during analyzing the ST-T segment displacement. This case has clearly proved that the significant T segment displacement is a basic factor for continuously elevated ST-T segment. Accordingly, the myocardial anaerobic metabolism was rising to some extent in rat models with myocardial ischemia reperfusion. Meanwhile, the oxygen tension dropped rapidly with a remarkable PD and significantly elevated ST-T. Within the first 30 minutes before coronary artery ligation in the rat models and 2 hours after reperfusion, ECG showed a significant increase in rats with coronary artery ligation in the model group, which indicated that resveratrol glucoside could effectively resist those adverse factors (Lu et al, 2014) of coronary artery ligation. Particularly, cardiac cells are usually irreversible after a rat model appears myocardial ischemia. However, these damages can be avoided or repaired within a short time. Therefore, resveratrol glucoside can really manage the damages and improve the survival opportunities. 6. CONCLUSION Most studies have shown that cardiac cells will be irreversible after severe myocardial ischemia lasting for 30 minutes. So an optimal treatment time for clinical treatment and rescue shall be managed in patients with myocardial ischemia. While in this study, we have found that the rat models experience significant myocardial fiber lesions with focal liquefied necrosis. However, during the intervention of resveratrol glucoside, the lesion areas were reduced to some extent and injury scope was gradually decreasing after using different doses of intervention. This suggests that the clinical effects of resveratrol glucoside are significant and it plays an important protective role from myocardial ischemia reperfusion injuries. Under this protective action, it can effectively prolong and shorten the process of the irreversible myocardial injuries after myocardial ischemia reperfusion, thereby allowing partial cardiac muscles safely going through the injury period caused by severe myocardial ischemia reperfusion. These cardiac muscles will survive due to established collateral circulation thereby limiting the extension of necrosis areas. Resveratrol glucoside can effectively reduce the serum activities and recover the ECG ST-T segment changes to normal after ischemia reperfusion via reducing MI scope induced by ischemia reperfusion in rats, thereby playing a role of anti-myocardial ischemia injuries. Therefore, its mechanism is closely associated with anti-lipid peroxidation. ACKNOWLEDGMENTS Study on research and development of cordyceps militaris functional food and rapid and exact determination of it s active ingredient. Project No: REFERENCES Dong L.Y., Fan Y.F., Shao X., Chen Z.W. (2011). The effect of vitrecin on induced apoptosis induced by myocardial ischemia reperfusion injury in rats, Chinese medicine, 33 (6), Fan H.T., Ding S.L., Lin H.S. (2013). Research progress of pharmacological research of Chinese traditional 346

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