Studies On Mitochondrial Mechanism Of Resveratrol Protection Against Myocardial Ischemia/Reperfusion Injury

Resveratrol is a polyphenolic compound which mainly lies in some popular food and herbal medicine, such as grape, peanut and Polygonum cuspidatum. In recent years, resveratrol was well-documented having good nutritional activities, such as cardioprotection, anti-aging and anti-cancer. Due to these, resveratrol becomes a hotspot both in molecular nutriology and pharmacology. Recently, many evidences have demonstrated that resveratrol can protect against myocardial ischemia/reperfusion (I/R) injury. However, the mechanism is not well-illustrated. Because of the importance of mitochondria to cardiomyocytes function, the possible mechanism of resveratrol’s cardioprotection was supposed relating to mitochondrial function. Mitochondria was not simply the powerplant of cardiocytes, but also important in maintaining the Ca2+ homeostasis and regulating cell apoptosis or necrosis. The function of mitochondria is tightly related to mitochondrial permeability transition pore (mPTP). mPTP opening is considered as a critical event in the transition from reversible to irreversible reperfusion injury. Voltage-dependent anion channel (VDAC), which located in the mitochondria outer membrane, functions as gatekeeper for the entry and exit of mitochondrial metabolites and ions. VDAC plays an important role in the regulation of mPTP opening, so we presume VDAC is the possible mitochondrial target of resveratrol during I/R injury.In this study, an in vitro I/R model was replicated on H9c2 cardiomyocyte by anoxia/reoxygenation (A/R) treatment. Gene clone and RNA interference (RNAi) were employed to explore the relationship of VDAC expression and the protective effects of resverastrol. Relative biochemical index, such as LDH, CPK, MDA, SOD and GSH-Px were determined, as well as Ca2+, ROS and Cl-, that were assayed by flow cytometry or spectrofluorometer, were collectively used to reveal the molecular mechanism of resveratrol protection. The main results obtained in the study are summarized as follow:1. The dose-effect relationship of resveratrol protection against A/R injury was investigated. Cell viability was detected by Methyl thiazolyl tetrazolium (MTT) method. The activity of LDH and CPK in culture medium, SOD, GSH-Px and MDA in cardiomyocytes were measured by a colorimetric method. The percentage of apoptosis was measured by annexin V-FITC/PI double staining with flow cytometry. Meanwhile, intracellular ROS, Ca2+, and mitochondria membrane potential were also measured by flow cytometry. mPTP opening was detected by spectrophotometer. The results show that pretreatment with resveratrol significantly suppressed the increase of LDH, CPK activity and the decrease of viability that resulted from A/R in a dose-dependent manner up to 50μM. Moreover, resveratrol reduced ROS generation and MDA content, increase the SOD, GSH-Px activity. With pretreatment of resveratrol, mitochondria membrane potential and mPTP was well preserved.2. VDAC1 expression was detected by western blot in H9c2 cells subjected to A/R injury with or without resveratrol pretreatment. We found that VDAC1 expression was up-regulated in A/R injury, which was inhibited in resveratrol group.3. RNAi was employed to further illustrate the role of VDAC1 in A/R-induced mPTP opening. The results showed that, with silencing VDAC1 expression, oxidative stress and Ca2+ overload were significantly attenuated, mitochondrial membrane potential was well preserved, mPTP opening was blocked, then the mitochondria-mediated apoptosis was inhibited, as demonstrated by the decrease of Cyt c release from mitochondria and the inactivation of caspase-3. These findings concluded that VDAC1 plays a vital role in modulating A/R-induced mPTP opening.4. A pFLAG-VDAC1 recombinant was constructed and transferred to H9c2, for demonstrating the relationship between VDAC1 overexpression and resveratrol protective effect. The results showed that up-regulated VDAC1 will attenuate resveratrol protective effects, which suggested that the cardioprotection against A/R injury of resveratrol is mediated by VDAC1.5. As a final, the possible regulation mechanism of mPTP opening via VDAC1 was depicted. Many evidences have demonstrated that the incidences of Cl-oscillation play an important role in I/R injury. Down-regulation of VDAC1 expression by shRNA or resveratrol decreased mitochondrial Cl- concentration significantly, resulting in attenuation of calcium overload and oxidative stress. It indicated that Cl- was a key factor of mPTP regulation and involved in the protective of resveratrol mediated by VDAC1.In summary, during I/R process, cytoplasmic Cl- flows into mitochondria through VDAC, subsequently inducing Ca2+ release, which is termed "chloride-induced calcium release (ClICR)". ClICR can induce Ca2+ overload and oxidative stress, resulting in mPTP opening and activating mitochondria-mediated apoptosis pathway. Resveratrol pretreatment reverse I/R injury via down-regulation of VDAC1.