| Background:Ischemic heart disease, also known as coronary atherosclerotic heart disease, has become a leading cause of death worldwide. Previous evidence has indicated that not only ischemia itself but also reperfusion contributes to the tissue injury sustained following myocardial ischemia, a phenomenon known as ischemia/reperfusion(IR)injury(IRI). Currently, there are a multitude of drugs that protect against myocardial IRI. However, an ideal pharmaceutical cardioprotective strategy has not been accepted because of the side effects, ethical problems and drug safety.Bakuchiol(BAK), a monoterpene phenol isolated from the seeds of Psoraleacorylifolia(Leguminosae), exerts organic protective effects in both the liver and the skin. Recent studies have demonstrated that BAK exerts a wide spectrum of pharmacologic effects, including anti-oxidative, anti-inflammatory, anti-aging, and anti-diabetic effects. However, oxidative stress, inflammation and diabetes are the primary elements of myocardial IRI, which suggests that BAK may play a promising role in attenuating IRI.Silent information regulator 1(SIRT1) is a histone deacetylase that depends on nicotinamide adenine dinucleotide(NAD+) and plays a key role in the longevity effects of calorie restriction; its cardioprotective effects are also well known. The activation of SIRT1 by IPC and resveratrol, a natural phenolic phytoalexin, reportedly attenuates myocardial IRI. Previous studies have demonstrated that resveratrol exerts cardioprotective effects by protecting cardiomyocytes from oxidative stress and improving mitochondrial function via the SIRT1 signaling pathway. Additionally,SIRT1 activates peroxisome proliferator-activated receptor-γ co-activator-1α(PGC-1α), which attenuates oxidative damage. Previous studies have indicated that the up-regulation of PGC-1α depends on SIRT1 and protects tissues in both cerebral ischemia and renal IRI. Mitochondrial dysfunction is a pathological consequence of IRI; maintaining mitochondrial function following ischemia may preserve organ function; SIRT1/PGC-1α activation following IRI accelerates mitochondrial biogenesis and dynamics and ameliorates mitochondrial damage, which contributes to the recovery of mitochondrial protein expression and function. BAK is an analogue of resveratrol and has a resveratrol-like structure. Thus, BAK likely attenuates myocardial IRI by maintaining mitochondrial function via SIRT1. However, the definitive role of SIRT1/PGC-1α signaling and its relationship with the cardioprotective effects of BAK in the setting of IRI have not been investigated.Therefore, this study was designed to evaluate the protective effects of BAK in the setting of myocardial IRI and to investigate its potential effects on both myocardial mitochondrial redox states and mitochondrial function. The involvement of SIRT1/PGC-1α signaling in the maintenance of mitochondrial function was also evaluated.Objective:1. To study whether Bak can fight against myocardial IRI.2. To study whether Bak exerts cardioprotection via the regulation of SIRT1 pathway.Materials:Healthy adult male Sprague-Dawley(SD) rats(weighing between 220 and 250 g)and newborn SD rats(1-2 days old) were obtained from the animal center of the Fourth Military Medical University.Methods:1.Preparation of the perfused isolated rat hearts;2.Cell culture, ischemia reperfusion(IR) treatment, and si RNA transfection;3.Myocardial apoptosis analyses;4.Myocardial necrosis analyses;5.Mitochondria isolation and oxidative damage analyses;6.Western blot analyses.Results:1. BAK significantly increased the levels of SIRT1 expression in the normal hearts in a dose-dependent manner(P<0.05).2. BAK treatment(0.25, 0.5, or 1 μM) significantly increased the functional recovery of post-ischemic hearts, as demonstrated by dose-dependent increases in the LVDP, +d P/dt max, CF, and HR values throughout the reperfusion period(P<0.05 vs. the IR group). BAK treatment(0.25, 0.5, or 1 μM) facilitated significant dose-dependent reductions in the apoptotic index(P<0.05 vs. the IR group). BAK treatment(0.25, 0.5, or 1 μM) also significantly reduced LDH and CK-MB release(P<0.05 vs. the IR group), respectively. These changes were most significant in response to 1 μM BAK.3. Bak also increased the levels of SOD, SDH and COX effectively in myocardial mitochondria subjected to IRI and decreased mitochondrial MDA significantly(P<0.05 vs. the IR group).4. BAK treatment significantly increased the levels of SIRT1, PGC-1α, and Bcl2 expression and decreased the level of Bax expression(P<0.05 vs. the IR group).These effects were most significant with 1 μM BAK.5. BAK(1 μM) treatment significantly increased LVDP following 60 min of reperfusion and decreased LDH and CK-MB release(P<0.05 vs. IR group),respectively.Sirtinol, a specific inhibitor of SIRT1 based on our previous research,reversed the protective effects of BAK, decreased LVDP and increased LDH and CK-MB release(P<0.05 vs. BAK+IR group).6. Sirtinol reversed the mitochondrial protective effects of BAK and decreased the SDH, COX, and SOD activities but increased MDA content(P<0.05 vs.BAK+IR group).7. Sirtinol not only effectively inhibited SIRT and PGC-1α expression(P<0.05)but also reversed the effect of BAK and decreased the levels of SIRT1, PGC-1α, and Bcl2 expression, respectively, and increased the levels of Bax, respectively(P<0.05 vs. BAK+IR group).8. The cardiomyocyte results were consistent with those that were obtained from the isolated rat. The cell viability was analyzed by the MTT assay. Pretreatment with BAK markedly increased cell viability following IR(vs. control si RNA+IR group, P<0.05). In addition, BAK pretreatment significantly decreased the apoptotic index(vs. control si RNA+IR group, P<0.05).The protective effect of BAK pretreatment was abolished by SIRT1 si RNA(vs. control si RNA+BAK+IR group,P<0.05). Significantly reduced levels of LDH and MDA were observed in the control si RNA+BAK+IR group compared with the control si RNA+IR group(P<0.05). In addition, BAK pretreatment significantly increased mitochondrial SOD(vs. control si RNA+IR group, P<0.05). SIRT1 si RNA pretreatment abolished most of the protective effect afforded by BAK pretreatment(P<0.05). Compared with the control group, control si RNA had no effect on the cell viability, apoptotic index, and LDH, SOD, and MDA release.9. BAK pretreatment significantly increased SIRT1, PGC-1α, and Bcl2 and decreased Bax expression(vs. the control si RNA+IR group, P<0.05) in IR-injured cardiomyocytes. However, the effect of BAK pretreatment on the expression of these proteins was abolished by SIRT1 si RNA(vs. control si RNA+BAK+IR group,P<0.05).Conclusions:1.Following 5 min of perfusion, BAK(0.25, 0.5, or 1 μM) treatment did not affect the LVDP. Additionally, BAK treatment did not affect the LDH or CK-MB release in the coronary effluent. Interestingly, BAK significantly increased the levels of SIRT1 expression in the normal hearts in a dose-dependent manner.2.BAK treatment exerts promising cardioprotective effects in the setting of IRI.3.BAK alleviates IRI-induced mitochondrial oxidative stress and cellular apoptosis by the activation of SIRT1/PGC-1α pathway, and further employs protective effect against myocardial IRI. |
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