| Background:Acute myocardial infarction is the most major cause to result in death in cardiovascular diseases. Nowadays the acknowledged and most effective therapeutic method of acute myocardial infarction is to recover the blood of the infracted coronary artery, and to relieve the state of myocardial anoxia, thus to alleviate the injury resulted from myocardial ischemia. The clinical therapeutic methods of acute myocardial infarction have made great progress, for example early thrombolysis, coronary artery bypass grafting, and percutaneous transluminal coronary angioplasty(PTCA) can quickly recover the blood of the infracted coronary artery to rescue the agonal cardiac muscle and to reduce the myocardial infarct size, finally to improve the clinical prognosis of patients. However, early reperfusion of the ischemic myocardium causes subsequent myocardial ischemia-reperfusion (I/R) injury. Thus it has become a pivotal issue to treat myocardial infarction that the blood supply of ischemic myocardium is recuperated as early as possible with the alleviation of reperfusion injury. Ischemic preconditioning (IP) is known as the best strategy to protect ischemic myocardium. However, during the clinical practice, because it is impossible to foresee the particular time of myocardial ischemia, which limits the clinical application of IP that must be carried out before the ischemic affair happens. The subsequent studies have found that ischcmic postconditioning (IPO) can also alleviate myocardial I/R injury. Furthermore, because the onset of reperfusion can be controlled and be foreseed, it is possible for IPO to achieve cardiovascular protection in the clinical. But IPO can only apply to the patients who will accept PTCA and the mechanical operation of IPO, with many times coronary artery blood blocked, may increase the risk of coronary artery distal embolization. Therefore, it is the new trend to explore drugs to replace the mechanical IPO in the study of I/R.Resveratrol (Res), a naturally occurring polyphenol phytoalexin, is abundant in a wide variety of plant species, such as grapes skin, polygonum cuspidatum, peanuts, veratrum nigrum and so on. Res has diverse biochemical and physiological actions, including anti-inflammatory, anti-oxidant, antiplatelet, vasodilation and antiapoptosis actions. Many studies have shown that Res accomplishes cardiovascular protection via preconditioning. However, there are few reports at home and abroad about whether Res can obtain cardiovascular protection via postconditioning. Moreover, Since Res is a poorly water soluble drug (<0.001mol/L), weakly absorbed after oral administration with quick metabolism in vivo and short half life, and unstable as it converts to the cis-form (a less active form) particularly on exposure to UV light, the bioactivity of free Res is not able to bring into full play.Nowadays there are not correlated reports at home and abroad that the efficient Res delivery by innovative pharmaceutical forms is applied in cardiovascular study. In this study, Res was loaded in the polymeric micelles to obtain controlled-release (Res-PEG-P), and to explore the effects of controlled-release resveratrol postconditioning on myocardial I/R injury in rat and the underlying molecular mechanism.Methods:Part one:PEG-MEMA-co-Boc-Cyst-MAAm)-b-PEG, a water-soluble amphiphilic block copolymer, was synthesized. Res was loaded in the polymeric micelles to obtain controlled-release resveratrol (Res-PEG-P). In vitro release of resveratrol from PEG-P micelles was measured. Primary cultures of neonatal rat cardiomyocytes were randomly distributed into five groups:control group, A/R group (cultured cardiomyocytes were subjected to3h anoxia/2h reoxygenation), A/R+Res group(cardiomyocytes were subjected to A/R,10μM Res was applied5min after reoxygenation), A/R+Res-PEG-P group(cardiomyocytes were subjected to A/R,10μM Res-PEG-P was applied5min after reoxygenation), and A/R+PEG-P group (cardiomyocytes were subjected to A/R,25mg/L PEG-P was applied5min after reoxygenation). In order to evaluate cardiomyocyte damage, cell viability, lactate dehydrogenase (LDH) release, caspase-3activity, and apoptosis were analyzed by the cell counting kit (CCK)-8assay, colorimetric method and flow cytometry, respectively. The mRNA and protein expression of Toll-like receptor4(TLR4) were detected by quantitative real-time PCR and western blot analysis. Nuclear factor-KB (NF-κB) p65protein and I-κBα protein levels were also examined by western blot analysis. The levels of proinflammatory cytokines in the culture medium were assessed by enzyme-linked immunosorbent assay.Part two:Male rats were randomly divided into five groups:sham group, I/R group(myocardium were subjected to30min ischemia followed by24h reperfusion), I/R+Res group (myocardium were subjected to I/R,10μM Res was applied5min after reperfusion), I/R+Res-PEG-P group (myocardium were subjected to I/R,10μM Res-PEG-P was applied5min after reperfusion), and I/R+PEG-P group (myocardium were subjected to I/R,25mg/L PEG-P was applied5min after reperfusion). Each group was ten rats. LDH release in blood serum, caspase-3activity, infarct size and apoptosis were analyzed by colorimetric method, Evans blue/TTC staining and histological TUNEL staining, respectively. The mRNA and protein expression of TLR4were detected by quantitative real-time PCR and western blot analysis. NF-KBp65protein and I-KBa protein levels were also examined by western blot analysis. The levels of proinflammatory cytokines in blood serum were assessed by enzyme-linked immunosorbent assay.Results:Part one:Compared with control group, in A/R group cell viability was significantly decreased, the levels of LDH in the culture medium of cardiomyocytes and caspase-3activity were significantly increased. Stimulation induced by A/R in cardiomyocytes led to enhanced apoptosis. Real-time RT-PCR and western blot analysis revealed that the expressions of TLR4mRNA and protein were significantly increased in cardiomyocytes undergoing A/R. Western blot analysis showed that A/R induced nuclear translocation of NF-κBp65protein in cardiomyocytes and the degradation of I-κBα protein. Cardiomyocytes subjected to A/R had an increase of TNF-a and IL-1β contentrations in the culture medium. Compared with A/R group, both Res and Res-PEG-P significantly prevented the loss of cardiomyocyte viability, the release of LDH and the activation of caspase-3. Treatment with both Res and Res-PEG-P showed a significant resistance in apoptosis in cardiomyocytes, a significant reduction of TLR4mRNA and protein expression, a markedly inhibition of NF-κB nuclear translocation and an increase of I-KBa protein levels. Treatment with both Res and Res-PEG-P produced a significant reduction of TNF-a and IL-1β concentrations in the culture medium. In addition, the effect of Res-PEG-P was better than that of Res. PEG-P had no significant influence on cardiomyocytes undergoing A/R.Part two:Compared with sham group, in I/R group the levels of LDH in blood serum, caspase-3activity, infarct size and the index of apoptosis were significantly increased, the expressions of TLR4mRNA and protein were significantly increased, the nuclear translocation of NF-KBp65protein and the degradation of I-KBa protein were markedly enhanced, the contentrations in blood serum of TNF-a and IL-1β were significantly increased. Compared with I/R group, both Res and Res-PEG-P significantly prevented the release of LDH and the activation of caspase-3, reduced infarct size and the index of apoptosis. Treatment with both Res and Res-PEG-P showed a significant reduction of TLR4mRNA and protein expression, a markedly inhibition of NF-κB nuclear translocation and an increase of I-κBα protein levels. Treatment with both Res and Res-PEG-P produced a significant reduction of TNF-α and IL-1β concentrations in blood serum. In addition, the effect of Res-PEG-P was better than that of Res. PEG-P had no significant influence on myocardium undergoing I/R.Conclusion:1. In vitro cultured cardiomyocytes were subjected to A/R, treatment with both Res and Res-PEG-P postconditoning improve cell survival and attenuate A/R-induced inflammatory response. The effect of Res-PEG-P is better than that of Res. This protection mechanism is possibly related to the TLR4/NF-κB signaling pathway.2. In vivo myocardium were undergone I/R, treatment with both Res and Res-PEG-P postconditoning provide myocardial protection, reduce infarct size, inhibt myocardial apoptosis and attenuate inflammatory response. The effect of Res-PEG-P is better than that of Res. This protection mechanism is possibly related to the TLR4/NF-κB signaling pathway.
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