Study Of The Role And Mechanism Of Vitamin D₃on Myocardial Ischemia Reperfusion Injury In Rats

Background:In the recent years, thrombolytic therapy or primary percutaneous coronary intervention is the most effective therapeutic strategy of acute myocardial infarction. However, the process of revascularization of the occluded coronary artery which induce myocardial infarction, restoration of coronary blood and reperfusion of the ischemic myocardium, a process termed myocardial reperfusion injury, may lead to additional myocardial damage. Typically, this process may cause four type phenomenons including myocardial stunning, no-reflow phenomenon, reperfusion arrhythmias and lethal reperfusion injury. The pathogenesis of reperfusion was not completely understood. Most researchers concluded that the mechanism of the reperfusion injury may be related with several factors, such as oxygen radicals generation, calcium loading, neutrophils infiltration, etc. It is possible that these factors may interact and promote each other, thereby leading to the myocardial death mediated by inflammation and apoptosis. It is an interesting research direction to look for the effective strategy to reduce or avoid myocardial ischemia and reperfusion injury during the course of acute myocardial infarction treatment.Vitamin D is a fat-soluble vitamin, which mainly includes vitamin D2and vitamin D3. The main physiological functions of vitamin D are associated with the metabolism regulation of calcium and phosphorus, maintaince of blood calcium and blood phosphate levels, thus promoting the normal growth and development of bone and teeth. Therefor vitamin D is one of the most important nutrients for growth and health maintenance in human and animal. In clinic, vitamin D is mainly used for the treatment of rickets. In the recent years, lots of studies show that vitamin D play a role in decreasing the risk of many chronic diseases, including cancers, autoimmune diseases, infectious diseases, diabetes, and cardiovascular diseases. One recent study shows that vitamin D can reduce the cortex injury caused by cerebral ischemia in rats and attuenuate the infarction area. However, whether vitamin D could protect myocardia during the process of myocardial ischemia and reperfusion is still unknown. In this study, through establishing the rat myocardial ischemia and reperfusion model and rat cardiomyocytes hypoxia and reoxygenation model, we aimed to investigate the effect of vitamin D3on myocardial ischemia and reperfusion injury, as well as elucidate its possible underlying mechanisms.Methods:Part one Wistar rats were randomly assigned into four groups, Sham group, Sham-VD3group, I/R group, I/R-VD3group. Animals in Sham-VD3group and I/R-VD3group receive vitamin D3at a dose of5μg/kg by oral gavage once a day for4weeks before operation. Myocardial ischemia and reperfusion injury model was carried as30minutes of ischemia followed by3hours of reperfusion. Commercial kits were used to detect the serum levels of CK and LDH. Evans blue and TTC double staining were used to detect infarct size, and PARP activity was tested by immunohistochemistry and Western blot.Part two Cardiomyocytes were cultured with stand method, CCK8assay was used to detect the cell viability after cardiomyocytes were pretreated with vitamin D3(0,1,10,100μM) or1,25-dihydroxy vitamin D3(0,0.01,0.1,1μM) for29hours. Cardiomyocytes were pretreated with vitamin D3(0,1,10,100μM) or1,25-dihydroxy vitamin D3(0,0.01,0.1,1μM) for24hours, followed by3hours of hypoxia and2hours of reoxygenation, the cell viability was detected with CCK8assay. Cardiomyocytes were pretreated with vitamin D3(0,1,10,100μM) or1,25-dihydroxy vitamin D3(0,0.01,0.1,1μM) with or without ketoconazole (10μM) for24hours, followed by3hours of hypoxia and2hours of reoxygenation, CCK8assay was used to detect the myocardial cell viability. Moreover, PARP activity was tested by immunofluorescence and Western blot.Part three Animal model and cell models were established as mentioned above. MPO activity was detected by using commercial kit. ELISA assay was used to detect the TNF-a and IL-6levels in the serum, the apoptosis of myocardial cells was test with TUNEL assay and Flowcytometry. NF-κB activity was tested by EMSA assay. Western Blot was used to detect the activities of iNOS and Caspase-3. The mRNA levels of iNOS, TNF-a and IL-6was detected by Real-time RT-PCR.Result:Part one Levels of CK and LDH in the serum were increased during myocardial ischemia and reperfusion. Vitamin D3could decrease the levels of CK and LDH and reduce myocardial infarction area. The activity of PARP was increased in the ischemic myocardia tissue during myocardial ischemia and reperfusion. Treatment of vitamin D3 could reduce PARP activity.Part two Vitamin D3and1,25-dihydroxy vitamin D3with different concentrations have not obvious cytotoxic effect on cardiomyocytes. Hypoxia and reoxygenation could reduce the survival rate of cardiomyocytes. Vitamin D3and1,25-dihydroxy vitamin D3with different concentrations can increase the survival rate of cardiomyocytes during hypoxia and reoxygenation. Ketoconazole can attenuate the protective effect of vitamin D3and1,25-dihydroxy vitamin D3on cardiomyocytes. Hypoxia and reoxygenation could enhance the activity of PARP in cardiomyocytes, while1,25-dihydroxy vitamin D3treatment could attenuate the PARP activity of cardiomyocytes during hypoxia and reoxygenation.Part three During myocardial ischemia and reperfusion, the activity of MPO and NF-κB were increased, the expression of iNOS was up-regulated, and the activity of Caspase-3was increased in the ischemic myocardia. The levels of TNF-α and IL-6in serum were increased during myocardial ischemia and reperfusion. Vitamin D3inhibited the activity of MPO, NF-κB and Caspase-3, reduced the expression of iNOS in the ischemic myocardia, and decreased the levels of TNF-α and IL-6in serum. Moreover, Vitamin D3reduced the apoptosis rate of cardiomyocytes during myocardial ischemia reperfusion.ConclusionVitamin D3could attenuate myocardial injury induced by ischemia and reperfusion. This protective effect may be attributed to the inhibition of PARP by1,25-dihydroxy vitamin D3, a metabolite of vitamin D3. The mechanism may be related with the attenuation of inflammation and apoptosis which are mediated by the suppression of PARP.