Protective Effects Of Melatonin On Renal Ischemia/Reperfusion-induced Remote Organs Injury And Its Underlying Mechanisms In Rats

Ischemia-reperfusion (I/R) injury is a general reaction, I/R injury of one organ can induce remote organs injury. Oxygen free radicals (OFR) are produced through an oxidizing agent or during corpuscular aerobic oxidation. Superoxide dismutase and catalase clear OFR quickly in vivo and lessen the impact of accumulating OFR in an organism's cells. Ischemia and anoxia directly decrease nourishment material and ATP within the cells, which result in increased OFR. This surge in OFR under these conditions exceeds the cells capacity to clear these harmful molecules. Unscavenged OFR can cause immediate damage and induce apoptosis.Melatonin (MT) is one of the most effective antioxidant that clears free oxygen radicals quickly. Previous studies confirmed that MT can protect heart,brain,liver,intestin and pancreas against I/R injury. At present, there is seldom report about the protective effects and mechanisms of melatonin on renal ischemia/reperfusion- induced remote organs injury . The present study observed that protective effects and mechanisms of melatonin on renal ischemia/ reperfusion injury (RIRI)-induced myocardium injury through use of biochemistry, immunohistochemistry and flow cytometer methods.PartⅠEffects of MT on oxygen free radical and apoptosis of myocardium in RIRI ratsObjective: To investigate the effects of MT on RIRI induced myocardium injury and its underlying mechanisms in rats .Methods: The model of RIRI was induced by bilateral clamping the renal artery and vein for 45 min followed by reperfusion and observing effects of melatonin in rats. 32 SD rats were randomly divided into sham, I/R, Vitamin C (VC) and MT groups. MT and VC group rats were given MT (10mg/kg) and Vitamin C (125 mg/kg) respectively by intraperitoneal injection at 30min before ischemia and 30min after reperfusion. IR group rats were inject the same doses solution in the same way. After reperfusion, each layer opened was closed with suture, including the peritoneum, abdominal wall muscle and skin. The rats in the sham group were treated identically except for the clamping. After 24 hours of reperfusion, the blood samples were taken for detecting contents of serum creatinine (Scr), serum urea nitrogen (SUN), activities of lactate dehydrogenase (LDH) and creatine kinase (CK). After blood was taken, the heart was excised for detection activity of superoxide dismutase (SOD), the content of malondialdehyde (MDA), histological examination, the expression of Bcl-2, Bax, (Immunohistology) and apoptosis rate (flow cytometry). Results: Renal and myocardium was impaired in RIRI rats:1 The contents of Scr and SUN, activities of LDH and CK in serum and MDA in the myocardium were significantly increased in I/R group compared to sham group (P<0.01); the activity of SOD was significantly decreased in I/R group compared to sham group (P<0.01); Compared with those of I/R group, contents of MDA in the myocardium, contents of Scr and BUN, activities of LDH and CK in serum were significantly decreased (P<0.01, P<0.05), but the activity of SOD were significantly increased in MT groups (P<0.05).2 Renal tubule epithelial cells showed signs of damage, especially proximal convoluted tubule in I/R group, in which the lumina was enlarged; also there were some cast and caduceus cells in many of the renal tubules. Chromatin was localized in the cell nucleus periphery; In the MT group, the nucleus appeared normal, and the few cast cells present were localized to the tubule cells. There was seldom inflammatory infiltration and apoptosis in I/R group and the hitomorphology was nomal in MT group in myocardium.3 The apoptotic rate of myocardium in I/R group was significantly higher than that of sham group (20.76±2.02% vs 2.66±0.53%, P<0.01). The apoptotic rates of myocardium in MT and Vc group were (13.40±0.96%) and (13.49±1.51% ) respectively. The percentage of apoptosis of myocardium cell in both of MT and VC group were significantly lower than that of I/R group (P<0.01). Compared with those of sham group, the expression of Bcl-2 and Bax in I/R group were markedly increased. In MT group, the expression of Bcl-2 were markedly increased but those of Bax were significantly decreased.Conclusion:1 MT has a protective effects on the RIRI induced myocardial injury in rats.2 It is considered that the protective effects may be related to clearing oxygeon free radicals quickly, restraining apoptosis through upregulating the expression of anti-apoptotic protein Bcl-2, downregulating the expression of pro-apoptotic protein Bax.PartⅡEffects of MT on OFR, NO and apoptosis of brain tissue in RIRI ratsObjective: To investigate the effects of MT on RIRI induced brain tissue injury and its underlying mechanisms in rats .Methods: The animal model of RIRI and experimental methods were made as same as describe in part 1. The brain tissue were taken for measured Nitric Oxide (NO) and inducible nitric oxide synthase (iNOS).Results: 1 The contents of Scr and SUN, MDA and NO in the tissue, the activity of iNOS were significantly increased in I/R group compared to sham group (P<0.01); the activity of SOD was significantly decreased in I/R group compared to sham group (P<0.01); Compared with those of I/R group, contents of MDA and NO in the tissue, contents of Scr and SUN, activities of iNOS were significantly decreased (P<0.01, P<0.05), but the activities of SOD were significantly increased in MT groups (P<0.05).2 Renal tubule epithelial cells showed signs of damage, especially in the proximal convoluted tubule in I/R group, in which the lumina was enlarged; also there were some cast and caduceus cells in many of the renal tubules. Chromatin was localized in the cell nucleus periphery; In the MT group, the nucleus appeared normal, and the few cast cells present were localized to the renal tubule cavity. Intercellular spaces of neurocytes were widen in I/R group. The neuronal degeneration wes obviously, and some of them were characteristics of apoptosis in I/R group.3 The apoptotic rate of neurocytes in I/R group was significantly higher than that of sham group (23.53±3.13% vs 3.24±0.71%, P<0.01). The apoptotic rates of neurocytes in MT and VC group were (13.81±2.01%) and (14.20±1.51% ) respectively. The percentage of apoptosis neurocytes in both of MT and VC group were significantly lower than that of I/R group (P<0.01). Compared with those of sham group, the expression of Bcl-2 and Bax in I/R group were markedly increased. In MT group, the expression of Bcl-2 were markedly increased but those of Bax were significantly decreased.Conclusion: 1 MT has a protective effects on the RIRIinduced brain tissue injury in rats. 2 It is considered that the protective effects is related to inhibit the activities of iNOS and decreasing NO in brain tissue, restraining apoptosis through upregulating the expression of anti-apoptotic protein Bcl-2, downregulating the expression of pro-apoptotic protein Bax.