| Objective: To study the mechanism of paraquart-induced renal injury and protective effect of melatonin (MT). Pathological changes, the expression changes of inducible nitric oxide synthase(iNOS)and Heme oxygenase-1(HO-1)were studied in renal of paraquart-poisoned rat.Methods: 182 adult healthy Spragne-Dawley(SD) rats (female and male in half) were randomly divided into 3 groups:⑴The control group (group A): 42 rats,⑵The paraquart poisoned group (group B): 70 rats, and⑶MT group (group C): 70 rats. The rats in group B and C were treated PQ (25mg/kg) intraperitoneally, the rats in group A were treated with the same dose of normal saline(NS)as that of group B and C; In 15 minutes, the rats in group C were given MT intraperitoneally at a dose of 10mg/kg·d and the rats in group A and B were treated intraperitoneally with the same dose of normal saline once a day as those in group C. At the 3rdh,6thh,12thh,1std,2ndd, 3rdd,5thd,six rats respectively from each group were measured for BUN and Cr taken away the blood from abdominal aorta. Part of the right kidney was stained with Hematoxylin-Eosin(HE)to observe its pathologic, the other part of right kidney was observed the expression of HO-1 and iNOS with immunohistochemistry. The upper pole of left kidney was used for evaluating the level of malondialdehyde(MDA), the activity of superoxide dismutase( SOD ) and glutathione peroxidase ( GSH-Px ) after homogenated, the inferior was sharply frozed in liquid nitrogen for evaluating the mRNA expression of HO-1 by Reverse Transcrip-tion-Polymerase Chain Reaction(RT-PCR).Results:①Clinical signs after poisoning: In group B and C all of the rats began to demonstrate the changes to different extent in their clinical signs in 30mins~2hrs, and were especially severe in the first three days, including the respiratory system, psychological system, neural system and digestive system, etc. However, the clinical changes of all the rats in group C were slighter than those in group B (especially in the respiratory system). Compared with group A, the weight of rats in group B decreased much more, and the weight in group C didn't lost much.②Pathologic changes: In group A, the tissue structure is clear without edema, vacuolar degeneration, cloudy swelling and necrosis. In group B, evident lesions in the tissue structure could be found in the renal tubule of cortical part, including cellular swelling, the narrow canula, the mesenchymal congestion and edema. These pathologic changes gradually became more severe, reached the peak on the 1st day, and did not relieved till the end of this study; Some glomerulus and medulla were also involved in. karyopycnosis and the disappeared cellular structure could be found in some severe cases; Compared with group B, the aforementioned pathological lesion was more palliative. The glomerulus, medulla injury and karyopyknosis were rarely observed in group C.③BUN and Cr: The levels of BUN in group B were much higher than that in group A on the 1st,2nd,3rd,4thand 5thday(P<0.05). In group C, the levels of BUN were obviously higher on the 3rd day than that in group A (P<0.05), but the levels of BUN in group C were lower than that in group B, there was a statistical significance between them (P<0.05). The levels of Cr were normal in all groups.④MDA: The levels of homogenate MDA in group B increased obviously at the 3rd hour, and reached the peak on the 2nd day, there was a significant difference between group A and group B (P<0.05), then it decreased slowly, it showed no difference between group A and group B on the 5th day(P>0.05). The levels of homogenate MDA in group C were higher compared with group A, but there was no significant difference between group A and group C(P>0.05). Compared with group B, the levels of MDA in group C increased more slowly, there was a significant difference from the 6th hour to the 2nd day (P<0.05), but no difference(P>0.05)on the 3rd day.⑤The activity of homogenate SOD in group B significantly decreased ,then ascended slowly, there was a statistical significance between group B and group A from the 3rd hour to the 3rd day(P<0.05). but it was still lower compared with group A on the 5th day (P>0.05). In group C, it significantly increased at the 12th hour, reached the peak on the 2nd day, and remained a high level, there was a statistical significance between group C and group A (P<0.05). Compared with group B, there was a statistical significance in all these time points(P<0.05).⑥The activity of homogenate GSH-Px in group B decreased at the 3rd hour, and there was a statistical significance between group B and group A (P<0.05). Then it ascended quickly, reached the peak on the 1st day, and remained the level. However it showed no difference between group A and group B(P>0.05). In group C, it significantly increased at the 3rd hour, and there was a statistical significance between group C and group A (P<0.05), it reached the peak at the 6th hour, and then remained the high level. Compared with group B, there was a statistical significance in the whole time points (P<0.05).⑦iNOS expression in renal tissue: In group A, there was no or weak expression; The expressions of iNOS in group B were observed at the 3rd hour in the kytoplasm of renal tubular epithelial cell and glomerular endothelial cell of cortical part. Immunohistochemistry score(IHS) was significantly higher at the 6th hour, and reached the peak on the 1st day and continued at a high level after that and there was a statistical difference between group B and group A (P<0.01). In group C, the expressions of iNOS were observed at the 3rd hour. IHS was significantly higher at the 12th hour, and there was a statistical difference between group C and group A (P<0.05). It continued at a low level after that. There was no difference between group C and group A on the 5th day (P>0.05). Compared with group B, IHS in group C were lower from the 6th hour to the 5th day(P<0.05).⑧H O-1 expression in renal tissue:In group A, there was no or weak expression. The expressions of HO-1 in group B were observed at the 3rd hour in the membrane and kytoplasm of renal tubular epithelial cell of cortical part. IHS was higher than that of group A (P<0.05), and reached the peak on the 1st day, and then decreased, it showed no difference between group B and group A(P>0.05). In group C, there showed positive expression at the 3rd hour. Compared with group B and group A, IHS was significantly higher (P<0.05). The expressions of HO-1 reached the peak on the 1st day, and then weakened, compared with group A, there was a statistical significance on the 5th day(P<0.05), but compared with group B, it showed no difference(P>0.05).⑨HO-1mRNA expression in renal tissue: there was no or weak expression of HO-1mRNA in group A; In group B, the expression of HO-1mRNA increased in the 3rd hour, and there was a statistical significance between group B and group A (P<0.05).It reached the peak on the 1st day, the relative expression level of HO-1mRNA was 2.28 times of that in group A, then it decreased, there was no difference between group B and group A on the 3rd day(P>0.05). In group C, the expression of HO-1mRNA significantly increased at the 3rd hour, there was a statistical significance between group C and group A (P<0.05), It reached the peak on the 1st day, its relative expression level was 3.03 times of that in group A. Then it showed decreased slowly, compared with group A, there was a statistical significance on the 5th day(P<0.05). Compared with group B, there was a statistical significance in the 6th hour(P<0.05). but it showed no difference between group C and group B on the 5th day(P>0.05).Conclusions: In this study, Injecting PQ in peritoneal is a reliable method of making a model of Renal induced. It can imitate the clinic of PQ poisoning well and is worth of more widespread use. The levels of homogenate MDA increased and the activity of SOD and GSH-Px decreased markedly in PQ poisoned rats. That is to say oxidative insult and the imbalance of oxidation-antioxidation system were the main mechanisms of PQ-induced renal injury. Meanwhile iNOS was involved in the procedures of renal injury. MT surely had an protective effect, which might be mediated by HO-1 and iNOS on the paraquart-induced renal injury, but its regulating path still needs a further exploration.
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