| Objective: Paraquat (PQ) has been widely used as a contact herbicide of organism heterocycle throughout the world. Along with the widespread use of paraquat in our country,PQ poisoning is becoming a more and more serious clinical problem. Although PQ is very effective as a herbicide, it is highly toxic to human beings and animals. It can be absorbed through skin,pneogaster or enteron,and then lead to acute poisoning. Its human lethal oral dose is very small, its progress is short. After entering body, PQ impairs the lung firstly, the renal and liver secondly. There is no special antidote for paraquat and effective way to reduce the toxicity of poison currently. PQ is excreted as the original form from the renal, therefore ,understand the excreted mechanism and renal injury mechanism is very important . Through observing the ultrastructure variation , determining content of BUN,Cr in serum and malonyldialdehyde(MDA), activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in renal tissue, the expressions of apoptotic regulatory proteins― mutant P53 and Fas/Fas-L in renal of the acute paraquat-induced rats by the method of immunohisto-chemistry, we try to explore the possible mechanism and the protective effect of Hepatocyte Growth-promoting Factors, (pHGF) on renal injury induced by PQ poisoning.Methods: One hundred and eighty adult healthy Sprague-Dawley (SD) rats (90 female, 90 male) were divided into three groups randomly.(1) Control group (group A): 60 rats, (2)Poisoned group (group B): 60 rats, (3) pHGF group (group C): 60 rats. Group B and C were treated intraperitoneally with 1ml of PQ (25mg/kg) diluted with normal saline. Group A rats were treated with the same dose of normal saline as group B and C. Group C rats were given intraperitoneally 1ml of pHGF at a dose of 1mg/kg diluted with normal saline ( once every 12h) immediately after the administration of the PQ untill be executed. Group B rats were treated intraperitoneally with the same way of normal saline (once 12h) as group C. Ten rats in group A,B,C were killed and thoracotomy by ether anaesthesi on 1stday, 3rdday, 5thday, 7thday, 10thday, 14thday respectively, then taken blood samples from abdominal aorta to detect serum levels of BUN,Cr. Next ,made left renal tissue samples into homogenate to measure activity of SOD,GSH-Px and MDA concentration. Part of the right renal tissue was stained with hematoxylin-eosin for pathological observation, the rest of it was remained to observe the expression of mutant P53 and Fas/Fas-L by the method of immunohistochemistry.Results:1 Clinical signs after poisoning: Rats in group B began to demonstrate the changes to different extent in their clinical signs in 30mins~2hrs, including the respiratory system, psychological system, neural system and digestive system, etc. Intoxication involves a combination of signs and symptoms that included lassitude, lethargy, restlessness, Walking unsteadily, tremor,dyspnea,appetite badly, hematuria, oliguria,anuria,especially severe in the first three days. Compared with Group B, rats in Group C demonstrated the intoxication manifestations were relieved obviously, especially the symptoms of dyspnea hematuria, oliguria and anuria.2 Serum BUN,Cr measurement:①Group A:the contents of BUN and Cr were normal;②Group B: BUN were manifest to advance on 1st day, 1st～5th day were significantly higher than that in group A, which gradually lessoned. BUN changes have statistical significance(P<0.05).③Group C:after pHGF treatment, 1st～3rd day the contents of BUN were lower than that in Group B, changes have statistical significance(P<0.05),and no statistical significance after 5th day between Group B and Group C(P>0.05). Cr changes have statistical significance(P<0.05) among Group A,B and C.3 SOD,GSH-Px,MDA measurement in renal tissue homogenate:⑴SOD measurement①Group B: the activity of renal tissue SOD in Group B were significantly lower than that in Group A on 1st,3rdday(P<0.05), and no statistical significance after 5th day between Group B and Group A(P>0.05);②Group C: Compared with that of Group B, the level of SOD activity were increased in Group C on 1st,3rd,5th,7thday, changes have statistical significance(P<0.05); Compared with that of Group A,the lower levels only kept on 1st day(P<0.05), changes have no statistical significance(P<0.05) after 3rd day.⑵GSH-Px measurement①Group B: the activity of renal tissue GSH-Px in Group B were significantly lower than that in Group A on 1st,3rdday(P<0.05);it increased gradually after 5th day, differences between Group B and Group A have no statistical significance (P>0.05).②Group C: the activity of GSH-Px in Group C remarkable increased, and was higher than that in Group B on 1st ,3rd day (P<0.05);But there were no statistical significance all the time point between Group C and Group A(P>0.05).(3)MDA measurement①Group B: the levels of MDA were higher than that in Group A on 1st～10th day(P<0.05);while on 14th day, there is no statistical significance between Group B and Group A(P>0.05).②Group C: whereas the increase of renal tissue MDA were remarkably inhibited in Group C,in which, the levels of renal tissue MDA on 1st～7th day were significantly lower than that in Group B(P<0.05);Compared with MDA in Group A ,the statistical significant higher levels of it in Group C only kept on 1st, 3rd day(P<0.05).4 Histological changes(HE staining): Group A: The structure of renal glomerulus and renal tubule are clear without edema, vacuolar degeneration, cloudy swelling and necrosis. Group B: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 ,edema and red cells within the glomerulus. These pathologic changes gradually became more severe in next two weeks. Group C: pHGF alleviated the hyperaemia and distension in enal glomerulus and accute injury in renal tubule.5 Immunohistochemistry (IH) staining:⑴Fas/Fas-L: In group A,there was only very weak expressions of Fas/Fas-L in the normal renal tissue. Fas/Fas-L can be observed in the cytoplasm of renal tubular epithelial cells. While In group B, the expressions of Fas/Fas-L were significantly higher than that in group A on the 1st day which could be observed in renal tubule epithelial cells mainly, changes have statistical significance(P<0.05) on every time point. In group C, pHGF can exhibit the express of Fas/Fas-L remarkably. Compared with both Group A and Group B,statistical significance kept on till 14th day(P<0.05).⑵Mutant P53: In group A, there was only very weak expressions of mutant P53 in the normal renal tissue. Mutant P53 can be observed in the nucleus of renal tubular epithelial cells. In group B, the expressions of mutant P53 were significantly higher than that in group A on the 1st day which could be observed in renal tubule epithelial cells mainly. The expressions of mutant P53 could be observed in the nucleus, changes have statistical significance(P<0.05) on every time point. In group C, pHGF can exhibit the express of mutant P53 remarkably. Compared with both Group A and Group B, statistical significance kept on from3rd day till 14th day(P<0.05).Conclusion: (1) PQ intraperitoneally poisoning can induce acute renal injury and principal display the damage of renal tubule endothelial cell; (2) 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;(3) The expressions of mutant P53 and Fas/Fas-L in renal tissue significant changed , indicate that apoptosis may be one of the mechanisms of PQ-induced renal injury; (4). pHGF treatment increased significantly the activity of SOD and GSH-Px in PQ poinsoning rats and decreased significantly to alleviate the renal injury; (5). pHGF may inhibit apoptosis induced by mutant P53 and Fas/Fas-L to alleviate the renal injury.
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