【作者】 曹宗迅；

【导师】 田英平； 石汉文；

【作者基本信息】 河北医科大学 ， 急诊医学， 2010， 硕士

【摘要】 目的:百草枯(paraquat, PQ)是目前世界范围内广泛使用的有机杂环类接触性除草剂。随着百草枯在我国农业上的广泛应用,PQ中毒也日趋增多。百草枯除草效果好,但对人畜均有较强毒性,可以经消化道吸收、皮肤接触及呼吸道吸入等途经进入体內，造成急性中毒，且中毒致死量小，病程发展快。百草枯进入人体后,可引起多个脏器的损害,其中以肺脏为主,其次是肾脏和肝脏。目前尚缺乏百草枯特效解毒剂以及有效降低毒物毒性的治疗手段。PQ主要以原形的形式经肾脏排出,因此了解其排泄机制及肾脏损伤机制非常重要。本研究通过观察大鼠PQ染毒后肾组织病理变化、测定血清肌酐(Cr)、尿素氮(BUN)的变化,及肾组织匀浆中超氧化物歧化酶(SOD)、谷胱甘肽-过氧化物酶(GSH-PX)的活力和丙二醛(MDA)的含量,并采用免疫组织化学方法观察肾组织中突变型P53蛋白、肿瘤坏死因子家族中Fas蛋白及其细胞表面受体Fas-L的表达,以及促肝细胞生长素(Hepatocyte Growth-promoting Factors, pHGF)对其表达的影响,进一步探讨急性百草枯中毒导致肾损伤的机制及促肝细胞生长素对PQ中毒大鼠肾损伤的治疗作用。方法:选用健康成年Spragne-Dawley(SD)大鼠180只,雌雄各半,随机分为3组:空白对照组(A组)60只、单纯染毒组(B组)60只、pHGF治疗组(C组)60只。B、C组PQ(25mg/kg)腹腔注射染毒,A组等量生理盐水腹腔注射,之后,C组立即给予pHGF 1mg/kg腹腔注射,每12h一次,最长用药时间为14天,B组给予与C组等量生理盐水腹腔注射。各组分别于1d、3d、5d、7d、10d、14d ,取10只大鼠,乙醚麻醉、腹主动脉取血处死,血液离心后取血清以备测定血BUN、Cr水平;留取左肾上极组织,制成10%肾组织匀浆测定MDA的含量,SOD及GSH-Px的活力;右肾用10%甲醛固定,行苏木精-伊红(hematoxylin-eosin,HE)染色观察病理学变化,免疫组化检测肾组织中P53、Fas /Fas-L的表达。结果:1大鼠中毒表现:于染毒30min～2hB组大鼠即出现不同程度的中毒症状,包括精神、神经、呼吸、消化和泌尿系统等方面,主要表现为倦怠、嗜睡、烦躁、走路不稳、头颤;呼吸困难、进食水减少、血尿少尿甚至无尿。症状以1～3d最明显,3d后症状逐渐减轻。C组经pHGF治疗组中毒表现较B组减轻,呼吸困难改善,血尿减轻,少尿及无尿时间缩短。2血BUN、Cr测定( table 1, Fig.53)A组:血BUN、Cr各时段均在正常范围内;B组:血BUN于第1天逐渐升高,1d、3d、5d各时间点与A组比较有统计学意义(P<0.05);5d后存活大鼠血BUN逐渐下降,7～14d时段逐渐恢复正常,与A组比较无统计学意义(P>0.05);C组:血BUN在1d、3d时段与A组比较具有统计学意义(P<0.05);5d、7d、10d、14d时段与A组比较无统计学意义(P>0.05); 1d、3d时段较B组降低,有统计学意义(P<0.05);5d、7d时段较B组降低,但无统计学意义(P>0.05);三组Cr变化比较均无统计学意义(P>0.05);3肾组织匀浆SOD、GSH-Px及MDA变化(table 2,Fig.54-56)3.1 SOD活性检测(Fig.55)B组:染毒后1d后明显下降,之后缓慢升高,与A组相比,染毒后1d、3d二者差异比较有统计学意义(P<0.05),5d、7d时虽明显低于A组,但不再有统计学意义(P>0.05),10d、14d时间点SOD活力逐渐增高,与A组比较无统计学意义(P>0.05);C组:促肝细胞生长素干预后SOD活力增加,染毒后1d、3d、5d、7d各时间点较B组升高,有统计学意义(P<0.05);10d、14d时间点与B组相比无统计学意义(P>0.05);与A组相比,1d差异有统计学意义(P<0.05),3d达高峰,之后维持在较高水平,5d开始呈下降趋势,3d、5d、7d、10d、14d与A组比较均无统计学意义(P>0.05);3.2 GSH-Px活性检测(Fig.56)B组:染毒后1d和3d时段低于A组(P<0.05),5d及以后时段肾组织GSH-Px活力逐渐升高,与A组比较无统计学意义(P>0.05);C组:促肝细胞生长素干预后GSH-Px活力增加,染毒后1d和3d时段较B组增高,差异有统计学意义(P<0.05),所有时段与A组比较均无统计学意义(P>0.05);3.3 MDA含量测定(Fig.54)B组: 1d、3d、5d、7d、10d时段均高于A组,差异有统计学意义(P<0.05),14d时段与A组相较无统计学意义(P>0.05);C组:促肝细胞生长素对大鼠肾组织MDA升高有抑制作用,1d和3d时段高于A组,差异有统计学意义(P<0.05),5d及以后各时段与A组相较均无统计学意义(P>0.05);1d、3d、5d、7d各时段较B组降低(P<0.05),10d及以后时段与B组相较无统计学意义(P>0.05);4肾组织HE染色结果A组(Fig.1):肾小球、肾小管及肾间质结构清晰,未见水肿、空泡变性、浊肿及坏死;B组((Fig.2-7):与A组相比,B组各组织结构清晰度下降,染毒后1d皮质部小管上皮细胞肿胀,小管管腔狭小,间质充血、水肿,肾小球内可见红细胞轻度增多,随时间延长而加重;C组((Fig.8-13):与B组相比,C组肾小球充血水肿减轻,肾小管上皮细胞损伤减轻,3d时即可见再生上皮细胞,肾间质可见炎性细胞浸润。5免疫组织化学检测结果( table 3, Fig.14-52)5.1 Fas蛋白在大鼠肾组织中的表达与分布A组(Fig.14):Fas蛋白无表达或有微弱表达,主要分布在肾小管上皮细胞的胞质中;B组(Fig.15-20):染毒后1d肾组织中Fas的表达即明显强于A组(P<0.05),主要分布在肾小管上皮细胞的胞浆中,其表达呈持续高水平,7d有所下降,至14d仍高于A组(P<0.05)。C组(Fig.21-26):促肝细胞生长素抑制了染毒大鼠肾组织中Fas的表达,与B组比较1d时段无统计学意义(P>0.05),3d至14d各时段则有统计学意义(P<0.05);与A组比较所有时段均有统计学意义(P<0.05)。5.2 Fas-L蛋白在大鼠肾组织中的表达与分布A组(Fig.27):Fas-L蛋白无表达或有微弱表达,主要分布在肾小管上皮细胞的胞质中;B组(Fig.28-33):染毒后1d肾组织中Fas-L的表达即明显强于A组(P<0.05),主要分布在肾小管上皮细胞的胞浆中,其表达呈持续高水平,10d有所下降,至14d仍高于A组(P<0.05)。C组(Fig.34-39):促肝细胞生长素抑制了染毒大鼠肾组织中Fas-L的表达,与B组比较1d时段无统计学意义(P>0.05),3d至14d各时段则有统计学意义(P<0.05);与A组比较所有时段均有统计学意义(P<0.05)。5.3突变型P53在大鼠肾组织中的表达与分布A组(Fig.40):突变型P53无表达或有微弱表达,主要分布在肾小管上皮细胞的胞核中。B组(Fig.41-46):大鼠在染毒1d肾组织中突变型P53的表达即明显强于A组(P<0.05),主要分布在肾小管上皮细胞的胞核中,其表达呈持续高水平,10d有所下降,至14d仍高于A组(P<0.05)。C组(Fig.47-52):促肝细胞生长素明显抑制了染毒大鼠肾组织中突变型P53的表达,与B组比较所有时段均有统计学意义(P<0.05),与A组比较所有时段均有统计学意义(P<0.05)。结论:1百草枯腹腔注射染毒可引起急性肾损伤,且主要表现为肾小管上皮细胞的损伤;2百草枯中毒大鼠体内MDA含量升高、SOD活力下降及GSH-Px活力先降后升,说明过氧化损伤及氧化-抗氧化系统失衡是造成肾损伤的重要机制之一;3突变型P53和Fas/Fas-L在染毒大鼠肾小管上皮细胞的表达发生了明显改变,说明细胞凋亡可能是百草枯中毒肾损伤的机制之一;4应用促肝细胞生长素治疗后,可以增加百草枯中毒大鼠肾组织中SOD及GSH-Px活力,降低MDA含量,从而减轻肾组织的过氧化损伤;5促肝细胞生长素可能通过抑制由突变型P53和Fas/Fas-L介导的细胞凋亡而减轻百草枯中毒所致的肾损伤。更多还原

【Abstract】 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.更多还原