乌司他丁联合维拉帕米对大鼠肝缺血再灌注损伤的保护作用

【作者】 高飞；

【导师】 赵浩亮；

【作者基本信息】 山西医科大学 ， 外科学， 2010， 硕士

【摘要】 研究背景：肝脏缺血再灌注损伤(ischemia-reperfusion injury, I/R injury)是肝胆外科中不可避免的一个病理生理现象。严重的腹腔感染、失血等原因导致的休克复苏后；肝脏的肿物进行肝部分切除时；严重的肝脏外伤及肝移植的时候,肝脏缺血再灌注损伤都是一个不容忽视的问题。搞清肝脏缺血再灌注损伤的机制,研究一些行之有效的方法或药物治疗,这个问题一直是许多学者及临床医生研究的热点与难点。目前研究认为,肝脏缺血再灌注损伤的主要机制是：氧自由基的损伤、钙超载及细胞因子损伤。乌司他丁和维拉帕米是针对这些机制中的代表性药物,两药单独运用均可以对肝脏的缺血再灌注损伤起到保护作用,但联合用药的效果如何尚未有明确报道。目的：本课题旨在研究乌司他丁联合维拉帕米对大鼠肝缺血再灌注损伤的保护作用。探讨两药在缺血再灌注损伤中的作用机制,以指导乌司他丁联合维拉帕米在临床中的应用。方法：采用大鼠在体肝缺血再灌注(I／R)模型。健康雄性Wistar大鼠40只,随机分成5组,每组8只：假手术组(A组),缺血再灌注组(B组),乌司他丁预处理组(C组),维拉帕米预处理组(D组),以及乌司他丁联合维拉帕米预处理组(E组)。A组：开腹后关腹；B组：开腹后采用Pringle法阻断入肝血流30min,后关腹；C组：开腹前30min腹腔注射乌司他丁,余处理同B组；D组：开腹前10min尾静脉注射维拉帕米,余处理同B组；E组：分别在开腹前30min腹腔注射乌司他丁和开腹前10min尾静脉注射维拉帕米,余处理同B组。检测再灌注6h后大鼠血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)、乳酸脱氢酶(LDH)、肿瘤坏死因子-α(TNF-α)、及白细胞介素-1β(IL-1β)的水平并取部分肝脏进行病理学观察。结果：1、谷丙转氨酶(ALT)、谷草转氨酶(AST)、乳酸脱氢酶(LDH)测定结果：与假手术组相比,缺血再灌注组ALT、AST、LDH含量明显增加,差别有统计学意义(P<0.01)；与缺血再灌注组相比,乌司他丁预处理组、维拉帕米预处理组,以及乌司他丁联合维拉帕米预处理组ALT、AST、LDH含量明显减少,差别有统计学意义(P<0.01)；乌司他丁预处理组、维拉帕米预处理组ALT、AST、LDH含量高于乌司他丁联合维拉帕米预处理组,差别有统计学意义(P<0.05)；乌司他丁预处理组与维拉帕米预处理组ALT、AST、LDH含量无明显差异(P>0.05)。2、肿瘤坏死因子-α(TNF-α)、及白细胞介素-1β(IL-1β)测定结果：与假手术组相比,缺血再灌注组TNF-α、IL-1β含量明显增加,差别有统计学意义(P<0.01)；与缺血再灌注组相比,乌司他丁预处理组、维拉帕米预处理组,以及乌司他丁联合维拉帕米预处理组TNF-α、IL-1β含量明显减少,差别有统计学意义(P<0.01)；乌司他丁预处理组、维拉帕米预处理组TNF-α、IL-1β含量高于乌司他丁联合维拉帕米预处理组,差别有统计学意义(P<0.05)；乌司他丁预处理组与维拉帕米预处理组TNF-α、IL-1β含量无明显差异(P＜0.05)。3、肝组织病理学观察结果：B组的病理学改变最严重,C组与D组的改变均较B组轻,E组的改变最轻。结论：1与假手术组相比,缺血再灌注组中TNF-α、IL-1β水平显著增高,进一步证实了TNF-α、IL-1β的水平增高与肝缺血再灌注损伤有着密切的联系。2与缺血再灌注组相比,乌司他丁和维拉帕米均可减轻肝脏组织损伤,对肝细胞起保护作用,乌司他丁联合维拉帕米对肝脏的保护作用优于单药。更多还原

【Abstract】 Background:The liver ischemia-reperfusion injury is inevitable pathophysiological phenomen-on in the liver and gallbladder surgery. When Shock, because of serious abdominal infection, bleeding and other reasons, has been the recovery; When the liver is cut partly because of liver tumor; When liver is damaged seriously, or is transplanted; The liver ischemia-reperfusion injury can not be ignored problem. To elaborate the mechanism of hepatic ischemia-reperfusion injury and investigate some effective methods or drug treatments, which has been a hot and difficult for many scholars and clinicians all the time. At present studies suggest that the main mechanisms of hepatic ischemia-reperfusion injury are:oxygen free radical damage, calcium overload and cell factor damage. Ulinastatin and verapamil are represent durgs aimed at these mechanisms. Two drugs used alone can play a protective role in the hepatic ischemia-reperfusion injury, however, the effect of combination therapy has not been clearly reported.Objective:The purpose of this topic is studying protective effect of ulinastatin combined with verapamil on liver ischemic-reperfusion injury in rats. Approaching ulinastatin and verapamil possible mechanism in ischemia-reperfusion injury, the purpose is to guide the ulinastatin comb-ined with verapamil at clinical application.Methods:Liver I/R model in vivo rats were established. Healthy male wistar rats (n=40) were randomly divided into five groups (n=8):sham-operation group (Group A), ischemia-reperfusion group (Group B), pretreatment with ulinastatin group (Group C), pretreatment with verapamil group (Group D),and pretreatment with ulinastatin combined with verapamil group (Group E). Group A:The abdomen was opened and then closed; Group B:Hepatic inflow was occluded for 30 minutes by pringlep’s maneuver, and then abdomen was closed; Group C:Ulinastatin was administered intraperitoneally 30 minutes before the experiment, the dealing was the same as group B; Group D:Intravenous verapamil was given 10 minutes before the experiment, the dealing was the same as group B; Group E:The methods was the same as group B, group C and group D. The concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), tumor necrosis factor-α(TNF-α), and interleukin-1β(IL-1β) in serum were measured, and the histopathological changes of liver were also observed.Result:1. The concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH):Compared with sham-operated group rats, the concentrat-ions of ALT, AST, LDH of ischemia-reperfusion group was significantly increased, the difference has statistics significance (P<0.01); Compared with ischemia-reperfusion group rats, the concentrations of ALT, AST, LDH of pretreatment with ulinastatin group, pretreatment with verapamil group and pretreatment with ulinastatin combined with verapamil group were significantly reduced, the difference has statistics significance (P<0.01); Compared with pretreatment with ulinastatin combined with verapamil group rats, the concentrations of ALT, AST, LDH of pretreatment with ulinastatin group, pretreatment with verapamil group was significantly increased, the difference has statistics significance (P<0.05); Compared with pretreatment with ulinastatin group rats, the concentrations of ALT, AST, LDH of pretreatment with verapamil group was not significantly different (P>0.05).2. The concentrations of tumor necrosis factor-α(TNF-α), and interleukin-1β(IL-1(3):Compared with sham-operated group rats, the concentrations of TNF-α, IL-1βof ischemia-reperfusion group was significantly increased, the difference has statistics significance (P<0.01); Compared with ischemia-reperfusion group rats, the concentrations of TNF-α, IL-1βof pretreatment with ulinastatin group, pretreatment with verapamil group and pretreatment with ulinastatin combined with verapamil group were significantly reduced, the difference has statistics significance (P<0.01); Compared with pretrea-tment with ulinastatin combined with verapamil group rats, the concentrations of TNF-a, IL-1βof pretreatment with ulinastatin group, pretreatment with verapamil group was significant-1y increased, the difference has statistics significance (P<0.05); Compared with pretreatment with ulinastatin group rats, the concentrations of TNF-a, IL-1βof pretreatment with verapamil group was not significantly different (P>0.05).3. the histopathological change of liver in group B was the severest, The change of group C and D were better than group B, The change of group E was the slightest.Conclusion:1. Compared with the sham-operated group, the level of TNF-α, IL-1βin ischemia-reperfusion group was significantly increased, further confirming that the increased level of TNF-α, IL-1βis closely linked to liver ischemic-reperfusion injury.2. Compared with ischemia-reperfusion group, both ulinastatin and verapami can reduce liver ischemia-reperfusion injury and protect the liver cells. Ulinastatin combined with verapamil has a better effect than ulinastatin or verapami.更多还原