依达拉奉对脑死亡状态下SD大鼠血清TNF-α、IL-6变化及肝脏NF-κB的表达的影响
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摘要
目的利用脑死亡供者提供器官进行移植成为缓解供体来源短缺的有效途径,而目前对于脑死亡状态对各个器官功能影响的机制,研究仍不深入。本实验旨在探讨脑死亡动物模型的建立和维持,观察依达拉奉对脑死亡状态下SD大鼠肝脏功能、形态,血清TNF-α、IL-6及肝脏核因子κB(NF-κB)蛋白表达的影响。
方法健康成年SD大鼠24只,雌雄不拘,体重220-280克,3-4月龄,随机分成四组(n=6),假手术组(C组);脑死亡组(B组);依达拉奉干预组1(I_1组,3mg/kgiv脑死亡诱导前给药);依达拉奉干预组2(I_2组,3mg/kgiv确认脑死亡后给药)。所有大鼠行气管切开后行气管内插管,颅骨钻孔置管,股、动静脉置管后,B组、I_1组、I_2组应用改进的缓慢间断颅内加压法建立脑死亡模型并在判定为脑死亡后维持6h,C组假手术后维持6h。于脑死亡诱导前(T_1),判定为脑死亡时(T_2,C组在假手术后1h采血),确认脑死亡后6h(T_3),股动脉采血测血清AST、ALT、IL-6、TNF-α水平。首次确认脑死亡6h后开腹取肝脏组织苏木素-伊红染色观察肝脏组织形态变化。免疫组化染色观察NF-κB蛋白的表达水平。
结果与C组比较,T_3时B组、I_1组、I_2组血清ALT、AST水平升高(P＜0.05);与B组比较,T_3时I_1组、I_2组血清ALT、AST水平升高幅度较小(P＜0.05);与T_1比较,T_3时B组、I_1组、I_2组ALT、AST水平明显升高(P＜0.05)。
与C组比较,B组、I_1组、I_2组血清TNF-α和IL-6水平升高(P＜0.05);与B组比较,I_1组、I_2组血清TNF-α和IL-6水平升高幅度较小(P＜0.05);与T_1比较,B组、I_1组、I_2组TNF-α和IL-6水平白T_2时即有升高,T_3时升高幅度较T_2时大(P＜0.05)。
肝脏NF-κB蛋白表达的改变:肝脏组织中NF-κB在C组表达弱且分散,B组、I_1组、I_2组与C组相比NF-κB表达均有增高(P＜0.05);B组表达明显强于I_1组、I_2组(P＜0.05)。
光镜下:C组肝细胞形态结构正常;B组肝细胞体积变大,胞浆疏松,部分细胞水肿明显;I_1、I_2组可见肝细胞轻度水肿,基本形态正常。
结论1.应用改进的缓慢间断颅内加压法并持续监测AAI制作SD大鼠脑死亡模型,成功率和可重复性高,更符合临床脑死亡的发展过程,经有效的呼吸和循环支持,脑死亡状态可稳定维持。
2.脑死亡可导致SD大鼠肝脏的损伤,随着脑死亡时间的延长可出现形态学改变。
3.脑死亡可导致SD大鼠血清炎症因子TNF-α、IL-6释放增加,肝脏组织NF-κB蛋白表达增加,使用依达拉奉干预后炎症因子TNF-α、IL-6释放减少,肝脏组织NF-κB蛋白表达降低。
4.依达拉奉对SD大鼠脑死亡肝损伤具有一定保护作用。
Objective To investigate the establishment and the maintenance of brain-dead model with SD rat.To evaluate the effects of edaravone on the change of hepatic function,morphology and serum TNF-αand IL-6 concentration and NF-κB protein expression of liver of SD rat brain-death state.
Materials and Methods Twenty-four 3-4 month healthy SD rats of either sex were randomly assigned to four study groups(n=6):sham(group C);brain death(group B);brain death with edaravone 1(3mg/kg iv before brain death induced,groupI_1)and brain death with edaravone 2(3mg/kg iv at brain death confirmed immediately,groupI_2).groupB、groupI_1、groupI_2 established brain death model and mechanically ventilated for 6 hours after brain death,group C mechanically ventilated for 6 hours after sham operation.Rats were at time before brain death induced(T_1),brain death confirmed(T_2,groupC at 1 hour after sham operation)and 6 hours after brain death(T_3),serum TNF-αand IL-6 concentration were determined. right lobe of liver was removed for morphologic examination by HE staining and determination of NF-κB protein expression by immnohistochemistry.
Result The serum ALT and AST concentration were significantly higher in group B,group I_1 and group I_2 than in group C at T_3(P＜0.05). edaravone treatment significantly attenuated the increase in serum ALT and AST concentration in group I_1 and group I_2(P＜0.05).
The serum TNF-αand IL-6 concentration were significantly increased in group B,group I_1 and group I_2(P＜0.05)and the concentration were lower in group I_1 and group I_2 than in group B(P＜0.05).
The expression of NF-κB protein of group B was significantly increased,the expression of group C was weak and scattered.Expression of NF-κB protein for group B,group I_1 and group I_2 was significantly increased than that for group C(P＜0.05),the expression of group I_1 and group I_2 was lower than that of group B(P＜0.05).
Morphological changes of hepatic tissues:in group C,hepatic tissues appearances were normal.In group B,obvious edema of partial cells. Liver injury was significantly attenuated in group I_1 and group I_2.
Conclusion 1.To establish brain-dead model by increasing intracranial pressure in a modified,slow and intermittent way with AAI monitoring in SD rat can effectively mimic the clinical brain death.With effective respiration and circulation support,the brain-dead state could be maintained.
2.Brain death may evoke hepatic morphological injury of SD rats.
3.Serum TNF-αand IL-6 concentration and t NF-κB protein expression in the liver tissues can be enhanced by the brain death.Serum TNF-αand IL-6 concentration and t NF-κB protein expression in the liver tissues were significantly attenuated in group I_1 and group I_2 by edaravone treatment.
4.edaravone has protection which can against the liver injury caused by brain death.
方法健康成年SD大鼠24只,雌雄不拘,体重220-280克,3-4月龄,随机分成四组(n=6),假手术组(C组);脑死亡组(B组);依达拉奉干预组1(I_1组,3mg/kgiv脑死亡诱导前给药);依达拉奉干预组2(I_2组,3mg/kgiv确认脑死亡后给药)。所有大鼠行气管切开后行气管内插管,颅骨钻孔置管,股、动静脉置管后,B组、I_1组、I_2组应用改进的缓慢间断颅内加压法建立脑死亡模型并在判定为脑死亡后维持6h,C组假手术后维持6h。于脑死亡诱导前(T_1),判定为脑死亡时(T_2,C组在假手术后1h采血),确认脑死亡后6h(T_3),股动脉采血测血清AST、ALT、IL-6、TNF-α水平。首次确认脑死亡6h后开腹取肝脏组织苏木素-伊红染色观察肝脏组织形态变化。免疫组化染色观察NF-κB蛋白的表达水平。
结果与C组比较,T_3时B组、I_1组、I_2组血清ALT、AST水平升高(P＜0.05);与B组比较,T_3时I_1组、I_2组血清ALT、AST水平升高幅度较小(P＜0.05);与T_1比较,T_3时B组、I_1组、I_2组ALT、AST水平明显升高(P＜0.05)。
与C组比较,B组、I_1组、I_2组血清TNF-α和IL-6水平升高(P＜0.05);与B组比较,I_1组、I_2组血清TNF-α和IL-6水平升高幅度较小(P＜0.05);与T_1比较,B组、I_1组、I_2组TNF-α和IL-6水平白T_2时即有升高,T_3时升高幅度较T_2时大(P＜0.05)。
肝脏NF-κB蛋白表达的改变:肝脏组织中NF-κB在C组表达弱且分散,B组、I_1组、I_2组与C组相比NF-κB表达均有增高(P＜0.05);B组表达明显强于I_1组、I_2组(P＜0.05)。
光镜下:C组肝细胞形态结构正常;B组肝细胞体积变大,胞浆疏松,部分细胞水肿明显;I_1、I_2组可见肝细胞轻度水肿,基本形态正常。
结论1.应用改进的缓慢间断颅内加压法并持续监测AAI制作SD大鼠脑死亡模型,成功率和可重复性高,更符合临床脑死亡的发展过程,经有效的呼吸和循环支持,脑死亡状态可稳定维持。
2.脑死亡可导致SD大鼠肝脏的损伤,随着脑死亡时间的延长可出现形态学改变。
3.脑死亡可导致SD大鼠血清炎症因子TNF-α、IL-6释放增加,肝脏组织NF-κB蛋白表达增加,使用依达拉奉干预后炎症因子TNF-α、IL-6释放减少,肝脏组织NF-κB蛋白表达降低。
4.依达拉奉对SD大鼠脑死亡肝损伤具有一定保护作用。
Objective To investigate the establishment and the maintenance of brain-dead model with SD rat.To evaluate the effects of edaravone on the change of hepatic function,morphology and serum TNF-αand IL-6 concentration and NF-κB protein expression of liver of SD rat brain-death state.
Materials and Methods Twenty-four 3-4 month healthy SD rats of either sex were randomly assigned to four study groups(n=6):sham(group C);brain death(group B);brain death with edaravone 1(3mg/kg iv before brain death induced,groupI_1)and brain death with edaravone 2(3mg/kg iv at brain death confirmed immediately,groupI_2).groupB、groupI_1、groupI_2 established brain death model and mechanically ventilated for 6 hours after brain death,group C mechanically ventilated for 6 hours after sham operation.Rats were at time before brain death induced(T_1),brain death confirmed(T_2,groupC at 1 hour after sham operation)and 6 hours after brain death(T_3),serum TNF-αand IL-6 concentration were determined. right lobe of liver was removed for morphologic examination by HE staining and determination of NF-κB protein expression by immnohistochemistry.
Result The serum ALT and AST concentration were significantly higher in group B,group I_1 and group I_2 than in group C at T_3(P＜0.05). edaravone treatment significantly attenuated the increase in serum ALT and AST concentration in group I_1 and group I_2(P＜0.05).
The serum TNF-αand IL-6 concentration were significantly increased in group B,group I_1 and group I_2(P＜0.05)and the concentration were lower in group I_1 and group I_2 than in group B(P＜0.05).
The expression of NF-κB protein of group B was significantly increased,the expression of group C was weak and scattered.Expression of NF-κB protein for group B,group I_1 and group I_2 was significantly increased than that for group C(P＜0.05),the expression of group I_1 and group I_2 was lower than that of group B(P＜0.05).
Morphological changes of hepatic tissues:in group C,hepatic tissues appearances were normal.In group B,obvious edema of partial cells. Liver injury was significantly attenuated in group I_1 and group I_2.
Conclusion 1.To establish brain-dead model by increasing intracranial pressure in a modified,slow and intermittent way with AAI monitoring in SD rat can effectively mimic the clinical brain death.With effective respiration and circulation support,the brain-dead state could be maintained.
2.Brain death may evoke hepatic morphological injury of SD rats.
3.Serum TNF-αand IL-6 concentration and t NF-κB protein expression in the liver tissues can be enhanced by the brain death.Serum TNF-αand IL-6 concentration and t NF-κB protein expression in the liver tissues were significantly attenuated in group I_1 and group I_2 by edaravone treatment.
4.edaravone has protection which can against the liver injury caused by brain death.
引文
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