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c-jun氨基末端激酶抑制剂SP600125对小鼠急性肝衰竭的保护作用
陈珑
2013-05-23
导师胡瑾华
专业内科学
授予单位北京大学
授予地点北京大学解放军302医院教学医院
学位硕士
关键词肝衰竭,急性 c-jun氨基末端激酶 SP600125 肝细胞凋亡
其他题名Protective effects of a c-jun N-terminal kinase inhibitor, SP600125, against D-galactosamine/lipopolysaccharide induced mice acute liver failure
分类号R575.3
摘要

目的 观察c-jun氨基末端激酶(c-jun N-terminal kinase, JNK)抑制剂SP600125对小鼠急性肝衰竭的保护作用,探讨JNK信号通路在肝衰竭发病机制中的作用。

方法 采用腹腔注射D-半乳糖胺(D-galactosamine, D-GalN) 400mg/kg体重)/脂多糖(LPS) 30μg/kg体重)致小鼠肝衰竭模型,将雄性C57/BL6小鼠随机分为实验组和对照组,实验组、对照组分别在造模前12h1h皮下注射溶于10%二甲基亚砜的JNK抑制剂SP60012575mg/kg体重),和等体积10%二甲基亚砜;通过蛋白免疫印迹(Western blot)和免疫组化方法检测比较小鼠肝组织磷酸化JNK(Phospho-JNK, p-JNK)的定量、定位表达情况;采用TUNEL染色观察肝组织中细胞凋亡,明确SP600125抑制JNK活化从而减少肝组织细胞凋亡的作用;进一步观察比较实验与对照小鼠的生存率、血清ALT水平及肝组织损伤程度阐明SP600125对急性肝衰竭小鼠的保护作用。

结果 1. SP600125能明显抑制D-GalN/LPS诱导的JNK激活。Western blot结果示,D-GalN/LPS造模后2h,实验组小鼠肝组织匀浆p-JNK表达量明显低于对照组(P=0.0007);免疫组化结果示,D-GalN/LPS造模后2h,实验组小鼠肝组织偶见p-JNK阳性细胞,对照组小鼠肝组织出现大量肝实质细胞表达p-JNK2. SP600125减轻D-GalN/LPS急性肝衰竭造模小鼠肝细胞凋亡。D-GalN/LPS给药后6h,实验组小鼠肝组织Caspase-3阳性细胞少见,对照组见满视野Caspase-3阳性细胞,实验组肝组织中TUNEL染色凋亡阳性细胞数明显低于对照组(t=9.743, P=0.000)。3. SP600125减轻D-GalN/LPS急性肝衰竭造模小鼠肝损伤程度。D-GalN/LPS造模后6h,实验组小鼠血清ALT水平显著低于对照(t=4.734, P=0.0015)。HE染色结果示对照组小鼠肝脏出现大片坏死,小叶内出血,小叶结构部分消失。实验组小鼠肝小叶结构完整,小叶内出血、坏死罕见。4. SP600125提高D-GalN/LPS诱导的小鼠急性肝衰竭生存率。实验组小鼠D-GalN/LPS给药后24小时生存率80%,显著高于对照组10%χ=5.225P=0.0223)。

结论 JNK抑制剂SP600125通过抑制JNK的激活,减少肝组织细胞凋亡、坏死,提高小鼠生存率。SP600125D-GalN/LPS诱导的小鼠急性肝衰竭的具有保护作用。

英文摘要

Objectives To investigate the role of JNK pathway in D-galactosamineD-GalN/lipopolysaccharideLPSinduced mice liver failure with an specific JNK inhibitor SP600125.

Methods 55 male C57/BL6 mice were divided into control group (30) and SP600125 group (25) and were given a intraperitoneal dose of D-GalN(400mg/kg body weight)/LPS(30μg/kg body weight). The control group and SP600125 group were given 10%DMSO (15ml/kg body weight) or SP600125 (75mg/kg body weight) subcutaneously 12h, 1h before D-GalN/LPS administration. Liver and serum sample were collected 0h, 2h, 4h, 6h after D-GalN/LPS administration. D-GalN/LPS induced mice JNK activation was detected by Werstern blot and immunohistochemistry for p-JNK. D-GalN/LPS induced mice liver cell apoptosis were detected by immunohistochemistry for Caspase-3 and TUNEL. Serum ALT was tested to assess liver injury. Survival rate of mice within 24h after D-GalN/LPS administration was observed.

Results Mice in the SP600125 group shows a significantly lower epression of p-JNK in western blot analysis 2h. JNK activity was diminished in SP600125 group mice 2h after D-GalN/LPS administration (observed in immunohistochemistry for p-JNK). Reduced Caspase-3 activity was observed 6h after D-GalN/LPS administration in SP600125 group mice (observed in immunohistochemistry for Caspase-3). Mice in SP600125 group shows significant lower TUNEL-positve cell count than mice in control group (t=9.743, P=0.000). Mice serum ALT elevation was attenuated 6h after D-GalN/LPS administration in SP600125 group (t=4.734, P=0.0015). SP600125 significantly improved mice survival within 24h after D-GalN/LPS administration (χ=5.225P=0.0223).

Conclusions SP600125 exerts its protective effects against D-GalN/LPS induced mice acute liver failure by suppressing liver cell apoptosis and necrosis.
语种中文
出处http://xuewei.bjmu.edu.cn/simpsearch.action?keyword=c-jun氨基末端激酶抑制剂SP600125对小鼠急性肝衰竭的保护作用&dbid=72
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文献类型学位论文
条目标识符http://ir.bjmu.edu.cn/handle/400002259/105424
专题北京大学解放军302医院教学医院
作者单位北京大学解放军302医院教学医院
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陈珑. c-jun氨基末端激酶抑制剂SP600125对小鼠急性肝衰竭的保护作用[D]. 北京大学解放军302医院教学医院. 北京大学,2013.
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