【作者】 牛利；

【导师】 许瑞龄；

【作者基本信息】 山西医科大学 ， 病理学与病理生理学， 2007， 硕士

【摘要】 目的:探讨热休克蛋白70(HSP70)对脂多糖(LPS)诱导的急性肝损伤发生及对肝脏信号转导的影响。方法:实验1.1.雄性昆明小鼠24只,给予亚砷酸钠(SA)8mg/kg腹腔注射,分别于3,6,9,12,24h(n=4)在戊巴比妥钠麻醉下开腹取肝,用Western blotting测定HSP70的表达高峰。1.2.雄性昆明小鼠66只,随机分为三组,(1)对照组:腹腔注射0.9%NaCl 0.2 ml;(2)LPS组:腹腔注射LPS 5mg/kg;(3)SA+LPS(SA)组:于前一晚8时给予腹腔注射SA8mg/kg,12h后即次日早8时腹腔注射LPS,剂量与LPS组相同。各组动物分别于注射生理盐水或LPS后0.5h(n=4)、1.5h(n=8)、6h(n=10),在戊巴比妥钠麻醉下经眼内眦静脉采血、开腹取肝,用于血浆ALT、肝脏MDA、TNF-α、GSH含量和GSH/GSSG测定。1.3.雄性昆明小鼠30只随机分为三组,处理同实验1.2观察其存活率。实验2.雄性昆明小鼠36只,随机分为两组,(1)对照组(n=18):腹腔注射0.9%NaCl 0.2 ml;(2)SA组(n=18):SA8mg/kg腹腔注射,所有动物均于12h后自小鼠尾静脉注入按1∶5稀释的印度墨汁,测定枯否细胞吞噬指数k及其校正值a。实验3.动物和分组同实验1.2,分别于生理盐水或LPS注射后0.5h(n=4)、1.5h(n=8)、6h(n=10),在戊巴比妥钠麻醉下开腹取肝脏,用Western blotting方法检测肝脏HSP70、NF-κB、I-κB、TLR4、CD14、IRAK-M、TNF-α、MEK1/2、ERK1/2、p38MAPK、STAT1、STAT3蛋白的表达及磷酸化水平。结果:SA预处理可诱导肝脏HSP70表达上调,明显减轻LPS所致急性肝损伤。经SA预处理后动物血浆ALT活性和肝脏MDA、GSSG含量下降,而GSH含量、GSH/GSSG比值则升高。肝匀浆的TNF-α测定结果表明,SA组TNF-α含量明显低于肝损伤组(p＜0.05)。HSP70表达上调可明显提高小鼠生存率与损伤组相比有统计学差异(p＜0.05)。SA预处理可减弱LPS诱导肝脏TLR4、CD14、NF-κB、及TNF-α的表达上调,增强I-κB蛋白表达上调及MEK1/2、ERK1/2、p38MAPK蛋白磷酸化。SA预处理后可减弱STAT1蛋白磷酸化的表达,但对STAT3蛋白磷酸化则无明显影响。结论:HSP70主要通过抑制LPS介导的NF-κB信号通路,使TNF-α释放减少,从而减轻LPS诱导的急性肝损伤。更多还原

【Abstract】 Subject:To investigate the effect of HSP70 on the LPS induced acute liver injury and LPS induced liver signal transduction.Methods:The experiment(Exp)1.1 SA 8mg/kg i.p.At 3,6,9,12,24h the liver was extirpated with carbrital anaesthesia and the proteins extracted from livers was assayed for the expression of HSP70.1.2 The Kunming mice were divided randomly into three groups.(1)control: 0.9%Sodium Chloride 0.2 ml,i.p..(2)LPS group:LPS 5mg/kg,i.p..(3)SA+LPS(SA)group:SA 8mg/kg,i.p.were administered on prenight 8pm then treated with i.p injection of LPS 5mg/kg,12h later.The blood was gathered from i.p.eye vein and liver was excised with carbrital anaesthesia after LPS or 0.9%Sodium Chloride injected at 0.Sh(n=4),1.5h(n=8)and 6.0h(n=10)for ALT, MDA,TNF-α,GSSG,GSH and GSH/GSSG assays for each groups.1.3.The animal and groups were same as Exp 1.2 in order to observe suvival rate o Exp 2.The experimental mice were divided randomly into two groups.(1)control:0.9%Sodium Chloride intraperitoneal injections.(2)SA. SA.intraperitoneal injections 8mg/kg.After 12h,India ink 1:5 diluted with germ free physiological saline was injected into the vail vein in each group rats.To calculate phagocytic index k and its correction a.Exp 3.The animal and groups were same as Exp 1.2(1)control:0.9%Sodium Chloride 0.2 ml,i.p..(2)LPS group:LPS 5mg/kg i.p..(3)SA+ LPS(SA)group:SA 8mg/kg,i.p.was administered on prenight(8pm),then treated with i.p injection of LPS 5mg/kg,12h later.The livers were excised in anaesthetic state by carbrital after LPS or 0.9%Sodium Chloride injected at 0.5h, 1.5h and 6h and the protein extracted from livers was assayed for the phosphorylation level of MEK1/2,ERK1/2,p38MAPK,STAT1,STAT3 and expression of TLR4,NF-κB,I-κB,CD14, IRAK-M and TNF-αby western blotting analysis.Results:HSP70 can expression and LPS-induced acute liver injury could attenuated significantly by SA pretreatment.The plasma ALT activity and liver MDA、GSSG contant were decreased remarkably by SA preteatment and the GSH and GSH/GSSG assays of liver was increased significantly.In LPS treatment groups,LPS stimulation increased plasma level of TNF-αexpression in the liver,in which the peak values of plasma TNF-αin SA group were much lower than LPS group(p＜0.05).HSP70 can increase survive rate of mice compare to LPS group(p＜0.05). CD14、TLR4、NF-κB、NF-αwere decreased significantly by SA pretreatment.And I-κB、p-MEK1/2,and p-ERK1/2、p-p38MAPK could increased.The phosphorylation of STAT1 was appeared to decreased after SA pretreatment,but the phosphorylation of STAT3 appear to no obviously change. Conclusion:SA pretreatment can induce expression of HSP70 of mice,partially inhibited LPS induced NF-κB signalling pathway and made TNF-αless releasing,to reduce LPS induced acute liver injury significantly.更多还原