内毒素致大鼠急性肝损伤的机制研究

The research on mechanism of acute hepatic injury induced by endotoxin in rats

目的:观察内毒素致急性肝损伤模型大鼠不同时间点的肝脏功能及血清内毒素含量、肝脏组织中白介素6(IL-6)表达的水平、蛋白Toll样受体4(Toll-like receptor 4,TLR4)、NFκB p65亚基及其磷酸化蛋白的相对表达情况,以及TLR4、IL-6 mRNA的相对表达情况,初步探讨内毒素致大鼠急性肝损伤的时效性变化及作用机制。方法:腹腔注射LPS 5mg/kg后,每0.5h测定一次动物肛温,采用断点法分别于造模后2、4、6、8、10h处死6只动物,剖取肝脏、脾脏、胸腺、肾上腺等组织,采用比色法测定采用肝脏功能指标谷丙转氨酶(ALT)、谷草转氨酶(AST)及碱性磷酸酶(ALP)的活性,采用鲎试剂动态比浊法测定各组动物血清内毒素的含量,采用ELISA检测受试动物肝脏组织匀浆中IL-6的含量,运用Western Blot检测肝脏中TLR4、NFκB p65亚基及其磷酸化蛋白的表达,采用QRT-PCR测定肝脏中TLR4、IL-6 mRNA的相对表达情况。结果:LPS刺激后0.5h大鼠体温升高;1~2h时体温降低;4h^10h体温在不断升高,其中在4~4.5h增幅明显;4h组大鼠肝脏系数及脾脏系数显著升高;与0h组相比,各组大鼠血清内毒素含量均显著升高(P<0.01),其中4h、6h、8h组大鼠血清内毒素含量为0h组的40倍以上;LPS刺激后各组大鼠血清ALT、AST、ALP含量均明显升高趋势,且在4h或6h时达到最高;4h组大鼠肝脏中IL-6的含量均明显高于0h组;4h组大鼠肝脏中TLR4及NFκBp65亚基磷酸化的相对表达量比0h组明显升高;8h组大鼠肝脏中TLR4 mRNA的相对表达量有一定的升高趋势,4h组、6h组、8h组、10h组大鼠肝脏中IL-6 mRNA的相对表达量均有升高,其中4h和6h组差异显著。结论:内毒素致急性肝损伤模型大鼠造模4h后IL-6分泌增加,4h组大鼠肝脏组织TLR4功能加强,转核因子NFκB p65亚基的磷酸化程度增加,表明IL-6、TLR4、NFκB p65亚基三者在内毒素致肝脏组织损伤中起重要作用,LPS致急性肝损伤的作用机制与其上调TLR4-NFκB炎症信号通路中TLR4蛋白及NFκBp65亚基的磷酸化水平、增加致炎因子IL-6的表达等环节有关。

Objective： To observe Liver function,the level of endotoxins in serum,the level of interleukin-6（ IL-6） expression,the relative expression of Toll-like receptor 4（ TLR4） protein and phosphorylated level NFκB p65 subunit,and the relative expression of TLR4 and IL-6mRNA in endotoxin-induced acute hepatic injury at different time points in rats. Then preliminary make studies on the mechanism of endotoxin-induced acute liver injury in rats. And explored the optimal sampling point for the LPS-induced acute liver injury model initially. Methods： After intraperitoneal injection of LPS,animals were tested rectal temperature once every 0. 5h; Every 6 rats were sacrificed at 2,4,6,8,10 h after injecting LPS,taken liver,spleen,thymus,and adrenal glands,and been cryopreserved; Liver function index,such as ALT,AST and ALP,were measured by using colorimetric indicator,the level of serum endotoxin was detected by using the kinetic turbidimetric LAL assay,the level of IL-6 in liver tissue was measured by Elisa; the expression of TLR4 protein,phosphorylation of NFκB p65 subunit in liver were detected by Western Blot; the relative expression of TLR4 and IL-6 mRNA were determinate using QRT-PCR. Results： 0. 5h after LPS stimulation,rats anal gentle rise,then the temperature hypothermia When 1 ~ 2h,and rise when 4h-10 h,which increased significantly in the4 ~ 4. 5h; the coefficient factor of liver and spleen in rats of was significantly higher than 0h group（ P 〈 0. 01 ~ 0. 05）; Compared with the 0h group,toxin levels in rats serum were significantly elevated（ P 〈 0. 01）,of which the content of 4h,6h,8h were 40 times more than 0h group at least,the activity of ALT,AST,ALP was significantly increased（ P 〈 0. 01 ~ 0. 05）,this index in 4h and6 h was the highest,the IL-6 level in liver of 4h group rats were significantly higher than 0h group（ P 〈 0. 01）; the relative expression ratio of TLR4 and phosphorylation of NFk B p65 subunit was significantly increased in 4h group rats liver（ P 〈 0. 05）; and the relative expression of TLR4 mRNA has a certain amount increasing trend in 8h group rats liver（ P 〉 0. 05）,the relative expression levels of IL-6 mRNA in 4h group,6h group,8h group,and10 h group rat livers were elevated（ P 〈 0. 05 ~ 0. 01）,which 4h and 6h groups had significant difference（ P 〈 0. 01）. Conclusion： IL-6 secretion rise in Endotoxin-induced acute liver injury rats model after modeling 4h,and TLR4 function of liver tissue strengthen and the degree of phosphorylation of transfer of nuclear factor NFκB p65 subunit increased,which indicating that IL-6,TLR4,NFκB p65 subunit play an important role in endotoxin-induced liver injury. The mechanism of LPS-induced acute liver injury was related. to raising TLR4 protein in TLR4-NFk B inflammation signaling pathway and increased phosphorylation levels of NFκBp65 subunit,and strengthen expression of pro-inflammatory cytokines IL-6 and other aspects.