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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。
AIM: To observe the liver function and serum endotoxin levels, the expression of interleukin-6 (IL-6), the level of Toll-like receptor 4 (Toll-like receptor 4) in liver tissue of rats with acute liver injury induced by endotoxin at different time points , TLR4), NFκB p65 subunit and phosphorylated protein, as well as the relative expression of TLR4 and IL-6 mRNA in rat model of acute liver injury induced by endotoxin. Methods: The rectal temperature was measured every 0.5h after intraperitoneal injection of 5mg / kg LPS, and 6 animals were sacrificed at 2, 4, 6, 8 and 10h after the model was established. The liver, spleen, thymus, Adrenal gland and other tissues. The activity of ALT, AST and ALP were measured by colorimetric method. The levels of serum endotoxin The content of IL-6 in liver homogenate of the tested animals was detected by ELISA. The expression of TLR4, NFκB p65 subunit and phosphorylated protein in liver were detected by Western Blot. The levels of TLR4, IL -6 mRNA relative expression. Results: The body temperature increased 0.5h after LPS stimulation, the body temperature decreased at 1 ~ 2h, the body temperature increased continuously at 4h ~ 10h, and the increase was obvious at 4 ~ 4.5h. The liver and spleen coefficients of rats in 4h group were significantly increased ; Compared with 0h group, the levels of serum endotoxin in rats in each group were significantly increased (P <0.01), and the levels of endotoxin in rats in 4h, 6h, 8h group were more than 40 times higher than those in 0h group; The contents of ALT, AST and ALP in serum of rats in group 4h increased significantly at 4h or 6h, and the content of IL-6 in liver of 4h group was significantly higher than that in 0h group. The levels of TLR4 And NFκBp65 subunit phosphorylation relative expression increased significantly than the 0h group; 8h group liver TLR4 mRNA relative expression increased to a certain extent, 4h group, 6h group, 8h group, 10h group rat liver IL-6 mRNA relative expression levels were increased, of which 4h and 6h group significant difference. CONCLUSION: IL-6 secretion is increased 4 h after acute liver injury induced by lipopolysaccharide in rats, and the function of TLR4 in liver tissue of 4 h group is enhanced. The phosphorylation of nuclear factor NFκB p65 subunit is increased, indicating that IL-6 and TLR4 , NFκB p65 subunits play an important role in endotoxin-induced hepatic tissue injury. The mechanism of LPS-induced acute liver injury is related to its up-regulation of TLR4 protein and NFκBp65 subunit phosphorylation in TLR4-NFκB signaling pathway, Factor IL-6 expression and other aspects.