TAK242阻断Toll样受体4通路在脓毒症中对肝脏起到保护作用

Inhibition of Toll-like receptor 4 pathway by TAK242 protects the liver in sepsis

目的探讨Toll样受体4(TLR4)特异性抑制剂TAK242对脓毒症大鼠模型肝脏的保护机制。方法将18只雄性SD大鼠按随机数字表法分为3组,每组6只,采用腹腔注射脂多糖(LPS)15 mg/kg制备脓毒症模型;TAK242干预组于制模前腹腔注射TAK242(5 mg/kg)进行预处理,脓毒症模型组和对照组则注射等量溶剂〔10%二甲基亚砜(DMSO)+90%玉米油〕。6 h后取大鼠腹主动脉血,采用酶联免疫吸附试验(ELISA)检测各组大鼠血清丙氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST)水平;处死大鼠取肝组织,采用蛋白质免疫印迹试验(Western blotting)检测各组大鼠肝组织TLR4、肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、天冬氨酸特异性半胱氨酸蛋白酶3(caspase-3)、核转录因子-κB p65及其磷酸化(NF-κB p65,p-NF-κB p65)的表达水平,采用免疫组织化学(免疫组化)染色观察肝组织NF-κB p65蛋白表达,采用苏木素-伊红(HE)染色评估肝细胞损伤情况。结果脓毒症模型组ALT和AST水平均较对照组显著升高〔ALT(μg/L):26.639±7.814比2.847±2.150,AST(μg/L):28.442±8.417比5.779±3.019,均P<0.01〕,TAK242干预组ALT和AST水平则较脓毒症模型组明显降低〔ALT(μg/L):7.269±3.398比26.639±7.814,AST(μg/L):3.580±3.115比28.442±8.417,均P<0.01〕。光镜下显示,脓毒症模型组肝细胞排列紊乱,细胞水肿明显,炎症细胞浸润增多;TAK242干预组肝细胞排列整齐,肝细胞水肿明显减轻,炎症细胞浸润减少。Western blotting结果显示,脓毒症模型组肝组织caspase-3蛋白表达较对照组明显升高(caspase-3/GAPDH:0.794±0.164比0.482±0.055,P<0.05),TAK242干预组caspase-3蛋白表达则较脓毒症模型组明显降低(caspase-3/GAPDH:0.482±0.056比0.794±0.164,P<0.05),说明TAK242可减轻脓毒症大鼠肝脏细胞凋亡。脓毒症模型组肝组织IL-6、TNF-α和TLR4蛋白表达及p-NF-κB p65/NF-κB p65比值均明显高于对照组(IL-6/GAPDH:1.442±0.204比1.019±0.024,TNF-α/GAPDH:1.089±0.098比0.806±0.005,TLR4/GAPDH:1.292±0.085比0.941±0.087,p-NF-κB p65/NF-κB p65比值:1.936±0.081比1.579±0.183,均P<0.05),TAK242干预组肝组织IL-6、TNF-α和TLR4蛋白表达及p-NF-κB p65/NF-κB p65比值均较脓毒症模型组明显降低(IL-6/GAPDH:1.035±0.042比1.442±0.204,TNF-α/GAPDH:0.572±0.096比1.089±0.098,TLR4/GAPDH:0.984±0.078比1.292±0.085,p-NF-κB p65/NF-κB p65比值:1.484±0.255比1.936±0.081,均P<0.05),说明LPS诱导的脓毒症可激活肝组织TLR4/NF-κB通路所介导的炎症反应,给予TAK242阻断TLR4通路后,TLR4/NF-κB通路的激活被抑制,从而减轻脓毒症大鼠肝组织的炎症反应。免疫组化染色显示,脓毒症模型组肝组织NF-κB p65阳性表达较对照组明显增加;TAK242干预组NF-κB p65阳性表达则较脓毒症模型组明显减少,细胞核内几乎无阳性表达。结论TAK242通过阻断肝脏TLR4/NF-κB通路可减轻脓毒症大鼠的肝功能损伤,起到保护肝脏的作用。

Objective To investigate the protective mechanism of TAK242,a specific inhibitor of Toll-like receptor 4(TLR4),on the liver of septic rats.Methods Eighteen male Sprague-Dawley(SD)rats were randomly divided into three groups(n=6 in each group).The septic model was established by intraperitoneal injection of lipopolysaccharide(LPS)15 mg/kg.The rats in the TAK242 intervention group received intraperitoneal injection of TAK242(5 mg/kg)before modeling,while the rats in the septic model group and the control group were injected with the same amount of solvent[10%dimethyl sulfoxide(DMSO)+90%corn oil].Six hours later,the blood of abdominal aorta was collected and the levels of serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were measured by enzyme linked immunosorbent assay(ELISA).The rats were sacrificed to obtain liver,the expression levels of TLR4,tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),cysteinyl aspartate-specific proteinase-3(caspase-3),nuclear factor-κB p65(NF-κB p65)and phosphorylated NF-κB p65(p-NF-κB p65)were detected by Western blotting.Immunohistochemical staining was used to observe NF-κB p65 protein expression in liver,and hepatocyte injury was assessed by hematoxylin-eosin(HE)staining.Results Serum ALT and AST levels in the septic model group were significantly higher than those in the control group[ALT(μg/L):26.639±7.814 vs.2.847±2.150,AST(μg/L):28.442±8.417 vs.5.779±3.019,both P<0.01].The ALT and AST levels in the TAK242 intervention group were significantly lower than those in septic model group[ALT(μg/L):7.269±3.398 vs.26.639±7.814,AST(μg/L):3.580±3.115 vs.28.442±8.417,both P<0.01].Light microscopy showed that the hepatocytes in the septic model group were disordered,with obvious cell edema and increased inflammatory cells infiltration;the hepatocytes in the TAK242 intervention group were more neatly arranged,with significantly reduced hepatocyte edema and reduced inflammatory cells infiltration.Western blotting results showed that caspase-3 protein expression in hepatic tissue of septic model group was significantly higher than that in the control group(caspase-3/GAPDH:0.794±0.164 vs.0.482±0.055,P<0.05),and caspase-3 protein expression in the TAK242 intervention group significantly decreased than that in the septic model group(caspase-3/GAPDH:0.482±0.056 vs.0.794±0.164,P<0.05),which indicated that TAK242 could attenuate hepatocytes apoptosis of septic rats.The expression of IL-6,TNF-αand TLR4 protein and the ratio of p-NF-κB p65/NF-κB p65 in hepatic tissue of septic model group were significantly higher than those in control group(IL-6/GAPDH:1.442±0.204 vs.1.019±0.024,TNF-α/GAPDH:1.089±0.098 vs.0.806±0.005,TLR4/GAPDH:1.292±0.085 vs.0.941±0.087,p-NF-κB p65/NF-κB p65 ratio:1.936±0.081 vs.1.579±0.183,all P<0.05),IL-6,TNF-αand TLR4 protein expression and p-NF-κB p65/NF-κB p65 ratio in the TAK242 intervention group were significantly lower than those in septic model group(IL-6/GAPDH:1.035±0.042 vs.1.442±0.204,TNF-α/GAPDH:0.572±0.096 vs.1.089±0.098,TLR4/GAPDH:0.984±0.078 vs.1.292±0.085,p-NF-κB p65/NF-κB p65 ratio:1.484±0.255 vs.1.936±0.081,all P<0.05),it is suggested that LPS-induced sepsis could activate the inflammatory response mediated by TLR4/NF-κB pathway in liver,and the activation of TLR4/NF-κB pathway was inhibited by TAK242 through the TLR4 pathway,therefore,the inflammation of liver in septic rats was reduced.Immunohistochemical staining showed that the positive expression of NF-κB p65 in liver was significantly increased in the septic model group compared with the control group;the positive expression of NF-κB p65 was significantly reduced in the TAK242 intervention group compared with the septic model group,and there was almost no positive expression in the nucleus.Conclusion TAK242 could reduce liver function injury and protect the liver by inhibition TLR4/NF-κB pathway in septic rats.