糖原合酶激酶3β抑制剂TDZD-8对急性肝衰竭小鼠肝组织炎症的保护作用研究

Protective effect of glycogen synthase kinase 3βinhibitor TDZD-8 on liver injuries in mice with D-Gal/LPS-induced acute liver failure

目的 探索糖原合酶激酶3β(GSK3β)抑制剂TDZD-8对急性肝衰竭(ALF)小鼠的保护作用。方法 将24只小鼠随机分为对照组、模型组和TDZD-8处理组,给予D-Gal和LPS腹腔注射,制备ALF模型,分别给予生理盐水或TDZD-8干预。采用ELISA法检测肝组织匀浆肿瘤坏死因子-α(TNF-α)和白介素-1β(IL-1β)水平,采用蛋白印迹法检测肝组织IL-18、IL-1β、UNC-51样激酶1(ULK1)、Beclin 1和P62蛋白表达。结果 模型组血清ALT、AST和TBIL水平分别为(3460.8±105.2)U/L、(2710.4±84.3)U/L和(93.8±1.1)μmol/L,显著高于对照组【分别为(28.1±4.2)U/L、(25.78±2.5)U/L和(3.4±0.4)μmol/L,P<0.05】,而TDZD-8处理组各指标均显著降低【分别为(370.1±7.1)U/L、(277.3±20.3)U/L和(35.1±0.8)μmol/L,P<0.05】;模型组肝组织匀浆TNF-α、IL-1β和血清无细胞游离(cf)-DNA水平分别为(3004.6±239.2)pg/mL、(469.6±56.7)pg/mL和(772.2±192.2)ng/mL,经TDZD-8处理后,各指标均显著降低【分别为(2353.3±284.7)pg/mL、(339.2±48.7)pg/mL和(180.0±15.4)ng/mL,P<0.05】;模型组肝组织IL-18和IL-1β相对表达量较对照组显著增强(P<0.05),而TDZD-8处理组小鼠蛋白相对表达量较模型组显著减弱(P<0.05);模型组肝组织ULK1和Beclin1相对表达量均显著低于对照组(P<0.05),而P62蛋白表达显著高于对照组(P<0.05),经TDZD-8处理,肝组织ULK1和Beclin1相对表达量显著高于模型组,而P62蛋白表现显著降低(P<0.05)。结论 在急性肝衰竭小鼠,抑制GSK3β活性能够上调自噬水平,改善肝组织炎症因子的释放,从而起到保护肝脏作用。

Objective The aim of this study was to investigate the protective effect of glycogen synthase kinase 3β(GSK3β)inhibitor TDZD-8 on liver injuries in mice with D-Gal/LPS-induced acute liver failure(ALF).Methods Twenty-four male mice were randomly divided into control,model and TDZD-8-intervened group,with eight in each.The model of ALF was established by intraperitoneal injection of D-Gal/LPS.Serum free DNA(cf-DNA)was detected by fluorescent dye,and liver homogenate tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)were assayed by ELISA.The hepatic expression of L-18,IL-1β,UNC-51-like kinase 1(ULK1),Beclin 1 and P62 were detected by Western blotting.Results Serum ALT,AST and total bilirubin levels in model group were(3460.8±105.2)U/L,(2710.4±84.3)U/L and(93.8±1.1)μmol/L,all significantly higher than[(28.1±4.2)U/L,(25.78±2.5)U/L and(3.4±0.4)μmol/L,respectively,P<0.05]in the control,while they all greatly decreased in TDZD-8-intervened group[(370.1±7.1)U/L,(277.3±20.3)U/L and(35.1±0.8)μmol/L,respectively,P<0.05];the liver homogenate TNF-α,IL-1βand serum cf-DNA levels in the model were(3004.6±239.2)pg/mL,(469.6±56.7)pg/mL and(772.2±192.2)ng/mL,while they all greatly decreased in TDZD-8-intervened group[(2353.3±284.7)pg/mL,(339.2±48.7)pg/mL and(180.0±15.4)ng/mL,respectively,P<0.05];the hepatic expression of IL-18 and IL-1βin the model intensified greatly as compared to in the control(P<0.05),while they both became weaker in TDZD-8-intervened group(P<0.05);the relative hepatic expression of ULK1 and Beclin1 obviously decreased(P<0.05),and the hepatic expression of P62 protein increased greatly(P<0.05)compared to in the control,while the ULK1 and Beclin1 expression greatly intensified,and the P62 expression became weaker(P<0.05)in TDZD-8-intervened mice.Conclusion The inhibition of GSK3βactivity in a mouse model of acute liver failure could protect the liver injuries,which might be related to the up-regulation of autophagy and the inhibition of inflammatory cytokine release.