糖原合酶激酶3β抑制剂TDZD-8对急性肝细胞损伤中自噬及NLRP3炎性小体的影响

Effects of Glycogen Synthase Kinase 3β Inhibitor TDZD-8 on NLRP3 Inflammasome and Autophagy in Acute Hepatocyte Injury

目的探讨糖原合酶激酶3β(glycogen synthase kinase 3β,GSK3β)抑制剂TDZD-8在急性肝细胞损伤衰竭模型中对细胞自噬及NOD样受体热蛋白结构域相关蛋白3(NOD-like receptor family pyrin domain-containing 3,NLRP3)炎性小体的影响。方法体外培养人正常肝细胞L02细胞,CCK-8法测定不同浓度TDZD-8对L02细胞活性的影响;将细胞分为3组(对照组、模型组、TDZD-8组),经处理后,试剂盒检测细胞上清液中游离DNA(cell-free DNA,cf-DNA)及乳酸脱氢酶(lactate dehydrogenase,LDH)含量;免疫荧光法检测3组细胞第9位丝氨酸磷酸化的GSK3β(p-GSK3β,Ser9)表达含量;Western blot法检测3组细胞中NLRP3、白细胞介素-18(interleukin-18,IL-18)、IL-1β、UNC-51样激酶1(UNC-51-like kinase 1,ULK-1)、Beclin1及p62蛋白水平变化。结果CCK-8结果显示,在TDZD-8浓度≤20μmol/L时药物对细胞活性无明显影响。3组细胞经处理后,与对照组比较,模型组p-GSK3β(Ser9)荧光强度明显减弱,GSK3β被激活,同时上清液中cf-DNA和LDH水平明显升高,NLRP3、IL-18和IL-1β蛋白水平也明显升高,而自噬相关蛋白ULK1和Beclin1蛋白水平明显下降,p62水平升高,自噬水平受到抑制(P<0.05)。经TDZD-8处理后,与模型组比较,细胞p-GSK3β(Ser9)荧光强度明显增强,上清液中cf-DNA和LDH水平显著降低,NLRP3、IL-18和IL-1β蛋白水平也明显降低,而自噬水平显著升高(P<0.05)。结论TDZD-8在急性肝细胞损伤衰竭的模型中能够抑制NLRP3炎性小体活化同时激活细胞自噬起到保护作用。

Objective To investigate the effect of glycogen synthase kinase 3β(GSK3β)inhibitor TDZD-8 on autophagy and NL-RP3 inflammasome in a model of acute hepatocyte injury and failure.Methods Human normal hepatocyte L02 cells were cultured in vitro,and the effect of different concentrations of TDZD-8 on the activity of L02 cells was determined by CCK-8method.The cells were divided into three groups(control group,model group and TDZD-8group),the kits were used to detect the content of cell-free DNA(cf-DNA)and lactate dehydrogenase(LDH)in the cell supernatant;the expression of GSK3β(p-GSK3β,Ser9)phosphorylated at the ninth serine in the three groups of cells was detected by immunofluorescence;Western blot was applied to detect NLRP3,interleukin-18(IL-18),IL-1β,UNC-51-like kinase 1(ULK-1),Beclin1 and p62 protein levels in the three groups of cells.Results The results of CCK-8 showed that the drug had no significant effect on cell viability when the concentration of TDZD-8 was less than or equal to 20μmol/L.After the three groups of cells were treated,compared with the control group,the fluorescence intensity of p-GSK3β(Ser9)in the model group was significantly weakened,GSK3βwas activated,and the levels of cf-DNA and LDH in the supernatant were significantly increased,the protein levels of NLRP3,IL-18 and IL-1βwere also significantly increased,while the protein levels of autophagy-related proteins ULK1 and Beclin1 were significantly decreased,the level of p62 was increased,and the level of autophagy was inhibited(P<0.05).After treatment with TDZD-8,compared with the model group,the fluorescence intensity of p-GSK3β(Ser9)in the cells was significantly enhanced,the levels of cf-DNA and LDH in the supernatant were significantly decreased,and the protein levels of NLRP3,IL-18 and IL-1βwere also significantly reduced,while the level of autophagy was significantly increased(P<0.05).Conclusion TDZD-8 can inhibit the activation of NLRP3 inflammasome and activate autophagy to play a protective role in the model of acute hepatocyte injury and failure.