TLR4调节炎症反应和自噬影响对乙酰氨基酚肝损伤后的肝细胞再生

TLR4 affects hepatocyte regeneration after acetaminophen-induced injury by modulating inflammatory response and autophagy

目的探讨Toll受体4(TLR4)在对乙酰氨基酚(APAP)诱导的人正常肝细胞(L02)损伤后肝细胞再生中的作用以及其可能机制。方法体外培养L02细胞,利用CCK-8法检测细胞活力,筛选出APAP最佳作用浓度和作用时间、TLR4抑制剂(TAK-242)作用浓度。Western blot法检测核因子-κB(NF-κB)、微管相关蛋白轻链3(LC3)、螯合体1(p62)、受体相互作用蛋白激酶1(RIP1)、受体相互作用蛋白激酶3(RIP3)、信号转导和转录激活因子3(STAT3)、磷酸化信号转导和转录激活因子3(p-STAT3)、增殖细胞核抗原(PCNA)、细胞周期蛋白D1(Cyclin D1)蛋白表达水平。qRT-PCR法检测TLR4、NF-κB、肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-6、IL-1β、PCNA、Cyclin D1、细胞增殖核抗原(Ki67)mRNA表达水平。结果根据CCK-8结果,选择APAP 5 mmol/L作用L02细胞24、36、48 h模拟体外肝脏损伤及损伤后再生模型,TAK-242100 nmol/L于APAP给药前2 h预处理抑制TLR4。与对照组相比,APAP 24 h组的NF-κB、RIP1、p-STAT3、PCNA、Cyclin D1蛋白水平和TNF-α、IL-1β、PCNA mRNA水平升高;APAP 36 h组的NF-κB、RIP1、RIP3、p-STAT3、PCNA、Cyclin D1蛋白水平和TLR4、NF-κB、TNF-α、IL-1β、PCNA、Cyclin D1 mRNA水平升高;APAP 48 h组的NF-κB、RIP1、p-STAT3、PCNA、Cyclin D1蛋白水平和TLR4、NF-κB、TNF-α、IL-1β、IL-6、PCNA、Cyclin D1、Ki67 mRNA水平升高。相较于同时间点APAP组,APAP+TAK-24224 h组和48 h组的NF-κB、RIP1、RIP3、p-STAT3、PCNA、Cyclin D1蛋白水平和TLR4、NF-κB、TNF-α、IL-1β、IL-6、PCNA、Cyclin D1、Ki67 mRNA水平明显降低;APAP+TAK-24236 h组的NF-κB、PCNA蛋白水平和TLR4、NF-κB、TNF-α、IL-1β、IL-6、PCNA、Ki67 mRNA水平也明显低于APAP 36 h组。与对照组相比,APAP组自噬激活,而APAP+TAK-242组自噬被抑制。结论TLR4可能影响TLR4/NF-κB通路,上调炎症因子和自噬水平,促进APAP诱导的L02细胞肝损伤后的肝细胞再生。

Objective To investigate the role of Toll-like receptor 4(TLR4)in hepatocyte regeneration after acetaminophen(APAP)-induced injury in human normal liver cell(L02)and its possible mechanism.Methods L02 cells were cultured in vitro,and cell viability was detected by CCK-8 assay.The optimal concentration and duration of APAP and the concentration of TLR4 inhibitor(TAK-242)were determined.The protein expression levels of nuclear factor-κB(NF-κB),microtubule-associated protein light chain 3(LC3),p62,receptor interacting protein kinase 1(RIP1),receptor interacting protein kinase 3(RIP3),signal transducer and activator of transcription 3(STAT3),phosphorylation of STAT3(p-STAT3),proliferating cell nuclear antigen(PCNA)and Cyclin D1 were detected by Western blot.The mRNA expression levels of TLR4,NF-κB,tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),interleukin-1β(IL-1β),PCNA,Cyclin D1 and Ki67 were detected by qRT-PCR.Results According to the results of CCK-8,L02 cells were treated with 5 mmol/L APAP for 24,36,48 h to simulate liver injury and regeneration model in vitro,and TAK-242100 nmol/L was pretreated 2 h before APAP to inhibit TLR4.Compared with the control group,the protein levels of NF-κB,RIP1,p-STAT3,PCNA,Cyclin D1 and the mRNA levels of TNF-α,IL-1βand PCNA increased in the APAP 24 h group;the protein levels of NF-κB,RIP1,RIP3,p-STAT3,PCNA,Cyclin D1 and the mRNA levels of TLR4,NF-κB,TNF-α,IL-1β,PCNA and Cyclin D1 increased in the APAP 36 h group;the protein levels of NF-κB,RIP1,p-STAT3,PCNA,Cyclin D1 and the mRNA levels of TLR4,NF-κB,TNF-α,IL-1β,IL-6,PCNA,Cyclin D1 and Ki67 increased in the APAP 48 h group.The protein levels of NF-κB,RIP1,RIP3,p-STAT3,PCNA,Cyclin D1 and the mRNA levels of TLR4,NF-κB,TNF-α,IL-1β,IL-6,PCNA,Cyclin D1,Ki67 significantly decreased in APAP+TAK-24224 h and 48 h group than the APAP group at the same time point;the protein levels of NF-κB,PCNA and the mRNA levels of TLR4,NF-κB,TNF-α,IL-1β,IL-6,PCNA and Ki67 in APAP+TAK-24236 h group were also significantly lower than those in APAP 36 h group.Compared with the control group,autophagy was activated in the APAP group,while autophagy was inhibited in the APAP+TAK-242 group.Conclusion TLR4 may affect the TLR4/NF-κB pathway,up-regulate the levels of inflammatory factors and autophagy,and promote hepatocyte regeneration after APAP-induced liver injury in L02 cells.