匹诺塞林缓解大鼠肝细胞系BRL-3A低氧/复氧损伤

摘要

目的 探讨匹诺塞林(PIN)对低氧/复氧(H/R)诱导的大鼠肝细胞损伤的保护作用及其可能机制.方法 将大鼠肝细胞(BRL-3A细胞系)分为正常对照组、PIN实验组、低氧复氧损伤模型组和PIN预处理组.CCK-8检测细胞存活率;Annexin V-FITC/PI双染法流式细胞计量术检测细胞凋亡;检测细胞培养液中谷丙转氨酶(ALT)活性;ELISA检测TNF-α和IL-1β含量;Western blot检测细胞中TLR4、IκB-α和NF-κB P65蛋白水平;RT-qPCR检测细胞中TLR4、IκB-α和NF-κB P65mRNA表达.结果 在H/R条件下细胞存活率明显降低(P<0.01),细胞凋亡率增高(P<0.001),ALT活性升高(P<0.01),IL-1β和TNF-α含量增多(P<0.01),TLR4和NF-κB P65蛋白与mRNA表达水平显著提高(P<0.01)而IκB-α降低(P<0.05);经PIN预处理后,细胞存活率显著提高(P<0.01),细胞凋亡率显著减小(P<0.001),ALT活性降低(P<0.01),IL-1β和TNF-α含量降低(P<0.01),TLR4和NF-κB P65蛋白与mRNA表达水平明显降低(P<0.01)而IκB-α升高(P<0.05).结论 PIN对H/R诱导的BRL-3A肝细胞的损伤具有保护作用,且该作用可能是通过TLR4/NF-κB信号通路实现的.%Objcetive To investigate protective effect of pinocembrin ( PIN)on hepatocytes induced by hypoxia/reoxygenation ( H/R) as well as its relationship to the TLR 4/NF-κB signaling pathway .Methods The cells were randomly divided into 4 groups: control group, PIN group, hypoxia/reoxygenation injury group and PIN pretreatment group .The cell viability was measured with CCK-8.The apoptosis rate was determined by Annexin V-FITC/PI staining.The activity of ALT was detected .ELISA was used to evaluate the contents of TNF-αand IL-β.The mRNA and protein expression level of TLR 4, IκB-αand NF-κB P65 in cells was observed by quantita-tive real-time PCR or Western blot .Results The H/R stimulation decreased cell survival rate and enhanced the apoptosis .The activity of ALT was increased .The contents of TNF-αand IL-1βwere significantly enhanced , and the expression level of TLR4 and NF-κB P65 was markedly increased while IκB-αdecreased.After pretreatment with PIN, the cell survival rate increased while the apoptosis rate decreased .The activity of ALT was decreased. TNF-αand IL-1βwere reduced significantly and the expression level of TLR 4 and NF-κB P65 were decreased while IκB-αincreased.Conclusions PIN has protective effects on hypoxia/reoxygenation injury, which might be mediated in part by TLR4/NF-κB signaling pathway .