锌指蛋白A20对LPS刺激时人退变髓核细胞衰老与凋亡的影响

Effects of zinc finger protein A20 on senescence and apoptosis of human degenerative nucleus pulposus cells stimulated by lipopolysaccharide

目的探究人退变髓核细胞中锌指蛋白A20对衰老及凋亡的影响。方法提取并培养人退变髓核细胞,小干扰RNA(siRNA)降低A20表达,脂多糖(lipopolysaccharide,LPS)作为炎症诱导因素,设置对照组、LPS组、siRNA-A20组、siRNA-A20+LPS组。通过Western blot检测A20、P21、P16、BAX、BID、BCL2、P65、p-P65蛋白表达量。细胞衰老β-半乳糖苷酶染色试剂盒检测β-半乳糖苷酶。应用AnnexinV-PI双标法及TUNEL染色检测细胞凋亡,观察锌指蛋白A20对髓核细胞衰老及凋亡的影响。应用咖啡酸苯乙酯(caffeic acid phenethyl ester,CAPE)进行挽救实验,检测A20、P21、P16、BAX、BID、BCL2、P65、P-P65蛋白表达量,探索下调锌指蛋白A20诱导细胞衰老及凋亡可能通路。结果与对照组相比,LPS组P21、P16蛋白表达量无明显变化,siRNA-A20组及siRNA-A20+LPS组P21、P16蛋白表达明显增加(P<0.05),β-半乳糖苷酶染色实验显示着色阳性细胞数显著增多(P<0.05)。Annexin V-PI双标法凋亡结果显示,单纯LPS刺激及单纯siRNA-A20处理组中细胞凋亡占比高于对照组(P<0.05),但低于siRNA-A20+LPS组(P<0.05),TUNEL染色结果与Annexin V-PI双标法结果一致(P<0.05)。凋亡相关蛋白BAX、BID在siRNA-A20+LPS中显著增加(P<0.05),BCL2则明显降低(P<0.05),P-P65蛋白表达量在siRNA-A20+LPS组明显上升(P<0.05)。与siRAN-A20+LPS组相比,应用CAPE预处理后P-P65、P21、P16、BID、BAX蛋白表达量明显下降(P<0.05),BCL2上升(P<0.05)。结论下调锌指蛋白A20,LPS过度激活NF-κB信号通路诱导人退变髓核细胞衰老与凋亡。

Objective To explore the effect of zinc finger protein A20 on senescence and apoptosis in human degenerative nucleus pulposus cells.Methods Human degenerative nucleus pulposus cells were extracted and cultured.Small interference RNA(siRNA)decreased the expression of A20.Lipopolysaccharide(LPS)was used as an inflammatory inducing factor.Control group,LPS group,siRNA-A20 group and siRNA-A20+LPS group were set up.The protein expression levels of A20,P21,P16,BAX,BID,BCL2,P65 and P-P65 were detected by Western blotting.β-galactosidase was detected by agingβ-galactosidase staining kit,and Annexin V-PI double labeling and TUNEL staining was employed to detect apoptosis and to explore the effect of zinc finger protein A20 on senescence and apoptosis of nucleus pulposus cells.Caffeic acid phenethyl ester(CAPE)was used in rescue experiment,and the protein expression levels of A20,P21,P16,BAX,BID,BCL2,P65 and P-P65 were detected to explore the possible pathway of cell senescence and apoptosis induced by down-regulation of zinc finger protein A20.Results Compared with the control group,there was no significant change in protein levels of P21 and P16 in LPS group,but P21 and P16 increased significantly in siRNA-A20 group and siRNA-A20+LPS group(P<0.05).β-galactosidase staining test showed that the number of positive cells increased significantly in siRNA-A20 treated groups(P<0.05).The proportion of apoptosis in LPS group and siRNA-A20 group was higher than that of control group(P<0.05),but lower than that of siRNA-A20+LPS group(P<0.05).The result of TUNEL staining was consistent with that of AnnexinV-PI double labeling(P<0.05).Apoptosis related protein BID and BAX increased(P<0.05),and BCL2 decreased in siRNA-A20+LPS group(P<0.05),while P-P65 increased significantly in siRNA-A20+LPS group(P<0.05).Compared with siRNA-A20+LPS group,through the pretreatment of CAPE,protein levels of P-P65,P21,P16,BID and BAX decreased(P<0.05),and the protein levels of BCL2 increased(P<0.05).Conclusion Through down-regulation of zinc finger protein A20,LPS induces senescence and apoptosis of human degenerative nucleus pulposus cells through NF-κB signal pathway.