缺氧环境下HIF-1α和CXCL6在髓核细胞中的特异性调控机制

Specific regulatory mechanisms of HIF-1g and CXCL6 in nucleus pulposus cels under hypoxic conditions

目的 探讨缺氧环境下缺氧诱导因子1α (HIF-1α)和细胞因子C-X-C基序趋化因子6(CXCL6)在髓核细胞中的特异性调控机制。方法 在缺氧条件下(2%O_(2))分别培养髓核细胞0、3、6、12、24 h,分别采用Western blot法、荧光定量PCR(qPCR)法及ELISA法检测缺氧条件下髓核细胞HIF-1α和CXCL6蛋白表达、mRNA表达及细胞上清液中CXCL6水平。将髓核细胞分为A组(正常氧培养组)、B组(2%O_(2)培养组)和C组(2%O_(2)+HIF-1α抑制剂KC7F2 50μmol/L培养),24 h后采用Western blot法、qPCR法及ELISA法检测缺氧条件下髓核细胞HIF-1α和CXCL6蛋白表达、mRNA表达及细胞上清液中CXCL6水平。将髓核细胞随机分为D组(正常氧培养)、E组(2%O_(2)培养)、F组(2%O_(2)培养+siNC)、H组(2%O_(2)培养+siCXCL6#1)和L组(2%O_(2)培养+siCXCL6#2),且E、F、H、L组同时接受NF-κB抑制剂吡咯烷二硫代氨基甲酸铵25μmol/L处理,24 h后采用Western blot法检测缺氧条件下沉默CXCL6表达对C-X-C基序趋化因子受体1(CXCR1)、CXCR2、Bax和Bcl-2蛋白表达的影响,采用流式细胞术检测五组细胞凋亡情况。结果 缺氧条件下,髓核细胞HIF-1α和CXCL6蛋白表达、mRNA表达及细胞上清液中CXCL6水平呈时间依赖性升高(P<0.05)。与A组比较,B组髓核细胞HIF-1α和CXCL6蛋白表达、mRNA表达及细胞上清液中CXCL6水平均升高(P<0.05);与B组比较,C组上述指标表达均降低(P<0.05)。流式细胞术检测显示,与E、F组相比,H、L组细胞凋亡率降低(P<0.05)。结论 缺氧诱导CXCL6通过NF-κB信号通路促进髓核细胞凋亡。

Objective To explore the specific regulatory mechanisms of hypoxia inducible factor 1α(HIF-1α)and C-X-C motif chemokine ligand 6(CXCL6)in nucleus pulposus cells under hypoxic conditions.Methods The nucleus pulposus cells were cultured under hypoxic conditions(2%O_(2))for O,3,6,12 and 24 hours.Western blot,fluorescence quantitative PCR(qPCR)and ELISA were used to detect the protein and mRNA expressions of HIF-1α,CXCL6 and CXCL6 levels in cell supernatant of nucleus pulposus cells under hypoxic conditions.The nucleus pulposus cells were divided into three groups of A(cultured with normal oxygen),B(cultured with 2%O_(2))and C(cultured with 2%O_(2)+HIF-1αinhibitor KC7F250μmol/L).Western blot,qPCR and ELISA were used to detect the protein and mRNA expressions of HIF-1,CXCL6 and CXCL6 in cell supernatant of the three groups.The nucleus pulposus cells were randomly divided into five groups of D(cultured with normal oxygen culture),E(cultured with 2%O_(2)),F(cultured with 2%O_(2)+siNC),H(cultured with 2%O_(2)+siCXCL6#1)and L(cultured with 2%O_(2)+siCXCL6#2),which were treated with NF-cB inhibitor PDTC 25μmol/L.Western blot was used to detect the effects of silencing CXCL6 expression on CXCR1,CXCR2,Bax and Bcl-2 expressions under hypoxic conditions,and flow cytometry was used to detect cell apoptosis of the five groups.Results The protein and mRNA expressions of CXCL6,HIF-lαand the level of CXCL6 in cell supernatant of nucleus pulposus cells were enhanced in a time-dependent manner under hypoxic conditions(P<0.05).Compared with group A,the protein and mRNA expressions of CXCL6,HIF-1αand the level of CXCL6 in cell supernatant were increased in group B(P<0.05).Compared with group B,the expressions of the above indicators in group C were decreased(P<0.05).Flow cytometry analysis showed that the apoptosis rates in groups of H and L were decreased compared with groups of E and F(P<0.05).Conclusion Hypoxia induces CXCL6 to promote apoptosis of nucleus pulposus cells through the NF-kB signaling pathway.