川芎嗪调控NF-κB信号通路对银屑病HaCaT细胞模型趋化因子和炎症因子表达影响

Effect of ligustrazine regulating NF-κB signaling pathway on expressions of chemokines and inflammatory factors in a HaCaT cell model of psoriasis

目的:探讨川芎嗪调控NF-κB信号通路对银屑病HaCaT细胞模型趋化因子和炎症因子表达的影响。方法:HaCaT细胞分为Control、Model(10 ng/ml的TNF-α和IFN-γ处理)、TMP-L(0.1 mg/ml的川芎嗪、10 ng/ml的TNF-α和IFN-γ处理)、TMP-M(0.2 mg/ml的川芎嗪、10 ng/ml的TNF-α和IFN-γ处理)、TMP-H(0.4 mg/ml的川芎嗪、10 ng/ml的TNF-α和IFN-γ处理)、TMP-M+PMA组(1μmol/L的NF-κB信号激活剂PMA、0.2 mg/ml的川芎嗪、10 ng/ml的TNF-α和IFN-γ处理)。qRT-PCR检测Eotaxin、RANTES、TARC mRNA表达,ELISA法检测IL-6、IL-4、IL-13含量,Western blot检测p65、IκBα蛋白表达。结果:与Control组相比,Model组HaCaT细胞模型中Eotaxin、RANTES、TARC mRNA表达水平升高(1.00±0.09 vs 3.69±0.25,1.00±0.10 vs4.51±0.28,1.00±0.11 vs 2.96±0.21,P<0.05),细胞分泌的IL-6、IL-4、IL-13增多[(96.32±8.87)pg/ml vs(275.14±26.31)pg/ml,(23.61±1.47)pg/ml vs(108.62±11.96)pg/ml,(5.58±0.42)pg/ml vs(26.35±2.47)pg/ml,P<0.05],p65蛋白表达水平升高(0.23±0.02 vs 0.86±0.08,P<0.05),IκBα蛋白表达水平降低(0.96±0.09 vs 0.25±0.04,P<0.05)。与Model组相比,TMP-L、TMP-M、TMP-H组HaCaT细胞模型中Eotaxin、RANTES、TARC mRNA表达水平依次降低(3.69±0.25 vs 2.75±0.14、2.23±0.16、1.40±0.11,4.51±0.28 vs 3.27±0.36、2.23±0.17、1.80±0.12,2.96±0.21 vs 2.03±0.14、1.62±0.12、1.14±0.11,P<0.05),细胞分泌的IL-6、IL-4、IL-13减少[(275.14±26.31) pg/ml vs (204.15±13.62) pg/ml、(162.01±14.81) pg/ml、(117.62±10.30) pg/ml,(108.62±11.96) pg/ml vs(80.43±7.79)pg/ml、(53.24±4.11)pg/ml、(31.20±2.26)pg/ml,(26.35±2.47)pg/ml vs(14.20±1.22)pg/ml、(10.66±1.04)pg/ml、(6.42±0.50)pg/ml,P<0.05],细胞中p65蛋白表达水平降低(0.86±0.08 vs 0.56±0.05、0.45±0.04、0.30±0.03,P<0.05),IκBα蛋白表达水平升高(0.25±0.04 vs 0.52±0.06、0.63±0.06、0.79±0.07,P<0.05)。与TMP-M组相比,TMP-M+PMA组HaCaT细胞模型中Eotaxin、RANTES、TARC mRNA水平升高(1.00±0.08 vs 1.86±0.12,1.00±0.10 vs 2.64±0.23,1.00±0.09 vs 1.56±0.13,P<0.05),细胞分泌的IL-6、IL-4、IL-13增多[(163.50±12.84)pg/ml vs(200.61±13.60)pg/ml,(52.87±5.39)pg/ml vs(86.48±6.92)pg/ml,(9.76±0.75)pg/ml vs(16.42±1.37)pg/ml,P<0.05],p65蛋白表达水平升高(0.45±0.06 vs 0.98±0.12,P<0.05),IκBα蛋白表达水平降低(0.60±0.07 vs 0.26±0.03,P<0.05)。结论:川芎嗪通过抑制NF-κB信号通路降低银屑病HaCaT细胞模型趋化因子表达和炎症因子分泌水平。

Objective:To investigate the effect of ligustrazine-regulated NF-κB signaling pathway on expressions of chemokines and inflammatory factors in a HaCaT cell model of psoriasis.Methods:HaCaT cells were divided into Control,Model(10 ng/ml TNF-αand IFN-γ treatment),TMP-L(0.1 mg/ml ligustrazine,10 ng/ml TNF-α and IFN-γ treatment),TMP-M(0.2 mg/ml ligustrazine,10 ng/ml TNF-α and IFN-γ treatment),TMP-H(0.4 mg/ml ligustrazine,10 ng/ml TNF-α and IFN-γ treatment),TMP-M+PMA group(treated with 1 μmol/L NF-κB signal activator PMA,0.2 mg/ml ligustrazine,10 ng/ml TNF-α and IFN-γ treatment). Expressions of Eotaxin,RANTES,TARC mRNA were detected by qRT-PCR,and IL-6,IL-4,IL-13 contents were detected by ELISA,Western blot detection of p65,IκBα protein expressions.Results:Compared with Control group,expression levels of Eotaxin,RANTES and TARC mRNA in HaCaT cell model of Model group were increased(1.00±0.09 vs 3.69±0.25,1.00±0.10 vs 4.51±0.28,1.00±0.11 vs 2.96±0.21,P<0.05),IL-6,IL-4 and IL-13 secreted by cells increased[(96.32±8.87)pg/ml vs(275.14±26.31)pg/ml,(23.61±1.47)pg/ml vs(108.62±11.96)pg/ml,(5.58±0.42)pg/ml vs(26.35±2.47)pg/ml,P<0.05],p65 protein expression level increased(0.23±0.02vs 0.86±0.08,P<0.05),IκBα protein expression level decreased(0.96±0.09 vs 0.25±0.04,P<0.05). Compared with Model group,expression levels of Eotaxin,RANTES and TARC mRNA in HaCaT cell model of TMP-L,TMP-M and TMP-H groups decreased sequentially(3.69±0.25 vs 2.75±0.14,2.23±0.16,1.40±0.11;4.51±0. 28 vs 3.27±0.36,2.23±0.17,1.80±0.12;2.96±0.21 vs 2.03±0.14,1.62±0.12,1.14±0.11,P<0.05),IL-6,IL-4,IL-13 secreted by cells reduced[(275.14±26.31)pg/ml vs(204.15±13.62)pg/ml,(162.01±14.81)pg/ml,(117.62±10.30)pg/ml;(108.62±11.96)pg/ml vs(80.43±7.79)pg/ml,(53.24±4.11)pg/ml,(31.20±2.26)pg/ml;(26.35±2.47)pg/ml vs(14.20±1.22)pg/ml,(10.66±1.04)pg/ml,(6.42±0.50)pg/ml,P<0.05],expression level of p65 protein in cells decreased(0.86±0.08 vs 0.56±0.05,0.45±0.04,0.30±0.03,P<0.05),and expression level of IκBα protein increased(0.25±0.04 vs 0.52±0.06,0.63±0.06,0.79±0.07,P<0.05). Compared with TMP-M group,levels of Eotaxin,RANTES and TARC mRNA in HaCaT cell model of TMP-M+PMA group increased(1.00±0.08 vs 1.86±0.12,1.00±0.10 vs 2.64±0.23,1.00±0.09 vs 1.56±0.13,P<0.05),IL-6,IL-4 and IL-13 secreted by cells increased[(163.50±12.84)pg/ml vs(200.61±13.60)pg/ml,(52.87±5.39)pg/ml vs(86.48±6.92)pg/ml,(9.76±0.75)pg/ml vs(16.42±1.37)pg/ml,P<0.05],expression level of p65 protein increased(0.45±0.06 vs0.98±0.12,P<0.05),expression level of IκBα protein decreased(0.60±0.07 vs 0.26±0.03,P<0.05).Conclusion:Ligustrazine could reduce expressions of chemokines and secretion of inflammatory factors in HaCaT cell model of psoriasis by inhibiting NF-κB signaling pathway.