中波紫外线对角质形成细胞NF-κB p65及其抑制因子IκBα表达的影响

Effects of ultraviolet B on the expressions of nuclear factor-κB p65 and its inhibitory factor IκBα in keratinocytes

目的 探讨不同剂量中波紫外线（UVB）照射对体外培养的人皮肤角质形成细胞NF-κB p65及其抑制因子IκBα表达的调控效应.方法 4.5、10及50 mJ/cm2UVB照射人角质形成细胞系及人原代角质形成细胞,设置未经照射的对照组,在照射后4h使用Western印迹法测定细胞总蛋白中IκBα及NF-κB p65的表达水平.并对人原代角质形成细胞进行50 mJ/cm2UVB照射,照射后继续培养2、4、8、12h,分别测定4个时间点IκBα及NF-κB p65的表达水平.蛋白条带密度分析使用Quantity One（R）4.6.8软件,分别计算IKKβ、IκBα、NF-κB p65以及磷酸化IKKα/β、IκBα、NF-κB p65和β肌动蛋白条带吸光度和面积的乘积,再分别计算IKKβ、IκBα和NF-κB p65与β肌动蛋白的比值,取3次实验结果.结果 4.5和10 mJ/cm2 UVB照射后4h,相对于对照组细胞,人角质形成细胞系及人原代角质形成细胞总蛋白中IKKβ、IκBα、NF-κB p65表达水平均无明显变化（P＞ 0.05）;50 mJ/cm2 UVB照射后4h,细胞系及原代角质形成细胞中IKKβ表达水平均无明显变化（P＞0.05）;原代角质形成细胞IκBα表达水平（0.173±0.055）较对照组细胞（0.462±0.142）显著降低（t=5.64,P＜ 0.05）,NF-κB p65表达水平（0.076±0.030）也较对照组细胞（0.120±0.034）降低（t=5.40,P＜0.05）;角质形成细胞IκBα表达水平（0.160±0.046）较对照组细胞（0.398±0.136）轻度下调（t=4.37,P＜0.05）,NF-κB p65的表达水平无明显变化（P＞0.05）.50 mJ/cm2 UVB照射原代角质形成细胞后2h,IκBα表达水平（0.140±0.034）相对于对照组细胞（0.208±0.031）开始下调（t=17.55,P＜ 0.05）,并随照射后时间延长下调效应更为显著;NF-κB p65的表达水平在2h和4h时较对照细胞无明显变化（P＞0.05）,8h时（1.162±0.345）较对照细胞（1.235±0.349）表达水平下调（t=36.67,P＜0.05）,12 h时（1.061±0.246）较对照细胞（1.390±0.226）仍下调（t=8.71,P＜0.05）,随着时间的推进下调效应无明显改变;磷酸化IKKα/β（ser176/180）、IKKβ、磷酸化IκBα（ser32）、磷酸化NF-κB p65（ser536）表达量均无明显改变（P＞0.05）.结论 高剂量（50 mJ/cm2）UVB照射可显著抑制人原代角质形成细胞IκBα表达,同时轻度抑制NF-κB p65蛋白的表达,提示可能存在UVB照射人原代角质形成细胞引起NF-κB p65活化的另一种通路.

Objective To investigate the regulatory effects of different doses of ultraviolet B （UVB） radiation on the expressions of nuclear factor （NF）-κB p65 and its inhibitory factor IκBα in human keratinocytes cultured in vitro.Methods A human keratinocyte cell line CRL-2310 and primary human keratinocytes were used in this study.Both CRL-2310 and primary human keratinocytes were irradiated with 4.5,10 and 50 mJ/cm2 UVB,with unirradiated cells serving as the control.Western blot was performed to determine the expression levels of total IκBα,NF-κB p65 and IKKβ1 proteins in the cells at 4 hours after the irradiation.Some primary human keratinocytes were irradiated with 50 mJ/cm2 UVB,and Western blot was conducted to measure the expression levels of total and phosphorylated IκBα,NF-κB p65 and IKKβ1 proteins in these cells at 2,4,8,and 12 hours after the irradiation respectively.The optical density of protein bands was analyzed by the Quantity One software （version 4.6.8）,the product of the optical density and area of each band was calculated for IKKβ,IκBα,NF-κB p65,β-actin as well as phosphorylated IKKβ,IκBα,and NF-κB p65 separately,and the expression levels of target proteins were expressed as the ratio of the product for the target proteins to that for β-actin.All experiments were repeated for three times,and the average expression levels were calculated for these proteins.Results No significant changes were observed in the CRL-2310 keratinocytes or the primary keratinocytes for the protein expression levels of total IKKβ,IκBα and NF-κB p65 at 4 hours after UVB radiation at 4.5 and 10 mJ/cm2,or for those of total IKKβ at 4 hours after UVB radiation at 50 mJ/cm2 compared with the unirradiated controls （all P 〉 0.05）.However,there was a statistical decrease in the expression levels of IκBα and NF-κB p65 in primary keratinocytes （0.173 ± 0.055 vs.0.462 ± 0.142,t =5.64,P 〈 0.05; 0.076 ± 0.030 vs.0.120 ± 0.034,t =5.40,P 〈 0.05）,as well as a slight reduction in the expression level of IκBα （0.160 ± 0.046 vs.0.398 ± 0.136,t =4.37,P 〈 0.05） with no statistical changes in the expression level of NF-κB p65 （P 〉 0.05） in CRL-2310 keratinocytes,at 4 hours after 50 mJ/cm2 UVB radiation when compared with the unirradiated controls.After irradiation with 50 mJ/cm2 UVB,the expression level of IκBα in primary keratinocytes began to decrease at 2 hours compared with the unirradiated keratinocytes （0.140 ± 0.034 vs.0.208 ± 0.031,t =17.55,P 〈 0.05）,and the degree of decrease increased over time; the expression level of NF-κB p65 showed no changes at 2 or 4 hours （both P 〉 0.05）,but a significant decrease at 8 and 12 hours （1.162 ± 0.345 vs.1.235 ± 0.349,t =36.67,P 〈 0.05; 1.061 ± 0.246 vs.1.390 ± 0.226,t =28.71,P 〈 0.05）,but remained unchanged afterwards.No significant differences were noted in the protein expression levels of IKKβ,phosphorylated IKKα/β （ser176/180）,phosphorylated IκBα （ser32） or phosphorylated NF-κB p65 （ser536） between the irradiated and unirradiated primary keratinocytes at the 4 time points （all P 〉 0.05）.Conclusions High dose of UVB radiation （50 mJ/cm2） can significantly suppress the protein expression of IκBα,but slightly decrease the protein expression of NF-κB p65 in human primary keratinocytes,suggesting that UVB radiation induces the activation of NF-κB p65 through another potential pathway.