蛋白激酶B和丝裂原活化蛋白激酶在烟酸保护的HaCaT细胞抵抗中波紫外线损伤中的作用

目的 探讨烟酸保护由UVB诱导的角质形成细胞损伤的胞内信号传导分子机制。 方法 UVB照射和烟酸处理HaCaT细胞，TUNEL法检测细胞凋亡，Western印迹检测蛋白激酶B（Akt）/丝裂原活化蛋白激酶（MAPK）通路相关蛋白Akt、P38、JNK、ERK1/2的磷酸化水平变化。ELISA检测细胞分泌内皮素1（ET-1）及碱性成纤维细胞生长因子（bFGF）的水平。结果 Western印迹结果表明，UVB照射和烟酸处理HaCaT细胞后，p-Akt、p-P38、p-JNK、p-ERK1/2蛋白在60 min内都显著激活（P < 0.01）。烟酸预处理后的HaCaT细胞再经UVB照射,可以发现p-Akt、p-P38、p-ERK1/2信号分子在2 h内激活更显著（P < 0.01）。３个抑制剂加UVB照射组较3个单独抑制剂组ET-1、bFGF表达降低，差异均有统计学意义，其中LY294002组、SB203580组ET-1、bFGF水平最低；烟酸预保护的抑制剂处理组HaCaT细胞在UVB照射后，LY294002和U0126组没有出现ET-1、bFGF水平回升，SB203580组bFGF水平出现回升。结论　Akt信号分子在烟酸保护的HaCaT细胞抵抗UVB损伤中起一定的调控作用。

Roles of protein kinase B and mitogen-activated protein kinase pathways in the protection by nicotinic acid against ultraviolet B-induced damage in keratinocytes

Objective To investigate the intracellular signal transduction pathways involved in the protective effect of nicotinic acid against ultraviolet B （UVB）-induced damage in human skin keratinocytes. Methods Cultured human keratinocyte HaCaT cells were divided into several groups to be treated with nicotinic acid, UVB irradiation, LY294002 （an inhibitor of Akt）, U0126 （an inhibitor of extracellular signal-regulated kinase （ERK）1/2）, SB203580 （an inhibitor of P38） alone or in combination for different durations. Then, Western blot was performed to quantify the phosphorylation levels of the protein kinase B （Akt）/MAPK pathway-associated proteins including Akt, P38, JNK and ERK1/2, MTT assay to evaluate the activity of HaCaT cells, enzyme-linked immunosorbent assay to determine the levels of endothelin-1 （ET-1） and basic fibroblast growth factor （bFGF） in the culture supernatant of HaCaT cells, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling （TUNEL） to evaluate the apoptosis in HaCaT cells. Results As Western blot showed, phosphorylated Akt, P38, JNK and ERK1/2 were markedly activated within 60 minutes after pretreatment with nicotinic acid and irradiation with UVB （all P < 0.01）, and the activation was more significant for phosphorylated Akt, P38, and ERK1/2 within 2 hours （all P < 0.01）. Nicotinic acid effectively suppressed the UVB-induced cell death and apoptosis in HaCaT cells. The levels of supernatant ET-1 and bFGF were significantly decreased in HaCaT cells treated with the above 3 inhibitors followed by UVB irradiation than in those treated with the inhibitors alone （all P < 0.05）, and nicotinic acid pretreatment only reversed the decrease in supernatant bFGF in HaCaT cells treated with SB203580 followed by UVB irradiation. Conclusion The Akt signaling pathway may play a regulatory role in the protection by nicotinic acid against UVB-induced damage in HaCaT cells.