CAJ | 학술논문

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2h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.
목적 탐토연산보호유UVB유도적각질형성세포손상적포내신호전도분자궤제.방법 UVB조사화연산처리HaCaT세포,TUNEL법검측세포조망,Western인적검측단백격매B(Akt)/사렬원활화단백격매(MAPK)통로상관단백Akt、P38、JNK、ERK1/2적린산화수평변화.ELISA검측세포분비내피소1(ET-1)급감성성섬유세포생장인자(bFGF)적수평.결과 Western인적결과표명,UVB조사화연산처리HaCaT세포후,p-Akt、p-P38、p-JNK、p-ERK1/2단백재60 min내도현저격활(P＜0.01).연산예처리후적HaCaT세포재경UVB조사,가이발현p-Akt、p-P38、p-ERK1/2신호분자재2h내격활경현저(P＜0.01).3개억제제가UVB조사조교3개단독억제제조ET-1、bFGF표체강저,차이균유통계학의의,기중LY294002조、SB203580조ET-1、bFGF수평최저;연산예보호적억제제처리조HaCaT세포재UVB조사후,LY294002화U0126조몰유출현ET-1、bFGF수평회승,SB203580조bFGF수평출현회승.결론 Akt신호분자재연산보호적HaCaT세포저항UVB손상중기일정적조공작용.
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 pathwayassociated 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.