摘要
目的探讨白藜芦醇对中波紫外线(UVB)照射后人原代角质形成细胞(HEK)凋亡和细胞周期的影响。方法0、25、50、100、150和200μmol/L白藜芦醇处理人原代角质形成细胞12 h,分别采用CCK-8、BrdU和LDH法检测白藜芦醇对细胞增殖活性和死亡的影响。分别采用50 mJ/cm^(2)UVB和100μmol/L白藜芦醇处理HEK细胞,分为正常对照组、白藜芦醇组、UVB组和UVB+白藜芦醇组,采用Western印迹检测凋亡相关蛋白多腺苷二磷酸核糖聚合酶(PARP)和胱天蛋白酶3(caspase-3)以及细胞周期相关蛋白(cyclin)B1和cyclin E1的表达水平,采用流式细胞仪检测细胞凋亡水平和细胞周期分布。多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验。结果0、25、50、100、150和200μmol/L白藜芦醇组HEK细胞增殖活性差异有统计学意义(F=46.52,P<0.001),不同浓度组均低于对照组(均P<0.001);100μmol/L白藜芦醇组细胞DNA合成活力(0.761±0.141)低于对照组(2.226±0.141,t=17.92,P<0.001);25、50、100和200μmol/L白藜芦醇组细胞死亡率差异无统计学意义(F=1.938,P>0.05)。UVB和白藜芦醇处理后,4组细胞凋亡率和G1期、G2期、S期的细胞比例以及PARP、caspase-3、cyclin B1、cyclin E1的表达水平差异均有统计学意义(均P<0.05)。UVB组细胞凋亡率(24.69%±3.54%)及PARP、caspase-3的剪切水平(2.190±0.349、0.090±0.016)均高于对照组和UVB+白藜芦醇组(4.00%±0.81%、0.926±0.040、0.024±0.019,均P<0.05);UVB组cyclin E1、cyclin B1蛋白水平(0.116±0.017、0.775±0.080)均低于正常对照组,UVB+白藜芦醇组cyclin E1表达水平(0.287±0.047)高于UVB组、cyclin B1表达水平(0.504±0.009)低于UVB组(均P<0.05);UVB组S期细胞比例低于对照组和UVB+白藜芦醇组,G1期、G2期细胞比例均高于UVB+白藜芦醇组(P<0.05)。结论白藜芦醇可以抑制HEK增殖活力,抑制紫外线照射诱导的细胞凋亡,调控细胞周期进程。
Objective To evaluate the effect of resveratrol on apoptosis and cell cycle of human epidermal keratinocytes(HEKs)after ultraviolet B(UVB)irradiation.Methods After 12-hour treatment with 0(control group),25,50,100,150 and 200μmol/L resveratrol,cell counting kit-8(CCK8)assay,bromodeoxyuridine(BrdU)assay and lactate dehydrogenase(LDH)assay were performed to evaluate the effect of resveratrol on proliferative activity and death of HEKs.Some HEKs were divided into 4 groups:normal control group receiving no treatment,resveratrol group treated with 100μmol/L resveratrol,UVB group irradiated with 50 mJ/cm^(2)UVB,and UVB+resveratrol group irradiated with 50 mJ/cm^(2)UVB followed by 12-hour treatment with 100μmol/L resveratrol.Western blot analysis was conducted to determine the expression of apoptosis-related proteins poly(ADP-ribose)polymerase(PARP)and caspase-3,as well as cell cycle-related proteins cyclin B1 and cyclin E1,and flow cytometry to detect the apoptosis and determine cell cycle distribution.One-way analysis of variance was used for comparisons among multiple groups,and least significant difference-t test for multiple comparisons.Results The proliferative activity of HEKs significantly differed among the 25-,50-,100-,150-,200-μmol/L resveratrol groups and control group(F=46.52,P<0.001),and was significantly lower in these resveratrol groups than in the control group(all P<0.001);the DNA synthesis activity of HEKs was significantly lower in the 100-μmol/L resveratrol group(0.761±0.141)than in the control group(2.226±0.141,t=17.92,P<0.001);there was no significant difference in cell death rate among the 25-,50-,100-and 200-μmol/L resveratrol groups(F=1.938,P>0.05).After treatment with or without UVB and/or resveratrol,there were significant differences in the apoptosis rate,proportion of cells at G1,G2 and S phases,expression of PARP,caspase-3,cyclin B1 and cyclin E1 among the 4 groups(all P<0.05).The UVB group showed significantly increased apoptosis rates(24.69%±3.54%)and cleavage levels of PARP and
作者
陈虹颖
陈旭
李莉
顾恒
Chen Hongying;Chen Xu;Li Li;Gu Heng(Department of Physical Therapy,Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs,Institute of Dermatology,Chinese Academy of Medical Sciences and Peking Union Medical College,Nanjing 210042,China)
出处
《中华皮肤科杂志》
CAS
CSCD
北大核心
2021年第5期408-413,共6页
Chinese Journal of Dermatology
基金
国家自然科学基金(81703153)
中国医学科学院医学与健康科技创新工程项目(2017-I2M-1-017)
南京市国家级临床医学中心培育计划项目(2019060001)。
