安石榴苷对中波紫外线诱导HaCaT细胞光损伤的预防作用研究  

Preventive effects of punicalagin against ultraviolet B-induced damage to human HaCaT keratinocytes

作　　者：杨明美 马月红[1] 李锁[1] 王仕忠[2] 郭盛华[1] 

机构地区：[1]南京医科大学附属常州第二人民医院皮肤科,江苏常州213003 [2]南京医科大学附属常州第二人民医院中心实验室,江苏常州213003

出　　处：《中华皮肤科杂志》2014年第7期481-485,共5页Chinese Journal of Dermatology

摘　　要：目的探讨安石榴苷对中波紫外线（UVB）诱导角质形成细胞损伤的保护机制。方法培养的HaCaT细胞分为空白对照组、安石榴苷组、UVB组、安石榴苷＋UVB组。噻唑蓝（MTY）法检测细胞增殖能力，Hoechst／碘化丙锭（PI）染色和流式细胞仪检测细胞凋亡，RT—PCR法测定金属基质蛋白酶1（MMP1）及其组织抑制因子1（TIMP1）mRNA表达水平，Western印迹检测丝裂原活化蛋白激酶（MAPK）通路相关蛋白P38、JNK、ERK的磷酸化水平变化。结果MTr试验示，10-40μmol／L安石榴苷对UVB诱导的HaCaT细胞损伤有较佳的预保护作用。UVB组HaCaT细胞强Hoechst和强PI双染细胞较空白对照组增多，安石榴苷＋UVB组较UVB组减少。流式细胞仪分析，UVB组凋亡细胞百分率（9．82％±0．11％）高于空白对照（1．24％±0．91％，P〈0．01），而安石榴苷（10、20、40μmol／L）＋UVB组凋亡细胞百分率（分别为6．38％±0．14％、5．24％±0．17％、3．77％±0．11％）较UVB组低，差异有统计学意义（均P〈0．01）。UVB组MMPlmRNA相对表达量（12．376±0．602）高于空白对照组（1．007±0．147，P〈0．01），而TIMPlmRNA相对表达量（0．103±0．006）低于空白对照组（1．006±0．139，P〈0．01），安石榴苷组MMPl及TIMPlmRNA与空白对照组比较，差异无统计学意义（均P〉0．05）。安石榴苷预处理的HaCaT细胞经30mJ／cmzUVB照射后MMPlmRNA相对表达量较UVB组降低（均P〈0．01），而TIMPlmRNA较UVB组升高（均P〈0．01）。Western印迹示，经UVB照射后，HaCaT细胞p-ERK、p-JNK及p-p38表达升高（均P〈0．01）。安石榴苷组HaCaT细胞p-ERK、P—JNK及p-p38表达没有明显改变（P〉0．05），而安石榴苷＋UVB组有不同程度下降（均P〈0．01）。结论安石榴苷对UVB引起HaCaT细胞损伤有一定的预防作用。Objective To investigate the mechanisms underlying the protection by punicalagin against ultraviolet B （UVB）-induced damage to keratinocytes. Methods Cultured human HaCaT keratinocytes were divided into several groups： blank control group receiving no treatment, punicalagin groups treated with various concentrations of punicalagin, UVB group irradiated with UVB at 30 mJ/cm2, combination groups pretreated with different concentrations of punicalagin followed by UVB radiation at 30 mJ/cm2. The concentrations of punicalagin were 5, 10, 20, 40 and 80 p, mol/L in the cell proliferation assay, 10, 20 and 40 p, mol/L in the other assays. After additional culture for different durations, methyl thiazolyl tetrazolium （MTI＇） assay was performed to evaluate the proliferation of HaCaT cells, Hoechst and propidium iodide （PI） staining as well as flow cytometry to detect the apoptosis in cells, reverse transcription-PCR to quantify the mRNA expressions of matrix metalloproteinase-1 （MMP1） and tissue inhibitor of metalloproteinase-1 （TIMP1） in HaCaT cells, Western blot to determine the phosphorylation levels of the mitogen-activated protein kinase （MAPK） pathway-related proteins including P38, JNK and ERK. Statistical analysis was carried out by t test, one-way analysis of variance, and Dunnett＇s t-test. Results As the MTY assay showed, punicalagin at 10 - 40 txmol/L showed stronger pre-protective effects against UVB- induced damage to HaCaT cells compared with punicalagin at the other concentrations. The number of cells highly positive for both Hoechst and PI staining was larger in the UVB group than that in the blank control group, but smaller in the combination groups than in the UVB group. The percentage of apoptotic cells increased significantly in the UVB group compared with the blank control group （9.82% ± 0.11% vs. 1.24% ± 0.91%, P 〈 0.01）, but decreased significantly in the three combination groups （punicalagin （10, 20 and 40μmol/L） ＋ UVB） compared with the UVB group （

关 键 词：角蛋白细胞 紫外线 基质金属蛋白酶1 细胞外信号调节MAP激酶类 安石榴苷 基质金 属蛋白酶抑制因子 

分 类 号：R285[医药卫生—中药学]