中华皮肤科杂志 ›› 2014, Vol. 47 ›› Issue (7): 481-485.

• 论著 • 上一篇    下一篇

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

杨明美1,马月红2,李锁3,4,王仕忠1,郭盛华5   

  1. 1. 南京医科大学附属常州第二人民医院
    2.
    3. 江苏省常州市第二人民医院
    4. 中国医学科学院皮肤病研究所
    5. 南京医科大学附属常州市第二人民医院皮肤科
  • 收稿日期:2013-10-11 修回日期:2013-11-16 发布日期:2014-07-01
  • 通讯作者: 郭盛华 E-mail:czeygsh@163.com

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

  • Received:2013-10-11 Revised:2013-11-16 Published:2014-07-01

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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（MTT）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 MTT试验示，10 ～ 40 μmol/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、40 μmol/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MMP1 mRNA相对表达量（12.376 ± 0.602）高于空白对照组（1.007 ± 0.147，P < 0.01），而TIMP1 mRNA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MMP1及TIMP1 mRNA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30 mJ/cm2 UVB照射后MMP1 mRNA相对表达量较UVB组降低（均P < 0.01），而TIMP1 mRNA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。

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

Abstract: Yang Mingmei *, Ma Yuehong, Li Suo, Wang Shizhong, Guo Shenghua. *Department of Dermatology, Changzhou No. 2 People′s Hospital Affiliated to Nanjing Medical University, Changzhou 213003, China Corresponding author: Guo Shenghua，Email: czeygsh@163.com 【Abstract】 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 μmol/L in the cell proliferation assay, 10, 20 and 40 μmol/L in the other assays. After additional culture for different durations, methyl thiazolyl tetrazolium （MTT） 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 MTT assay showed, punicalagin at 10 － 40 μmol/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 40 μmol/L） + UVB） compared with the UVB group （6.38% ± 0.14%, 5.24% ± 0.17% and 3.77% ± 0.11% vs. 9.82% ± 0.11%, all P < 0.01）. The expression of MMP1 mRNA was significantly higher, but that of TIMP1 mRNA was significantly lower in the UVB group than in the blank control group （both P < 0.01）, whereas no statistically significant difference was observed in the expression of MMP1 or TIMP1 mRNA between the punicalagin groups and blank control group（all P > 0.05）. The pretreatment with punicalagin significantly reduced the expression level of MMP1 mRNA （P < 0.01）, but elevated that of TIMP1 mRNA （P < 0.01） in the combination groups compared with the UVB group. As Western blot showed, the phosphorylation levels of P38, JNK and ERK were markedly increased in the UVB group （all P < 0.01）, but experienced no significant changes in the punicalagin groups （all P > 0.05） compared with the blank control group, and decreased to different degrees in the combination groups compared with the UVB group （all P < 0.01）. Conclusion Punicalagin has a pre-protective effect on UVB-induced damage to HaCaT cells.

Key words: Keratinocytes, Ultraviolet rays, Matrix metalloproteinase 1, Extracellular signal-regulated MAP kinases, Punicalagin, Matrix metalloproteinase inhibitory factor

引用本文

杨明美 马月红 李锁 王仕忠 郭盛华. 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[J]. 中华皮肤科杂志, 2014,47(7):481-485. doi: