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