Inhibitory effect of tetramethylpyrazine on ultraviolet A-induced senescence and matrix metalloproteinase-1 and-3 mRNA expressions in human dermal fibroblasts / 中华皮肤科杂志

ABSTRACT

Objective To explore the inhibitory effect of tetramethylpyrazine (TMP) on ultraviolet A-induced senescence as well as matrix metalloproteinase-1 (MMP-1) and-3 (MMP-3) mRNA expressions in human dermal fibroblasts (HDFs).Methods HDFs were isolated from the prepuce by enzymatic digestion, and subjected to primary culture.Cultured HDFs were randomly divided into several groups control group cultured in high-glucose DMEM medium and receiving no treatment, three TMP groups treated with 20, 50 and 100 mg/L TMP respectively, UVA group receiving UVA radiation alone, UVA + TMP groups pretreated with 20, 50 and 100 mg/L TMP respectively for different durations followed by UVA radiation.UVA radiation was given once daily for 5 consecutive days.The 55th passage HDFs served as the P55 group (senescence control group).Subsequently, CCK-8 assay was performed to evaluate the proliferative activity of HDFs in vitro, optical microscopy to observe the morphologic changes of HDFs after UVA radiation, β-galactosidase staining to estimate the senescence in HDFs, and real-time fluorescence-based quantitative PCR to quantify the mRNA expressions of MMP-1 and MMP-3 in HDFs.Statistical analysis was carried out by one-way analysis of variance (ANOVA) followed by least significant difference (LSD)-t test or Dunnett's T3 test.Results Compared with the control group, the proliferation of HDFs was significantly but transiently inhibited in vitro after the treatment with 100 mg/L TMP for 48 hours (P ＜ 0.05), but showed no significant changes after the treatment with 20 or 50 mg/L TMP for 24, 48 or 72 hours or after the treatment with 100 mg/L TMP for 24 or 72 hours (all P ＜ 0.05).The pretreatments with TMP of 20, 50 and 100 mg/L for 24, 48 and 72 hours all promoted the proliferation of HDFs to a certain degree in the UVA + TMP groups compared with the UVA group, with significant differences in cellular proliferative activity among the UVA group, UVA + TMP groups and control group at 24, 48 and 72 hours (F =17.451,15.231, 23.535, all P ＜ 0.01).Compared with the UVA group, the proliferative activity of HDFs was significantly increased in UVA + 100-mg/L TMP group at 24, 48, 72 hours, UVA + 50-mg/L TMP group at 24 and 72 hours and UVA + 20-mg/L TMP group at 72 hours.After repetitive UVA radiation, HDFs in the UVA group experienced an increase in cell volume, granule acount, and β-galactosidase expression, which was similar to the changes in the P55 group, while the pretreatments with 20, 50 and 100 mg/L TMP for 24 hours suppressed these UVA-induced changes in HDFs.The percentage of β-galactosidase-positive HDFs was 68.417％ ± 1.181％ in the UVA group, 58.167％ ± 5.620％ in the UVA + 20-mg/L TMP group, 45.167％ ± 5.502％ in the UVA + 50-mg/L TMP group, 43.000％ ± 2.000％ in the UVA + 100-mg/L TMP group, 33.667％ ± 5.865％ in the control group, and 76.000％ ± 6.557％ in the P55 group, with significant differences among these groups (F =45.918, P ＜ 0.01).Furthermore, the UVA group significantly differed from the UVA + TMP groups and control group in the percentage of β-galactosidase-positive HDFs and mRNA expressions of MMP-1 and MMP-3 (all P ＜ 0.05).Conclusion TMP can protect HDFs against senescence induced by repetitive UVA radiation, and down-regulate the mRNA expressions of MMP-1 and MMP-3 during senescence.