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.