Abstract:

Zhao Minling, Liu Zhongrong, Chen Hulin, Zhu Yingjie, Yan Miaomiao, Fan Xiuzhen. Department of Dermatology, Guangzhou General Hospital of Guangzhou Military Command of PLA, Guangzhou 510010, China Corresponding author: Liu Zhongrong, Email: pfklzr@163.com 【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.