川芎嗪对UVA诱导人皮肤成纤维细胞衰老的拮抗作用及MMP-1、MMP-3mRNA表达影响

目的探讨川芎嗪对长波紫外线（UVA）诱导人皮肤成纤维细胞（HDF）衰老的拮抗作用及对基质金属蛋白酶（MMP）-1和MMP-3mRNA表达的影响。方法酶消化法分离HDF进行原代培养，用不同浓度川芎嗪分别作用UVA多次照射前后的HDF。CCK-8法检测川芎嗪作用24、48、72h或川芎嗪预处理24h进行多次UVA照射的各组HDF体外增殖情况。光学显微镜下观察经多次UVA照射的各组细胞形态变化及β半乳糖苷酶染色情况，实时荧光定量PCR检测各组细胞内MMP-1和MMP-3mRNA相对表达量。结果浓度为20、50mg／L的川芎嗪作用HDF24、48、72h对细胞的体外增殖活性无促进或抑制作用，但100mg／L作用48h对HDF出现短暂抑制作用，与未给药组比较细胞增殖活性差异有统计学意义（P〈0．05）。20、50、100mg／L的川芎嗪预处理HDF24、48、72h对经多次UVA照射的HDF体外增殖均有一定的促进作用，各组间细胞增殖活性在3个时间点差异有统计学意义（F值分别为17．451，15．231，23．535，均P〈0．01）。多次UVA照射后HDF形态出现体积变大、颗粒增加及β半乳糖苷酶表达增加等衰老现象，接近复制性衰老的HDF（P55组）。而20、50、100mg/L的川芎嗪预处理HDF24h可减轻这种衰老现象，其中UVA组β半乳糖苷酶阳性率（68．417±1．181）％，UVA＋川芎嗪20mg／L组（58．167±5．620）％，UVA＋川芎嗪50mg／L组（45．167±5．502）％，UVA＋川芎嗪100mg／L组（43．000±2．000）％，未照射组（33．667±5．865）％，P55组（76．000±6．557）％，各组间差异有统计学意义（F=45．918，P〈0．01），且UVA＋各浓度川芎嗪组、未照射组与UVA组比较差异有统计学意义（均P〈0．05）。川芎嗪可降低多次UVA照射诱导的HDF表达MMP-1和MMP-3mRNA，且UVA＋各浓度川芎嗪组、未照射组与UVA组比较差异有统计学意义（均P〈0．05）。结论川芎嗪对多次UVA照射诱导的HDF衰老有拮抗作用，并能降低HDF衰老过程中MMP-1和MMP-3mRNA的表达。

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.