芒果苷抑制中波紫外线诱导人成纤维细胞提前衰老的相关研究

【摘要】 目的 研究芒果苷是否对反复亚毒性剂量中波紫外线（UVB）诱导的人二倍体成纤维细胞的早衰起抑制作用及其可能的机制。 方法 实验分成空白对照组（不做处理），UVB-应激诱导的提前衰老（SIPS）组（单纯照光），芒果苷4.0 mg/L组（单纯加药），UVB + 芒果苷1.0 mg/L组、UVB + 芒果苷2.0 mg/L组、UVB + 芒果苷4.0 mg/L组（UVB照射联合药物处理）。成纤维细胞经5次UVB 10 mJ/cm2照射后，用细胞计数法（CCK8法）检测细胞增殖活性，β半乳糖苷酶染色（SA-β-Ga1）计算衰老细胞百分比，流式细胞仪测定细胞周期，蛋白印迹检测衰老相关蛋白p53、p21、p16含量，实时荧光定量PCR（RT-PCR）检测基质金属蛋白酶1（MMP1）、基质金属蛋白酶3（MMP3）、Ⅰ型胶原、Ⅲ型胶原mRNA和衰老相关基因p53、p21、p16 mRNA表达。采用单因素方差分析进行数据统计分析。 结果 UVB + 芒果苷1.0 mg/L组、UVB + 芒果苷2.0 mg/L组、UVB + 芒果苷4.0 mg/L组以及UVB-SIPS组细胞增殖活性（A450）依次为0.322 9 ± 0.011 3、0.336 1 ± 0.016 3、0.342 6 ± 0.014 4、0.288 2 ± 0.020 7（F = 110.08，P < 0.05），β半乳糖苷酶染色阳性率依次为（88.83 ± 4.54）%、（46.33 ± 5.51）%、（32.17 ± 6.05）%、（93.67 ± 3.75）%（F = 283.54，P < 0.05；与UVB-SIPS组比较，除UVB + 芒果苷1.0 mg/L组外，其他两组均P < 0.05）；G1期细胞阻滞率依次为（72.19 ± 3.42）%、（60.99 ± 2.70）%、（49.80 ± 2.10）%、（82.09 ± 0.89）%（F = 156.01，P < 0.05）。与UVB-SIPS组相比，UVB + 各浓度芒果苷组细胞MMP1和MMP3 mRNA表达降低（F = 69.41、106.41，均P < 0.05），Ⅰ型胶原和Ⅲ型胶原mRNA表达增加（F = 66.41、46.81，除UVB + 芒果苷1.0 mg/L组P > 0.05外，其他各组均P < 0.05），衰老相关基因p53、p21、p16 mRNA表达降低（F = 265.60、151.82、329.85，均P < 0.05），衰老相关蛋白p53、p21和p16的合成减少（F = 160.51、158.53、75.38，均P < 0.05），各指标检测值的变化与芒果苷浓度之间呈一定依赖性。 结论 芒果苷可能通过下调p53、p21、p16基因的表达来抑制UVB诱导的人二倍体成纤维细胞的早衰。

Mangiferin inhibits ultraviolet B-induced premature senescence in human fibroblasts: an experiment study

Hu Yanyan*, Zhao Hongwei, Zhou Bingrong, Fang Xiaobo, Luo Dan, Wu Zhiyun, Yin Huibin, Wu Wei, Zhang Jiaan. *Department of Dermatology and Venereology, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China Corresponding author: Luo Dan, Email: daniluo2013@njmu.edu.cn 【Abstract】 Objective To clarify whether mangiferin inhibits stress-induced premature senescence （SIPS） in human diploid fibroblasts （HDFs） induced by repeated exposure to a sub-cytotoxic dose of ultraviolet B （UVB）, and to investigate the mechanism underlying the effect of UVB. Methods HDFs were isolated from the circumcised foreskin of healthy males, and subjected to a primary culture and 6 － 12 passages of subculture. Then, the HDFs were divided into six groups, i.e. blank control group receiving no treatment, UVB-SIPS group receiving UVB irradiation only, three combination groups receiving UVB irradiation and post-irradiation treatment with mangiferin at 1.0, 2.0 and 4.0 mg/L respectively, and mangiferin group treated with mangiferin 4.0 mg/L only. Irradiation was performed once a day for five consecutive days, and mangiferin treatment was given immediately after each irradiation. Cell counting kit-8 （CCK-8） was used to evaluate the proliferative activity of cells, and β-galactosidase （SA-β-Gal） staining to estimate the degree of premature senescence in cells, flow cytometry to detect cell cycle, Western blot to quantify the protein expressions of senescence-related proteins p53, p21 and p16, real time-PCR to determine the mRNA expression levels of matrix metalloproteinase （MMP1）, MMP3, collagen types Ⅰand Ⅲ, p53, p21 and p16, at 72 hours after the last irradiation. One-way analysis of variance was used for statistical analysis. Results The proliferative activity （expressed as the absorbance value at 450 nm） of HDFs was 0.322 9 ± 0.011 3, 0.336 1 ± 0.016 3, 0.342 6 ± 0.014 4 and 0.288 2 ± 0.020 7 respectively in the UVB + mangiferin 1.0 mg/L group, UVB + mangiferin 2.0 mg/L group, UVB + mangiferin 4.0 mg/L group, and UVB-SIPS group respectively （F = 110.08，P < 0.05）, with the percentage of SA-β-Gal-positive cells being （88.83 ± 4.54）%, （46.33 ± 5.51）%, （32.17 ± 6.05）% and （93.67 ± 3.75）% respectively （F = 283.54, P < 0.05）, and the proportion of cells in the G1 phase being （72.19 ± 3.42）%, （60.99 ± 2.70）%, （49.80 ± 2.10）% and （82.09 ± 0.89）% respectively （F = 156.01, P < 0.05）. Compared with the UVB-SIPS group, the three combination groups showed significantly decreased expression levels of MMP1 and MMP3 mRNAs （F = 69.41, 106.41, respectively, both P < 0.05） as well as p53, p21 and p16 mRNAs （F = 265.60, 151.82, 329.85, respectively, all P < 0.05） and proteins （F = 160.51, 158.53, 75.38, respectively, all P < 0.05）, but increased expression levels of COL1a1 and COL3a1 mRNAs （F = 66.41, 46.81, respectively, both P < 0.05）. In these combination groups, the changes in the above parameters were dependent on the concentration of mangiferin to a degree. Conclusion Mangiferin may inhibit UVB-induced premature senescence in HDFs via downregulating the expressions of p53, p21 and p16 genes.