二甲双胍抑制UVA诱导的人皮肤成纤维细胞的光老化效应

Repression effect of metformin on photo-damage of human skin fibroblasts induced by UVA

目的研究二甲双胍(MF)对长波紫外线(UVA)诱导的人皮肤成纤维细胞(HSF)光老化效应的抑制作用及相关机制。方法体外培养人皮肤成纤维细胞,分为对照组(control)、UVA组、UVA+MF组。以CCK-8法检测细胞增殖;对各组细胞进行细胞衰老β-半乳糖苷酶染色;以荧光探针DCF-DA染色流式细胞仪检测细胞内ROS水平;real-time PCR检测衰老相关基因MMP1、MMP3的mRNA相对表达量;Western blot检测蛋白MMP1、MMP3、SOD1和SOD2的表达。结果 0.01 mmol/L二甲双胍对HSF增殖无显著影响,0.1和1 mmol/L二甲双胍则显著抑制HSF的细胞增殖(P<0.05)。与对照组相比,UVA组β-半乳糖苷酶染色阳性率显著增高(P<0.01);ROS水平显著升高(P<0.05);MMP1和MMP3 mRNA相对表达量显著增加(P<0.01);MMP1和MMP3蛋白表达量显著增多(P<0.01);SOD1、SOD2蛋白表达显著减少(P<0.01)。与UVA组相比,UVA+MF组β-半乳糖苷酶染色阳性率显著降低(P<0.05);ROS水平显著下降(P<0.05);MMP1和MMP3的mRNA相对表达量显著降低(P<0.05);蛋白MMP1和MMP3表达量显著降低(P<0.05);蛋白SOD1表达显著增加(P<0.01);SOD2表达量增加。结论二甲双胍可通过抑制细胞内ROS水平和相关基质金属蛋白酶的表达,提高细胞抗氧化能力,从而抑制UVA诱导的皮肤成纤维细胞的光老化效应。

Objective To determine the inhibitory effect and mechanism of metformin（ MF） on photo-damage of human skin fibroblasts（ HSF） induced by UVA. Methods Human skin fibroblasts were randomly divided into control group,UVA group and UVA＋MF group. The proliferation of HSF was detected by CCK-8 assay kit. SA-β-gal staining was performed to evaluate the senescence state. The level of ROS was examined by fluorescence probe DCF-DA staining using flow cytometry. Real-time PCR was used to determine mRNA expression of senescence-associated signals of MMP1 and MMP3. The protein expression of MMP1,MMP3,SOD1 and SOD2 were measured by Western blot. Results To the proliferation of HSF,0. 01 mmol/L Metformin had no significant effect,but 0. 1 and1 mmol/L Metformin depressed significantly（ P〈0. 05）. Compared with the Control group,it showed that UVA irradiation increased the positive rate of SA-β-gal staining（ P〈0. 01）,the level of ROS（ P〈0. 05）,mRNA and protein expression of MMP1 and MMP3 significantly（ P〈0. 01）; Also decreased the expression of SOD1 and SOD2（ P0. 01）. Compared with the UVA group,it showed that metformin decreased the positive rate of SA-β-gal staining（ P0. 05）,the level of ROS（ P0. 05）,mRNA and protein expression of MMP1 and MMP3 significantly（ P0. 05）; Also increased the expression of SOD1（ P〈0. 01） and SOD2. Conclusions Metfomin can inhibit photodamage of human skin fibroblasts induced by UVA via decreasing ROS and metal matrix protease generation,also the improvement of cellular antioxidant capacity.