研究黑豆多肽(BSP)对UVB诱导的人皮肤成纤维细胞活性和胶原分泌的影响。使用MTT法测试不同质量浓度的黑豆多肽对人皮肤成纤维细胞的细胞毒性。将人皮肤成纤维细胞分为正常对照组(NC)、UVB组、UVB+100,200,400,800 μg/mL BSP组。使用MTT法和EdU染色法测试细胞活力。采用ELISA法测定细胞培养上清中HA和HYP含量。RT-qPCT检测COL1A1、COL3A1、MMP-1和MMP-3 mRNA相对水平。使用DCFH-DA探针检测ROS。按照试剂盒说明测定氧化应激指标(SOD、CAT、GSH-Px和MDA)水平。使用Western blotting检测Nrf2(细胞核)和Keap1蛋白表达。结果表明,质量浓度0,100,200,400和800 μg/mL的黑豆多肽不影响人皮肤成纤维细胞的细胞活力(
F=0.597,
P<0.668)。与UVB组比较,UVB+100,200,400,800 μg/mL BSP组的相对细胞活力和EdU阳性率逐渐升高,HA和HYP含量逐渐升高,COL1A1和COL3A1 mRNA相对水平升高,MMP-1和MMP-3 mRNA相对水平降低,ROS相对荧光强度和MDA水平降低,SOD、CAT和GSH-Px水平升高,Nrf2(细胞核)蛋白相对水平升高,Keap1蛋白相对水平降低(
P<0.05)。黑豆多肽可提高UVB诱导的人皮肤成纤维细胞活力,促进胶原分泌,通过激活Nrf2/Keap1信号通路抑制氧化应激,黑豆多肽有望成为抗衰老化妆品或美白化妆品的添加剂。
The study aimed to investigate the effects of black soybean peptide (BSP) on the activity and collagen secretion of human skin fibroblasts induced by UVB. The cytotoxicity of different concentrations of black soybean peptide (0, 100, 200, 400, 800 μg/mL) on human skin fibroblasts was tested by MTT method. Human skin fibroblasts were divided into normal control group (NC), UVB group, and UVB+100, 200, 400, 800 μg/mL BSP groups. The cells in NC group and UVB group were cultured in normal medium. The cells in UVB+100, 200, 400, 800 μg/mL BSP groups were cultured for 24 h with 100, 200, 400, and 800 μg/mL black soybean peptide, respectively. MTT method and EdU staining method were used to test the cell viability. The contents of HA and HYP in the culture supernatant of cells were determined by ELISA method. The relative levels of COL1A1, COL3A1, MMP-1 and MMP-3 mRNA were detected by RT-qPCT. DCFH-DA probe was used to detect ROS. The levels of oxidative stress indexes (SOD, CAT, GSH-Px and MDA) were measured according to the kit instructions. The expression of Nrf2 (nucleus) and Keap1 protein was detected by western blotting. The black soybean peptide at the doses of 0, 100, 200, 400, 800 μg/mL does not affect the cell viability of human skin fibroblasts (F=0.597, P<0.668). Compared with UVB group, the relative cell viability and EdU positive rate, the content of HA and HYP, the relative levels of COL1A1 and COL3A1 mRNA, the levels of SOD, CAT and GSH-Px, and the relative levels of Nrf2 (nucleus) protein of UVB+100, 200, 400, 800 μg/mL BSP groups gradually increase, while the relative levels of MMP-1 and MMP-3 mRNA, the relative fluorescence intensity and MDA level of ROS decreased, and the relative level of Keap1 protein decreased (P<0.05). Black bean peptide can increase the vitality of human skin fibroblasts induced by UVB, promote collagen secretion, and inhibit oxidative stress by activating the Nrf2/Keap1 signaling pathway. Black bean peptide is expected to become additives in anti-aging cosmetics or whitening cosmetics.