热灭活植物乳杆菌ATCC 8014对UVB暴露皮肤细胞的抗光老化和抗黑色素生成作用研究  

Anti-photoaging and anti-melanogenesis effects of heat-inactivated Lactobacillus plantarum ATCC 8014 on UVB-exposed skin cells

作　　者：龚淼 徐京 郑斌 闻正顺 宋燕 张晓芳 孟令婷 GONG Miao;XU Jing;ZHENG Bin;WEN Zhengshun;SONG Yan;ZHANG Xiaofang;MENG Lingting(College of Food and Pharmacy,Zhejiang Ocean University,Zhoushan 316022,Zhejiang,China)

机构地区：[1]浙江海洋大学食品与药学学院,浙江舟山316022

出　　处：《微生物学报》2023年第7期2681-2698,共18页Acta Microbiologica Sinica

基　　金：浙江省教育厅省属高校基本科研业务费(2021J011);浙江海洋大学人才引进科研基金(11135090621,11135090320)。

摘　　要：【目的】中波紫外(ultraviolet B,UVB)照射是导致皮肤光老化形成和色素沉着的主要环境原因之一。植物乳杆菌是一种广泛研究的益生菌。本研究利用UVB照射皮肤细胞[正常人皮肤成纤维细胞(normal human dermal fibroblast,NHDF)和小鼠B16F10黑色素瘤细胞],探讨热灭活植物乳杆菌(Lactobacillus plantarum,LP)ATCC 8014的抗光老化和抗黑色素生成作用。【方法】以暴露于UVB的NHDF细胞和小鼠B16F10黑色素瘤细胞为研究对象,用甲基噻唑基四唑(methyl thiazolyl tetrazolium,MTT)法、末端脱氧核苷酸转移酶dUTP缺口末端标记(terminal-deoxynucleoitidyl transferase dUTP nick end labeling,TUNEL)染色分析和2’,7’-二氯荧光素二乙酸酯(2’,7’-dichlorofluorescin diacetate DCFH-DA)染色分析实验检测2组细胞的细胞活力、DNA损伤及相对活性氧(reactive oxygen species,ROS)水平;利用酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)法检测NHDF细胞中的I型胶原蛋白水平;通过NaOH裂解法和多巴氧化法对B16F10细胞黑色素生成和酪氨酸酶活性进行测定;通过qRT-PCR分析和Western blotting分析检测光老化和黑色素生成相关基因和蛋白表达水平变化。【结果】(1)LP抑制UVB诱导的NHDF和B16F10细胞毒性,这与减少ROS介导的细胞DNA损伤有关;(2)LP下调基质金属蛋白酶(matrix metalloproteinase,MMP)-1、MMP-3和MMP-9(而不是MMP-2)mRNA水平[与抑制细胞外信号调节激酶(extracellular signal-regulated kinase,ERK),p38(而不是JNK)/c-Fos(而不是c-Jun)信号通路有关],增加I型前胶原(procollagen type-1 alpha 1,COL1A1)蛋白水平,从而提高I型胶原蛋白含量;(3)LP作为自噬诱导剂(增加LC3-Ⅱ和Beclin 1蛋白水平以及LC3-Ⅱ/LC3-Ⅰ比率)抑制酪氨酸酶、酪氨酸相关蛋白(tyrosinase-related protein,TYRP)-1和TYRP-2活性和/或表达(与压制PKA/CREB/MITF信号通路有关),从而降低黑色素含量。【结论】LP在UVB暴露的皮肤细胞中具有潜在的抗光老�[Objective]Ultraviolet B(UVB)irradiation is one of the main environmental causes of skin photoaging and hyperpigmentation.Lactobacillus plantarum is a well-studied species of probiotics that can benefit human health.The present study investigated the anti-photoaging and anti-melanogenesis effects of heat-inactivated L.plantarum(LP)ATCC 8014 on the skin cells(normal human dermal fibroblast(NHDF)cells and B16F10 murine melanoma cells)exposed to UVB irradiation.[Methods]The viability,DNA damage,and reactive oxygen species(ROS)levels of NHDF and B16F10 cells were determined by methyl thiazolyl tetrazolium(MTT)assay,terminal-deoxynucleoitidyl transferase dUTP nick end labeling(TUNEL)assay,and 2′,7′-dichlorofluorescin diacetate(DCFH-DA),respectively.The level of collagen I in NHDF cells was determined by enzyme-linked immunosorbent assay(ELISA).The melanin production and tyrosinase activity in B16F10 cells were measured by NaOH lysis method and L-DOPA oxidation method,respectively.qRT-PCR and Western blotting were employed to measure the expression levels of genes and proteins associated with photoaging and melanin production.[Results](1)LP inhibited UVB-induced cytotoxicity by reducing the ROS-mediated DNA damage in NHDF and B16F10 cells;(2)LP down-regulated the mRNA levels of matrix metalloproteinase(MMP)-1,MMP-3,and MMP-9(rather than MMP-2),relating to inhibit the extracellular signal-regulated kinase(ERK),p38(rather than JNK)/c-Fos(rather than c-Jun)signaling pathway.It up-regulated the protein level of procollagen type-1 alpha 1(COL1A1),thereby increasing the content of type I collagen;(3)LP as an autophagy inducer(up-regulating the protein levels of LC3-II and Beclin 1 and increasing the LC3-II/LC3-I ratio)inhibited the activities and/or expression of tyrosinase,tyrosinase-related protein(TYRP)-1,and TYRP-2 by inhibiting the PKA/CREB/MITF signaling pathway,thereby decreasing the melanin content.[Conclusion]LP has potential anti-photoaging and anti-melanogenesis effects on the skin cells exposed to UVB.

关 键 词：热灭活植物乳杆菌ATCC 8014 抗光老化 抗黑色素生成 中波紫外(UVB) 皮肤细胞 

分 类 号：TS201.3[轻工技术与工程—食品科学]