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

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化和抗黑色素生成作用。

Abstract:

[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.