評估乳酸菌裂解液對角質細胞抗光老化之效用

Evaluation of the Anti-Photoaging Effects of Lactic Acid Bacteria Lysates on Keratinocytes

李嬋娟(Ly Thuyen Quyen)

指導教授 ： 鄭 達 智

國立屏東科技大學/國際學院/熱帶農業暨國際合作系/碩士(2022年)

https://doi.org/10.6346/NPUST202200038

摘要

皮膚直接暴露於陽光下且長期接受紫外線（UV）的輻射，會導致皮膚光老化而造成皮膚的損傷。一些益生菌已被證實對皮膚具有抗光老化的作用。近年來，人們將乳酸菌（LAB）應用於化妝品的興趣正逐漸在擴大，許多研究已證明乳酸菌對皮膚健康的有益影響。其中，一些乳酸菌被認為對皮膚具有抗炎的特性，能夠改善皮膚的健康。然而，LAB改善皮膚健康的確切機制，還沒有得到很完善的解釋，許多LAB菌株還沒有被研究。在本研究中，旨在從泡菜中分離出LAB，並評估其裂解液對初級人類角質細胞（HaCaT）接受UVB照射後的抗光老化作用。本研究從市販的泡菜產品中，分離出八個形態類似於LAB的菌落。其中RVQK3與其他七個分離株相比，其生長速度明顯地較快，因此被選作為研究的對象。在通過溶血試驗，確認RVQK3的安全性後，接著研究RVQK3裂解液對皮膚的抗光老化作用，包括：保濕、抗皺和抗炎。以不同濃度的裂解液處理HaCaT細胞，培育24小時後，通過細胞存活率分析（MTT），顯示裂解物對兩個細胞株（HaCaT細胞和NIH3T3細胞）的毒性。以無毒劑量的UVB照射HaCaT細胞，經MTT檢測，確認UVB照射後細胞的存活率。抗皺效果則以透過與皺紋有關的基因：如裂解液對基質金屬蛋白酶（MMPs）活性的影響來證明。而皮膚保溼的效果，則以透過裂解液對人類透明質酸合成酶1（HAS1）基因表達的影響來證實。而抗炎的效果則以透過裂解液對促炎細胞激素基因，如IL-6、IL-8和TNFα表達的影響來證實。所有基因的表達，都是以反轉錄定量聚合酶鏈反應法（RT-qPCR）進行分析。結果顯示，應用生物資訊學和親緣關係樹鑒定，泡菜產品中的RVQK3與植物乳桿菌最接近，因此稱為植物乳桿菌RVQK3。酶活性測試結果顯示，植物乳桿菌RVQK3沒有蛋白酶、脂肪酶和纖維素酶的活性。溶血試驗證實，RVQK3菌株不具溶血性，所以該菌株對紅血球是安全的。使用攪拌珠研磨破碎法提取植物乳桿菌RVQK3的裂解液，RVQK3經0、2、4、6和8分鐘的研磨，以研磨4分鐘具有顯著較高的溶解性蛋白質。植物乳桿菌RVQK3裂解液被發現對HaCaT和NIH3T3細胞無毒性的濃度分別為2000 g/mL和400 g/mL。當以裂解液處理HaCaT細胞，並暴露在20 mJ/cm2的UVB下時，裂解液沒有抑制促發炎基因表現的作用。結論：植物乳桿菌RVQK3裂解液，可能不具有抗光老化之作用。

關鍵字

溶菌產物 ； 益生菌 ； 光老化 ； UVB ； 皮膚

並列摘要

Photoaging is caused by chronic exposure to ultraviolet (UV) radiation by directly contacting the sunlight and results in skin damage. Some probiotic bacteria have been confirmed with the anti-photoaging effects. In recent years, the interest in using probiotics from lactic acid bacteria (LAB) for cosmetic products is expanding. Numerous studies have demonstrated that LAB can benefit skin health, in which some LAB have been remarked with anti-inflammatory properties and are capable of enhancing skin health. However, the precise mechanisms by which LAB improves skin health are not yet well-explained, and many other LAB strains are not yet studied. In this study, we aimed to isolate a potent Lactic Acid Bacteria (LAB) from Kimchi and to evaluate the anti-photoaging effects of that LAB lysate in the UVB irradiated primary human keratinocyte (HaCaT) cells. Eight colonies with morphology similar to the LAB group were isolated from a commercial Kimchi product. Only the isolate named RVQK3 was selected for further experiment due to its significant growth faster compared to the other seven isolates. After confirming the safety of this bacterium by hemolytic assay, the anti-photoaging effects including moisturizing, anti-wrinkle, and anti-inflammatory of RVQK3 lysate was then examined. HaCaT cells were treated with different lysates concentrations. After 24 h of incubation, the toxicity of lysate was indicated through the MTT assays on two cell lines including HaCaT cells and NIH3T3 cells. The nontoxic doses were used to analyze the effects of UV-B irradiation-induced on HaCaT cells. The MTT assay to confirm the viability of cells after the UVB irradiation process was also conducted. The anti-wrinkle effects were demonstrated by the regulation of wrinkle-related genes - matrix metalloproteinases (MMPs); the skin hydration factor as Hyaluronic acid was confirmed by the expression of human hyaluronan synthase 1 (HAS1) genes. Whereas the anti-inflammatory was expressed by the expression of pro‑inflammatory cytokines genes such as IL-6, IL-8, and TNFα. All genes expression were analyzed via the reverse transcription-quantitative polymerase chain reaction method (RT-qPCR). The results demonstrated that RVQK3 from kimchi product was identified through the BLAST tool and phylogenetic tree as closest to Lactobacillus plantarum and then called Lactobacillus plantarum RVQK3. The enzymatic activities tests indicated that L. plantarum RVQK3 fail to demonstrate enzymatic activities of protease, lipase, and cellulase. This bacteria strain was confirmed as safe by a hemolysis test. The lysate of L. plantarum RVQK3 was extracted using the bead beating method at different beating periods including 0, 2, 4, 6, and 8 min for optimal protein level and quality of lysate. The optimal process was recorded at 4 min of beating, which showed significantly higher protein level but the shortest time compared to the other treatments. L. plantarum RVQK3 lysate was found as non-toxic to the HaCaT and NIH3T3 cells at concentrations up to 2000 g/mL and 400 g/mL, respectively. While treating the HaCaT cells with lysate and exposure to UVB at 20 mJ/cm2, the lysate did not show the expected expressions on the tested genes. Therefore, we suggest conducting an experiment on UVB-irradiation-induced HaCaT cells again to confirm the result of this study.

並列關鍵字

Lysate ； Probiotic ； Photoaging ； UVB ； Skin

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