乳酸菌應用於預防異位性皮膚炎之研究

Abstract

異位性皮膚炎 (atopic dermatitis, AD) 是一種會復發的過敏性皮膚皮疾病，常導致嚴重的搔癢和皮膚病變。根據國際兒童氣喘過敏研究統計，AD影響全球20%兒童和3%成年人。目前，已知AD的致病機制與過度表現的第二型輔助性T細胞 (type 2 T helper cells, Th2 cells) 有關。雖然有許多藥物可以對AD進行治療，但這些藥物可能會引起副作用。近幾年來，許多研究顯示益生菌具有調節免疫系統、改善腸道屏障和穩定腸道菌相等功能，可能可以做為減輕AD的替代方法。因此本研究第一部分欲擬探討益生菌對於減緩AD症狀之潛力，並且開發最適化培養基和評估益生菌特性。 經體外試驗與小鼠巨噬細胞株RAW 264.7共培養後，發現Lactobacillus johnsonii J2和Lactiplantibacillus plantarum K1顯著刺激巨噬細胞分泌細胞激素腫瘤壞死因子-α (tumor necrosis factor, TNF-α) 和介白素 (interleukin, IL)-10 (P < 0.05)。接續探討Lb. johnsonii J2和Lb. plantarum K1改善由1-氯-2,4-二硝基苯 (1-chloro-2,4-dinitrobenzene, DNCB) 和塵螨蛋白萃取物 (house dust mite extracts, HDM extracts) 誘導AD BALB/c小鼠模型之潛力。結果顯示，Lb. johnsonii J2和Lb. plantarum K1改善AD的皮膚症狀。在皮膚組織分析中，益生菌兩處理組發炎程度皆顯著降低 (P < 0.05)。不過，雖然Lb. johnsonii J2和Lb. plantarum K1無法有效降低血液免疫球蛋白 (immunoglobulin, Ig) E和平衡IgG1/IgG2a比例。但在脾臟中，Lb. johnsonii J2顯著提升第一型輔助性T細胞 (type 1 T helper cells, Th1 cells) 細胞激素IL-2的濃度並顯著調降Th2細胞激素IL-4濃度 (P < 0.05)，而Lb. plantarum K1則顯著提升Th1細胞激素IL-2的濃度並顯著調降Th2細胞激素IL-4與IL-5和第十七型輔助型T細胞 (type 17 T helper cells, Th17 cells) 細胞激素IL-17的濃度 (P < 0.05)，因此兩株菌株具有平衡Th1/Th2/Th17免疫系統之功能。進一步探討腸道菌相和短鏈脂肪酸 (short chain fatty acids, SCFAs)，Lb. johnsonii J2可顯著提升雙歧桿菌屬 (Bifidobacterium) 和降低大腸桿菌 (Escherichia coli) 數量 (P < 0.05)，不過SCFAs濃度未顯著提升，而Lb. plantarum K1可顯著提升Bifidobacterium和乳桿菌屬 (Lactobacillus) 菌量 (P < 0.05)，且在大腸 (colon) 與直腸 (rectum) 中顯著提升乙酸 (acetic acid) 的濃度 (P < 0.05)。綜上述，服用Lb. johnsonii J2和Lb. plantarum K1可減緩AD的症狀。 本研究室先前試驗另外篩選出兩株具有改善AD的菌株Lactococcus lactis subsp. cremoris和Lacticaseibacillus paracasei subsp. paracasei，為完備此兩株菌株後續量產開發作準備，第二部分試驗則針對此兩株菌株，使用不同比例的碳源、氮源和礦物質開發Lc. lactis subsp. cremoris和Lb. paracasei subsp. paracasei最適化培養基。結果顯示，本次試驗的配方無法得到反應曲面方程式，因此尚未尋求到Lc. lactis subsp. cremoris和Lb. paracasei subsp. paracasei最佳培養基配方，不過利用不同培養基培養後與小鼠巨噬細胞株RAW 264.7共培養發現，培養基配方的改變不會影響菌株在細胞存活率和刺激巨噬細胞釋放細胞激素TNF-α與IL-10的能力。後續接著探討益生菌此兩株菌抗菌及抗生素敏感性特性，在抗菌試驗中，Lc. lactis subsp. cremoris和Lb. paracasei subsp. paracasei皆能顯著抑制仙人掌桿菌 (Bacillus cereus) 的生長 (P < 0.05)。此外，在抗生素敏感性試驗中，Lb. paracasei subsp. paracasei 對青黴素(penicillin)、四環素 (tetracycline) 和萬古黴素 (vancomycin) 具有耐藥不敏感性。 綜合以上結果，在進行菌株篩選的體外試驗以及動物試驗評估後，可開發菌株最適化培養基，以在大量生產菌株時也能降低生產成本，並評估益生菌特性，藉此提升菌株商業上的價值，以增加益生菌在保健食品市場上的競爭力。

Atopic dermatitis (AD) is a chronic and relapsing inflammatory skin disease accompanied with severe itching and skin lesions. According to statistics from the International Study of Asthma and Allergies in Childhood (ISAAC), AD affects up to 20% of children and 3% of adults worldwide. Up to present, underlying pathogenesis of AD is known to be associated with the overexpression of type 2 T helper (Th2) cells. Although therapeutic medicines have been used for AD treatment, the side effects limit the usage. Many studies have demonstrated that probiotics can exert immunomodulatory effect, improve epithelial barrier function and normalize the composition of gut microbiota, which might provide an alternation treatment to alleviate AD. Thus, the aim of this study was assessing the effect of probiotics to alleviate the symptoms of AD, optimizing medium for production of probiotics and evaluating the properties of probiotics. First, Lactobacillus johnsonii J2 and Lactiplantibacillus plantarum K1 were selected due to their stimulating abilities in tumor necrosis factor (TNF)-α and Interleukin (IL)-10 when co-culturing with RAW 264.7 in vitro (P < 0.05). Further, in vivo study also showed that both Lb. johnsonii-treated and Lb. plantarum-treated groups improved the symptoms of AD with reduction of skin inflammation using 2,4-dinitrochlorobenzene (DNCB) and house dust mite (HDM)-induced BALB/c mice model. We then investigated the possible mechanism. The oral administration of Lb. johnsonii J2 and Lb. plantarum K1 had no significantly reduced serum IgE and IgG1/IgG2a ratio. However, Lb. johnsonii J2 significantly increased IL-2 and reduced IL-4 in spleen. Lb. plantarum K1 also significantly increased IL-2 and reduced IL-4, IL-5 and IL-17 in spleen (P < 0.05). Moreover, microbiota analysis indicated that the Lb. johnsonii-treated mice significantly increased Bifidobacterium and reduced Escherichia coli (P < 0.05), while the Lb. plantarum-treated mice significantly increased Lactobacillus and Bifidobacterium. Otherwise, it was found that the concentration of acetic acid was also upregulated in L. plantarum-treated group than AD group (P < 0.05). As a result, both Lb. johnsonii J2 and Lb. plantarum K1 might have potential to attenuate the symptoms of AD. In our previous study, we also islated another two anti-AD probiotics, Lactococcus lactis subsp. cremoris and Lacticaseibacillus paracasei subsp. paracasei. To complete the process for possible commercialization, thus, in the second part, we maximized the yield of Lc. lactis subsp. cremoris and Lb. paracasei subsp. paracasei by the response surface methodology (RSM). Unfortunately, the optimal medium cound not be found by RSM due to unproper upper and lower bounds of carbon sources, nitrogen sources and minerals. However, we still wanted to know if the ingrdients of medium affected the functional properties. The cell viability of MTT assay and two cytokines (TNF-α and IL-10) analysis showed that different media didn’t affect immunomodulatory activities of Lc. lactis subsp. cremoris and Lb. paracasei subsp. paracasei. Additionally, antimicrobial activity and antibiotic susceptibility testing were also performed. Both strains could inhibit Bacillus cereus. Lb. paracasei subsp. paracasei were also resistant to penicillin, tetracycline and vancomycin in antibiotic susceptibility testing (P < 0.05). In conclusion, through selecting the potential probiotic strains which could alleviate the symptoms of AD, developing the most cost-effective medium to increase the yield of probiotics, and evaluating the characteristics of probiotics could provide a potential for commercilization.