篩選改善異位性皮膚炎症狀之潛力菌株以發展寵物益生菌產品

Abstract

犬異位性皮膚炎為臨床常見之第一型免疫失衡疾病，其主要成因為遺傳及環境交互作用所造成，當患病犬隻接觸過敏原後會增加免疫球蛋白 (immunoglobulin, Ig) E及第二型輔助T細胞 (type 2 T helper cell, Th2) 相關細胞激素生成，進而誘發過敏症狀產生。目前，已有許多藥物可以減緩過敏症狀但無法進行根治，且多數對動物體會產生副作用，故尋找適當替代方法改善寵物及宿主生活品質為當務之急。過去幾十年，已有許多研究指出益生菌可以透過刺激第一型輔助T細胞 (type 1 T helper cell, Th1) 或是調節型T細胞 (regulatory T cell, Treg) 相關反應來改善過敏症狀。因此，本次試驗欲從蒙古傳統發酵乳製品篩選具改善過敏症狀之乳酸菌，以做為日後寵物益生菌之使用。 將本研究室先前自蒙古發酵乳所篩之菌株凍管，利用塗盤法及革蘭氏染色法篩選出61株菌做為本次候選菌株。在In vitro 研究中，候選菌株與小鼠巨噬細胞株RAW 264.7共培養24小時後，進行細胞存活率與細胞激素分析，結果顯示其中57株菌可以維持細胞存活率於80%以上，且10株菌於刺激RAW 264.7分泌腫瘤壞死因子 (tumor necrosis factor, TNF)-α及介白素 (interleukin, IL) 10有較好效果，其中以MP01可以顯著刺激兩種細胞激素生成 (P < 0.05)，而MP02亦有刺激趨勢。透過分子鑑定技術，確認MP01及MP02分別屬於Lactococcus lactis及 Lactobacillus paracasei。隨後將2株潛力菌株與小鼠腹水細胞進行共培養以再確認其免疫調節能力，結果顯示2株菌於活菌及死菌狀況下皆可刺激免疫細胞生成TNF-α及IL10，且具有濃度依賴性 (dose-dependent) 現象。基於上述結果，最終使用2 株潛力菌株以混菌方式運用於in-vivo實驗以評估其改善功效。 本次研究使用塵蟎萃取物 (house dust mite extration, HDM) 做為過敏原以誘導異位性皮膚炎 (atopic dermatitis, AD) 小鼠模型，將小鼠隨機分為以下組別：操作控制組 (PC)、誘導傷害組 (NC)、商業競品組 (MC)、低劑量活菌 (LL)、低劑量死菌 (LD)、高劑量活菌 (HL) 及高劑量死菌 (HD)。相較於NC組別，潛力菌組別 (LL、LD、HL及HD組) 有改善AD症狀趨勢，此外，潛力菌組之血清總IgE及HDM-specific IgE濃度亦有顯著降低的結果。在腸繫膜淋巴結細胞激素分析部分，潛力菌組亦可以刺激生成Treg相關細胞激素 (IL2及IL10)，但是在Peyer’s patches中CD4+ CD25+ T cell之數目，各組間並無顯著差異。另外，血清細胞激素分析結果顯示潛力菌組有降低AD相關細胞激素 (IL6) 及提升Treg相關細胞激素之趨勢，且可以顯著降低脾臟中Th1 及Th2 cell比例 (P < 0.05)。 綜合以上結果，給予潛力菌株可以減緩AD症狀、降低血清IgE濃度、增加Treg相關免疫反應及降低全身性免疫反應。然而，未來仍需更多研究以深入探討潛力菌之免疫調節機制。

Canine atopic dermatitis (CAD) is regards as one of the type one hypersensitivity immune disorder in veterinary practice, caused by the interaction of genetic and environmental factors. The immune disorder increases the production of immunglobulin (Ig) E and type 2 T helper cell (Th) -related cytokines. Nowadays, several treatments can reduce pain efficiently, but most of them might cause some side-effects. Therefore, it is necessary to find alternatives to improve patients’ life quality. Over the past few decades, many studies indicated that probiotics could improve the allergic symptoms through stimulating type 1 T helper cell (Th1)-/regulatory T (Treg)-relative immune response. Thus, the aim of this study was to select lactic acid bacteria from Mongolian traditional fermented milk with anti-allergic effect. By culture-based methods and Gram-staining, we isolated 61 strains from various kinds of Mongolian fermented milk products. In vitro studies, the candidate strains (2 × 104 CFU/mL) were co-cultured with the murine RAW 264.7 cell line for 24 hours and determined the cell viability and proinflammatory cytokines. The MTT results revealed that 57 candidates could maintain the cell viability over 80%. The further cytokines results indicated that 10 isolated strains could upregulate the concentration of tumor necrosis factor (TNF)-α, and regulatory T (Treg) cytokine (interleukin (IL) 10). Among the potential strains, MP01 could significantly stimulate RAW 264.7 to produce Th1- and Treg- related cytokines (P < 0.05) and MP02 could also enhance more cytokine production than others. Through 16S rRNA and housekeeping gene analysis, MP01 and MP02 are belonged to Lactococcus lactics and Lactobacillus paracasei, respectively. In order to confirm the immune-regulatory function, the selected two strains were co-cultured with murine peritoneal cells. The results indicated that both strains could provoke the cytokine production in live- and dead- forms in dosage-dependent. Based on the results, we selected Lc. lactics and Lb. paracasei as our potential probiotics mixture components for the further in-vivo study. In house dust mite (HDM)-induced atopic dermatitis (AD) mouse model, mice were assigned randomly to seven groups: positive control (PC), negative control (NC), market products (MC), low-dosage live bacteria (LL), low-dosage dead bacteria (LD), high-dosage live bacteria (HL), and high-dosage dead bacteria (HD) group. Compared with NC group, the probiotic treatments could relieve the AD symptoms. Moreover, potential probiotic mixture groups (LL, LD, HL, and HD) could significantly decrease total and HDM-specific IgE concentration (P < 0.05). In mesenteric lymph nodes (MLN) cytokine analysis, potential probiotic mixture groups could stimulate the production of Treg-related cytokines (IL2 and IL10). However, there was no significantly different on the population of CD4+ CD25+ T cell in Peyer’s patches. In serum cytokine analysis, probiotic treatments could reduce the production of AD-related cytokines (IL6) and increase the Treg-related cytokines (IL10 and IL2) concentration. Furthermore, potential probiotic mixture groups could also decrease the population of Th1 and Th2 cell in spleen. In conclusion, administration of potential probiotic mixture groups appeared to relieve the classic AD signs, decrease serum IgE concentration, increase the Treg-related immune response and reduce the systemic immune response. The additional studies are necessary to consider the possible mechanism of the immune-regulatory function.