目的 制备唾液乳杆菌与蛹虫草或铁皮石斛共培提取物，研究其对慢性皮肤湿疹的抗炎作用。方法 以加入蛹虫草或铁皮石斛的MRS培养基培养唾液乳杆菌，培养后菌体通过发酵、高压破碎、离心制备药液。取小鼠随机分为对照组、模型组、阳性药组、唾液乳杆菌与蛹虫草共培提取物组、唾液乳杆菌与铁皮石斛共培提取物组，建立小鼠湿疹模型，并测量耳部肿胀程度、计算双耳质量差及背部皮肤厚度的变化。以RAW264.7细胞和HaCaT细胞作为炎症细胞模型，采用脂多糖（lipopolysaccharide，LPS）诱导炎症反应，评估唾液乳杆菌与蛹虫草或铁皮石斛共培提取物调节细胞中白细胞介素-1β（interleukin-1β，IL-1β）、一氧化氮（nitric oxide，NO）和白细胞介素-10（interleukin-10，IL-10）及皮肤炎症生物标志物水平的作用。测定RAW264.7细胞在各种提取物处理下的吞噬能力。选择MatTek人体皮肤敏感性测试来研究唾液乳杆菌与蛹虫草/铁皮石斛共培养提取物的过敏反应。结果 唾液乳杆菌与蛹虫草或铁皮石斛共培组小鼠的双耳质量差和背部皮肤厚度均显著低于模型组（P＜0.01、0.001）。同时，在唾液乳杆菌与蛹虫草或铁皮石斛共培提取物的处理下，LPS诱导的RAW264.7细胞中的炎症介质如IL-1β、NO和抗炎因子IL-10的产生受到显著调节（P＜0.05、0.01、0.001），细胞的吞噬率显著降低（P＜0.01、0.001）。HaCaT细胞在唾液乳杆菌与蛹虫草或铁皮石斛共培提取物处理下，皮肤炎症生物标志物I型胶原α1蛋白（alpha-1 type I collagen，COL1A1）、I型胶原α2蛋白（alpha-2 type I collagen，COL1A2）、III型胶原α1蛋白（alpha-1 type III collagen，COL3A1）的mRNA表达水平显著上调（P＜0.05、0.01）。唾液乳杆菌与蛹虫草或铁皮石斛共培养的提取物对人体皮肤无过敏反应。结论 唾液乳杆菌和蛹虫草或铁皮石斛共培提取物对慢性皮肤湿疹和皮炎具有抗炎和治疗作用，可作为抗炎、抗过敏护肤品的原料，具有较大的应用前景。

Objective To study the anti-inflammatory effect of the co-cultured extracts of Lactobacillus salivarius and Yongchongcao[Cordyceps militaris (L.) Link] or Tiepishihu (Dendrobium officinale Kimura et Migo) on chronic skin eczema dermatitis.Methods L. salivarius was cultured in MRS medium with C. militaris or D. officinale. Then, the bacterial cells were fermented, crushed under high pressure, and centrifuged to prepare liquid medicine. Mice were randomly divided into control group, model group, positive drug group, co-cultured extract group of L. salivarius and C. militaris, and co-cultured extract group of L. salivarius and D. officinale. The eczema model of mice was established, and the degree of ear swelling, both ear quality difference and back skin thickness were measured. RAW264.7 cells and HaCaT cells were used as inflammatory cell models, and lipopolysaccharide (LPS) was used to induced the inflammatory responses. Effects of the co-cultured extracts of L. salivarius and C. militaris or D. officinale on the levels of interleukin-1β (IL-1β), nitric oxide (NO), interleukin-10 (IL-10) and skin inflammation biomarkersin in cells was evaluated. In addition, the phagocytic capacity of RAW264.7 cells treated with various extracts was determined. The MatTek human skin sensitivity test was selected to reveal the allergenic effect of the co-cultured extracts of L. salivarius and C. militaris or D. officinale.Results The difference in weight between left and right ears and dorsal skin thickness of mice in co-cultured extracts of L. salivarius and C. militaris or D. officinale were significantly lower than that in the model group (P < 0.01, 0.001). In parallel the production of inflammatory mediators (IL-1β and NO) and anti-inflammatory factor IL-10 were markedly regulated in cultured in RAW264.7 cells under the treatment of the co-cultured extracts of L. salivarius and C. militaris or D. officinale (P < 0.05, 0.01, 0.001). The rate of phagocytosis of LPS-induced RAW264.7 cells under the treatment was significantly reduced (P < 0.01, 0.001). HaCaT cells were treated with the co-cultured extracts of L. salivarius and C. militaris or D. officinale, the mRNA expression levels of the biomarkers of skin inflammation alpha-1 type I collagen (COL1A1), alpha-2 type I collagen (COL1A2), alpha-1 type III collagen (COL3A1) were significantly up-regulated (P < 0.05, 0.01). The co-cultured extracts of L. salivarius and C. militaris or D. officinale did not show allergy to human skin. Conclusion The co-cultured extracts of L. salivarius and C. militaris or D. officinale has anti-inflammatory and therapeutic effects on chronic skin eczema and dermatitis. They can be used as a raw material for anti-inflammatory and anti-allergic skin care products and has a strong application prospect.

R285

港特区创新科技署项目(UIM/385)