D-柠檬烯抑制NF-κB通路介导COPD小鼠的炎症和免疫反应

D-limonene interferes with inflammatory and immune responses in COPD mice by inhibiting the NF-κB pathway

为探讨泻肺清肝饮活性成分D-柠檬烯对慢性阻塞性肺疾病(chronic obstructive pulmonary disease,COPD)小鼠炎症和免疫反应的影响及其机制,将36只6周龄的雄性C57BL/6J小鼠随机分为3组:对照组、模型组和D-柠檬烯组,每组12只。通过LPS和烟熏诱导COPD小鼠模型,D-柠檬烯组小鼠从造模第2天开始每天按照100 mg/kg的剂量给予腹腔注射D-柠檬烯,持续给药至第28天。观察各组小鼠的毛发、行动以及精神状态,并记录小鼠体质量,H-E染色观察小鼠肺组织病理变化,Western blotting检测小鼠肺组织中NF-κB、p-NF-κB蛋白的表达,ELISA检测血清以及支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)中IL-6、TNF-α、IL-1β和IL-10的水平,FACS检测外周血中Th17/Treg比值。结果显示,与对照组比较,模型组小鼠体质量减轻,精神萎靡颓废,毛色暗淡,脱毛,活动度降低;肺泡结构紊乱,支气管壁上皮增生,肺组织周围有大量炎症细胞浸润;肺组织中p-NF-κB蛋白表达水平,血清及BALF中IL-6、TNF-α、IL-1β水平和外周血中Th17/Treg比值均显著升高(P<0.01),血清及BALF中IL-10水平显著降低(P<0.01)。D-柠檬烯用药后显著逆转了上述变化(P<0.01)。该研究提示,D-柠檬烯通过抑制NF-κB通路来恢复Th17/Treg平衡,降低炎性因子的表达,对COPD小鼠具有较好的治疗效果。

To investigate the effects and mechanism of D-limonene,the active ingredient of Xiefeiqingganyin,on inflammatory and immune responses in mice with chronic obstructive pulmonary disease(COPD),366-week-old male C57BL/6J mice were randomly divided into three groups:control group,model group,and D-limonene group,with 12 mice in each group.The COPD model were established by administration of LPS and smoking.Mice in D-limonene group were intraperitoneally injected with 100 mg/kg D-limonene daily from the 2nd day of modeling until the 28th day.The hair,movement,and mental state of mice in each group were observed,and the body mass was recorded.Pathological changes of mouse lung tissues were observed by H-E staining.Western blotting was used to detect the expression of NF-κB and p-NF-κB protein in lung tissues,and ELISA was used to detect the levels of IL-6,TNF-α,IL-1β,and IL-10 in serum and bronchoalveolar lavage fluid(BALF).FACS was used to detect the ratio of Th17/Treg in peripheral blood.The results showed that compared to control group,mice in model group showed more severe symptoms including weight loss,listless and decadent,dull coat and hair loss,reduced activity,abnormal alveolar structure,hyperplasia of bronchial epithelium,and increased infiltration of inflammatory cells around the lung tissues.In addition,the expression of p-NF-κB protein in lung tissue,the levels of IL-6,TNF-α,and IL-1βin serum and BALF,and the ratio of Th17/Treg in peripheral blood were all significantly increased(P<0.01),while IL-10 in serum and BALF was significantly decreased(P<0.01).These changes were significantly reversed after D-limonene administration(P<0.01).Altogether,this study suggests that D-limonene has a good therapeutic effect on COPD mice by restoring the Th17/Treg balance and reducing the expressions of inflammatory factors by inhibiting NF-κB pathway.