摘要
目的:探讨芹菜素介导Toll样受体4(TLR4)-肿瘤坏死因子受体相关蛋白6(TRAF6)轴对肺纤维化(Pulmonary fibrosis,PF)大鼠的改善及对肺泡巨噬细胞(AMs)自噬的影响。方法:SPF级健康雄性Wistar大鼠随机分为正常对照组、模型对照组、芹菜素0.02、0.04 g/kg组及地塞米松0.003 g/kg组。气管内滴注博莱霉素0.05 g/kg制备PF大鼠模型,次日给药,1次/d,连续给药14 d。HE染色评估肺组织炎症及病理变化;Masson染色检测肺组织胶原蛋白沉积以评估肺纤维化情况;免疫组化染色检测肺组织胶原蛋白Ⅰ(ColⅠ)及α-平滑肌肌动蛋白(α-SMA)阳性表达;ELISA法检测大鼠血清肿瘤坏死因子-α(TNF-α)、白介素6(IL-6)、IL-1β含量;比色分析法检测肺组织羟脯氨酸(HYP)含量;分离支气管肺泡灌洗液(BALF)中AMs并培养,透射电镜观察AMs自噬体形成情况;Western blot检测AMs中TLR4、髓样分化因子(MyD88)、TRAF6、核转录因子-κBp65(NF-κBp65)、p-NF-κBp65、Beclin1、微管相关蛋白1A/1B-轻链3(LC3)、泛素样结合蛋白P62(P62)蛋白相对表达水平。结果:成功构建PF大鼠模型,与正常对照组比较,模型对照组大鼠肺组织病理显著损伤,见肺泡结构紊乱,肺泡壁增厚,炎性细胞广泛浸润;胶原纤维严重沉积,支气管及血管周围纤维化区域ColⅠ及肺间质、肺泡上皮α-SMA强阳性表达,血清TNF-α、IL-6、IL-1β含量及肺组织中HYP含量升高,AMs中自噬体数量增加,TLR4、MyD88、TRAF6、Beclin1蛋白相对表达水平及p-P65/P65、LC3Ⅱ/LC3Ⅰ均升高,P62蛋白相对表达下调(P<0.05或P<0.01);与模型对照组比较,芹菜素各剂量组及地塞米松组大鼠肺组织病理损伤明显好转,胶原纤维沉积减少,肺组织中ColⅠ及α-SMA阳性表达减弱,血清TNF-α、IL-6、IL-1β含量及肺组织中HYP含量降低,AMs中自噬体数量减少,TLR4、MyD88、TRAF6、Beclin1蛋白相对表达水平及p-p65/p65、LC3Ⅱ/LC3Ⅰ均降低,P62蛋白相对表达水平上调(P<0.05或P<0.01
Objective:To investigate the effects of apigenin-mediated Toll-like receptor 4(TLR4)-tumor necrosis factor receptor-associated protein 6(TRAF6)axis on pulmonary fibrosis(PF)and autophagy of alveolar macrophages(AMs)in rats.Methods:Healthy male Wistar rats of SPF grade were randomly divided into a normal control group,a model control group,two apigenin groups(0.02 and 0.04 g/kg),and a dexamethasone group(0.003 g/kg).The PF model was induced in rats by intratracheal instillation of 0.05 g/kg bleomycin,followed by drug intervention the next day,once a day for 14 days.HE staining was used to evaluate lung inflammation and pathological changes.Masson staining was used to detect collagen deposition in lung tissues to evaluate PF.Immunohistochemical staining was used to detect the positive expressions of collagen Ⅰ(ColⅠ)andα-smooth muscle actin(α-SMA).Serum levels of tumor necrosis factor-α(TNF-α),interleukin 6(IL-6),and interleukin 1β(IL-1β)were determined by ELISA.Colorimetric analysis was used to detect the content of hydroxyproline(HYP)in lung tissues.AMs in bronchoalveolar lavage fluid(BALF)were isolated and cultured,and the formation of AMs autophagosomes was observed by transmission electron microscopy.The relative expression levels of TLR4,myeloid differentiation factor 88(MyD88),TRAF6,nuclear factor-κBp65(NF-κBp65),p-NF-κBp65,Beclin1,microtubule-associated protein 1 A/1 B-light chain 3(LC3),and ubiquitin-like binding protein P62(P62)in AMs were detected by Western blot.Results:The PF model was properly induced in rats.Compared with the normal control group,the model control group showed pathologically damaged lung tissues(such as alveolar structure disorder,alveolar wall thickening,and extensive infiltration of inflammatory cells),severe collagen fiber deposition,strong positive expression of ColⅠ in bronchial and perivascular fibrosis areas and α-SMA in interstitial and alveolar epithelial regions,increased serum levels of TNF-α,IL-6,and IL-1β and HYP content in lung tissues,increased number
作者
白辉辉
李彬
张珊珊
曹晶
Bai Huihui;Li Bin;Zhang Shanshan;Cao Jing(TCM Teaching and Research Section,Department of Medicine,Henan Medical College,Zhengzhou 451191;Respiratory Department,the First Affiliated Hospital of Henan University of Chinese Medicine,Zhengzhou 450000)
出处
《中药药理与临床》
CAS
CSCD
北大核心
2022年第6期90-95,共6页
Pharmacology and Clinics of Chinese Materia Medica
基金
河南省中医药科学研究专项课题(编号:20-21ZY1018)。
