维替泊芬在实验性哮喘气道重塑中的作用及其机制  

Effect and mechanism of verteporfin on airway remodeling of experimental asthma

作　　者：刘敏[1] 王艺颖 简鼎 李媛媛[1] 张光莉[1] 罗征秀[1] Liu Min;Wang Yi-Ying;Jian Ding;Li Yuan-Yuan;Zhang Guang-Li;Luo Zheng-Xiu(Department of Respiratory,Affiliated Children's Hospital of Chongqing Medical University/Key Laboratory of Child Developmentaldiseases of Ministry of Education/National Clinical Research Center for Children's Health and Disorders/China-International Science andTechnology Cooperation Base for Child Development and Critical Disorders/Chongqing Key Laboratory of Pediatrics,Chongqing 400014,China)

机构地区：[1]重庆医科大学附属儿童医院呼吸科/儿童发育疾病研究教育部重点实验室/国家儿童健康与疾病临床医学研究中心/儿童发育重大疾病国家国际科技合作基地/儿科学重庆市重点实验室,重庆400014

出　　处：《解放军医学杂志》2022年第8期745-755,共11页Medical Journal of Chinese People's Liberation Army

基　　金：重庆英才计划(CQYC2020030393);重庆市自然科学基金(cstc2020jcyj-msxmX0677);重庆市渝中区科委项目(20200156)。

摘　　要：目的 探讨维替泊芬(Vp)在实验性哮喘气道重塑中的作用及其机制。方法 (1)动物实验:将12只雌性BALB/c小鼠分为对照组、实验性哮喘组与Vp干预组,每组4只。实验性哮喘组小鼠使用屋尘螨提取液(HDM)滴鼻建立哮喘模型,Vp干预组在HDM激发前腹腔注射Vp进行干预,对照组给予等量生理盐水。收集小鼠肺组织及支气管肺泡灌洗液(BALF),采用HE染色、Masson染色、过碘酸希夫(PAS)染色观察肺组织病理变化,瑞氏-吉姆萨染色计数BALF中的炎性细胞;采用免疫组化检测肺组织中Yes相关蛋白(YAP)、磷酸化Yes相关蛋白(p-YAP)的表达情况,RT-PCR、Western bloing检测肺组织中YAP、p-YAP、骨桥蛋白(OPN)、平滑肌肌球蛋白重链(SMMHC)的表达情况。(2)细胞实验:将人气道平滑肌细胞(HASMC)分为对照组(加入等量生理盐水)、HDM组(加入50μg/ml HDM作用24 h)与HDM+Vp组(加入0.05 mg/L Vp刺激2 h后,加入50μg/ml HDM作用24 h),采用CCK-8法检测细胞增殖能力,流式细胞术检测细胞凋亡率,Western blotting检测YAP、OPN、SMMHC蛋白表达情况。结果 (1)动物实验:实验性哮喘组肺组织炎症较对照组加重,支气管基底膜、气道平滑肌及支气管管壁明显增厚[(1.13±0.38)μm vs.(0.79±0.36)μm,(6.49±2.36)μm vs.(4.56±1.52)μm,(33.85±5.95)μmvs.(22.08±3.30)μm,P<0.05],气道周围胶原纤维沉积面积、PAS染色阳性面积明显增加(5.85%±2.35%vs. 0.36%±0.12%,28.81%±5.89%vs. 13.57%±2.08%,P<0.01),肺组织中YAP、OPN mRNA和蛋白表达水平明显升高(P<0.05或P<0.01),p-YAP/YAP比值、SMMHC mRNA和蛋白表达水平明显降低(P<0.05或P<0.01)。与实验性哮喘组比较,Vp干预组小鼠肺组织炎症减轻,支气管基底膜、气道平滑肌及支气管管壁明显变薄[(0.93±0.27)μm、(4.99±1.75)μm、(26.59±2.76)μm,P<0.05],气道周围胶原纤维沉积面积、PAS染色阳性面积明显减少(2.14%±0.89%、17.92%±1.89%,P<0.05),肺组织YAP、OPN mRNA和蛋白表达水平明显降低(Objective To investigate the effect and mechanism of verteporfin(Vp)in airway remodeling of experimental asthma.Methods(1)Animal experiment:Twelve female BALB/c mice were divided into 3 groups(4 each):control group experimental asthma group and Vp intervention asthma group.Mice in experimental asthma group were treated by intranasal delivery of house dust mite(HDM)extract,in Vp intervention group were treated with intraperitoneal injection of Vp before HDM stimulation,and in control group was treated with the same amount of normal saline.Lung tissues and bronchoalveolar lavage fluid(BALF)of mice were collected,HE staining,Masson staining and Periodic Acid-Schiff(PAS)staining were used to observe the pathological changes of lung tissue,Wright-Giemsa staining was used to count the inflammatory cells in BALF,and the expressions of Yes-associated protein(YAP)and phosphorylated Yes-associated protein(p-YAP)in lung tissues were detected with immunohistochemistry,RT-PCR and Western blotting were performed to detect the expressions of YAP,p-YAP,osteopontin(OPN)and smooth muscle myosin heavy chain(SMMHC).(2)Cell experiment:Human airway smooth muscle cells(HASMC)were divided into control group(treated with the same amount of normal saline),HDM group(treated with 50μg/ml HDM for 24 hours)and HDM+Vp group(treated with 0.05 mg/L Vp for 2 hours,and then with 50μg/ml HDM for 24 hours).The cell proliferation of HASMC was detected by CCK-8.The apoptosis rate of HASMC were detected by flow cytometry.The protein levels of YAP,OPN and SMMHC in cells were detected by Western blotting.Results(1)Animal experiment:Compared with control group,the lung inflammation was aggravated,the bronchial basement membrane,airway smooth muscle(ASM)and airway wall were thickened in experimental asthma group[(1.13±0.38)μm vs.(0.79±0.36)μm,(6.49±2.36)μm vs.(4.56±1.52)μm,(33.85±5.95)μm vs.(22.08±3.30)μm,P<0.05].The deposition proportion of collagen fiber around airway and PAS staining area increased(5.85%±2.35%vs.0.36%±0.12%,28.81%±5.89%

关 键 词：Hippo/YAP信号通路 维替泊芬 哮喘 气道重塑 

分 类 号：R563.1[医药卫生—呼吸系统]