摘要
目的探讨小儿止哮平喘颗粒对卵清蛋白(OVA)诱导的哮喘小鼠气道重塑的干预作用,及其通过调控巨噬细胞迁移抑制因子(MIF)并抑制磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)信号通路的潜在机制。方法将96只SPF级BALB/c雄性小鼠随机分为空白组,模型组,止哮平喘低、中、高剂量组(2.05、4.10、8.20 g/kg),腺相关病毒(AAV)阴性对照shRNA组(AAV NC shRNA组),MIF基因沉默组(AAV MIF shRNA组),PI3K/Akt抑制剂LY294002组(1 mg/kg),每组12只。通过OVA致敏与激发构建哮喘模型,检测气道阻力、支气管肺泡灌洗液(BALF)中炎症细胞(嗜酸性粒细胞、巨噬细胞等)占比,定量分析血清及BALF中OVA-免疫球蛋白E、白细胞介素(IL)-1β、IL-6、肿瘤坏死因子-α、γ干扰素含量。采用HE、Masson、PAS染色法评估气道壁厚度与支气管周围膜周长比值(Wat/Phm)、平滑肌面积与支气管周围膜周长比值(Wam/Phm)、胶原沉积及杯状细胞化生程度。通过蛋白质印迹法、免疫荧光及实时荧光定量聚合酶链反应,分别检测肺组织中MIF蛋白和mRNA表达、PI3K/Akt通路活化指标及细胞周期相关蛋白[周期蛋白依赖性激酶(CDK)6、周期蛋白(Cyclin)D1、Cyclin D3、p21]表达变化。结果与模型组比较,小儿止哮平喘颗粒中、高剂量组气道阻力降低(P<0.05),BALF中嗜酸性粒细胞与巨噬细胞比例失衡得到改善,血清及BALF中炎症因子水平降低(P<0.05);病理观察显示,小儿止哮平喘颗粒中、高剂量组气道上皮损伤、杯状细胞增生及胶原纤维沉积减轻,Wat/Phm和Wam/Phm降低(P<0.05),效果与AAV MIF shRNA组及LY294002组相近;分子机制研究表明,小儿止哮平喘颗粒中、高剂量组MIF蛋白和mRNA表达抑制(P<0.05),同时Akt磷酸化水平下调(P<0.05),p21上调,Cyclin D1、Cyclin D3及CDK6的表达下降(P<0.05)。结论小儿止哮平喘颗粒可通过抑制MIF的表达,并抑制PI3K/Akt信号通路活化,调控细胞周期进程,从而减轻OVA诱导的哮喘小鼠气道炎症,改善气道重塑。
Objective To investigate the intervention effect of Xiao′er Zhixiao Pingchuan Granules(XEZXPCG)on ovalbumin(OVA)-induced airway remodeling in asthmatic mice and its potential mechanism by regulating macrophage migration inhibitory factor(MIF)and inhibiting the phosphoinositide 3-kinase(PI3K)/protein kinase B(Akt)signaling pathway.Methods A total of 96 SPF-grade male BALB/c mice were randomly divided into blank,model,XEZXPCG low/medium/high-dose groups(2.05,4.10,and 8.20 g/kg),adeno-associated virus(AAV)NC shRNA,AAV MIF shRNA(MIF gene silencing),and LY294002(PI3K/Akt inhibitor,1 mg/kg)groups(12 mice in each group).Asthma models were established through OVA sensitization and challenge.Airway resistance and the proportions of inflammatory cells(eosinophils and macrophages)in bronchoalveolar lavage fluid(BALF)were detected.Serum inflammatory factor(OVA-IgE,interleukin[IL]-1β,IL-6,tumor necrosis factor-alpha,and interferon-gamma)levels and BALF were quantified.Hematoxylin and eosin,Masson,and periodic acid-Schiff staining were used to evaluate airway wall thickness(Wat/Phm),smooth muscle area(Wam/Phm),collagen deposition,and goblet cell metaplasia.Western blotting,immunofluorescence,and real-time fluorescence-qPCR were used to detect MIF protein and mRNA expressions,as well as activation markers of the PI3K/Akt pathway and cell cycle-related proteins(including cyclin-dependant kinase 6[CDK6],Cyclin D1,Cyclin D3,and p21),in lung tissues.Results Compared to the model group,a XEZXPCG medium or high-dose significantly reduced airway resistance(P<0.05),improved the imbalance of eosinophil and macrophage proportions in BALF,and decreased inflammatory factor levels in serum and BALF(P<0.05).XEZXPCG medium or high-dose alleviated airway epithelial damage,goblet cell hyperplasia,and collagen fiber deposition,and reduced the Wat/Phm and Wam/Phm(P<0.05),with effects comparable to those of the AAV MIF shRNA and LY294002 groups.XEZXPCG medium and high-inhibited MIF protein/mRNA expression(P<0.05),downregulated Akt phosphorylation(P<0.05),upregulated p21 protein expression,and downregulated Cyclin D1,Cyclin D3,and CDK6 expressions(P<0.05).Conclusion XEZXPCG alleviates airway inflammation and improves airway remodeling in OVA-induced asthmatic mice by inhibiting MIF expression,downregulating the PI3K/Akt signaling pathway,and regulating cell cycle progression.XEZXPCG enhances airway remodeling through MIF-mediated PI3K/Akt pathway regulation.
作者
陈恂
安子萌
李敏
万通
黄静
冀晓华
燕晓茹
CHEN Xun;AN Zimeng;LI Min;WAN Tong;HUANG Jing;JI Xiaohua;YAN Xiaoru(Xiyuan Hospital,China Academy of Chinese Medical Sciences,Beijing 100091,China)
出处
《北京中医药大学学报》
北大核心
2025年第10期1365-1376,共12页
Journal of Beijing University of Traditional Chinese Medicine
基金
国家自然科学基金青年科学基金项目(No.82104934)
中国中医科学院科技创新工程项目(No.CI2021A02508)
中国中医科学院优秀青年科技人才培养专项(No.ZZ15-YQ-007)
中国中医科学院西苑医院师承项目(No.0203070)。
关键词
气道重塑
儿童哮喘
巨噬细胞迁移抑制因子
磷脂酰肌醇3激酶/蛋白激酶B信号通路
小儿止哮平喘颗粒
小鼠
airway remodeling
childhood asthma
macrophage migration inhibitory factor
phosphoinositide 3-kinase/protein kinase B signaling pathway
Xiao′er Zhixiao Pingchuan Granules
mice
