Lu Yao1, Siqi Wang2, Pan Wei2, Kaifan Bao2, Weiyuan Yuan2, Xiaotong Wang2, Jie Zheng3, Min Hong4. 1. Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medical, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China; Department of Biotherapy, Bayi Hospital Affiliated Nanjing University of Chinese Medicine, Nanjing 210001, China. 2. Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medical, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China. 3. Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medical, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China; Department of Pharmacology, School of Medicine and Life Sciences, Nanjing University of Chinese Medicine, Nanjing 210023, China. 4. Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medical, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China. Electronic address: hongmin72@126.com.
Abstract
BACKGROUND: Long-term exposure to aeroallergens such as house dust mite (HDM) could result in airway inflammation and airway remodeling, characteristic features of allergic asthma. Huangqi-Fangfeng (HF), an important "couplet medicines" of Yu-Ping-Feng-San (YPFS), mediates allergen-induced airway inflammation in mice, but its role in the airway remodeling is not known. PURPOSE: To evaluate the effects of HF on airway remodeling of allergic asthma in a murine model and to investigate the underlying mechanisms in vivo and in vitro. METHODS: The main components of HF were analyzed by HPLC. The HDM-induced asthma mice model was established to study the effects of HF on airway inflammation and airway remodeling in vivo. Enhanced pause (Penh) index value was used as an indicator of airway hyper-reactivity. Bronchoalveolar lavage fluid (BALF) was processed for differential cell counting and determination of cytokines production. The lungs were fixed in 4% paraformaldehyde for histological examination after staining with H&E, trichrome and IHC. Production of interleukin (IL)-4, IL-5, IL-13, and transforming growth factor beta-1 (TGF-β1) in BALF and lung tissues, IgE in serum were measured by ELISAs. Expression of epithelial markers and mesenchymal markers were detected by immunohistochemistry and western blots. The effects of HF and its components on epithelial-mesenchymal transition (EMT) were detected in human bronchial epithelial cells (16HBE) treated with TGF-β1 and HDM. RESULTS: The main components of Huangqi-Fangfeng detected by HPLC were Calycosin, Formononetin and Cimifugin. In HDM-induced allergic asthma mice model, respiratory exposure to HDM lead to airway hyperresponsiveness and thickening of the smooth muscle layer in the airway. TGF-β1 levels increased in mice airways while epithelial cells lost expression of E-cadherin and gained expression of the mesenchymal proteins N-cadherin, α-SMA and collagen І. These changes were relieved by treatment with HF. Furthermore, restored epithelial markers expression treated with individual components were also detectable in 16HBE cells. CONCLUSION: These results demonstrated that Huangqi-Fangfeng protected against allergic airway remodeling through inhibiting epithelial-mesenchymal transition process in mice via regulating epithelial derived TGF-β1.
BACKGROUND: Long-term exposure to aeroallergens such as house dust mite (HDM) could result in airway inflammation and airway remodeling, characteristic features of allergic asthma. Huangqi-Fangfeng (HF), an important "couplet medicines" of Yu-Ping-Feng-San (YPFS), mediates allergen-induced airway inflammation in mice, but its role in the airway remodeling is not known. PURPOSE: To evaluate the effects of HF on airway remodeling of allergic asthma in a murine model and to investigate the underlying mechanisms in vivo and in vitro. METHODS: The main components of HF were analyzed by HPLC. The HDM-induced asthmamice model was established to study the effects of HF on airway inflammation and airway remodeling in vivo. Enhanced pause (Penh) index value was used as an indicator of airway hyper-reactivity. Bronchoalveolar lavage fluid (BALF) was processed for differential cell counting and determination of cytokines production. The lungs were fixed in 4% paraformaldehyde for histological examination after staining with H&E, trichrome and IHC. Production of interleukin (IL)-4, IL-5, IL-13, and transforming growth factor beta-1 (TGF-β1) in BALF and lung tissues, IgE in serum were measured by ELISAs. Expression of epithelial markers and mesenchymal markers were detected by immunohistochemistry and western blots. The effects of HF and its components on epithelial-mesenchymal transition (EMT) were detected in human bronchial epithelial cells (16HBE) treated with TGF-β1 and HDM. RESULTS: The main components of Huangqi-Fangfeng detected by HPLC were Calycosin, Formononetin and Cimifugin. In HDM-induced allergic asthmamice model, respiratory exposure to HDM lead to airway hyperresponsiveness and thickening of the smooth muscle layer in the airway. TGF-β1 levels increased in mice airways while epithelial cells lost expression of E-cadherin and gained expression of the mesenchymal proteins N-cadherin, α-SMA and collagen І. These changes were relieved by treatment with HF. Furthermore, restored epithelial markers expression treated with individual components were also detectable in 16HBE cells. CONCLUSION: These results demonstrated that Huangqi-Fangfeng protected against allergic airway remodeling through inhibiting epithelial-mesenchymal transition process in mice via regulating epithelial derived TGF-β1.