Regulation And Mechanism Of Autophagy In OVA And HDM Induced Asthma Airway Inflammation

Bronchial asthma (asthma for short), one of the most common chronic airway inflammatory diseases, has been a serious lite-threatening to human health. At present,300million individuals are affected by asthma worldwide, of which there are at least30millioin patients in China. Althouth the incidence of this disease is not that high, its fatality rate keeps rather high. Asthma is a disease primarily based on airway local infiltration by eosinophils, mast cells and T lymphocytes, main clinical manifestations of which are airway hyperresponsiveness, mucus hypersecretion and reversible airflow limitation. Traditional allergic asthma airway inflammation is characterized by increased levels of Th2cells, eosinophils, activated mast cells and multiple cytokines largely secreted by Th2cells (including IL-4, IL-5, IL-13and so on). In recent years some studies have shown that some new cell types like Th17cell, Treg cell and DC cell and new cytokines such as IL-25, IL-33and TSLP play crucial roles in development of asthma. However, the molecular mechanism of asthma remains largely unclear.As a significant defence and protection mechanism in organism, autophagy plays an important role in maintaining homeostasis and adaptting the change of micro environment. In some specific environment, for instance, starvation, lack of growth factors or oxygen, a kind of double-membrane constraction, namely autophagosome, generates in cells. Autophagosome could grasp the damaged cytoplasmic organoids including mitochondria, endoplasmic reticulum and Golgi apparatus, and the denatured protein, which then combine to form autolysosome with lysosome. The autolysosome digests and degrades kinds of cytoplasmic organoids and biological macromolecules with the help of proteolytic enzymes in lysosome, providing enegy for the synthesis of new biological macromolecules. Autophagy (including the generation of autophagosome, seizing impaired cytoplasimc organoids and biological macromolecules) is regulated by varieties of factors called Atg (autophagy-related) family. As a member of that family, Beclin-1(also called Atg6) plays a key role in the generation of autophagosome, meanwhile LC3B (a homologous protein of Atg8) is vital in seizing impaired cytoplasimc organoids and biological macromolecules. The ratio of the lipid LC3BⅡ to LC3BⅠ (noumenon of LC3B) has been widely known as the standard detection of autophagy by Western Blot. Besides, currently there have been over30atg genes and proteins found related to the molecular mechanisms of autophagy.In spite of that autophagy helps to maintain a stable internal environment, in particular cases, massive consuming of cytoplasmic organoids and macromolecules will cause cells to die, which is called autophagic death. Therefore, in different conditions, autophagy could protect cell survival as well as promote cell death. And it plays different roles in different diseases. At the moment, more and more studies indicate that autophage takes an important part in kinds of respiratory system diseases, for example, chronical obstructive pulmonary disease (COPD), pulmonary arterial hypertension, acute lung injury and cystic fibrosis. Nevertheless, studies about the role of autophagy in chronical asthma are ralely reported.My research is divided into two parts:one is to illuminate the regulations and mechanisms of autophagy in OVA-induced asthma airway inflammation, basing on the asthma model of LC3B-/-mice (autophagy related gene knock-out mice), the other is to elucidate roles of autophagy in HDM-induced asthma airway infammation, basing on the asthma model of LC3B-/-mice and bone marrow transplanation technology. Part I Regulation of autophagy in OVA-induced asthma airway inflammationObjective:To investigate the regulation roles and mechanisms of autophagy in OVA-induced asthma airway inflammation.Methods:In vivo, male C57BL/6mice and the same age male LC3B-/-mice are respectivily randomly divided into two groups:NS group (WT/NS and LC3B/NS) and OVA group (WT/OVA and LC3B/OVA). In OVA group, mice were sensitized and challenged with ovabumin (OVA). In NS group, mice were sensitized and challenged with isodose normal saline (NS) replacing OVA. Mice were sacrificed24hours after the final OVA challenge, and the total number of inflammatory cells in bronchoalveolar fluid (BALF), the number and the ratio of eosinophils in BALF were accessed. Lungs were processed for histologic staining (HE) to analyzed airway inflammatory infiltrate and also for periodic acid-schiff staining to observe mucus secretion. The mRNA expression of lung tissue was analyzed to detect the expression of some inflammatory factors. The secretion of inflammatory factors in BALF was detected by enzyme-linked immunosorbent assay (ELISA). In vitro, human lung epithelial cells (BEASE-2B) were treated with IL-13. The IL-25mRNA expression was detected by Q-PCR, then autophage-related gene was silented by si-RNA and IL-25induction was analyzed. Human bronchial epithelial cells (HBE) were treated with OVA, Western Blot of LC3B protein was analyzed.Results:In vivo, OVA-induecd asthma airway inflammation was apparently relieved after LC3B was knocked out. The total number of inflammatory cells, the ratio and the total number of eosinophils in BALF decreased evidently. Lung inflammation infiltrate was improved. The secretion of mucus was down-regulated. The secretion of inflammatory fators in BALF and the expression of inflammatory factors in lung tissue were down-regulated as well. In vitro, the expression of IL-25was up-regulated in BEASE-2B cells treated with IL-13, while expression of IL-25was down-regulated in BEASE-2B cells whose autophagy-related genes were silented after treatment with IL-13. Furthermore, the expression of LC3B protein increased in HBE cells after treatment with OVA.Conclusion:OVA-induced airway inflammation was relieved in autophagy-related gene LC3B knock-out mice potentially through inhibiting the secretion of inflammatory fators like IL-25and IL-33to alleviate airway epithelial injuries.Part Ⅱ Regulation of autophagy in HDM-induced asthma airway inflammationObjective:To investigate the regulation roles and mechanisms of autophagy in HDM-induced asthma airway inflammation.Methods:Male C57BL/6mice and the same age male LC3B-/-mice are respectively randomly divided into two groups:NS group (WT/NS and LC3B/NS) and HDM group (WT/HDM and LC3B/HDM). In HDM group, mice were sensitized and challenged with house dust mite extract (HDM) in way of airway intratracheally. In NS group, mice were sensitized and challenged with isodose normal saline (NS) replacing HDM. Mice were sacrificed48hours after the final HDM challenge, and the total number of inflammatory cells in bronchoalveolar fluid (BALF), the number and the ratio of eosinophils in BALF were accessed. The mRNA expression of lung tissue was analyzed to detect the expression of some inflammatory factors. The secretion of inflammatory factors in BALF was detected by enzyme-linked immunosorbent assay (ELISA). Bone marrow of LC3B-/-mice was transplanted to WT mice, and the phenotypes of asthma were detected in bone marrow LC3B knock-down mice. Afterwards, bone marrow of WT mice were transplanted back to LC3B-/-mice and observed whether the phenotype of asthma was relieved in LC3B-/-mice.Results:HDM-induecd asthma airway inflammation was apparently relieved after LC3B was knocked out. The total number of inflammatory cells, the ratio and the total number of eosinophils in BALF decreased evidently. The secretion of inflammatory fators in BALF and the expression of inflammatory factors in lung tissue were down-regulated as well. Transplanting bone marrow of WT mice to LC3B-/-mice significantly decreased the HDM-induced asthma airway inflammation, while transplanting bone marrow of LC3B-/-mice to WT mice (equals to bone marrow LC3B knock-out) exerted very minor protective effect.Conclusion:HDM-induced airway inflammation was relieved in autophagy-related gene LC3B knock-out mice primarily depending on airway epithelial cells but not on bone marrow.