The Regulatory Mechanisms Of Total Flavonoids From Sea Buckthorn On Airway Inflammation And Mucus Secretion In “Lounitepo”(chronic Bronchitis)

Chronic bronchitis is a very common respiratory inflammatory disease,which characterized by coughing,sputuming and wheezing.According to the four medical records,it belongs to the category of “Lounitepo” or peigen type pulmonary disease in the traditional Tibetan medicine theory,which means peigen and peigen mucus in the trachea and bronchus is significantly increased,with peigen mucus accumulation,airflow silting or obstruction,induced cough,sputum and wheezing.In Tibetan medicine,“long,chiba and peigen” are considered to be three types of energy and substances.When the three elements affected by abnormal dietary,behavioral and other external factors,the dynamic balance of three elements that have been broken,It causes the weakness of long and chiba,the overabundance of peigen,which induced peigen type pulmonary disease.According to the above theory and viewpoint,we selects the representative Tibetan medicine seabuckthorn,which has the effects of clearing lung heat,promoting blood circulation and curing peigen were selected for pharmacodynamic study.The Tibetan medicine Seabuckthorn is a dried ripe fruit of the plant Hippophae rhamnoides L,which called “?????” in Tibetan,and it is recorded in Yue wang yao zhen firstly.four medical records also recorded and affirmed that it was an main drug for the treatment of respiratory diseases.In the classic book of Tibetan medicine,Jing Zhu Materia Medica recorded that Seabuckthorn tastes acidic and cold in nature,curing the lung diseases and peigen,expelling phlegm,promoting blood circulation.Seabuckthorn as the most important conponent in this prescription of Wu wei sha ji san is an empirical formula for respiration disease,which has been used for many years with remarkable curative effect.Modern pharmacological studies have shown that the main active constituents of seabuckthorn is flavonoids with antibacterial,antiviral,antiinflammatory,antitussive,expectorant,antispasmodic,anti-allergic and so on,however,the effect of TFSB on respiratory inflammatory disease(chronic bronchitis)is not fully known.In this study,We made a hypothesis that the total flavonoids of seabuckthorn(TFSB)regulates airway inflammation and mucus secretion,and the pharmacodynamic and mechanism of TFSB in treating chronic bronchitis were discussed.Objective1.Through depthly the nature and characteristics of Peigen,the physiological and pathological relationship between Peigen and chronic bronchitis are discussed comprehensively.2.To predict and select the active components and core targets of TFSB in the treatment of chronic bronchitis.3.To conduct pharmacodynamic experiments on TFSB in the treatment of chronic bronchitis,and to explore its molecular mechanism of anti-inflammatory and inhibition of mucin secretion.Method1.Digging into the classical books of Tibetan medicine Four Medical Codes,Yue wang yao zhen,Gongzhu Tibetan Medical Records,etc,using Lounitepo or peigen type pulmonary disease as keywords,using CNKI to search the relevant literatures,to summarize,combine and analyze those information,to determine the pathogenesis and treatment principles of chronic bronchitis.2.TCMSP was used to search the effective components of TFSB.PharmMapper network service system,BATMAN-TCM network pharmacology research platform,DrugBank and OMIM database were all used to predict and match effective components of TFSB on the potential target of chronic bronchitis.GO enrichment and KEGG pathway annotation analysis were conducted by MAS 3.0,Cytoscape3.5.1 software was used to establish TFSB's component-target-pathway network for the treatment of chronic bronchitis.3.Based on the previous network pharmacological analysis results,the core components were selected as candidate ligand molecules and applied to ligand preparation through Ligprep wizard to form ligand 3D conformation.The structure of the core target was retrieved from the protein database,and the original PDB structure was modified by the Maestro protein preparation guide.Molecular docking was performed by GLIDE method.The output GScore was calculated by kcal/mol,and the specific ligand-protein interaction pattern was analyzed by standard visual analysis.4.HBE16 were stimulated with LPS/CSE to establish an in vitro bronchitis model.The cytotoxic effect of TFSB on HBE16 cells was examined by CCK8 method.RTqPCR was used to detect the effect of TFSB on the expression of IL-1?,IL-6,CXCL1,COX2 and MUC5 AC in LPS/CSE-induced HBE16 cells.The effects of TFSB on the expression of IL-1?,IL-6,CXCL1,PGE2 and MUC5 AC in HSE16 cells induced by LPS/CSE were detected by ELISA and WB,respectively.In the mechanism research,WB was used to detect the effect of TFSB on LPS/CSE-induced ERK,Akt and PKC? activation in HBE16 cells.5.The in vivo model of chronic bronchitis was established by LPS nasal spray combined with cigarette smoke(CS)exposure.After intervention by TFSB,the number of macrophages,neutrophils and total cells in the alveolar lavage fluid of mice was measured.HE staining was used to observe the pathological changes of lung tissue.Immunohistochemistry was used to detect the expression of IL-1?,IL-6,COX2,CXCL1 and MUC5 AC in lung tissue.Result1."Lounitepo" is the main pathogenesis of peigen disorder in the theory of three causes of Tibetan medicine.Among them,"peigen mucus increases,thickens,poor ventilation,and decreased lung function" is the main contradiction in the occurrence and development of this disease.In clinical treatment,we should grasp the two aspects of "increased peigen mucus and decreased lung function".Flexible use of drugs for nourishing Long and lung,curing peigen and relieving cough and phlegm can achieve good results.2.The relationship between 13 compounds,30 target proteins,64 pathways and chronic bronchitis were visualized through network pharmacology.Among Fc epsilon RI,VEGF and MAPK pathways are the core pathways of the network,and MAPK1,PIK3 CG,AKT1,PKCa are the core targets of the network.These targets play an important role in the prevention and treatment of chronic bronchitis through protein amino acid phosphorylation,signal transduction and molecular functions such as protein binding and transferase activity regulation.3.Molecular docking verification of core components and targets obtained by network pharmacology analysis showed that the quercetin,isorhamnetin,the main components of TFSB,were effectively combined with core targets.For MAPK1,quercetin and isorhamnetin showed binding energy values ranging between-7.28 and-7.859 kcal/mol.For PIK3 CG,the binding energy values of quercetin and isorhamnetin were-8.427 and-9.415 kcal/mol,respectively.4.TFSB remarkably inhibited LPS/CSE-induced expression of IL-1?,IL-6,CXCL1,and MUC5 AC at both mRNA and protein levels in HBE16 bronchial epithelial cells.TFSB also decreased the production of PGE 2 through inhibition the expression of COX2 in LPS/CSE-stimulated HBE16 cells..Mechanistically,TFSB blocked LPS/CSE-induced activation of ERK,Akt,and PKC?5.In vivo experiments showed that bronchoalveolar fluid(BALF)and histological analyses revealed that LPS/cigarette smoke(CS)exposure-induced elevated cell numbers of neutrophils and macrophages in BALF,inflammatory cell infiltration and airway remodeling were remarkably attenuated by TFSB in mice.Immunohistochemical results also confirmed that TFSB decreased the expression of IL-1?,IL-6,COX2,CXCL1,and MUC5 AC in LPS/CS-exposed mice.Conclusion1.From the classical theory of Tibetan medicine,it is deeply recognized that peigen mucus growth is an important factor leads to the pathogenesis of chronic bronchitis,and provide experimental basis for the pharmacodynamics of treating chronic bronchitis from bacon mucus.2.TFSB exerts a potent protective effect against LPS/CS-induced chronic bronchitis through inhibit the expression of IL-1?,IL-6,COX2,PGE2 and MUC5 AC induced by LPS/CS by inhibiting ERK,PI3K/Akt and PKC signaling pathways.