Study On The Effect Of Theaflavins On MUC5AC Of Inflammation Airway And Its Signal Transduction Mechanism

Chronic obstructive pulmonary disease (COPD) are common diseases in respiratory medicine. Its incidence was increasing year by year, it is the main cause for chronic diseases and death. Among all those pathological changes of COPD, airway mucus hypersecretion is a very important clinicopathologic feature. Excessive secretion of mucus retention in the airway, further exacerbating the already narrow airway obstruction, directly leads to the deterioration of illness and even death. The basic structure of airway mucus hypersecretion is considered to be a wide range of epithelial goblet cell metaplasia and submucosal bronchial gland hyperplasia caused by chronic airway inflammation, but in small airways, its mucus hypersecretion is mainly contributed by the hyperplasia, metaplasia of goblet cells as its lack of the basic structure of bronchial gland hyperplasia.With the depth of airway mucus studies, more than 20 different types of mucin genes have been found. Mucin (mucin, MUC) 5AC is major secretory mucin of airway, it is highly expressed in human airway epithelial goblet cells as well as a pathological increase in the major mucin phenotype. Much stimulating factor may promote mucus secretion, including a variety of inflammatory mediators such as: IL-9, IL-1, TNF-α, etc. Pathogenic microorganisms and their products, Cellular proteins such as human neutrophil elastase (HNE)and epidermal growth factor receptor ligand; reactive oxygen species; air pollutants, they all can activation-related transcription factor binding to mucin5AC gene promoter on the corresponding site to promote transcription through a variety of their own known signal transduction pathway, causing the increase of mucin synthesis. HNE is a highly potential agent to promote mucus secretion, it has a strong effect on promoting the synthesis and secretion of MUC5AC. It can increase mucus through a variety of ways such as the EGFR(epidermal growth factor receptor) signaling pathway, oxidant-dependent mechanism, victoria A acid receptor mechanism and a direct increase in MUC5ACmRNA stability etc. The EGFR pathway is one of the most important signaling pathways. Studies have shown that epidermal growth factor receptor is a receptor tyrosine kinase, it is in the living center of the signal transduction pathway of the airway mucus hypersecretion. Therefore, drugs of inhibition of mucus hypersecretion can be able to effectively prevent and mitigate the course of COPD.Theaflavins (TFs) are a class of flavonoids extracted from the black tea. It has a wide range of biological activities, including anti-inflammatory, anti-allergic, anti-oxidation, scavenging free radicals, anti-bacterial, anti-viral and anti-tumor effects. Although its mechanism is not entirely clear, but it's derived from plants with little drug side effects. Therefore in recent years, both at home and abroad, people are studying its pharmacological effects, and intend to use it in clinical. Studies indicate that the biological effect of theaflavins is very beneficial for prevention and treatment of lung diseases, it may become an effective way to treat lung diseases. As theaflavins can significantly inhibit the occurrence and development of tumors, and can also induce tumor cell apoptosis, it is called green cancer biology protective agents by foreign literature. Although anti-tumor EGFR blockers currently used has a downward effect for mucus hypersecretion, they are too expensive to conventional application. Whether theaflavins can regulate mucus secretion, and the intensity of the monomer as well as its role in the way they work have not been reported yet. Therefore, this study constructed tectonic model of inflammatory mucus hypersecretion by using HNE to stimulate A549 cells, selected theaflavins and its monomer to intervene, then applied various technologies to detect its impact on MUC5AC, explored its roles and ways by the determination of the transduction factor in each cell, thus laid the foundation of drug's efficacy for the development of theaflavins at the prevention and treatment of COPD.Experiment is divided into three parts:Part one:Objective: Firstly, the use of resin separation purification to purify crude theaflavins to achieve the purity requirements of the experiment. And then use polyamide column chromatography to separate theaflavins to receive the monomer theaflavins. Then purified theaflavins added to cells stimulated by HNE A549 to intervente, testing its impact on MUC5AC.Methods: Resin separation purification method being used to purify 40% purity theaflavins (TFs) crude, so that more than 80% purity theaflavins can be obtained. Then Polyamide column chromatography followed by separating theaflavins 80% purity to receive more than 80% purity of theaflavin (TF1), theaflavin-3/3'-gallate (TF2), and theaflavin-3,3'-digallate (TF3). Then thawed, cultured human lung adenocarcinoma cell line A549, Stimulation with 50nM of HNE, setting up HNE-induced MUC5AC high-expression cell model. Firstly, cells are divided into 6 groups: TFs 0mg/L group,TFs 50 mg/L group,TFs 100 mg/L group,TFs 200 mg/L group,TFs 300 mg/L group,and TFs 400 mg/L group. Using MTT absorption method (MTT method) detected TFs on cell activity. After determining the role of dose, cells were divided into 4 groups to conduct experiments, separately as follows:①the control group;②HNE (50 nM) stimulation;③TFs (concentration, respectively 50,100,200 mg/L) and④epidermal growth factor receptor (EGFR) inhibitor AG1478 (5μM) in intervention group . Reverse transcription - polymerase chain reaction (RT-PCR) detection of changes in MUC5ACmRNA; Enzyme-linked immunosorbent assay (ELISA) quantitative analysis of differences in MUC5AC protein content; cell-immunochemistry and laser confocal microscopy technology provided direct observation of further MUC5AC protein expression changes.Result: MTT results showed that TFs's impact on A549 cell viability dose-dependently. HNE stimulation of MUC5AC gene transcription and protein expression level was significantly higher than that of the control group, the difference has statistical significance (P <0.01). Cell immunofluorescence showed MUC5AC protein located in the cytoplasm of A549 cells, the expression of HNE stimulation is apparently stronger than that of control group. In each TFs treatment group, MUC5AC gene transcription and protein expression were significantly weaker than those of HNE stimulation group, and in a dose-dependent manner. Cell immunofluorescence- laser confocal microscopy results also further confirmed this result.Conclusion: The influence of theaflavins category on A549 cell activity increased with the concentration gradually enhanced. Theaflavins can inhibit the HNE-induced MUC5AC gene transcription and protein overexpression enhanced synthesis in a dose-dependent manner. It has been confirmed that theaflavins can lower mucin level, with value that can be developed.Part Two:Objective: Comparing the three types of monomer theaflavins TF1, TF2, TF3 at different concentrations of human neutrophil elastase (HNE)-induced secretion of MUC5AC in the role of high intensity, and TF3 at different time changes in the expression of MUC5AC.Methods: Firstly, cells were still divided into 6 groups: 0 mg/L group, 50 mg/L group, 100 mg/L group, 200 mg/L group, 300 mg/L group, 400 mg/L group.)Detected TF1, TF2, TF3 on cell activity by using MTT absorption method (MTT method). Cells were divided into 4 groups afterwards:①control group;②HNE (50 nM) stimulation;③TF1, TF2, TF3 (concentration, respectively 50,100,200 mg/L) in intervention group and④epidermal growth factor receptor (EGFR) inhibitor AG1478 (5μM) in intervention group. Reverse transcription - polymerase chain reaction (RT-PCR) detection of changes in MUC5ACmRNA; Enzyme-linked immunosorbent assay (ELISA) quantitative analysis of MUC5AC protein content differences.Results: MTT results showed that the impact of TF1, TF2, TF3 on the A549 cell viability is still a dose-dependent, and TF3 can damage cells more seriously than the TF1, TF2. RT-PCR and protein quantitative analysis results showed that if pretreatment is given to TF1, TF2, TF3, comparing with HNE stimulation group, the two indicators were significantly reduced (both P <0.01), and dose is a dose-related, TF3 role to one of the most obvious. There was a positive correlation between the role of TF3 time and the decrease of mucin 5AC. The blocker AG1478 group had stronger effect on decrease of the mucus than theaflavins in each group (P <0.01). Conclusion: Theaflavins monomers: TF1, TF2, TF3 are all able toinhibit HNE-induced A549 cells MUC5AC in the state of high expression. Their inhibition has a positive correlation with the concentration of dose and time, of which the strongest downward effect is TF3.Part Three:Objective: Discussion theaflavins of HNE-induced MUC5AC expression in the role of high cell transduction pathway.Methods: Taking TF3 of final concentration of 200 mg/L as research subjects, The A549 cells were divided into 5 groups: The control group, HNE treatment group, TF3 group, AG1478 group and TF3 + AG1478 group. Reverse transcription - polymerase chain reaction (RT-PCR) being used to detecte changes of MUC5ACmRNA, EGFRmRNA in each group, Western immunoblotting (Western blot) being used to detecte protein expression of EGFR, P-EGFR, P-ERK1/2, P-p38 and P-JNK, Enzyme-linked immunosorbent assay to observe the changes in protein expression of MUC5AC, cell-immunochemistry and laser confocal microscopy technology provided direct observation of protein expression changes in MUC5AC and P-EGFR.Results: After the intervention of TF3 , RT-PCR determination of MUC5ACmRNA and EGFRmRNA were significantly reduced compared with HNE stimulation. The results of western blot also indicated that corresponding EGFR and P-EGFR protein expression significantly decreased (all P <0.01); P-ERK1/2 and P-p38 increased after be stimulated by HNE, and the former is more significant. No significant differences can be observed for P-JNK. P-ERK1/2 and P-p38 were reduced to varying degrees (the former P <0.01; latter P <0.05) after the intervention of theaflavins category and AG1478.Conclusion: Theaflavins can realize the impact on downstream channels and thereby exert its inhibition of inflammatory airway mucus hypersecretion formation through reducing EGFR levels and the activation of EGFR, partly to deterring EGFR signal transduction pathway, and extracellular signal-regulated kinase (extracellular signal-regulated kinase, ERK) .