| Objective:This study is aimed to investigate effects of Gubenfangxiaoyin on of ADAM33, ETS-1, MMPs/TIMPs and airway remodeling in asthma models with molecular biotechnology to explore the possible mechanisms.Methods:Experiment in vivoChronic asthma murine model was developed by ovalbumin (OVA) sensitization and airway challenge and respiratory syncytial virus (RSV) infection. Treatments with Montelukast Sodium or GBFXY were maintained for four weeks. The blood, bronchoalveolar lavage fluid (BALF), and lung tissue of mice in each group were collected. Pathological and morphological changes of. lungs were examined by hematoxylin and eosin staining, periodic acid-Schiff staining and Masson’s trichrome staining. Enzyme linked immunosorbent assay was used to measure the levels of serum immunoglobulin (Ig) E, Interlukin (IL)-12, transforming growth factor (TGF) β1 in BALF and IL-1β in lung tissue. mRNA expressions of IL-6, IL-13, IL-1β, TGFβ1, ADAM33, ETS-1, MMP-2, MMP-9, TIMP-1, TIMP-2 in lung tissue were determined by real-time reverse transcription polymerase chain reaction (real-time RT-PCR). Western Blot was conducted to measure the relative expression of ADAM33, ETS-1, MMP-2, MMP-9, TIMP-1, TIMP-2 protein. Immunohistochemistry was performed to confirm the expressions and localization of ADAM33, ETS-1, MMP-2, MMP-9, TIMP-1, TIMP-2 protein in lung tissue.Experiment in vitroSerum containing GBFXY was prepared according to a serum pharmacology method. Human fetal lung fibroblasts (HFLFs) were stimulated with IL-4 and incubated with serum containing GBFXY or Montelukast for 24 h, respectively. Fibroblast proliferation was measured by MTT assay. mRNA and protein expressions of ADAM33, ETS-1, MMP-2, MMP-9, TIMP-1, TIMP-2 in HFLFs were determined by real-time RT-PCR and Western Blot assay. Immunofluroscence was performed to confirm the expressions and localization of ADAM33, ETS-1, MMP-2, MMP-9, TIMP-1, TIMP-2 protein in HFLFs.Results:1. Effect of GBFXY on pulmonary inflammation, mucus secretion and collagen deposition in mice with asthmaCompared to the control group, pathological changes of lung tissue from BALB/c mice in model group was significant such as inflammatory cells infiltration around bronchus, alveolar epithelial cell shedding, hyperplasia of fibrous connective tissue; goblet cell metaplasia, mucus secretion, and collagen deposition. GBFXY and Montelukast improved these pathological changes and no difference existed between them.2. Effect of GBFXY serum on proliferation of HFLFsIL-4 augmented the proliferation of HFLFs in a concentration-dependent manner range from 0 ng/mL to 50 ng/mL. Serum containing GBFXY or Montelukast exerted inhibitory effect of fibroblast proliferation in a concentration-dependent manner range from 5% to 20%.3. Effect of GBFXY on expressions of asthma susceptibility gene ADAM33Expressions of ADAM33 mRNA and protein in lung tissue of BALB/c mice in model group were significantly higher than in control group. When compared to model group, expressions of ADAM33 mRNA and protein in lung tissue of BALB/c mice in Montelukast group and four GBFXY groups were significantly down-regulated. ADAM33protein mainly located on airway wall.With IL-4-stimulation, expressions of ADAM33 mRNA and protein in HFLFs in model group were significantly elevated. ADAM33 mRNA and protein in HFLFs in Montelukast and GBFXY groups were significantly down-regulated with no significance in GBFXY group. ADAM33 protein mainly located on cell membrane and in cytoplasm.4. Effect of GBFXY on expressions of transcription factor ETS-1Expressions of ETS-1 mRNA and protein in lung tissue of BALB/c mice in model group were significantly higher than in control group. When compared to model group, expressions of ETS-1 mRNA and protein in lung tissue of BALB/c mice in Montelukast group and four GBFXY groups were significantly down-regulated. ETS-1 protein mainly located on airway wall.With IL-4-stimulation, expressions of ETS-1 mRNA and protein in HFLFs in model group were significantly elevated. ETS-1 mRNA and protein in HFLFs in Montelukast and GBFXY groups were significantly down-regulated. ETS-1 protein mainly located on cell membrane and in cytoplasm.5.Effect of GBFXY on Matrix metalloproteinases and tissue inhibitors of metalloprotein asesExpressions of MMP-2, TIMP-2, MMP-9, TIMP-1 mRNA and protein in lung tissue of BALB/c mice in model group were significantly higher than those in control group. When compared to model group, expressions of MMP-2, TIMP-2, MMP-9, TIMP-1 mRNA and protein in Montelukast group and four GBFXY groups were significantly down-regulated and no difference was found in TIMP-2 protein in GBFXYH group. Protein of MMP-2, TIMP-2, and TIMP-1 mainly located on airway wall and MMP-9 protein located in lung.MMP-2/TIMP-2 ratio in lung tissue of BALB/c mice in model group was significantly lower than in control group at mRNA level; When compared with model group, MMP-2/TIMP-2 ratio was decreased in Montelukast group and elevated in four GBFXY groups. MMP-9/TIMP-1 ratio in lung tissue of BALB/c mice in model group was significantly lower than in control group at mRNA level; When compared with model group, MMP-9/TIMP-1 ratio was elevated in Montelukast, GBFXYM, GBFXYH groups.With IL-4-stimulation, expressions of MMP-2, TIMP-2, MMP-9, TIMP-1 mRNA and protein in HFLFs in model group were significantly higher than in control group. When compared to model group, expressions of MMP-2, MMP-9, TIMP-1 mRNA and protein in HFLFs were significantly down-regulated with no difference in TIMP-2 mRNA in Montelukast group.With IL-4-stimulation, MMP-9/TIMP-1 and MMP-2/TIMP-2 ratios were significantly elevated at mRNA levels. Serum containing GBFXY or Montelukast reduced MMP-9/TIMP-1 ratio and MMP-2/TIMP-2 ratio. MMP-2, MMP-9, TIMP-1 and TIMP-2 protein mainly located on cell membrane and in cytoplasm.6. Effect of GBFXY on asthma associated cytokinesThe average concentration of serum total IgE and TGFβ1 in BALF and IL-1β in lung tissue of BALB/c mice in model group were significantly higher than those in control group; the average concentration of serum total IgE and TGFβ1 in BALF and IL-1β in lung tissue in Montelukast and four GBFXY groups were significantly decreased. The average concentration of IL-12 in BALF of BALB/c mice in model group was significantly lower than that of control group, when compared to the model group, average concentration of IL-12 in Montelukast group was significantly increased and average concentration of IL-12 of four GBFXY groups were higher than that of model group and lower than that of Montelukast group with no significance. mRNA expressions of IL-6, IL-13, IL-1β, TGFβ1 in lung tissue of BALB/c mice in model group was significantly higher than those of control group. When compared with model group, mRNA expressions of IL-6, IL-13, IL-1β, TGFβ1 in lung tissue of BALB/c mice in Montelukast group and four GBFXY groups were significantly decreased.Conclusion:1.GBFXY can obviously alleviate the pathological changes of lung tissue of asthma model mice; reduce inflammatory cells infiltration around bronchus, alveolar epithelial cell shedding, hyperplasia of fibrous connective tissue; reduce goblet cell metaplasia, mucus secretion, and the deposition of collagen.2.Chronic asthma murine model was developed by ovalbumin sensitization and airway challenge and respiratory syncytial virus infection. Excessive expression of ADAM33 was found in BALB/c mice exposed to OVA and RSV and HFLFs exposed to IL-4, and GBFXY can down-regulate expression of ADAM33 and reduce the susceptibility to asthma. It may be one of the mechanisms that GBFXY can improve the constitution of children with asthma.3.Both exposure to OVA and RSV and IL-4 stimulation can induce expression of the transcription factor ETS-1, promote the transcription of MMP-2 and MMP-9, increase the expressions of MMP-2 and MMP-9 which stimulate the production of endogenous tissue inhibitor TIMP-2 and TIMP-1, lead to imbalance between MMPs and TIMPs resulting in ECM deposition and collagen fiber hyperplasia, leading to airway repairing and remodeling. GBFXY can reduce the expression of ETS-1, transcription of MMP-2 and MMP-9 gene, reduce the expressions of MMP-2, MMP-9, TIMP-1 and TIMP-2, regulate the balance between MMP-2/TIMP-2 and MMP-9/TIMP-1, and reduce the expression of profibrotic cytokine TGFβ1 in order to abolish excessive production and deposition of ECM, diminish goblet cell mucus secretion, reduce collagen deposition and delay of airway remodeling process. Early administration of GBFXY can also prevent airway remodeling.GBFXY can prolong asthma remission duration and reduce the frequency of asthma attack targeting against ETS-1 and specific MMPs and TIMPs.4. Exposure to OVA and RSV can promote the production of IgE from B lymphocytes, and also promote the secretion and release of cytokines IL-1β, IL-6 and IL-13 from T lymphocytes, activation of inflammatory cells, release of inflammatory mediators. GBFXY could reduce the IgE level in serum and inhibit secretion of some cytokines like IL-1β, IL-6, IL-13 and reduce airway inflammation. |
