| BackgroundBronchial asthma (asthma) is a chronic airway inflammatory disease of respiratory system. Currently, it is defined as a chronic inflammatory disorder of the airways in which many cells (eosinophils, mast cells, T lymphocytes, neutrophils, epithelia cells) and cellular elements play a role. The frequent occurrence of injury and repair initiated by chronic inflammation could lead to the structural changes in airway, collectively termed as airway remodeling. Airway remodeling is characterized by airway wall thickening, subepithelial fibrosis, mucous metaplasia, fibroblast hyperplasia, myocyte hyperplasia and hypertrophy, epithelial hyperplasia, increased vascularity and angiogenesis. Generally, airway remodeling is thought to contribute to airway hyperresponsiveness and reversible or irreversible airflow limitation. As a member of matrix metalloproteinases (MMPs), MMP-9 could degrade most of components in extracellular matrix (ECM), and its activity is specifically inhibited by tissue inhibitor of metalloproteinase-1 (TIMP-1). The imbalance of MMP-9/TIMP-1 could disrupt the normal turnover of ECM, and may participate in airway inflammation and airway remodeling.Glucocorticoids are the first line thrapy for the control of asthma, they are capable of decreasing the number and activation of inflammatory cells in airway, inhibiting the inflammatory mediators synthesized by inflammatory cells and structural cells and eliciting apoptosis in inflammatory cells. The long-term inhalation of steroids is safeand beneficial to the improvement of pulmonary function, but it remains uncertain whether airway remodeling could be prevented or inhibited by steroids. It is worthwhile to investigate when the airway remodeling occurs in the course of asthma, how both the MMP-9 and TIMP-1 express in airway remodeling and the modulation by steroids as well as whether the early treatment of steroids could prevent airway remodeling. Objective1. To observe the changes of airway inflammation and remodeling in a murine model of chronic asthma,2. To observe the effect of early treatment with dexamethasone(DEX) on the occurrence and development of airway inflammation and remodeling,3. To investigate the protein and mRNA levels of MMP-9 and TIMP-1 in the airway and lung tissue of murine model of asthma,4. To investigate the modulation effect of DEX on the protein and mRNA levels of MMP-9 and TIMP-1.Methods77 Balb/c mice were randomly divided into three groups: the saline/saline group, the OVA/OVA group and OVA/DEX treated group. On the day 0 and 7 the asthmatic group and DEX treated group were sensitized by ovalbumin (OVA), and from the day 14 onward were challenged with OVA two days a time, thrice a weekss. The DEX treated mice were treated with intraperitoneal injection (i.p) of DEX (1.5mg/kg) 30 min before the OVA challenge every time. Simultaneously, all the mice were given 50mg/kg BrdU (i.p) 30 min before OVA challenge twice a weekss. On Day 28(2w), 42(4w) and 70(8w), three groups of mice were sacrificed. The lung sections were sectioned with HE, Masson PAS staining, and immunohistochemistry for BrdU, MMP-9 and TIMP-1. The parameters including the number of inflammatory cell per unit airway, perimeter of basement membrane (Pbm), area of the mucous cell layer (WAmuc), smooth muscle area (WAm), inner way area (WAi), collagen area (Wcol), goblet cell percentage andBrdU-positive cell per mm Pbm, mean optical density of MMP-9,TIMP-1 were measured by MetaMorph image analysis software. The content of OVA-specific IgE in serum was determined by ELISA. The expression level of MMP-9, TIMP-1, Collagenlll, CollagenlV mRNA were assessed by semiquantitative RT-PCR. Results1. The effect of OVA and DEX on the number of inflammatory cells The number of inflammatory cells per unit airway increased in OVA/OVA groups after 2, 4 and 8 weekss of OVA challenge compared with saline/saline groups (all P<0.01), while the number of inflammatory cells in OVA/DEX groups was less than OVA/OVA groups (all PO.01), but greater than saline/saline groups (all P<0.01). The number of inflammatory cells did not increase further in each group after 4 and 8 weekss of OVA or saline challenge compared with 2 weeks (P>0.05).2. The effect of OVA and DEX on the level of OVA-specific IgE The level of OVA-specific IgE in sera increased in OVA/OVA groups after 2, 4 and 8 weeks of OVA challenge compared with saline/saline groups (all P<0.01), while the level of OVA-specific IgE in OVA/DEX groups was lower than OVA/OVA groups (all P<0.01), but higher than saline/saline groups (all P<0.01). The level of OVA-specific IgE did not increase further in each group after 4 and 8 weeks of OVA or saline challenge compared with 2 weeks (all P>0.05).3. The effect of OVA and DEX on the parameters of airway structure WAmuc/PbnK Wam/Pbm and WAi/Pbm increased in OVA/OVA groups after 2,4 and 8 weeks of OVA challenge compared with saline/saline groups(all P<0.01). Wamuc/Pbm and Wam/Pbm in OVA/DEX group did not increased, but Wai/Pbm increased after 2 weeks of OVA challenge compared with that of saline/saline group (P<0.05). WAmuc/Pbm, Wam/Pbm and WAi/Pbm did not increase further in each group after 4 and 8 weeks of OVA or saline challenge compared with 2 weeks (all P>0.05).4. The effect of OVA and DEX on the collagen deposition Wcol/Pbm increased inOVA/OVA groups after 2, 4 and 8 weeks of OVA challenge compared with saline/saline groups (PO.01), while Wcol/Pbm in OVA/DEX groups was decreased compared with OVA/OVA group, but increased compared with saline/saline groups. Wcol/Pbm in OVA/OVA groups increased further after 8 weeks of OVA challenge compared with 2 weeks of OVA challenge (PO.01). However, Wcol/Pbm in saline/saline and OVA/DEX groups did not increase further after 4 and 8 weeks of saline or OVA challenge compared with 2 weeks (P>0.05).5. The effect of OVA and DEX on the goblet cell score The goblet cell score was zero in saline/saline groups, while it increased in other groups and it was greater in OVA/OVA groups than OVA/DEX groups after 2, 4 and 8 weeks of saline or OVA challenge (P<0.05, PO.01, PO.01). The goblet score did not increase further in OVA/OVA groups, but decreased further in OVA/DEX groups after 4 and 8 weeks of OVA challenge compared with 2 weeks (PO.05, P<0.01).6. The effect of OVA and DEX on the number of BrdlT cell The number of BrdU+ cell/1 mm basement membrane (Bm) increased in OVA/OVA groups after 2, 4 and 8 weeks of OVA challenge compared with saline/saline group(P<0.01),and it was greater than OVA/DEX groups(P<0.05). The number of BrdU+ cell/1 mm Bm increased further in OVA/OVA and OVA/DEX groups after 4 and 8 weeks of OVA challenge compared with 2 weeks (all P <0.05), and it was also increased saline/saline group after 8 weeks of saline challenge compared with 2 weeks (PO.05).7. The effect of OVA and DEX on the expression of MMP-9,TIMP-1 The mean optical density (MOD) of MMP-9 increased after 2, 4 and 8 weeks of OVA challenge in OVA/OVA groups compared with saline/saline groups (all P O.01), and it was greater in OVA/DEX groups than saline/saline groups (all P O.01) and less than OVA/OVA groups (PO.05,PO.01,PO.01). The MOD of MMP-9 increased further in OVA/OVA groups after 4 and 8 weeks of OVA challenge compared with 2 weeks (PO.01,PO.05). The MOD of TIMP-1 increased after 2, 4 and 8 weeks of OVA challenge in OVA/OVAgroups compared with saline/saline groups (P<0.05,P<0.01,P<0.01), and it was decreased in OVA/DEX group after 8 weeks of OVA challenge compared with OVA/OVA group (P<0.01). Moreover, the MOD of TIMP-1 in OVA/OVA group after 8 weeks of OVA challenge increased further compared with 2 weeks(P<0.05). However, the MOD of MMP-9 and TIMP-1 did not increase or decrease after 4 and 8 weeks of saline or OVA challenge in saline/saline and OVA/DEX group compared with 2 weeks (P>0.05).8. The effect of OVA and DEX on the mRNA expression of MMP-9 and TIMP-1 The mRNA levels of MMP-9 and TIMP-1 increased in OVA/OVA groups after 2, 4 and 8 weeks of OVA challenge compared with saline/saline groups (all P <0.01). The mRNA level of MMP-9 decreased in OVA/DEX groups after 2, 4 and 8 weeks of OVA challenge compared with OVA/OVA groups (P<0.05,P<0.01,P<0.01), while the mRNA level of TIMP-1 decreased in OVA/DEX groups after 4 and 8 weeks of OVA challenge (P<0.05,P<0.01). The mRNA level of MMP-9 in OVA/OVA groups increased after 4 and 8 weeks of OVA challenge compared with 2 weeks (P<0.01, P<0.01), while the mRNA level of TIMP-1 did not increased accordingly. The mRNA levels of MMP-9 and TIMP-1 did not increase or decrease in saline/saline or OVA/DEX groups after 4 and 8 weeks of saline or OVA challenge compared with 2 week.9. The effect of OVA and DEX on the mRNA expression of Collagen III ,Collagen IV The mRNA level of Collagen III increased in OVA/OVA groups after 2, 4 and 8 weeks of OVA challenge compared with saline/saline groups (all P <0.01), and the level of Collagen III in OVA/OVA group after 8 weeks of OVA challenge was higher than OVA/DEX group (P<0.01). The mRNA level of Collagen IV increased in OVA/OVA groups after 4 and 8 weeks of OVA challenge compared with saline/saline groups (all PO.01), while the level was not statistically different between OVA/OVA group and OVA/DEX group. The mRNA levels of Collagen III and Collagen IV increased further in OVA/OVA groups after 8 weeks of OVA challenge compared with 2 weeks... |
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