The Regulation Of Melatonin And Luzindole On Asthmatic Inflammation, Airway Remodeling And The Dynamic Expression Of MMP-9/TIMP-1 In Chronic Asthma Mouse Model

Backgroud:Asthma is characterized by chronic airway inflammation and airway structural changes including progressive subepithelial fibrosis, myofibroblast accumulation, airway smooth muscle hyperplasia and hypertrophy, mucous gland and goblet cell hyperplasia, which results in increased airway wall thickening . Airway remodeling was recognized as an indepent paralleled mechanism with airway remodeling. Oxidative stress played an important role in the pathogenisis of airway remodeling. MMP-9, which is synthetized and secreted by inflammatory cells, can degrade a wide range of ECM and possesses a particular potent collagenolytic activity against type IV collagen, the major component of airway basement membrane, suggesting a pivotal role for MMP-9 in promoting airway remodeling in asthma. TIMP-1 is an endogenous inhibitor for MMP-9, which can specifically inhibit MMP-9 activity through binding with both the MMP-9 and PRO-MMP-9. The imbalance of MMP-9/TIMP-1 will disrupt the normal turnover of ECM, and may participate in airway inflammation and remodeling. Pineal gland is an important physiological source of melatonin and believed to be involved in the regulation of the human immune system and the mechanism in asthma.Objective:1.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 melatonin and dexamethasone 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 modulated effect of melatonin and dexamethasone on the protein and mRNA levels of MMP-9 and TIMP-1.5. To investigate the mechanism of the effect of early treatment with melatonin and dexamethasone on the occurrence and development of airway inflammation and remodeling.6. To investigate the mechanism of the modulation of melatonin and dexamethasone to MMP-9 and TIMP-1, to observe the effect occurs at transcriptional or at post- transcriptional level.Method:106 male BALB/c mice were maintained in a laminar flow holding unit and housed in cages on autoclaved bedding in an air-conditioned room on a 12-h light/dark cycle for 1 wk prior to study for the duration of the experiments. Irradiated food and autoclaved water were provided. The procedure used in this study was previously described by Haile S et al with a slight modification. On the day 0 and 7, the mice of control group were intraperitoneally injected ( i.p.) with 0.4 ml of saline , the other mice were i.p. with 0.4ml of the suspension of ovalbumin (OVA) adsorbed to potassium aluminum sulfate (OVA 100 ug, potassium aluminiun sulfate 1.6mg dissolve in 0.4 ml of saline). In order to suppress endogenous melatonin, we intraperitoneal injected the mice with luzindole at 0, 7 day of sensitization. From 14 days on, the mice of control group were challenged with aerosolized saline, the mice of other groups with aerosolized OVA for 30 min/day on 3 days/week in a whole-body inhalation exposure chamber. The aerosol was produced by an ultrasonic nebulizer. All the mice were i.p. with Brdu 50mg/kg 30 min before inhalation for twice a week. At the same time, the mice of control and asthma group were i.p. with saline, while melatonin group with melatonin (10mg/kg), dexamethasone group with dexamethasone (1.5mg/kg), and luzindole group with Luzindole (30 mg/kg). The mice of luzindole group were sacrificed 24 hours after the last exposure at 2 weeks; while the mice of other groups were sacrificed 24 hours after the last exposure at 2, 4, 8 weeks, respectively. Numbers of inflammatory cells aside the airway were counted in hematoxylin and eosin -stained sections of the trachea and standardized by a unit of 2200 um2. The area of blue collagen 20um beneath subepithelial basement membrane was measured by MetaMorph image analysis software, and then standardized by Pbm, recording as Wcol/ Pbm. Mucus-secreting goblet cells were quantified in intrapulmonary airways in sections stained with Schiff-PAS. Immunohistochemistry of Brdu, MMP-9 and TIMP-1 were performed. The expression of MMP -9 and TIMP-1 was assessed semiquantitatively by immunopositive area per unit area by using metamorph analysis software. Semi-quantitive RT-PCR was used to evaluate the mRNA level. One-way analysis of variance was conducted using SPSS 11.5 for Windows. The level of significance was set at P < 0.05.Results:10 mice died in the whole experiment(4 died because of improper performance , the other were agnogenic death). There was no obvious death caused by medicine . 96 mice were observed.1.The number of inflammatory cells in airway and the parameters of airway wall remodeling in each group.The number of inflammatory cells per unit was larger in asthma. Melatonin and dexamethasone group than control group (P<0.01); That in asthma group were larger than in melatonin and dexamethasone group (P<0.01); That in Luzindole group was larger than in asthma group (P<0.01); while there was no significant difference between melatonin and dexamethasone group (P>0.05). Moreover, the differences in three groups between 2, 4 and 8 weeks'OVA exposure were not statistically significant (P>0.05).The WAmuc/Pbm, WAm/Pbm and WAi/Pbm were significantly higher in asthma group than control group, melatonin and dexamethasone group(P<0.01); Those in melatonin and dexamethasone group were higher than control group (P<0.01); While there is no significant difference between melatonin and dexamethasone, asthma and Luzindole group. The differences of WAmuc/Pbm,WAm/Pbm and WAi/Pbm in three groups between 2,4 and 8 weeks'OVA exposure were not statistically significant(P>0.05).2.The collagen area and the score of Goblet cells.There is a significant increase in Wcol/Pbm in asthma, melatonin and dexamethasone group compared with control group (P<0.01). Wcol/Pbm in asthma group was larger than melatonin and dexamethasone group (P<0.01). There is no significant difference between melatonin and dexamethasone, Luzindole and asthma group(all P>0.05). Wcol/Pbm was not statistically different in2,4, 8 weeks'OVA exposure in asthma group(P<0.01).Goblet cell score was zero in control group.That in asthma group was higher than melatonin and dexamethasone group (P<0.01); That in Luzindole group were higher than asthma group (P<0.01); while there was no significant difference between group melatonin and dexamethasone. The goblet cell scores were lower in melatonin and dexamethasone group after 4 and 8 weeks OVA exposure than after 2 weeks'(P<0.05).3. The number of Brdu Immunohistochemical positive cells per um Pbm. The number of BrdU-positive cells per um Pbm was larger in asthma, melatonin and dexamethasone group than in control group (P<0.01); That in asthma group was larger than melatonin and dexamethasone group (P<0.01); While that in Luzindole group was larger than asthma group (P<0.01). The number of BrdU-positive cells in control group was greater after 4 and 8 weeks'OVA exposure than 2 weeks'(P<0.01). The number of BrdU-positive cells in asthma, melatonin and dexamethasone group was greater after 4 and 8 weeks'OVA exposure than 2 weeks'(P<0.05). 4. The PA/UA of MMP-9 and TIMP-1.Immunohistochemical analysis revealed that there was a significant increased immunoreactivity of MMP-9 in asthma group compared with control group respectively at 2, 4, 8 weeks (all P<0.01). In melatonin treated animals, the PA / UA for MMP-9 were decreased significantly compared with asthmatic mice (P<0.01).That was significantly diminished in dexamethasone group compared with asthma group at 4 and 8 weeks (P<0.01). The PA / UA of Luzindole treated group was enlarged in comparison with asthma group, but it was not statistically significant(P>0.05).The PA/UA for TIMP-1 was reduced in OVA sensitized animals compared with control mice; the treatment of melatonin and dexamethasone enlarged the mean immunopositive area of TIMP-1; and so did the luzindole treatment, although none of them was statistically significant (all p>0.05). At 4 weeks, the production of TIMP-1 was increased in asthma group and dexamethasone group compared with control group (P<0.01), and decreased by melatonin treatment compared with asthma group (P<0.01). At 8 weeks, the secretion of TIMP-1 was decreased compared with control group (P<0.01), which could be upregulated by melatonin and dexamethasone treatment(P<0.01).5. The mRNA level of MMP-9, TIMP-1, collagenⅢand collagenⅣ.We detected that, under asthmatic condition, the MMP-9 mRNA level in all groups were dramatically increased (P<0.01 compared to control group). The preadministration of melatonin could attenuate it to normal level, while dexamethasone showed this effect at 2 and 4 weeks (P<0.01 compare to each control group). The mean integrated intensity ratio of MMP-9 toβ-actin was higher in luzindole group, although the difference was not significant(P>0.05).The mRNA level of TIMP-1 was only increased in asthma group at 2 and 4 weeks. melatonin could not restore this increase at 4 weeks (P<0.05 compared with control group). There was no difference among each group at 8 weeks.Increases of collagenⅢmRNA level were detected in asthma group( P<0.01 at 2 and 4 weeks, P<0.05 at 8 weeks, compared with control mice). A suppression induced by melatonin was observed at 2 (P<0.05), 4 (P<0.01), and 8(P<0.05) weeks compared to control mice, while the administration of dexamethasone induced a change in comparison with asthma group at 2 and 4 weeks (P<0.01).The mRNA levels of collagenⅣwere increased in untreated asthmatic groups (P<0.01 at 2 and 8 weeks, P<0.05 at 4 weeks, compared with control mice). Both melatonin and dexamethasone could suppress the transcription of collagenⅣgene(P<0.05 at 2 and 4 weeks, P<0.01 at 8 weeks compared to control mice). The mRNA level of collagenⅣin Luzindole group was higher than control group(P<0.01), while there was no significant difference in collagenⅢand collagenⅣbetween asthma group and Luzindole treated group.Conclusions:1. Allergen (OVA) sensitization and repeatedly challenge on mice could lead to some pathological changes in the airway that mimic airway inflammation and remodeling events seen in asthmatic patients. Airway inflammation and remodeling could occur in the early phase of asthma, and both of them do not become more pronounced with the extended OVA challenge.2. Melatonin and dexamethasone could inhibit the airway inflammation and remodeling to some extent.3.The increased level of MMP-9 after allergen challenge disrupts the normal ratio of MMP-9/TIMP-1, leads to the imbalance of MMP-9 and TIMP-1, and contribute to airway inflammation and remodeling. 4. The novel findings of this study are attributed to the attenuation of the MMP-9 both at protein and mRNA levels by pretreatment with melatonin. The finding suggests one of the MMP 9-mediated pathways for melatonin's action in asthma .5.the present study show the regulatory role of melatonin on the induction of MMP-9 in asthma during protection against OVA-induced chronic asthma occurs at transcriptional level , however, as to TIMP-1, it occurs at post-transcriptional level.