Chitosan Aerosolin Inhalation Alleviates Lipopolysaccharide-induced Pulmonary Fibrosis In Rats

Acute respiratory distress syndrome (ARDS) is a common perioperativeischemic/hypoxic injury and the acute inflammatory process may develop into chronicinflammatory process which is characteristed with a major pathophysiologic changes ofpulmonary fibrosis (PF). The lung apoptosis of epithelial cells, excessive proliferation andabnormal differentiation of lung fibroblasts (Fbs) and increased collagen synthesis mayplay a vital role in the course of PF. Medical chitosan is a polyamino glucose which canselectively promote epithelial, endothelial cell growth and inhibit Fbs growth, promotingtissue restoration.In this study, a rat model of PF was established by intratracheal injection oflipopolysaccharide (LPS). Chitosan was nebulized to rats after the daily intratrachealinstillation of LPS for the first3days until the experiment was end. We analyzed the effectof chitosan aerosol inhalation on LPS-induced pulmonary remodelling and fibrosis byhematoxylin-eosin staining (HE), Masson staining and the determination of thehydroxyproline content，and then to see whether chitosan aerosol inhalation has effect onthe expression intensities of matrix metalloproteinase-3(MMP-3) and tissue inhibitor ofmetalloproteinase-1(TIMP-1) which are associated with pulmonary fibrosis by Westernblots analysis. Trying to provide a new experimental ideas for clinical pulmonary fibrosistreatment.PART ONE The model establishment in rats with pulmonary fibrosisObjectives:Histopathology were determined to evaluate rats lung inflammatory reaction andfibrosis at different time point after daily intratracheal instillation of LPS for the first3days.Methods:Thirty SD rats were randomly divided into five groups-Control group (C group), LPSgroup: LPS3group (L3group), LPS7group (L7group), LPS14group (L14group), LPS28group (L28group).C group (n=6): rats received daily intratracheal instillation of sterile0.9%saline(0.2ml/kg) for the first3days, and were sacrificed on the3day after the first instillation.L3group (n=6): rats received daily intratracheal instillation of LPS (5mg/kg,0.2ml/kg) for the first3days, and were sacrificed on the3day after the first instillation. L7group (n=6): rats received daily intratracheal instillation of LPS (5mg/kg,0.2ml/kg) for the first3days, and were sacrificed on the7day after the first instillation.L14group (n=6): rats received daily intratracheal instillation of LPS (5mg/kg,0.2ml/kg) for the first3days, and were sacrificed on the14day after the first instillation.L28group (n=6): rats received daily intratracheal instillation of LPS (5mg/kg,0.2ml/kg) for the first3days, and were sacrificed on the28day after the first instillation.The left lung tissues were sectioned and stained with HE staining, Masson staining,and then use the Ashcroft score to grade the extent of lung inflammatory reaction andfibrosis at different time point after daily intratracheal instillation of LPS for the first3days.Results:HE and Masson staining showed that rats in the C group had no pulmonary restrcturechanges. Rats in the L3group had the typical symptoms of ARDS, with markedrecruitment of interstitial edema, neutrophils, alveolar haemorrhage, severe pulmonarylesions. Rats in the L7group, the excessive proliferation of fibroblastic cells, fibrogenesisand active collagen secretion in the pulmonary interstitium, and in the L14group, thenumber of inflammatory cells were decrease but had extensive fibrogenesis and collagendeposition. While rats in the L28group we can see remarkably collagen deposition anddistortion of the lung structure in the lungs. The fibrotic changes in the lung were evaluatedusing the Ashcroft score to grade the extent of lung fibrosis. The scores of the L group (L3,L7, L14, L28) were significantly higher than that of the C group, L28group＞L14group＞L7group＞L3group, the comparison of any two group was considered statistically significant(P <0.05).Conclusion:On the3day of the establishment of a rat model of pulmonary fibrosis showed acuteinflammatory response, interstitial pulmonary fibrosis began to appear on the14day, andwe can see remarkably collagen deposition and distortion of the lung structure in the lungson the28day, all above conform with ARDS inflammatory response and fibrosisprocesses. PART TWO Chitosan aerosolin inhalation alleviates Lipopolysaccharide-induced pulmonary fibrosis in ratsObjectives:Histopathology and the hydroxyproline content were determined to evaluate the effectof chitosan aerosol inhalation on lipopolysaccharide-induced pulmonary fibrosis in rats,and then to see whether chitosan aerosol inhalation has effect on the expression intensitiesof MMP-3and TIMP-1which are associated with pulmonary fibrosis by Western blotsanalysis.Methods:Twenty four SD rats were randomly divided into four groups: Control group (C group,n=6), LPS group (L group, n=6), Chitosan group (Ch group, n=6), and LPS+Chitosangroup (LCh group, n=6).C group: rats received daily intratracheal instillation of sterile0.9%saline(0.2ml/kg) for the first3days, followed by nebulization with sterile0.9%saline for two30mins (a30min rest between two deliveries) for the duration of the experiment.L group: rats received daily intratracheal instillation of LPS (5mg/kg,0.2ml/kg)for the first3days, followed by nebulization with sterile0.9%saline for two30mins in thesame manner as the controls.Ch group: rats were treated as for those in C group except that the sterile0.9%saline was replaced by Chitosan solution.LCh group: rats were treated as for those in LPS group except that the sterile0.9%saline was replaced by Chitosan solution.All the animals were sacrificed on the28day. The left lung tissues were sectioned andstained with HE and Masson staining to investigate the degree of fibrosis. To estimate totalcollagen deposition, the hydroxyproline content of the upper lobe of right lung wasmeasured according to the protocol of a commercial hydroxyproline detection kit. Themiddle lobe of right lung were sectioned and the expression intensities of MMP-3andTIMP-1were determined by Western blot analysis.Results:No pulmonary histopathological changes were found in the C group and Ch group,whereas remarkably induced collagen deposition and distortion of the lung structure in thelungs appeared in the L group, which demonstrated by HE and Masson staining. Compared with the L group, LPS-induced pulmonary fibrosis was effectively ameliorated in the LChgroup. The fibrotic changes in the lung were evaluated using the Ashcroft score to gradethe extent of lung fibrosis. The scores of the LCh group were significantly lower than thatof the L group on the28day after LPS instillation (P <0.05).The collagen content of lungs was evaluated using the hydroxyproline assay.Compared with the C group, hydroxyproline levels were significantly increased in the Lgroup on the28day (P <0.01). In the LCh group, the hydroxyproline content wassignificantly reduced compared with the L group on the28day (P <0.01). Althoughdecreased, hydroxyproline levels in the LCh group were still significantly higher than thatof the C group (P <0.01).The expression of MMP-3and TIMP-1in the L group was significantly higher thanthat of the C group on the28day (P <0.01). Compared with the L group, the proteinexpressions of MMP-3and TIMP-1in the LCh group was significantly decreased (P <0.01).Conclusion:Chitosan aerosol inhalation ameliorates LPS-induced pulmonary fibrosis in rats andreduces the hydroxyproline content which indicates the decrease of collagen deposition inthe lungs. The antifibrotic efficacy of chitosan inhalation may be mediated through itspotentially regulatory effects on MMP-3and TIMP-1.