| Many acute and chronic lung disorders with variable degrees of pulmonary inflammation and fibrosis are collectively referred to as interstitial lung diseases?ILDs?or diffuse parenchymal lung diseases.Idiopathic pulmonary fibrosis?or cryptogenic fibrosing alveolitis??IPF or CFA?is one of several idiopathic interstitial pneumonias.Treatment options recommended by the American Thoracic Society?ATS?and the European Respiratory Society?ERS?include corticosteroids and immunosuppressive agents?azathioprine and cyclophosphamide?alone or in combination.But physicians lack enough evidence of the efficiency of the present treatment.Recent studies have confirmed that carboxyamidotriazole?CAI?has not only anti tumor but also anti-inflammatory effect.Some scholars at home and abroad have confirmed that CAI can reduce the IL-1 beta,TNF-alpha and IL-6 pro-inflammatory cytokines infiltration,inhibit synovial hyperplasia,has therapeutic effect on adjuvant arthritis in rats.Other scholars have also reported that CAI has anti-inflammatory analgesic effect.A scholar has reported the therapeutic effect of carboxyamidotriazole on bleomycin induced pulmonary fibrosis in mice from the perspective of some cytokines in inhibiting the formation of pulmonary fibrosis,and made a preliminary discussion on the treatment of pulmonary fibrosis with carboxyamidotriazole.The cytokine network is large and complex,and there are many signal pathways,and which signal pathways are used by carboxyamidotriazole to prevent fibrosis? In additiom to the cytokines detected in this article,does carboxyamidotriazole regulate other cytokines to prevent pulmonary fibrosis? How much of the carboxyamidotriazole dose can be the best anti-fibrosis? No relevant reports have been reported at home and abroad.From these problems,the experiment was designed to further explore the role of carboxyamidotriazole against pulmonary fibrosis in mice.Experiment OneObjectives To look for a reasonable and reliable method and dosage for mice pulmonary fibrosis model induced by bleomycin by comparison of different ways of administration of bleomycin.Methods 20 mice were divided into five groups randomly.Endotracheal group: a single intratracheal instillation of bleomycin 3.5mg/kg.Tail vein group-1: a single intravenous injection of bleomycin 80 mg·kg-1?0.2ml?;tail vein group-2: a single intravenous injection of bleomycin 150 mg·kg-1?0.2ml?;tail vein group-3: a single intravenous injection of bleomycin 300 mg·kg-1?0.2ml?;blank control group?blank group?: single tail vein injection of saline 0.2ml.Observation index: the general condition of mice,mice mortality rate,lung appearance,pathological section of lung tissue,lung hydroxyproline content determination.Results 1.28 day mortality rate from high to low was tail vein-3 group?75%?,tail vein-2 group and endotracheal group?both are 50%?,tail vein-1 group?25%?,and blank group?no death?.T 2.Degree of pulmonary fibrosis in lung tissue pathological specimens?HE staining?from heavy to light was tail vein-3 group,tail vein-2 group and endotracheal group,tail vein-1 group.Lung tissue pathological specimens?HE staining?in blank group was normal.3.The content of hydroxyproline in lung tissue of mice from high to low was the tail vein-3 group?0.621?g/mg?,endotracheal group?0.592 ± 0.019 g/mg?,tail vein-2 group?0.569 ± 0.015 g/mg?,tail vein-1 group?0.512±0.012?g/mg?and blank group?0.439±0.009?g/mg?.Conclusion Tail intravenous injection of bleomycin(150 mg·kg-1)induced pulmonary fibrosis model was made with simple method,good repeatability,low mortality rate,uniforom distribution in the pleural.Experiment TwoObjectives To look for a reasonable and reliable dosage of carboxyamidotriazole for the therapeutic effect to mice pulmonary fibrosis model induced by bleomycin.Methods 50 mice were divided into five groups randomly.Blank control group?blank group?: After a one-time tail vein injection of N.S.0.2ml,mice were given PEG400 solution 0.1ml/10 g by gavage once daily for 14 days;The bleomycin group?BLM group?: After a one-time tail vein injection of bleomycin 150 mg/kg?0.2ml?,mice were given PEG400 solution 0.1ml/10 g by gavage once daily for 14 days;Carboxyamidotriazole group-1?CAI-1group?: after a one-time tail vein injection of bleomycin 150 mg/kg?0.2ml?,mice were given carboxyamidotriazole solution 0.1ml/10g?10mg/kg?by gavage once daily for 14 days;Carboxyamidotriazole group-2?CAI-2 group?: after a one-time tail vein injection of bleomycin 150 mg/kg?0.2ml?,mice were given carboxyamidotriazole solution 0.1ml/10g?20mg/kg?by gavage once daily for 14 days;Carboxyamidotriazole group-3?CAI-3 group?: after a one-time tail vein injection of bleomycin 150 mg·kg-1?0.2ml?,mice were given carboxyamidotriazole solution 0.1ml/10g?40mg/kg?by gavage once daily for 14 days;Observation index: the general condition of mice,survival analysis of mice,pathological section of lung tissue,lung hydroxyproline content determination,TGF-beta 1 and IFN-gamma content determination in lung homogenate.Results 1.Weight The weight of each group was compared as follows: On day 7 the weight of each group was significantly lower than the blank group?P<0.05?.Three CAI groups and BLM group had similar body weight?P>0.05?.On day 14 the weight of each group was still lower than the blank group?P<0.05?.Except BLM group,mice weight in three CAI groups had begun to increase steadily and CAI-3 group weight gained obviously.There were significant differences between weignt of mice in BLM group and that of mice in CAI-3 group?P<0.05?.On day 28,except BLM group,mice weight in three CAI groups had increased steadily?P<0.05?,CAI-3 group mice increased the most.2.Lung coefficient The lung coefficient of each group was compared as follows: On day 28 the lung coefficient of each group was significantly higher than the blank group?P<0.05?.BLM group had the highest lung coefficient,the lowest in CAI-3 group.The mice lung coefficient in CAI-2 group and CAI-3 group was statistically significant compared with BLM group?P<0.05?.There were significant differences between lung coefficient of mice in CAI-1 group and that of mice in CAI-3 group?P<0.05?.3.28 day mortality rate from high to low was BLM group and CAI-1 group?both are 75%?,CAI-2 group?40%?,CAI-3 group?30%?,and blank group?no death?.The median survival time was opposite with the former.From the survival curve,survival curves of BLM group,CAI-2 group and CAI-3 group were significantly separated.Among the three treatment groups,the first 14 days were partially overlapping,and the survival curves were separated from each other after 14 days.4.On day 28 degree of pulmonary fibrosis in lung tissue pathological specimens?HE staining?from heavy to light was BLM group,CAI-1 group,CAI-2 group,and CAI-3 group.Lung tissue pathological specimens?HE staining?in blank group was normal.After ranking the pathological sections of lung tissue,verage rank's comparisons in the all groups were statistically significant?P<0.05?.5.On day 28 the content of hydroxyproline and TGF-?1 in mice lung homogenate from high to low was BLM group,CAI-1 group,CAI-2 group,CAI-3 group and blank group.The content of IFN-? in mice lung homogenate was CAI-3 group,CAI-2 group,CAI-1 group,BLM group,and blank group.comparisons in the all groups were statistically significant?P<0.05?.Conclusions The better dosage of intragastric infusion of CAI that can reduce lung injury induced by bleomycin in mice is 40 mg/kg.The mechanism of action is related to the effects of CAI on cytokines such as TGF-beta 1 and gamma-IFN.Experiment ThreeObjectives Research on the mechanism of carboxyamidotriazole against pulmonary fibrosis of mice based on TGF-?1 /smad signal tranmission pathway,and to detect the cytokines that may be regulated in the process of anti-fibrosis.Methods 50 mice were divided into three groups randomly.Blank control group?blank group?: After a one-time tail vein injection of N.S.0.2ml,mice were given PEG400 solution 0.1ml/10 g by gavage once daily for 14 days;The bleomycin group?BLM group?: After a one-time tail vein injection of bleomycin 150 mg/kg?0.2ml?,mice were given PEG400 solution 0.1ml/10 g by gavage once daily for 14 days;Carboxyamidotriazole group?CAI group?: after a onetime tail vein injection of bleomycin 150 mg/kg?0.2ml?,mice were given carboxyamidotriazole solution 0.1 ml/10g?40 mg/kg?by gavage once daily for 14 days.Observation index: the general condition of mice,survival analysis of mice,pathological section of lung tissue,collagen staining of lung tissue,lung hydroxyproline content determination,TGF-?1?IFN-?,MMP-9 and TIMP-1 content determination in lung homogenate,the expression of smad2 and smad3 in the immunohistochemical staining of lung tissue.Results 1.Weight On day 7 the weight of mice in BLM group and CAI group was significantly lower than the blank group?P<0.05?.There were no significant differences between the weight of mice in BLM group and that of mice in CAI group?P>0.05?.On day 14 the weight of mice in BLM group and CAI group was still lower than the blank group?P<0.05?.Mice weight in CAI group had begun to increase steadily.There were significant differences between the weight of mice in BLM group and that of mice in CAI group?P<0.05?.On day 28 the weight of mice in BLM group and CAI group was still lower than the blank group?P<0.05?.Mice weight in CAI group had increased steadily.There were significant differences between the weight of mice in BLM group and that of mice in CAI group?P<0.05?.2.Lung coefficient On day 28 the lung coefficient of mice in BLM group and CAI group was significantly higher than the blank group?P<0.05?.The lung coefficient of mice in BLM group was the highest.There were significant differences between the lung coefficient of mice in BLM group and that of mice in CAI group?P<0.05?.3.Survival Nalysis On day 28 mortality rate from high to low was BLM group?50%?,CAI group?30%?,and blank group?no death?.The survival curves of blank group,BLM group and CAI group were significantly separated after 14 days.4.On day 28 degree of pulmonary fibrosis in lung tissue pathological specimens?HE staining?from heavy to light was BLM group,CAI group,blank group.Lung tissue pathological specimens?HE staining?in blank group was normal.After ranking the pathological sections of lung tissue,verage rank's comparisons in the all groups were statistically significant?P<0.05?.5.Masson staining On day 28 collagen deposition in mice lung tissue of BLM group was significantly lower than that of CAI group.After collagen staining in lung tissue of mice,the percentage of collagenous fibers in the area of the lung tissue was statistically significant in the all groups?P<0.05?.6.On day 28 the content of hydroxyproline and TGF-?1 in mice lung homogenate from high to low was BLM group,CAI group,and blank group.The content of IFN-? in mice lung homogenate from high to low was CAI group,BLM groupand blank group?P<0.05?.Comparisons of the content of hydroxyproline and TGF-?1 in mice lung homogenate between each group have significant difference?P<0.05?.On day 28 the contents of MMP-9 in mice lung homogenate were?325.560±34.210 ng/ml??in blank group?,?362.512±62.231 ng/ml??in BLM group?,and?349.751±45.391 ng/ml??in CAI group?respectively.Comparisons of the content of MMP-9 in mice lung homogenate between each group have no significant difference?P>0.05?.On day 28 the contents of TIMP-1 in mice lung homogenate were?72.824±5.530 ng/ml??in control group?,?369.250±57.508 ng/ml??in BLM group?,and?246.208±20.572 ng/ml??in CAI group?respectively.Comparisons of the content of hydroxyproline and TIMP-1 in mice lung homogenate between each group have significant difference?P<0.05?.7.On day 28 tested the expressions of TGF-?1,smad2 and smad3 in lung tissue by immunohistochemistry method,to discuss the CAI's mechanism of anti-lung fibrosis through regulating TGF-?1/smad signal path.In blank group,there was no obvious or weak expression of TGF-?1,Smad2 and Smad3 in lung tissue.In BLM,there was a large number of expression in lung tissue,and the expression in CAI group was significantly reduced.The gray value of the positive coloring of three groups of immunohistochemical sections was measured by image analysis system in the all groups were statistically significant?P<0.05?.8.On day 28 tested the expressions of TGF-?1,smad2 and smad3 in lung tissue by Western blot method.The expression trend of TGF-?1 protein in mice showed that compared with the blank group,the expression of TGF-?1 increased in BLM group and CAI group,and CAI group was significantly lower than that in BLM group,and there was a significant difference between each groups?P < 0.05?.The expression trend of Smad2 and Smad3 protein was similar to that of TGF-?1.Compared with blank group,the expression of Smad2 and Smad3 protein in BLM group and CAI group increased,while CAI group decreased significantly compared with BLM group,and there was a significant difference between each groups?P<0.05?.Conclusion Carboxyamidotriazole can effectively improve pulmonary fibrosis in model mice,and regulate the content of IFN-??TGF-?1?MMP-9 ? TIMP-1 in lung tissue homogenate,inhibit the excessive of TGF-?1,smad2 and smad3 in lung tissues.Carboxyamidotriazole can effectively regulation the fibrosis induced by TGF-?1/smad singal pathway and thus play a role in anti-fibrosis. |
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