Establishment Of Murine Pulmonary Fibrosis Model Induced By Repetitive Intratracheal Administration Of Bleomycin And Observation Of The ABT263 Treatment Effect Of On Lung Fibrosis

Pulmonary fibrosis is a progressive and fatal diffuse interstitial lung disease characterized by inflammation and extracellular matrix deposition.It includes a heterogeneous group of lung disorders characterized by the progressive and irreversible destruction of lung architecture caused by scar formation that ultimately leads to organ malfunction,disruption of gas exchange,and death from respiratory failure.Idiopathic pulmonary fibrosis(IPF),the most common form of the idiopathic interstitial pneumonias,is a chronic,progressive,irreversible,and usually lethal lung disease of unknown cause.IPF occurs in middle-aged and elderly adults(median age at diagnosis 66 years,range 55-75 years),and has a life expectancy of 2-6 years after diagnosis.To better understand the pathogenesis of lung fibrotic disorders,multiple animal models have been developed,include exposure to bleomycin,silica,or fluorescein isothiocyanate(FITC),and irradiation.However,the mouse bleomycin model has garnered the most attention,perhaps because it is a well characterized and clinically relevant model of pulmonary fibrosis.There are two models of bleomycin induced pulmonary fibrosis in mice:the single-dose model and the repetitive dose model.In order to explore the difference between the single-dose model and the repetitive dose model,C57BL/6 mice(male)were randomly divided into three groups:control group,single dose group,repetitive dose group.The single dose mice were administered bleomycin at 3.5 mg/Kg,the repeated dose mice were given bleomycin 0.04 mg in a total volume of 100ul of sterile saline every other week.At the different time points(2nd week,4th week,8th week),the organ index(lung,kidney,thymus,spleen),peripheral blood cells,the content of hydroxyproline in lung tissue was detected;lungs tissues were fixed by formalin for HE staining and Masson staining,moreover,the frozen sections of lung tissues were prepared for senescence-associatedβ-galactosidase staining.The pathological results showed pathological score were the highest at 2nd week and decreased at 4th week in the single dose mice,while were relatively stable in the repetitive dose mice,suggests that the lung injury of repetitive dose mice persistent;and the content of hydroxyproline in lung tissue of the repetitive dose mice were higher than that of the single dose mice.Meanwhile,the two group of mice have little change in the body weight and the peripheral blood cells count,compared with the control group,the results showed that systemic reaction of the two kinds of model was not obvious,but lung damage in the repetitive dose model was heavier than that in the single dose model.Therefore,in order to establish a mouse model of pulmonary fibrosis by repetitive intratracheal administration of the bleomycin,the C57BL/6 mice were randomly divided into two groups:control group and model group,the mice in model group were given bleomycin(0.4 mg/mL)every other week with eight administrations in total,the lung coefficient,the HE staining,the Masson staining,and senescence-associated β-galactosidase staining were observed at different times.The results showed that lung fibrosis is induced by repetitive bleomycin intratracheal administration,this model provides substantial lung architectural distortion,further more,senescence cells may be involved in the pulmonary fibrosis.ABT263 is a specific inhibitor of the anti-apoptotic proteins BCL-2 and BCL-xL,the previous studies have found that ABT263 has selective toxicity to senescent cells.In the second part of the thesis,we first established A549 cell aging model induced by BLM,and found that the aging A549 cells were more sensitive to the cytotoxicity of ABT263,the result suggested that ABT263 could selectively kill the senescent cells.To determine whether ABT263 is senolytic in vivo and observe the therapeutic effect of ABT-263 on pulmonary fibrosis induced by repetitive intratracheal administration of the bleomycin,male C57BL/6 mice were divided into control group(Ctrl),ABT administration group(ABT),model group(BLM)and treatment group(BLM+ABT),after model mice were given eighth biweekly dose,ABT administration group(ABT)and treatment group(BLM+ABT)were administered ABT-263 at 50mg/kg,i.g for 5days,repeated administered another 5days after 2weeks interval.After two courses of treatment,mouse parenchyma lung injury was evaluated by HE staining,Msson staning,hydroxyproline content measurement,and β-galactosidase expression assay.The expression of BCL-2 in lung tissue of lung tissue was detected by immunofluorescence.The results showed that in treatment group,senescent cells of lung tissue were reduced and the grade of lung pathology score were decreased,these suggested that ABT-263 might play a role in the treatment and mitigation of BLM induced pulmonary fibrosis by inhibiting Bcl-2.In conclusion,mouse model of pulmonary fibrosis was established by repetitive intratracheal administration of the bleomycin,and ABT263 may have a certain therapeutic effect on lung fibrosis.