| Research background and purpose:Research background: Pulmonary fibrosis(PF)is a fibrotic disease whose lesion is confined to lungs and the etiology is not clear.The pathogenesis of PF is still not clear.At present,most PF patients are treated with pirfenidone,nidanib,oxygen therapy,invasive / non-invasive mechanical ventilation,and lung transplantation.Wildly application of these methods are limited because of their high costs,low efficacy,the shortage of transplant donors and poor compliance of patients.Therefore,it is of great significance to actively explore the pathogenesis of pulmonary fibrosis and develop new intervention drugs.Amifostine(AMI),as a membrane protectant used in radiotherapy for patients with various malignant tumors,can reduce lung tissue damage and pulmonary fibrosis.However,there is little research on AMI’s treatment for pulmonary fibrosis induced by bleomycin.Objective: To observe the protective effect of amifostine on pulmonary fibrosis in mice through an animal model of bleomycin-induced pulmonary fibrosis in mice,and to explore the mechanism and intervention targets of amifostine in treating pulmonary fibrosis.Materials and Methods:1.Establishment of experimental animal groups and animal models: 120 males,healthy SD mice,animal groups: blank group,vehicle group,model group,drug group,intervention group,randomized grouping,mice of each group They are all 24.200 ul bleomycin hydrochloride solution(5mg / kg)was slowly injected into the trachea of the model group。200ul bleomycin solution(5mg / kg)was injected into the trachea of the intervention group。And 200 ul amimi was injected into the trachea of the drug group.Futing injection(40mg / kg),the mice in the vehicle group were injected with 200 ul sterile normal saline into the trachea,and the blank group was not treated.After modeling,the mice in the daily intervention group were injected with 200 ul amifostine injection intraperitoneally;the mice in the drug group were injected with 200 ul amifostine injection intraperitoneally,and the mice in the vehicle group and the model group were injected with 200 ul sterile saline each day.2.Observe and record the changesof diet,activity,respiration and weight of mice during the experiment.Lung tissues of 8mice were collected in batches,and the appearance,volume,and elasticity of mice in each group were observed on days 7,14,and 28 of the experiment。Changes in lung weight and lung coefficient of mice in each group were compared.3.Comparison of the degree of fibrosis: Compare the degree of fibrosis(HE staining,masson staining,and Ashcroft score)of mice in each group on days 7,14,and 28 after modeling.4.Study on the mechanism of amifostine intervention: Immunohistochemistry was used to compare the expressions of TGF-β1,Smad-2,E-Cad,and α-SMA in the lung tissues of mice in each group on days 7,14,and 28 after modeling.Western blot was used to compare the expression of TGF-β1,Smad-2,E-Cad,and α-SMA proteins on day 28 of mice in each group.5.Drug safety research: observe the effects of amifostine on the diet,water intake,activity,normal lung tissue,liver function,and kidney function of mice.Results:1.General situation of mice: Blank group,vehicle group,and drug group: The three groups of mice showed no abnormal behaviors in diet,water intake,and activities during the whole experiment.The respiratory rate was normal,weight increased,and no hair loss occurred.Lung coefficient did not increase or decrease significantly.Model group: During the whole experimental period,the mice’s diet,water intake gradually decreased,activity decreased,appetite and appetite gradually worsened,hair loss,breathing increased,weight decreased significantly,and lung coefficient gradually increased.Intervention group: The breathing,response,weight,eating,drinking,and lung coefficient of the mice were worse than those of the blank group and better than the model group.Macroscopic observation of lung tissue: blank group,vehicle group,and drug group: Dissected lung tissues of mice were normal and without shrinking,the tissue was pink to the naked eye,full touch,no bleeding points on the surface,and good elasticity.Model group: smaller in size,the surface of the touch tissue are not smooth,the elasticity are poor,and the texture are hard.Intervention group: The volume of lung tissue of the mice are slightly reduced,and the surface are dark red.The part of the tissue are not smooth,the elasticity are slightly poor,and the texture is slightly hard.The overall visual status of the lung tissue was better than the model group and worse than the other three groups.2.Comparison of various lung tissue fibrosis:(1)HE staining: blank group,vehicle group,drug group: mice lung tissue was clearly visible at all time points(7 days,14 days,28 days)and the structure of the alveolar cavity were intact,no widening of the alveolar septum,no inflammatory cell infiltration,and no fibrin deposition were seen.Model group: On the 7th day,the lung tissues are dominated by inflammatory changes and inflammatory cells exudate;as time progresses,the inflammation of the alveolar tissue intensifies,and the degree of fibrosis becomes heavier.The more collagen fibers,the more severe the damage to the lung tissue,and the consolidation of some lung tissues was the most severe on the 28 th day.Intervention group: On the seventh day,inflammatory cells(mainly neutrophils)exuded in the lung tissue;with the progress of time,the number of inflammatory cells and the extent of fibrosis became more and more obvious,but the overall lung tissue condition was better than The model group,inferior to the other three groups.(2)Masson staining: blank group,vehicle group,and drug group: The results of masson staining on the 7th,14 th,and 28 th days of the experiment show that No damage to the lung tissue of the three groups of mice at each time point(7 days,14 days,28 days),no inflammatory cell infiltration and fibrosis,no blue collagen fibers in alveolar cavity.Model group: On the seventh day of the experiment,a few inflammatory cells and blue collagen fibers were seen in the alveolar lung tissue of the mice.Over time,the more alveolar destruction,the heavier the lung tissue inflammation,the heavier the fibrosis,the more obvious the lung tissue destruction,the most severe on the 28 th day,compared with the above three groups at the node pair,there are differences(P <0.05).Intervention group: On the 7th day,a small amount of inflammatory cell infiltration and blue collagen fibers were seen in the alveolar cavity of the mice.With the progress of time,lung tissue destruction,inflammatory cells,and collagen fibers also showed an upward trend.The degree of inflammation and fibrosis of lung tissue was better than the model group at each time point,and worse than the other three groups.There were differences(P <0.05).(3)Ashroft scores: The three groups of mice(blank group,vehicle group,and drug group)had no significant difference in Ashroft scores on day 7,14,and 28(P> 0.05).Compared with the blank group,the vehicle group,and the drug group at each time point,the model group’s Ashroft score increased gradually,and there were differences(P <0.05).The Ashroft score of the intervention group was lower than that of the model group at each node and higher than the other three groups,and there are a difference(P<0.05).3.Intervention mechanism:(1)Pathway protein expression: The experimental immune results on the 7th,14 th,and 28 th days of the experiment showed that TGF-β1 and Smad-2 were low in the blank group,vehicle group,and drug group,and the model group Compared with the blank group,the vehicle group,and the drug group at each time point,TGF-β1 and Smad-2 in the model group increased gradually,and there were differences(P <0.05);TGF-β1 and Smad-2 in the intervention group The expression at each time point was lower than the model group,higher than the above three groups,and there were differences(P <0.05).The Western-Blot-free experiments showed that the blank group,the vehicle group,and the drug the expression of TGF-β1 and Smad-2protein was low in the group.The TGF-β1 and Smad-2 protein in the model group increased gradually compared with the blank group,the vehicle group,and the drug group.<0.05);The expression of TGF-β1 and Smad-2 protein in the intervention group was lower than that of the model group,and higher than that of the other three groups,and there were differences(P <0.05).(2)Epithelial-mesenchymal transition-related proteins: The experimental immune results on the 7th,14 th,and 28 th days of the experiment showed that the E-Cad expression was high in the blank group,the vehicle group,and the drug group,and the α-SMA was low.E-Cad expression in the blank group,the vehicle group,and the drug group was reduced at each time point compared to the model group,while the expression of α-SMA gradually increased,and there were differences(P <0.05);E-Cad in the intervention group was different in each group.Time points were higher than the model group and lower than the above three groups(P<0.05);α-SMA expression at each time point was lower than the model group and higher than the other three groups(P <0.05);the results of Western blot-free experiments showed that the E-Cad protein was highly expressed in the blank group,the vehicle group,and the drug group,and the α-SMA protein was low.The model group and the blank group,the vehicle group,and the drug group were paired Compared with the model group,the expression of E-Cad protein was gradually reduced,and the expression of α-SMA protein was gradually increased,which was different(P <0.05).The E-Cad protein in the intervention group was higher than the model group,and lower than the above.There are a difference between the three groups(P <0.05);α-SMA protein was lower than the model group and higher than He said three groups,there are differences(P <0.05)。4.Results of drug safety: Amifostine has no effect on diet,water intake and activity of mice,has no damage to normal lung tissue,and has no effect on liver and kidney function.It shows that amifostine(40mg / kg)is a safe dose for treating pulmonary fibrosis.Conclusion1.Mouse lung tissue fibrosis model can be achieved by tracheal infusion of bleomycin.2.Amifostine delays pulmonary fibrosis by reducing inflammation and inhibiting collagen deposition.3.Amifostine down-regulates TGF-β1 / Smad-2,inhibits EMT,and delays pulmonary fibrosis.4.Amifostine delays pulmonary fibrosis and has high drug safety,which is worthy of clinical application. |
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