| Objective To explore the effect of autophagy activity and its possible mechanism in rat models of pulmonary fibrosis induced by bleomycin,and provide experimental basis for elucidating the pathogenesis of pulmonary fibrosis.Methods 12 male rats were randomly assigned into control group and experimental group,6 in each group.Three rats were placed in the atomizing device for 15 minutes with3-4ml atomized liquid(calculated according to body weight)at a time,atomized once a day for 3 consecutive days.Experimental group received bleomycin(1mg/ml)inhalation at a dose of 5mg/kg,while the control group received saline inhalation.Glucose tolerance test was performed on the 25th day after aerosol treatment.All rats were sacrificed on the 28h day,and serum,BALF(bronchoalveolar lavage fluid)and lung tissue were collected.Serum insulin levels and AMPK concentration in BALF were measured by enzyme-linked immunosorbent assay(ELISA).Hematoxylin-eosin staining(HE)was used to observe the pathological changes of lung tissue.Collagen fibers were evaluated by Masson staining.The expression of?-SMA,autophagy markers(p62 and LC3?/?),signaling pathway related proteins and its regulatory proteins(p-Akt,p-mTOR,mTOR,PTEN and OTUD3)were assessed with Western blot.Results(1)Body weight of rats in both groups showed continuous growth pattern.No significant difference was found between these two groups at each time point(P>0.05).(2)Serum glucose levels at each time point,the area under the glucose tolerance curve and fasting serum insulin levels were similar between the experimental group and the control group(P>0.05).(3)Anatomy of lung tissue:The lung tissue of the control group was pink,glossy,soft and springy.On the contrary,lung tissue of experimental group was dark,lackluster,stiff and partially hydropic with sporadic old hemorrhagic spots on its surface.(4)The lung weight and lung coefficient of the experimental group were higher than the control group(P<0.05).(5)HE staining of lung tissue:Lung tissue morphology of the control group was normal,its alveolar cavity was clear,and there was no or mild alveolar inflammation.In the experimental group,the structure of the lung tissue was destroyed,the alveolar septum was significantly thickened and deformed,and there was large consolidation of lung parenchyma with infiltration of inflammatory cells in the alveolar space.(6)Masson staining of lung tissue:The blue area of experimental group was significantly larger than the control group(21.77±5.92 mm~2 vs.12.24±3.44 mm~2,P<0.05).The stained area of the control group was concentrated around trachea and vessels,while the stained area of the experimental group could be further detected in the lung mesenchyme,presented as flake form.(7)Pulmonary fibrosis related indicators:the expression of?-SMA of the experimental group was higher than the control group(P<0.05).(8)Autophagy activity related indicators:The p62 level of the experimental group was significantly higher than the control group(P<0.05).LC3?/?ratio of the experimental group was lower than the control group(P<0.05).(9)Autophagy regulation mechanism:The expressions of p-Akt and p-mTOR of the experimental group were higher than the control group(P<0.05).The expressions of PTEN and OTUD3 of the experimental group were lower than the control group(P<0.05).There was no significant difference in the expression of mTOR between these two groups(P>0.05).The AMPK content in the BALF of the experimental group was significantly lower than that in the control group(P<0.05).Conclusions(1)Inhalation of bleomycin can induce pulmonary fibrosis in rats.(2)Autophagy activity was inhibited in rat models of pulmonary fibrosis induced by bleomycin.(3)The abnormal activation of Akt/mTOR signaling pathway and the decrease of AMPK expression may be related to the inhibition of autophagy activity.(4)The decreased expression of OTUD3 may lead to the decreased level of PTEN,which result in the decrease of its inhibitory effect on the Akt/mTOR signaling pathway. |
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