| ObjectiveIn this study,bleomycin-induced pulmonary fibrosis rats were treated by Smad3 specific inhibitor(Smad3,SIS3)and then observed the degree of pulmonary fibrosis and the expression of transforming growth factor-β1(TGF-β1)and Smad3 in these rats.To investigate the role of TGF-β1/Smad3 signaling pathway in endothelial cell mesenchymal transition(EndMT)in pulmonary fibrosis rats,we use immunohistochemistry technique to observe the expression of endothelial cell phenotype(PECAM-1/CD31)and interstitial cell phenotype(α-SMA)in rats.At the same time,we explored whether SIS3 has the effect of reducing pulmonary fibrosis.Methods96 healthy adult male Sprague-Dawley(SD)rats,which were 8-10 weeks old and(250±50)g weight were randomly divided into four groups,24 of each group.Group 1:saline control group(NS group);Group 2: bleomycin group(BLM group);Group 3:SIS3 intervention group(SIS3 group);Group 4: dimethyl sulfoxide solvent group(DMSO group).BLM group,SIS3 group and DMSO group rats were injected intratracheally with bleomycin solution(5mg/kg,0.3ml)to establish pulmonary fibrosis models and NS group rats were injected with equal volume of normal saline in the same way.SIS3 group rats were injected intraperitoneally with SIS3(2.5ug/g,1ml)for intervention,BLM group rats were injected with equal volume of normal saline and DMSO group rats were injected with equal volume of DMSO in the same way.Eight rats were randomly sacrificed on the 7th day,14 th day and 28 th day after modeling.We used hematoxylin-eosin(HE)staining to observed the lung tissue structure and inflammation in each groups.Masson staining,Ashcroft score,and hydroxyproline(HYP)content,which were used to observe the degree of pulmonary fibrosis.The immunohistochemistry of TGF-β1,Smad3,CD31 and α-SMA were detected in each group for judging the degree of endothelial-mesenchymal transition(EndMT)and investigating the role of TGF-β1 / Smad3 signaling pathway in it.Results(1)HE staining: NS group: On the 7th day,14 th day,28 th day,this group rats alveolar structure were normal.BLM group: On the 7th day,14 th day,28 th day the rats lung tissues can see congestion,hemorrhage,collagen fibers exudate,the range of lesion increased,alveolar structure was gradually replaced by fibrous tissue,and the alveolar cavity gradually disappeared.SIS3 group: On the 7th days,alveolar structure was not significantly damaged.Lung tissues can see that a small amount of congestion,hemorrhage,a small amount of inflammatory cell infiltration,alveolar septum slightly thicker.On the 14 th day and the 28 th day,the alveolar structure survived,the alveolar septum widened,the inflammatory reaction and the fiber exudation were lower than those in the BLM group rats,and the degree of fibrosis was mild.DMSO group: On the7 th day,14 th day,28 th day;this group rats lung histopathological changed similar to BLM group.(2)Masson staining: NS group: On the 7th day,14 th day,28 th day,this group rats alveolar structure was normal,no significant blue collagen deposition.BLM group: On the 7th day,alveolar structure was normal,a large number of inflammatory cells in the alveolar cavities and a small amount of blue collagen deposited in it.On the 14 th day and 28 th day,alveolar structure damaged gradually increased,alveolar septum gradually thickened,and a large amount of blue collagen was filled in it.Compared with NS group,the area of blue stained collagen was obviously increased(P <0.05).BLM group rats lung tissue Masson staining difference was significant at three time points(P<0.05).SIS3 Group: On the 7th day,the rats alveolar structure was destroyed and a small amount of blue collagen deposition in it.On the 14 th day,the rats alveolar structure was destroyed extensively and the area of blue collagen was increased,part of rats alveolar structure was retained.On the 28 th day,compared with the NS group,the area of blue collagen increased significantly(P <0.05),but it was lower than that in the BLM group(P <0.05).SIS3 group rats lung tissue Masson staining differences were significantat at three time points(P <0.05).DMSO group: Masson staining was similar to BLM group at day 7,day 14 and day 28(P> 0.05).(3)HYP determination: NS group: On the 7th day,14 th day,28 th day,HYP content of this group ratswas similarly(P> 0.05).BLM group: On the 7th day,14 th day,28 th day,the levels of HYP of this group rats increased gradually with the degree of pulmonary fibrosis.Comparing this with NS group increased significantly(P <0.05).The difference of HYP content in BLM group was significant at three time points(P> 0.05).SIS3 Group: On the 7th day,14 th day,28 th day,the levels of HYP of this group rats increased gradually.The content of HYP was between NS group and BLM group,which was significantly higher than that of NS group(P <0.05),but significantly lower than that of BLM group(P <0.05).The difference of HYP content in SIS3 group was meaningful at three time points(P <0.05).DMSO group: Compared with BLM group,this gourp rats HYP content was similarly,the difference was not statistically significant(P> 0.05).(4)Ashcroft score: NS group: On day 7,day 14 and day 28,the Ashcroft scores of this group rats were similar(P <0.05).BLM group: Compared with NS group,the Ashcroft score was increased at day 7,and this increased more obviously at day 14 and28,the difference was statistically significant(P<0.05).The difference of Ashcroft score in BLM group was significant at three time points(P> 0.05).SIS3 group: Ashcroft score of this group was between NS group and BLM group,which was higher than that of NS group(P <0.05),but lower than that of BLM group significantly(P <0.05).The difference of Ashcroft score of SIS3 group was meaningful at three time points(P<0.05).DMSO group: Compared with BLM group,Ashcroft score of this gourp was similarly,the difference was not statistically significant(P> 0.05).(5)TGF-β1,Smad3 immunohistochemistry: NS group: On the 7th day,14 th day and 28 th day,the structures of lung were normaly,and TGF-β1 and Smad3 were weakly expressed.BLM group: With the gradual increased pulmonary fibrosis,TGF-β1 and Smad3 expression gradually increased,on 7th day,14 th day and 28 tn day.Compared with the NS group,the difference was meaningful(P <0.05).The difference of TGF-β1and Smad3 positive expression in BLM group was significant at three time points(P<0.05).SIS3 group: The expression trend of TGF-β1 and Smad3 was similar to that of BLM group,which was higher than that of NS group(P <0.05),but it was less than BLM group(P <0.05).The difference of TGF-β1 and Smad3 positive expression in SIS3 group was significant at three time points(P <0.05).DMSO group: The positive expression of TGF-β1 and Smad3 was similar to that of BLM group,the difference wasnot statistically significant(P> 0.05).(6)CD31 immunohistochemistry: NS group: On the 7th day,14 th day and 28 th day,the pulmonary vascular morphology was normal,and the positive expression area of CD31 was brown.BLM group: On the 7th day,the pulmonary vessel wall was slightly thickened and the positive expression area of CD31 was found.On the 14 th day and 28 th day,the pulmonary vascular wall gradually thickened,pulmonary vascular malformation and CD31 positive expression area were significantly decreased,which was significant compared with the NS group(P <0.05).The differences of CD31 positive expression in BLM group rats was meaningful at three time points(P <0.05).SIS3 group: The expression trend of CD31 was similar to that of BLM group,which was smaller than NS group and greater than BLM group,this difference was statistically significant(P <0.05).The differences of CD31 positive expression in SIS3 group rats was meaningful at three time points(P <0.05).DMSO group: The positive expression of CD31 was similar to that of BLM group,the difference was not statistically significant(P> 0.05).(7)α-SMA immunohistochemistry: NS group: On day 7,day 14 and day 28,pulmonary vascular morphology was normaly,and α-SMA was very weakly positive expression.BLM group: On 7th day,the pulmonary vascular wall was slightly thickened and a small amount of α-SMA was expression.Compared with NS group,the vessel wall gradually thickened,deformed,α-SMA positive expression area was significantly increased,on the 14 th day and 28 th day(P <0.05).The difference ofα-SMA positive expression in BLM group was significant at three time points(P <0.05).SIS3 group: The expression trend of α-SMA was similar to that of BLM group,which was greater than NS group and lower than BLM group,and this difference was statistically significant(P <0.05).The difference of α-SMA positive expression in SIS3 group was significant at three time points(P <0.05).DMSO group: The positive expression of α-SMA was similar to that of BLM group,the difference was not statistically significant(P> 0.05).Conclusion(1)EndMT involved in the formation of pulmonary fibrosis.(2)TGF-β1/Smad3 signal pathway may play an important role in EndMT.(3)SIS3 attenuates pulmonary fibrosis by inhibiting Smad3 protein expression and inhibiting the process of EndMT. |
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