Study On The Role Of SB431542 Inhibiting TGF-β/Smad3 Signaling Pathway In Silicosis Fibrosis

Objective To investigate the effect of transforming growth factorβ1（TGF-β1）typeⅠreceptor specific inhibitor SB431542 on TGF-β/Smad3 signaling pathway in silicosis fibrosis in rats;to establish an in vitro model of silicosis and to explore the effect of SB431542 on silica（SiO₂）Effect of dust-induced epithelial-mesenchymal transition（EMT）on human normal lung epithelial cells（Beas-2B）.Methods 1.Forty healthy male SPF SD rats were divided into 4 groups according to the random number table method:physiological saline control group,model group,SB431542 inhibitor group,and SB431542 inhibitor control group,10rats in each group,5 days after the completion of the grouping Monitor rat weight once.One week after the adaptive breeding,except for the normal saline control group,the other three groups were injected with free silica（SiO₂）dust suspension1 mL（50 mg/mL）in a single trachea by non-exposed trachea injection;7,30 days,SB431542 inhibitor group rats were injected intraperitoneally（5 mg/kg）SB431542;SB431542 inhibitor control group was intraperitoneally injected（5mg/kg）SB431542 cosolvent at the same time;physiological saline control group was injected intratracheally Saline（5 mg/kg）.The material was collected on the60th day after the dust staining.The right upper lobe lung tissue was used for pathologicalexaminationandimmunohistochemistry（IHC）.Immunohistochemistry of smooth muscle actin（α-SMA）and fibronectin（FN）proteins;upper left lobe tissue was used for real-time fluorescent quantitative polymerase chain reaction（qRT-PCR）to detect FN,Ⅰ,andⅢtypes Collagen（COLⅠ,COLⅢ）,α-SMA and E-cadherin mRNA levels;right lower lobe lung tissue was detected by Western blot for protein FN,COLⅠ,COLⅢ,phosphate Protein expression levels of Smad3（p-Smad3）,Smad3,α-SMA and E-cadherin.2.Monocyte-macrophage（THP-1）adherence was induced by 100 ng/ml phorbol ester（PMA）.Oil red O staining was used to detect whether the induction was successful.CCK8 method was used to detect the effect of silica（SiO₂）dust on the proliferation of THP-1 cells.THP-1 macrophage/Beas-2B cell co-culture system was established and divided into four groups:control group,model group,SB431542 inhibitor group and SB431542 inhibitor control group.Except control group,the other three groups were stimulated THP-1 macrophage by adding SiO₂dust with concentration of 80μg/cm² in Transwell upper chamber and placed in culture.After 24 hours of incubation,10μmol/L SB431542 was added to the SB431542 inhibitor group,and the same amount of inhibitor cosolvent was added to the SB431542 inhibitor control group.Beas-2B cells in Transwell’s lower chamber were collected after 24 hours of incubation.Real-time fluorescent quantitative PCR（qPCR）was used to detect the expression of Smad3,α-SMA and E-cadherin.Western blot was used to detect the expression of TGF-βin THP-1macrophages stimulated by SiO₂ dust,and the expression levels of Smad3,phosphorylated Smad3,α-SMA,Vimentin,E-cadherin related to fibrosis.Results 1.Analysis of variance using the initial body weight of the rats before dust exposure as a covariate,the results showed that after adjusting the initial body weight as a covariate,the mean weight adjustments of the control group and the dust exposure group on day 5 were not equal（F=11.502,P=0.000）,the weight of the control group was significantly higher than that of the dust group.After 10 days of dust exposure,the weight of the control group and the dust group showed an upward trend.Compared with the normal saline control group,the lung tissue of the model group showed that the lung tissue was grayish-white with large numbers of silicon nodules,which was hard.The results of HE staining showed nodular structures of various sizes,which were fused with each other.Pulmonary interstitial fibrosis,partial alveolar septal rupture,and emphysema changes.Electron microscopy results showed that the osmophiles of the lung tissue of the model group were damaged,the mitochondrial ridges were dissolved and broken,and the microvilli were consolidated and squeezed.Lysosomal swelling and enlargement,increased number of typeⅠand typeⅡalveolar cells,nuclear shrinkage,tissue structure vacuole-like,collagen fibers increased,endothelial cells proliferated,blood vessels were squeezed and deformed,Sirius scar staining results showed that the model group had fibrous hyperplasia.It can be seen that typeⅠand typeⅢcollagen fibers dyed bright red and green are thicker and thicker than the control group,and are intertwined in a rope-like manner.The results of immunohistochemistry can show that the expression ofα-SMA and FN proteins in the model group is significantly higher.In the control group,qPCR and Western blot results showed that the expression levels of FN,COLⅠ,COLⅢ,p-Smad3,Smad3,andα-SMA mRNA and protein in lung tissues increased,and the expression of E-cadherin mRNA and protein The level was decreased,and the differences were statistically significant（P<0.05）.Comparing the SB431542inhibitor group with the SB431542 inhibitor control group,the lung tissue of the SB431542 inhibitor group can be seen dark red with no visible silicon nodules on the surface.Soft,HE staining results showed that the lung tissue structure was basically complete,no obvious nodules were seen,alveolar septal rupture,fusion with each other,emphysema-like changes,a small amount of exudate was seen in the alveolar cavity and bronchioles,and the electron microscope results showed Less dust deposits in lung tissue,swollen mitochondria,few mitochondrial ridges,damaged phytosomes and cytoplasm,Sirius scar staining results showed a small amount of red type I collagen fibers,which can be seen by immunohistochemical results The expression intensity ofα-SMA and FN proteins decreased,and qPCR and Western blot results showed that the expression levels of FN,COLⅠ,COLⅢ,p-Smad3,Smad3 andα-SMA mRNA and protein in lung tissue were reduced,and the expression levels of E-cadherin mRNA and protein The difference was statistically significant（P<0.05）;There was no significant difference in pathological observation between the model group and the SB431542 inhibitor control group,and no statistically significant difference in mRNA and protein expression levels of each factor detected by qPCR and Western blot（P>0.05）.2.Oil red O staining showed that after THP-1 was induced to adhere to macrophages from suspension cells,the cells changed from round to ellipse,a few cells extended pseudopodia,showed long spindle type,and the nucleus was stained red.Western blot showed that the expression of TGF-βprotein increased with the increase of dust concentration,which was in proportion to 0ug/cm².There was significant difference in the expression of TGF-βprotein among different concentration groups（P<0.05）.CCK8 results showed that cell viability decreased with the increase of dust dosage.When the concentration of SiO₂ dust was80ug/cm²,the cell viability decreased obviously.Transwell co-culture results showed that compared with the control group,the expression levels of Smad3,p-Smad3,α-SMA,Vimentin mRNA and protein in the model group were increased,while the expression levels of E-cadherin mRNA and protein were decreased（P<0.05）.Compared with the control group of SB431542 inhibitor,the expression levels of Smad3,p-Smad3,α-SMA,Vimentin mRNA and protein in the SB431542 inhibitor group were significantly increased（P<0.05）.The expression levels of E-cadherin mRNA and protein were significantly higher than those of the model group（P<0.05）.Compared with the model group,the expression levels of factor genes and proteins in the SB431542 inhibitor control group had no statistical significance（P>0.05）.Conclusion SiO₂ dust induces silicosis fibrosis through the TGF-β/Smad3signaling pathway.SB431542 may reduce the deposition of extracellular matrix（ECM）by inhibiting this signaling pathway,inhibit the EMT transformation process,and change the downstream fibrosis factors FN,COLⅠ,COLⅢ,α-SMA,Vimentin and E-cadherin expression,and then interfere with silicosis fibrosis.