Ambroxol Glucocorticoids On The Rat Fetal Lung Bone Morphogenetic Protein Signaling Pathway

PartⅠ:Effects of antenatal ambroxol and glucocorticoid on rat fetal lung morphogenesisObjective:To explore the effects of ambroxol with glucocorticoid (dexamethasone and betamethasone)administered antenatally on lung morphogenesis of rat embryo.Methods:Eighteen pregnant rats were randomly devided into 6 groups (n=3):the ones treated with ambroxol,1-day dexamethasone(1D-DEX), 1-day betamethasone(1D-BEX),3-day dexamethasone(3D-DEX),3-day betamethasone(3D-BEX)and 9g/L saline antenatal.The pregnant rats were injected with 0.2mg/(kg·d)of dexamethasone or betamethasone, 100mg/(kg·d)of ambroxol and saline respectively.The tissues were stained with hematoxylin and eosin(H&E)for morphometric measurements.The histologic structures of fetal rat lungs were observed with light microscope,transmission electron microscope and were assessed by image analysis.Results:Compared with those of controls,fetal rat lung injected with ambroxol,3D-DEX,3D-BEX,1D-BEX showed larger alveolar space, larger respiratory thecal perimeter(P＜0.05)and thinner alveolar walls (P＜0.01)but not that of 1D-DEX injected rats.Fetal lung in the rats treated with 3D-BEX had larger pulmonary alveoli,larger respiratory thecal perimeter and thinner alveolar septum(P＜0.01)than those of 3D-DEX.Under transmission electron microscopy,lamellar body and cellular organs in endochylema of typeⅡalveolar cell of the fetal rat lung treated with ambroxol and glucocorticoid were more common compared with those of the controls,except for thaoseof the 1D-DEX.Obvious changes were identified in the fetal lungs of the 3D-BEX and 3D-DEX treated animals.Conclusion:Ambroxol,low dose of dexamethasone and betamethasone can promote the development of rats fetal lung,and betamethasone will be better than those of the dexamethasone and ambroxol.Multi-dose of glucocorticoid has prior effect to single one.PartⅡ:Effects of antenatal ambroxol and glucocorticoid on signal transduction of bone morphogenetic protein in the fetal lung of ratsObjective:To investigate the role of antenatal ambroxol and glucocorticoid(dexamethasone and betamethasone)on bone morphogenetic protein(BMP)signal transduction of the rat fetal lung.Methods:Eighteen pregnant rats were randomly divided into six groups with 3 rats in each group:rats treated with ambroxol,1-day dexamethasone(1D-DEX),1-day betamethasone(1D-BEX),3-day dexamethasone(3D-DEX),3-day betamethasone(3D-BEX)and saline (controls)antenatal,followed cesarean section on the 19th day of gestation.The mRNA level of BMP4,BMPR-Ⅱ,Smad1 and ATF-2 of 6 fetal rat lungs in each pregnant rat were analyzed by reverse transcriptase polymerase chain reaction(RT-PCR).The expression of BMP4, BMPR-Ⅱ,Smad1 and ATF-2 antigen expression in embryo lung were assessed by immunohistochemistry stain.The expression of BMP4 and BMPR-Ⅱwere detected by Western blotting. Result:(1)BMP4 mRNA expression was up-regulated in the rats treated with 1D-BEX,3D-BEX and 3D-DEX compared with controls(P＜0.05). No significant difference was found in the 1D-DEX and ambroxol treated rats compared with the controls(P＞0.05).Results of the immunohistochemistry and Western blotting demonstrated that the expression of BMP4 protein was significantly increased in the 1D-BEX, 3D-BEX and 3D-DEX treated animals(P＜0.01).While no change could be found in the fetal lung of ambroxol and 1D-DEX treated anmimals. The BMP4 protein levels in ambroxol and 1D-DEX treated animals were not notably difference compared with that of controls(P＞0.05).(2)BMPR-ⅡmRNA expression was significant higher in the glucocorticoid treated rats(P＜0.01).Results of western blotting and immunohistochemistry analysis manifested that glucocorticoid treated group had a higher BMPR-Ⅱexpression than that of the normal control(P＜0.01),while the change in the ambroxol treated rats was not significant (P＞0.05).(3)The levels of Smad1 mRNA in the fetal lung of glucocorticoid treated rats were obviously higher than those of the normal controls(P＜0.05). Phospho-Smad1(pSmad1)protein expression of those of 1D-BEX, 3D-BEX and 3D-DEX treated rats were higher than the controls(P＜0.01),while the change in those of ambroxol and 1D-DEX treated rats was not significant(P＞0.05).(4)The difference of the expression of ATF-2 mRNA was not statistically difference between those of the ambroxol and glucocorticoid treated rats and the normal controls(P＞0.05).The protein expression of ATF-2 of those of 1D-EX,3D-BEX and 3D-DEX injected rats exceeded the controls(P＜0.01).Conclusion:Betamethasone and dexamethasone may have an effects in the regulation on BMP signal transduction in the rat fetal lung. Up-regulation of BMP4,BMPR-Ⅱand Smad1 might be one of critical factors for the glucocorticoid-induced maturity of fetal lung.