Based On The Relationship Between MiRNA-21, Let-7d And Autophagy In Idiopathic Pulmonary Fibrosis, The Intervention Effects Of Astragalus Components Were Studied

Idiopathic pulmonary fibrosis(IPF)is chronic,progressive diffuse interstitial lung diseases(ILD)caused by unknown causes.In interstitial lung disease,IPF has a high incidence rate,with short lifetime and relatively poor prognosis.It is Accounts for about 20%of ILD,and the 5 years survival rate is less than 50%.In present study,it showed that the repetitive alveolar epithelial cells(AEC)injury is the most important cause which leading to the origination of IPF.At prensent,it consensus that:epithelial mesenchymal transdifferentiation(EMT)induced by continuous alveolar epithelial injury is most closely related to the occurrence of IPF.In which,high expression of TGF-beta 1 induced by the multiple factors and excessive activation of smad molecular signal is the most important molecular mechanism for the EMT.The up-regulation of microRNA-21 and the down-regulation let-7 are also closely related to the IPF.microRNA-21 and let-7d directly or indirectly regulated the TGF-beta 1/smad signal which relates to EMT.Idiopathic pulmonary fibrosis belongs to "impotent" lung "lung bi" category in traditional Chinese medicine,with the basic pathogenesis of qi deficiency blood stasis and the treatment is given priority to yiqi huoxue.To TCM clinical treatment of IPF,astragalus is the most widely used as yiqi pills,tonifying qi and yang,gu table stop sweat,invigorate the circulation of qi and blood.Astragalus membranaceus mainly includes the composition of astragalus polysaccharides,Astragaloside,astragalus flavone.According to research,the composition of astragalus polysaccharides,Astragaloside,astragalus flavone are thought to have a anti-fibrosis effect.The study of the effect of astragalus composition on pulmonary fibrosis are limited to one unit with different conditions,make a difficulty to the compare the treatment effects of different ingredients on pulmonary fibrosis.Basing on the imbalances microrna-21 and the Let-7 d and EMT induced by TGF-beta l with down-regulation autophagy,our study explore intervention effect of astragalus polysaccharide,astragaloside and astragalus flavone on IPF.Methods&ResultsmethodsAnimal groups:90 C57BL/6 mice were randomly divided into sham operation group,model group and astragalus polysaccharides Intervention group,Astragaloside Intervention group,astragalus flavone group,astragalus astragalus flavone combined Astragaloside Intervention group.The mice of the model group and drug treatment were one-time Intratracheal instillation with BLM,and same amount of saline for sham operation group.Treatment animals were gavage with corresponding drugs and same amount of saline for sham operation group for 28 days.animals in each group were randomly divided into two groups Further.One group were operated for BALF(bronchoalveolar lavage fluid),which were used to cell count and cytological classification,and another group were used to HE and Masson staining to observe organization form and collagen expression level,immunohistochemical:TGF-beta 1,TNF alpha,Molecular experiment analysis:realtime PCR of miRNA-21 and let-7d,western bloting detection TGF?-1,TNF alpha beta,LC3 ?/?,p62,beclin-1,smad3,p-smad3,?-SMA and E-cadherin.To illustrate the relation of autophagy and the expression of miRNA-21 and let-7d,A549 cell line induced by TGF-? and 3-MA was treated by Astraloside,formononetin,Isorhamnetin,kaempferol,Calycosin,Quercetin with 12.5?mol/L.Total protein were collect and LC3 ?/?,p62,beclin-1,smad3,p-smad3,?-SMA and E-cadherin were detected by western-bloting.Results1.Effect of Ingredient in Astragalus on Inflammatory reaction Compared with sham goup,The inflammatory cells in model group alveolar lavage flood,inflammatory cell(P<0.05)and macrophages(P<0.01)increased significantly.Astragalus polysaccharides,astragaloside can significantly reduce inflammatory cells(P<0.05),Astragalus Total flavonoids with same effect(P<0.01)with a significantly significant difference to Astragalus polysaccharides,astragaloside.2.Effect of Ingredient in Astragalus on pathomorphology of lung tissue HE staining showed that BLM cause lung tissue inflammation cells infiltration,astragalus polysaccharides,astragaloside can reduce the amount of inflammatory cells infiltration.astragalus flavone significantly restrain the level of tissue inflammation,superior to astragalus polysaccharides group,astragaloside group.Masson staining showed that BLM induced lung tissue collagen hyperplasia,lung tissue structure disorder.astragalus flavone can significantly lower model lung tissue collagen deposition,and the effect.3.Effect of Ingredient in Astragalus on expression of TGF-beta 1 and TNF alpha The immunohistochemical results show that,in the model group,strongly expression of TGF-beta 1 and TNF alpha in alveolar space,especialy in peripheral pulmonary nodules and rare in the formation of pulmonary nodules.Compared with model group,astragaloside(P<0.01)and astragalus flavone group(P<0.01)can significantly reduce the TGF-beta 1 and TNF alpha.Western-bloting analysis also showed that:three kinds of ingredients are reducing TGF beta-1 and TNF alpha expression(P<0.05),but the astragalus flavone group was more significant(P<0.01)comparing to astragaloside and astragalus polysaccharides.4.Effect of Ingredient in Astragalus on Expression of miRNA-21and let-7d Realtime-PCR analysis suggest:in pulmonary fibrosis animal model induced by BLM the expression of miRNA-21 increased(P<0.05),while the let-7d expression decreased(P<0.05),astragalus flavone can reverse the expression of micrornas-21(P<0.01),but no obvious influence on the let-7d.5.Effect of Ingredient in Astragalus on autophagy activity in pulmary tissue It showed that LC3?/,Beclin 1 were decreased and P62 accumulate significantly in pulmonary fibrosis model induced by BLM.it prove that autophagy level was significantly inhibited;Compared with model group,both astragalus flavone and Astragaloside can significantly improve the LC3?/? and Beclin 1 and inhibited P62((P<0.01).6.Effect of Ingredient in Astragalus on expression of Smad signal protein and marker protein related to EMT.In the process pulmonary fibrosis,smad3 phosphorylation level increased(P<0.01),while smad7 decreased(P<0.01),that reinforcing the espressiong of alpha-SMA(P<0.01)with a lower E-cadherin expression.Activated downstream signal of smad3 phosphorylation promote the occurrence of EMT.With the intervention of Astragaloside and astragalus flavone,smad7 raised with a lower smad3 phosphorylation levels,which inhibited the expression of alpha-SMA decrease,with a higher E-cadherin.It show that astragalus flavone and Astragaloside inactivated TGF-beta 1 downstream signal,and restrain the occurrence of EMT.7.Effect of Ingredient in Astragalus on autophagy and micromas-21 and Let-7d,in the A549 cell indued by TGF-beta 1 and 3-MA Compared with the control group,expression of LC3 ?/? and beclin 1 decreased(P<0.01),as that p62 increased(P<0.01),while the miRNA-21 and Let-7 d expression were significantly lower(P<0.05),in modle group induced by TGF-beta 1.Inhibitor 3-MA can further enhance the miRNA-21 and inhibite the Let-7d.Compared with model group,quercetin and astragaloside reversed the inhibition on autophagy by 3-MA,with higher LC3 II and lower p62(P<0.01).It show significant inhibition on micromas-21,without effect on Let-7d.Other ingredients has no effect on both miRNA-21 and let-7d.Smad7,the target protein of miRNA-21,decreased and smad3 phosphorylation level enhancement,while autophagy activity weakened.After joining autophagy inhibitor 3-MA,smad7 cut,and smad3 phosphorylation level and a-SMA expression further increase.Astragaloside and quercetin improve cell autophagy activity and inhibited micrornas-21,with improving of the expression of smad7 and droping smad3 phosphorylation levels.It show EMT inhibtion.8.Effect of Ingredient in Astragalus on PI3K/AKT/Mtor/P70S6K signalIn A549 cell induced by TGF-beta 1,the p-PI3K,p-AKT,p-mTOR,p-70 s6k are increased,and LC3 II decreased.It show significant inhibition on the activity of cell autophagy through the PI3K/AKT/mTOR signal,chich can be reversed by quercetin s and astragaloside,except for PI3K.Conclusion:1 LAstragalus flavone can better inhibit lung inflammationthe proliferation and collagen deposition,alleviating development of pulmonary fibrosis.2.In the development of Pulmonary fibrosis induced BLM,the expression of miRNA-21 increased and Let-7d decreased.Astragalus flavone can obviously inhibit expression of micrornas-21 without effect on Let-7d.3.Astragalus flavone and Astragaloside inhibited EMT through TGF-betal/smad signal,which may be related to the activity of cell autophagy,as which Astragalus flavone was superior to Astragaloside.4.with the treatment of TGF-betal on alveolar cells,expression of smad7 was down-regulated,smad2/3 signal was excessive activated and promoted the EMT,which may be associated with increased expression of miRNA-21 and the decreased expression of let-7d.Improve cell autophagy activity can lead to cut expression of miRAN-21,and a rise in the let-7d.Cell autophagy involved regulation on the expression of the miRAN-21 and let-7d.5.Astragaloside and quercetin anti-pulmonary fibrosis is not only by cutting the expression of TGF-beta 1,still can regulated AKT/mTOR signal independently.