The Mechanism Of Compound Kushen Injection In Preventing And Treating Radiation-induced Pulmonary Injury Based On Epithelial Mesenchymal Transition

Objective:Radiation-induced pulmonary injury is the most common complication of thoracic radiotherapy,the mechanism about previous studies mainly focused on the production and the effect of oxygen free radical and inflammatory cytokines,and the phenomenon and mechanism of epithelial mesenchymal transition(EMT)following radiation-induced pulmonary injury has not attract enough attention.Recently,a number of studies have shown that compound kushen injection has a certain inhibitory effect on epithelial mesenchymal transition.This project ims to establish a model of radiation-induced pulmonary injury in mouse,and to intervene with different concentrations of compound kushen injection,then to evaluate the damage degree of lung tissue,measure EMT related phenotypic markers,growth factors(such as TGF-?1)cytokines(such as TNF-?),and changes EMT messenger mRNA transcription level and the resulting TGF-beta 1/Smads and Wnt/beta–catenin signaling interaction changes.In this study,the effect of compound kushen injection on the epithelial mesenchymal transition pathway of in mice with radiation-induced pulmonary injury was observed to clarify the mechanism of compound kushen injection in preventing and treating radiation-induced pulmonary injury based on epithelial mesenchymal transition,so as to provide a reliable experimental basis for the clinical application of compound kushen injection in preventing and treating radiation-induced pulmonary injury.Method:1.Theoretical discussion:Through the retrospective analysis of traditional Chinese medicine and Western medicine on the knowledge of radiation-induced pulmonary injury,to explores the source flow of heat toxin and collaterals stasis correlation theory and analyzes its typical representative chinese patent medicine,compound kushen injection.2.Experimental study:194 male C57bl/6 mice with specific pathogen free,was randomly divided into 10 groups:Normal Control group,Model group,Low dose group of compound kushen injection,Middle dose group of compound kushen injection,High dose group of compound kushen injection,SB431542group,XAV-939group,Compoundkusheninjection+SB431542group,Compound kushen injection+XAV-939 group,Dexamethasone injection group.The C57bl/6 mice model of radiation-induced lung injury was successfully established by single 20 Gy bilateral lung irradiation using XStrahl Precision Radiotherapy Research Platform for Small Animals(SARRP).The levels of tumor necrosis factor-?(TNF-?)and transforming growth factor-?1(TGF-?1)in plasma of C57bl/6 mice were measured by enzyme-linked immunosorbent assay(ELISA)at week 4,8weeks.Real-Time PCR absolute quantification method was used to detect and analyze the content of TGF-beta 1,Smad3,GSK-3beta and beta-catenin mRNA.Western blot was used to detect the expression of TGF-beta1,Smad3,GSK-3beta and beta-catenin protein in mice lung tissue which was injured by radiation.Results:Results 1.The results of experiment one showed that C57bl/6 mice model of radiation-induced lung injury was successfully established by single 20 Gy bilateral lung irradiation using XStrahl Precision Radiation Research Platform for Small Animals(SARRP).Compound kushen injection can increase the lung coefficient of mice with radiation lung injury,and reduce the pathological changes of mice with radiation lung injury,and reduce the degree of pathological damage of radiation lung injury,and improve the general conditions of mice,including hair color,hair removal,food intake,activity and weight.Results 2.2.1 HE staining results:blank control group:the alveolar wall of lung tissue was thinner,alveolar structure was clear,capillaries were intact,no congestion and edema,no inflammatory cell infiltration,no obvious pathological changes were observed at all time points;model group:exudative lesions were predominant in the first two weeks after irradiation,a small amount of inflammatory cell infiltration was observed in alveolar cavity,and acute pulmonary interstitial edema and congestion appeared.At the 4th week,pulmonary interstitial edema and peribronchial inflammatory cell infiltration were observed,alveolar structure was destroyed,some alveoli atrophied,inflammatory cell infiltration decreased at the 6th week,pulmonary interstitial edema gradually subsided,alveolar septum gradually widened,alveolar cavity shrank,fibroblasts and collagen proliferation appeared,inflammatory cells decreased at the 8th week,pulmonary interstitial edema further increased.The alveolar septum widened,alveolar structure destroyed,some alveolar collapse accompanied by atelectasis,and a few spindles formed fibroblasts.At the 12th week,alveolar dilatation was aggravated,collagen proliferation was obvious,gas exchange space was significantly reduced,alveolar collapse was more obvious and fibrosis was observed in mice.In low,medium and high dose groups,exudative lesions were milder in the 2nd week,pulmonary interstitial edema,congestion and inflammatory cell infiltration were significantly reduced compared with model group.At week 4,a little inflammatory cell infiltration and mild pulmonary interstitial edema were observed;at week 8,inflammatory exudation,mild congestion of alveolar wall capillaries,mild pulmonary interstitial edema,partial collapse of alveoli and slight thickening of alveolar wall were observed,but the above-mentioned manifestations were significantly alleviated compared with the model group;at week 12,a little fibroblasts,mild thickening of alveolar wall and some pulmonary fibrosis were observed,but all of them were obvious.Compared with the model group,the time points of dexamethasone injection group and the high dose group of compound Sophora flavescens injection had no significant difference;the time points of signal pathway inhibitor group 1(SB431542,SB group)and signal pathway inhibitor group 2(XAV-939,XAV group)were in the middle and high dose group of compound Sophora flavescens injection,but there was no significant difference.At the 2nd and 4th weeks,the exudative lesions were mild,interstitial edema,congestion and inflammatory cell infiltration were significantly reduced in the group of compound matrine injection+signal pathway inhibitor group 1(CKI+SB),compound matrine injection+signal pathway inhibitor group 2(CKI+XAV),compared with the group of single drug intervention;inflammatory exudation,mild alveolar wall capillary congestion,interstitial pulmonary interstitial edema were observed at the 8th week,and interstitial pulmonary infiltration was significantly reduced.Mild edema,partial collapse of alveoli and slightly thickening of alveolar wall were observed in the 12th week,but the above-mentioned manifestations were significantly less than those in the single drug intervention group.A few fibroblasts were observed in the 12th week,alveolar wall was slightly thickened and some pulmonary fibrosis was observed,but they were significantly less than those in the single drug intervention group.2.2 Masson staining showed that collagen fibers were blue,muscle fibers were red and nuclei were blue-black.Normal group:less collagen fibers,more muscle fibers and more nuclei;model group:with the prolongation of modeling time,collagen fibers gradually increased and a large number of collagen fibers were visible;compared with model group,the staining of collagen fibers in each concentration group of compound Sophora flavescens injection was significantly inhibited;the staining of collagen fibers in signal pathway inhibitor SB431542 group and XAV-939 group was also more obvious.Compared with model group,the amount of collagen fibers was significantly reduced,which was similar to that of medium and high dose group of compound Sophora flavescens injection.The amount of collagen fibers in compound Sophora flavescens injection+SB431542 group,compound Sophora flavescens injection+XAV-939 group was significantly reduced at each time point,and the staining of collagen fibers was more significantly inhibited than that in single drug intervention group.Radiation pneumonia and radiation pulmonary fibrosis often occur at the same time.As time goes on,radiation pneumonia gradually reduces and the degree of radiation pulmonary fibrosis gradually aggravates.In this study,radiation pneumonia is the main disease in 4 weeks,radiation pneumonia gradually reduces and the degree of radiation pulmonary fibrosis is aggravated in 4 to 8 weeks,radiation pulmonary fibrosis is the main disease in 12 weeks,after compound matrine injection.Radioactive pneumonia and radiation pulmonary fibrosis were significantly inhibited after single or combined interventions of ejection and signal pathway inhibitors.The intervention effect of medium and high dose groups of Compound Sophora Flavescens Injection was better than that of low dose group of Compound Sophora Flavescens Injection,which was similar to that of signal pathway inhibitor group,and inferior to that of Compound Sophora Flavescens Injection+SB431542 group and Compound Sophora Flavescens Injection+XAV-939 group.2.3 The results of transmission electron microscopy showed that in the blank control group,the nucleus was large,oval,collagen fibers were not seen,the structure was intact,the alveolar cavity was relatively clean,the alveolar septum was not widened and thickened,the surface of alveolar epithelial cells was globular,the number of Golgi bodies was large,occasionally macrophages and red blood cells were seen;in the model group,the nucleus was shrinking,a large number of collagen fibers could be seen,and striated changes could be seen.Mitochondrial swelling of alveolar epithelial cells was obvious,with more active substances,less cytoplasm,more serious vacuoles,more mega-fine cells,accompanied by fibrocyte production;in each concentration group of Compound Sophora flavescens injection,the nucleus size was between normal group and model group at each time point,the number of collagen fibers was medium,the mitochondrial swelling of alveolar epithelial cells was mild,and it was in chronic inflammation.Repair phase.Signaling pathway inhibitor SB431542 group and XAV-939 group:At each time point,the nucleus size was between the normal group and the model group,and the number of collagen fibers was similar to that in the medium and high dose groups of compound matrine injection.Compound Sophora Flavescens Injection+SB431542 group,Compound Sophora Flavescens Injection+XAV-939 group,two combined groups:at each time point,the nucleus size was between the normal group and the model group,the number of collagen fibers was significantly reduced compared with the single drug intervention group,and the mitochondrial swelling of alveolar epithelial cells was also significantly reduced compared with the single drug intervention group,which was in the stage of chronic inflammation repair.Results 3.3.1 E-cadheren protein expression in lung tissue of mice in each group:E-cadheren protein was mainly expressed in cell membrane,cytoplasm and cell junction.Immunohistochemical results showed that E-cadheren protein showed brown or brown or light yellow granules in the cytoplasm and membrane of lung tissue.The expression rate of E-cadheren in lung tissue of C57bl/6 mice in blank control group was higher than that in model group;the expression rate of E-cadheren in lung tissue of C57bl/6 mice in model group was very low;the expression rate of E-cadheren in lung tissue of C57bl/6 mice in each dose group of Compound Sophora flavescens injection was between model group and blank control group at different time points,and the expression rate of E-cadheren was medium;the signal pathway inhibitor SB431542 group and XAV-939 group C57.The expression rate of E-cadheren in lung tissue of bl/6 mice at different time points was also between model group and blank control group.The expression rate of E-cadheren was similar to that of medium and high dose group of compound Sophora flavescens injection.The expression rate of E-cadheren in lung tissue of C57bl/6 mice in combination group of compound Sophora flavescens injection+SB431542group and compound Sophora flavescens injection+XAV-939 group was between model group and blank pair.However,the expression of E-cadheren in the control group was significantly higher than that in the single drug intervention group,which was closer to the blank control group.3.2 Vimentin protein expression in lung tissues of mice in each group:Vimentin protein was mainly expressed in the cytoplasm,often attached to the nucleus,endoplasmic reticulum and mitochondria.Immunohistochemical results showed that Vimentin protein was brown or brown or yellowish granules in the cytoplasm of lung tissue.The expression rate of Vimentin in lung tissue of C57bl/6 mice in blank control group was very low;the expression rate of Vimentin in lung tissue of C57bl/6 mice in model group was higher;the expression rate of Vimentin in lung tissue of C57bl/6 mice in each dose group of Compound Sophora Flavescens Injection was between model group and blank control group at each time point,and the expression rate of Vimentin was medium;the expression rate of C57bl/6 in signal pathway inhibitor SB431542 group and XAV-939 group was small.The expression rate of Vimentin in rat lung tissue at different time points was also between the model group and the blank control group,and the expression rate of Vimentin was similar to that in the medium and high dose group of Compound Sophora Flavescens Injection;the expression rate of Vimentin in C57bl/6 mice lung tissue at different time points in the combination group of Compound Sophora Flavescens Injection+SB431542 group,Compound Sophora Flavescens Injection+XAV-939 group,though between the model group and the blank control group.However,the expression rate of Vimentin was significantly lower than that of single drug intervention group.Results 4.Compared with the blank control group,the serum levels of TGF-beta 1and TNF-alpha in the model group increased significantly in the 4th and 8th weeks(P<0.01);compared with the model group,the serum levels of TGF-beta 1 and TNF-alpha decreased significantly in the 4th and 8th weeks(P<0.01,P<0.05),except for the 8th week in the low-dose group of compound kushen injection(P>0.05).Compared with the medium dose group of compound kushen injection,the serum levels of TGF-beta 1 and TNF-alpha in mice of compound kushen injection+signaling pathway inhibitor group 1(CKI+SB)decreased significantly at the 8th week(P<0.05,P<0.01),while those of compound kushen injection+signaling pathway inhibitor group 2(CKI+XAV)were smaller.The content of serum TNF-a in mice decreased significantly at the 4th week(P<0.05).The content of serum TGF-beta 1 and TNF-a in mice in low dose group of compound kushen injection increased significantly at the 4th week(P<0.05).There was no significant difference in the content of serum TGF-beta 1 and TNF-a in mice in other intervention groups at the 4th and 8th week(P>0.05).Compared with the high dose group of compound kushen injection,the content of the serum levels of TGF-beta 1and TNF-alpha in low dose group of compound kushen injection increased significantly at the 4th week(P<0.05,P<0.01),while the serum levels of TGF-beta 1 in low dose group of compound kushen injection increased significantly at the 8th week(P<0.05,P<0.01).The serum levels of TGF-beta1 in group of compound kushen injection+CKI+SB decreased significantly at the 4th week.(P<0.05,P<0.01),the serum TNF-alpha content of mice in CKI+SB group decreased significantly at the 8th week(P<0.05),and there was no significant difference in the serum TGF-beta 1 content of mice in the other intervention groups at the 4th and 8th weeks(P>0.05);compared with the dexamethasone injection group,the serum TNF-alpha content of mice in the low dose group of compound kushen injection decreased significantly at the8th week(P>0.05).The serum levels of TGF-beta 1 and TNF-alpha increased significantly in the 4th week(P<0.05,P<0.01).The serum levels of TGF-beta1 in the low dose group of Compound kushen injection increased significantly in the 8th week(P<0.05,P<0.01).The serum levels of TGF-beta 1 in the group of Compound kushen injection+signal pathway inhibitor 1(CKI+SB)decreased significantly in the 4th week(P<0.05),and the rest of mice.There was no significant difference in serum TGF-beta 1 level between the intervention group and the control group at the 4th week and 8th weeks(~?P>0.05).Results 5.5.1 Real e time RT-PCR Results:Compared with the control group,the expression levels of TGF-beta 1,Smad3,beta-catenin and GSK-3 beta mRNA in the model group increased significantly at the 4th and 8th weeks(P<0.01);compared with the model group,the expression levels of TGF-beta 1,Smad3,beta-catenin and GSK-3 beta mRNA in the CKI-H group,DXM group,CKI+SB group,CKI+XAV group decreased in different degrees at the 4th and8th weeks(P<0.01,P<0.05).Compared with CKI-H group,the expression levels of TGF-beta 1,Smad3,beta-catenin and GSK-3 beta mRNA in CKI-L group?CKI-M group?DXM group,SB group,XAV group at the 4th and 8th weeks did not change significantly(P>0.05),while the expression levels of TGF-beta,Smad3,beta-catenin and GSK-3 beta mRNA in CKI+SB group,CKI+XAV group at the 4th and 8th weeks changed significantly(P<0.05,P<0.01);Compared with DXM group,the expression levels of TGF-beta 1,Smad3,beta-catenin and GSK-3 beta mRNA in CKI-L group?CKI-M groups?CKI-H group,SB group and XAV group were not significantly changed at 4and 8 weeks(~?P>0.05).The expression levels of TGF-beta 1,Smad3,beta-catenin and GSK-3 beta mRNA in CKI+SB group,CKI+XAV group were significantly changed at 4 and 8 weeks(P<0.05,P<0.01).5.2 Westernt bolt Results:Compared with the control group,the expression levels of TGF-beta 1,Smad3,beta-catenin and GSK-3 beta protein in the model group increased significantly in the 4th and 8th weeks(P<0.01);compared with the model group,the expression levels of TGF-beta 1,Smad3,beta-catenin and GSK-3 beta protein in the CKI-H group,DXM group,CKI+SB group,CKI+XAV group decreased in varying degrees in the 4th and8th weeks.(P<0.01,P<0.05);Compared with CKI-H group,the expression levels of TGF-beta 1,Smad3,beta-catenin and GSK-3 beta protein in CKI-L group,CKI-M group,DXM group,SB group,XAV group at the 4th and 8th week did not change significantly(P>0.05),while the expression levels of TGF-beta 1,Smad3,beta-catenin and GSK-3 beta protein in CKI+SB group,CKI+XAV group at the 4th and 8th week changed significantly(P<0.05,P<0.01).Compared with DXM group,the expression levels of TGF-beta,Smad3,beta-catenin and GSK-3 beta protein in CKI-L group,CKI-M group,CKI-H group,SB group,XAV group did not change significantly(P>0.05).The expression levels of TGF-beta,Smad3,beta-catenin and GSK-3 beta protein in CKI+XAV group changed significantly(P<0.05,P<0.01).5.3 Immunofluorescence results showed that TGF-beta 1 protein was mainly expressed in the interstitium.Smad3 protein is mainly expressed in the nucleus and cytoplasm.GSK-3 beta protein is mainly expressed in the nucleus,cytoplasm and cell membrane.The expression of beta-catenin protein was mainly localized in the nucleus,cytoplasm and cell membrane.Immunofluorescence results of TGF-beta 1 protein showed that TGF-beta 1,Smad3,beta-catenin and GSK-3 beta protein were red granules with different depths in pulmonary interstitium.The expression rates of TGF-beta 1,Smad3,beta-catenin and GSK-3beta in lung tissues of C57bl/6 mice in blank control group were very low;the expression rates of TGF-beta 1,Smad3,beta-catenin and GSK-3beta in lung tissues of C57bl/6 mice in model group were higher;the expression rates of TGF-beta 1 in lung tissues of C57bl/6 mice in each dose group of Compound kushen injection were between model group and blank control group,and the expression rates of TGF-beta-beta-beta-Smad3,beta-cat-The expression rates of TGF-beta,Smad3,beta-catenin and GSK-3beta in lung tissue of C57bl/6 mice in SB431542 group and XAV-939group were also intermediate between model group and blank control group.The expression rates of TGF-beta,Smad3,beta-catenin and GSK-3beta were similar to those in medium and high dose group of compound kushen injection.The expression rates of TGF-beta 1,Smad3,beta-catenin and GSK-3beta in lung tissue of C57bl/6 mice in SB431542 group and XAV-939 group were between model group and blank control group,but the expression rate of TGF-beta 1 was significantly lower than that in single drug intervention group.Conclusion:The pathogenesis of radiation-induced pulmonary injury is complex.Epithelial-mesenchymal transition(EIT)after radiation-induced lung injury has attracted much attention in recent years.Therefore,basing on the theory of epithelial-mesenchymal transition and heat toxin collateral stasis in traditional Chinese medicine(TCM),we chose the compound kushen injection to intervene in radiation-induced pulmonary injury in mice,and achieved good radioprotective effects.The possible mechanisms are as follows:1.Compound kushen injection can reduce radiation-induced pulmonary injury in C57bl/6 mouse by lowering the levels of EMT-related TNF-?and TGF-?1 in the plasma;2.The expression of EMT related genes in TGF-beta/Smads and Wnt/beta-catenin signaling pathway can be reduced by compound kushen injection.3.Compound kushen injection may be used to prevent and treatradiation-induced pulmonary injury by reducing the expression of EMT-related channel proteins in TGF-beta/Smads and Wnt/beta-catenin signaling pathways.