The Effect Of Bushen Huoxue Tiaoying Huatan Recipe On Homing Of Bone Mesenchymal Stem Cells In Asthma Based On MiRNA139-5p Regulating Notch Pathway

1 ObjectiveTo observe the effect of Bushen Huoxue Tiaoying Huatan Recipe-Yanghe Pingchuan Granules on the homing of asthmatic bone mesenchymal stem cells(BMSCs),to explore the possible mechanism of miRNA139-5p regulating Notch pathway on the homing of asthmatic BMSCs,in order to reveal the molecular mechanism of Yanghe Pingchuan granules promoting the homing of BMSCs to inhibit airway inflammation in asthma.2 Methods2.1 In vivo2.1.1 Experimental groupsEighty-four SPF SD rats were randomly divided into 7 groups:normal control(NC)group,model control(MC)group,BMSCs implantation(BMSCs)group,BMSCs implantation+Notch inhibitor(BMSCs+N inhibitor)Group,BMSCs implantation+Yanghe Pingchuan granules intervention(BMSCs Yanghe)group,BMSCs implantation+Notch inhibitor+Yanghe Pingchuan granules intervention(BMSCs Yanghe+N inhibitor)group,BMSCs implantation+Dexamethasone intervention(BMSCs DXM)group.The chronic asthma rat model was replicated.2.1.2 Intervention methodsBMSCs were extracted by the whole bone marrow adherence method.Except for the NC and MC group,homologous BMSCs(1×106 cells/mL)were injected into each group via tail vein an hour before the initial challenge.From the 21st day,each group was given drug intervention.NC,MC,BMSCs group were treated with normal saline(1mL·100mg-1),once a day.BMSCs+N inhibitor group were injected with DAPT(10mg·kg-1),once every other day,and were treated with normal saline(1mL·100mg-1),once a day.BMSCs Yanghe group were treated with Yanghe Pingchuan granules solution(7.74g·kg-1),once a day.BMSCs+DXM group were treated with dexamethasone suspension(0.125mg·kg-1)an hour before challenge,once a day.BMSCs Yanghe+N inhibitor group were injected with DAPT(10mg·kg-1)once every other day,and were treated with Yanghe Pingchuan granules solution(7.74g·kg-1),once a day.On the day of challenge,treatment was given an hour before the challenge.The above intervention lasted for 6 weeks.2.1.3 Evaluation indexThe general activity of rats was observed.The expression of fluorescent labeled BMSCs in the lung tissue of asthmatic rats was observed by flow cytometry.The lung histopathology was observed by He staining.The inflammatory cells in BALF were classified and counted were evaluated by Wright-Giemsa staining.The expressions of Th1,Th2 related cytokines and chemokines in rat serum were detected by ELISA.The expressions of miR139-5p and Notch pathway related genes in lung tissue were detected by RT-qPCR.The expression of Notch pathway related proteins in lung tissue was detected by Western blot.2.2 In vitro2.2.1 Cell construction and siRNA screeningThe rat bronchial epithelial cells were obtained by scrubbing after digestion.The containing serum of Yanghe Pingchuan granules is made,and then the optimal concentration and action time are screened out.The Notchl siRNA was designed and constructed,and select the best interference plasmid.2.2.2 Experimental groups and intervention methodsThe experiment was divided into 6 groups.Blank control(blank)group:Normal rat bronchial epithelial cells were cultured with 10%blank serum.Model control(model)group:Bronchial epithelial cells were obtained from asthmatic rats were cultured with 10%blank serum.BMSCs group:Bronchial epithelial cells of asthmatic rats were intervened by BMSCs.siRNA group:Bronchial epithelial cells of asthmatic rats were transfected with Notchl siRNA.Yanghe Pingchuan Granules(Yanghe)group:Bronchial epithelial cells of asthmatic rats were cultured with the best concentration of Yanghe Pingchuan Granules containing serum.BMSCs+Yanghe Pingchuan Granules(BMSCs Yanghe)Group:Bronchial epithelial cells of asthmatic rats were intervened by BMSCs and cultured with Yanghe Pingchuan Granules containing serum.2.2.3 Evaluation indexDouble luciferase reporter assay was used to confirm whether there was a binding site between miR139-5p and Notch1.The expression of Th1/Th2 cytokines in bronchial epithelial cells was measured by ELISA.The expressions of Thl/Th2 cytokines and specific transcription factors,miR139-5p,CXCR4,SDF-1 and Notch pathway related genes were detected by RT-qPCR.The expressions of CXCR4,SDF-1 and Notch pathway related proteins in bronchial epithelial cells were detected by immunofluorescence staining.The expressions of CXCR4,SDF-1 and Notch pathway related proteins in bronchial epithelial cells were detected by Western blotting.3 Results3.1 In vivo3.1.1 Tracing of BMSCsThe expression of CFSE in lung tissue increased gradually with time,reached the highest value at 72 h,then decreased gradually,and reached the lowest value at 144h.It shows that allogeneic BMSCs can migrate to the lung tissue after being injected into the asthmatic rat model via the tail vein.3.1.2 Histopathological observation of lung tissueThe lung tissue structure of rats in the NC group is complete,without obvious inflammation.The lung tissue structure of MC group was severely damaged,with bronchial epithelial shedding and inflammatory cell infiltration.The smooth muscle of the tube wall is thickened with a large amount of mucus-like substance inside.In the BMSCs group,there were more folds in the bronchial mucosa and inflammatory cells gathered in the surrounding lung tissue.The bronchial lumen stenosis and epithelial shedding in the BMSCs+N inhibitor group and the BMSCs Yanghe group were improved compared with the MC group,and the lung tissue structure destruction and inflammatory cell infiltration were alleviated.The lung tissue structure of BMSCs Yanghe+N inhibitor groups was less damaged.Tissue hyperplasia and inflammation are not obvious.The bronchial lumen of the BMSCs DXM group was still regular,and there was less inflammatory cell infiltration around the lung tissue.3.1.3 Comparison results between groups Comparison between MC group and NC groupCompared with NC group,the expressions of N,EOS and M in BALF of MC group increased,while the expression of MΦ decreased significantly(P<0.01).The expression of IFN-γ in serum decreased,as was the expression of IL-4,IL-13,CXCR3,CCR4 and CCR7 increased(P<0.01).The expressions of CXCR4,T-bet mRNA/protein and miR139-5p in lung tissue were all increased,and the expressions of GATA-3 mRNA/protein,Notchl,Jaggedl,and RBP-J mRNA/protein decreased(P<0.05,P<0.01).Comparison between Yanghe intervention group and MC groupCompared with MC group,the expression of N,EOS,M in BALF of BMSCs Yanghe group and BMSCs Yanghe+N inhibitor group decreased,while the expression of MΦ increased(P<0.01).The expression of serum IFN-γ increased,and the expression of IL-4,IL-13,CXCR3,CCR4,and CCR7 decreased(P<0.05,P<0.01).The expressions of CXCR4,T-bet mRNA/protein and miR139-5p in lung tissue were all increased,and the expressions of GATA-3 mRNA/protein,Notch1,Jagged1,and RBP-J mRNA/protein decreased(P<0.05,P<0.01).Comparison between Yanghe intervention group and BMSCs groupCompared with the BMSCs group,the percentage of N and M in BALF of the BMSCs Yanghe group,BMSCs Yanghe+N inhibitor group decreased,and MΦincreased(P<0.01).The expressions of serum IL-4 decreased,while IFN-y increased(P<0.05).The expressions of T-bet mRNA/protein,miR139-5p,CXCR4,SDF-1 increased,GATA-3,Notch1,and the expression of RBP-J mRNA/protein decreased in Lung tissue(P<0.05,P<0.01).3.1.4 Correlation analysisCorrelation analysis of CXCR4,SDF-1,inflammatory cells,cytokines,and Notch pathwayCXCR4 was positively correlated with IFN-y,T-bet protein,miR139-5p,and SDF-1 was positively correlated with T-bet mRNA(P<0.05).CXCR4 is negatively correlated with EOS,IL-4,CCR4,GATA-3 mRNA,Notchl,SDF-1 was negatively correlated with M,CXCR3,Notch1,Hes1(P<0.05).Correlation analysis of cytokines/chemokines and Notch pathwayIFN-y was positively correlated with T-bet and miR139-5p(P<0.05).IL-4 was positively correlated with Notch1 mRNA(P<0.05).IL-13 was positively correlated with Jaggedl(P<0.05).CCR4 is positively correlated with Notchl and Jagged1.IFN-γwas negatively correlated with Notch1 mRNA(P<0.05).IL-4 was negatively correlated with miR139-5p(P<0.05).Correlation analysis of miR139-5p,Th1/Th2 factors,and Notch pathwayT-bet mRNA was positively correlated with miR139-5p(P<0.05).GATA-3 mRNA was positively correlated with Notch1mRNA and RBP-JmRNA(P<0.05).T-bet was negatively correlated with Notch1 mRNA(P<0.05).miR139-5p was negatively correlated with Notch1 mRNA,Notchl and Hesl(P<0.05).3.2 In vitro3.2.1 Dual luciferase reportThe results showed that,compared with the Notch1+NC group,the luciferase activity of the miR139-5p mimic group treated with Notch1 3’-UTR was significantly decreased.When the potential binding site of miR-139-5p is mutated,the luciferase activity is not significantly reduced.3.2.2 The changes of Model groupCompared with blank group,the expression of IL-5,IL-13,miR139-5p,and GATA-3 of bronchial epithelial cells increased significantly,as was CXCR4,SDF-1,Notch1,Jagged 1,RBP-J,Hes1mRNA/protein,while the expression of IFN-y,IFN-y mRNA,Th1/Th2,and T-bet protein of model group decreased(P<0.01).3.2.3 The changes of Yanghe intervention groupCompared with model group,the expression of CXCR4,SDF-1 mRNA/protein,IFN-y,Thl/Th2,miR139-5p,and T-bet of bronchial epithelial cells in Yanghe group and BMSCs Yanghe group increased,while the expression of Notch 1,Jagged 1,RBP-J,Hes1mRNA/protein,IL-5,IL-13,and GATA-3 decreased(P<0.05,P<0.01).Compared with BMSCs group,IL-13 of BMSCs Yanghe group decreased,as was RBP-J,Hes1mRNA and Notch1,Jagged,and Hesl protein in bronchial epithelial cells,while thr expression of SDF-1,GATA-3 protein increased(P<0.05).3.2.4 Correlation analysisCorrelation analysis of CXCR4,SDF-1 and Thl/Th2 cells,miR139-5p,Notch pathwayCXCR4 mRNA was positively correlated with IFN-y(P<0.01).CXCR4 was positively correlated with miR139-5p(P<0.05).CXCR4 mRNA was negatively correlated with Notch1 mRNA(P<0.01).SDF-1 was negatively correlated with IL-4 and NICD mRNA(P<0.05).SDF-1 mRNA was negatively correlated with Notch1 mRNA and Jagged 1 mRNA(P<0.05).Correlation analysis of Thl/Th2 cells,miR139-5p and Notch pathwayIFN-y was negatively correlated with Hesl mRNA and Notchl(P<0.01 or P<0.05).miR139-5p was negatively correlated with Hesl mRNA,Notchl and Jaggedl(P<0.05).IL-4 was positively correlated with Jagged1 mRNA and RBP-J(P<0.05).4 Conclusion1.There are many kinds of inflammatory cell infiltration in chronic asthma rat model.The expressions of Th2-related inflammatory factors/chemokines and specific transcription factors in lung tissue and serum were abnormally increased,while the expression levels of Thl-related factors decreased,Thl/Th2 balance shifts to Th2.2.Allogeneic BMSCs can migrate to asthmatic lung tissue,regulate Thl/Th2 cell imbalance,and improve asthma airway inflammation.After the combined intervention of Bushen Huoxue Tiaoying Huatan Recipe,the improvement of asthma airway inflammation was better than that of BMSCs alone.3.Bushen Huoxue Tiaoying Huatan Recipe can up-regulate the expression of miR139-5p in asthmatic lung tissue.Acting on Notch1,the key target of Notch signaling pathway,miR139-5p can inhibit the overactivation of the Notch signaling pathway,and promote the homing ability of asthmatic BMSCs.4.Bushen Huoxue tiaoying Huatan Recipe can promote BMSCs homing in asthmatic lung tissue,thus strengthening its regulatory effect on airway inflammation.The mechanism may be related to its targeting of miR139-5p/Notch signal axis,and regulating the expression of CXCR4 and SDF-1.