Effect And Mechanism Of Rat Bone Marrow Mesenchymal Stem Cells And Exosomes On Airway Remodeling Caused By Asthma

Objective:Airway remodeling is one of the most important pathological features of asthma,and it is also a difficult problem in asthma treatment.EMT of airway epithelium can cause a decline in the barrier function of the epithelial cells which is the origin and core of airway hyperresponsiveness and airway remodeling.In recent years,multiple studies have confirmed that mesenchymal stem cells reinfusion therapy can inhibit the progress of airway remodeling caused by asthma,but the specific mechanism is not clear.Some reseaches have pointed out that the use of exosomes secreted by stem cells can achieve similar effects to stem cells during infusion therapy.Exosomes contain proteins,DNA,RNA and lipids.MicroRNA is a single-stranded small-molecule RNA with a length of 20-25 nucleotides.It can degrade mRNA by pairing with the specific sequence of the 3'-UTR region of the target gene.Therefore,we hypothesized that one highly expressed miRNA in MSC-derived exosomes could inhibit EMT-related signaling pathways.In this study,we injected rat BMMSCs or BMMSC-derived exosomes into asthma model rats,which both can inhibit EMT-related pathways.Then reseach the effect ofMSC-derived exosomal miRNA on EMT-related signaling pathways.Try to explain part of the mechanisms by which MSCs improve airway remodeling.Methods:1.Isolation and identification of rat BMMSCs and BMMSC-derived exosomes1.1 Utilizing the characteristics of fast attachment and high proliferation ability of BMMSCs,high-purity rat BMMSCs were obtained through fluid exchange and passage.1.2 CD34,CD45,CD90,and CD105 of P3 BMMSCs were detected by flowcytometric.1.3 Adipogenic,osteogenic and chondrogenic differentiation of P3 BMMSCs to confirm differentiation ability.1.4 Exosomes were extracted from serum-free culture supernatant of BMMSCs by immunomagnetic bead method.1.5 The morphology and size of the exosomes were detected by transmission electron microscope.1.6 Detect the expression of the marker protein in the exosomes by WesternBlot.2.Study on the mechanism of rat BMMSCs and BMMSC-derived exosomes affecting airway remodeling caused by asthma through the Wnt/?-catenin pathway.2.1 Rat bone marrow mesenchymal stem cells and exosomes were labeled with PHK67fluorescent labeling kit.After labeling,the labeling effect was observed under a fluorescent microscope2.2 Establishment of a bronchial asthma model rat:4-week-old SD rats were injected intraperitoneally with 1 ml OVA+aluminum hydroxide emulsion.On the 7th day,intraperitoneal injection of 1 ml OVA solution to sensitize.On 14th to 19th days,5%OVA was used to stimulate the model rats in a transparent closed container for 30 minutes.Tail vein injections were performed 1h before the first challenge.The rats were anesthetized 1h after the last challenge for subsequent collection operations(the control group replaced the sensitizing solution and the nebulizing challenge solution with an equal volume of1×PBS).2.3 Experimental groups:Experimental rats were divided into 6 groups of 5 rats each.Control group:using 1×PBS for sensitization,stimulation and infusion.Model group:OVA+aluminum hydroxide sensitization,6-day OVA atomization excitation.Model+MSCs group:model rats,5×10~6BMMSCs were injected intravenously before nebulization challenge.Model+Exo group:model rats,50ugBMMSC-derived exosomes were injected intravenously before nebulization challenge.Model+MSCs+BML-284group:model rats,5×10~6BMMSCs were injected intravenously before nebulization challenge,and then injected with 1mg BML-284.Model+Exo+BML-284 group:model rats,50ugBMMSC-derived exosomes were injected intravenously before nebulization challenge,and then injected with 1mg BML-284.2.4 Collect BALF of each group of rats,and then analysis the number of total cells,lymphocytes,eosinophils and neutrophils.2.5 Lung tissue sections were stained with HE and Masson,and analyzed the pathological conditions of each group.2.6 Frozen sections of lung tissue were observed by a fluorescence microscope to see the migration of reinfused stem cells and exosomes to the lungs.2.7 Realtome-PCR was used to detect the changes of the mRNA expression levels of E-cadherin,Vimentin,Wnt,Frizzled,GSK-3?,?-catenin,LEF1,Snail1,and c-Myc in the lung tissue of each group.2.8 WB was used to detect the changes of the mRNA expression levels of E-cadherin,Vimentin,Wnt,Frizzled,GSK-3?,?-catenin,LEF1,Snail1,and c-Myc in the lung tissue of each group.3.Effect of MSCl-derived exosomal MicroRNA on asthmatic airway remodeling through inhibition of sonic hedgehog signaling pathway3.1The rat miRNA chip was used to determine the types of miRNAs which is highly expressed in rat BMMSC-derived exosomes.3.2 Bioinformatics analysis finds target genes of miRNAs,which is highly expressed in rat BMMSC-derived exosomes,in EMT-related signaling pathways.3.3 Using Dual-Luciferase Reporter Assay System to determine the interference effect of miR-212-3p on its predicted target gene Shh.3.4 Cell experiment3.4.1 Isolation and culture of primary rat bronchial epithelial cells.3.4.2 Immunofluorescence identification of primary rat bronchial epithelial cells.3.4.3 Primary rat bronchial epithelial cells were grouped into 8 groups:Control group:cells were transfected with miR-212-3p NC and cultured in normal medium.TGF-?1group:cells were transfected with mi R-212-3p NC,cultured in medium withTGF-?1.TGF-?1+MSC group:cells transfected with mi R-212-3p NC and co-culture with MSCs using medium containing TGF-?1;TGF-?1+Exo group:Cells were transfected with miR-212-3p NC and co-cultured with MSC exosomes using medium containing TGF-?1.TGF-?1+miR-212-3p minics group:cells were transfected with miR-212-3p minics and cultured in medium withTGF-?1.TGF-?1+miR-212-3p inhibitor group:cells are transfected with miR-212-3p inhibitor and cultured in medium with TGF-?1.TGF-?1+MSC+miR-212-3p inhibitor group:cells were transfected with miR-212-3p inhibitor and co-cultured with MSCs in medium containing TGF-?1.TGF-?1+Exo+miR-212-3p inhibitor group:cells were transfected with miR-212-3p inhibitor,co-culture with MSC exosomes in medium containing TGF-?1.3.4.4 Afer 72h,all 8 groups of cells extract total protein and detect EMT and Shh related proteins:E-cadherin,Shh,Gli1,c-Myc.3.5 animal experiment3.5.1 Experimental groups:Experimental rats were divided into 6 groups of 5 rats each.Control group:using 1×PBS for sensitization,stimulation and infusion.Model group:OVA+aluminum hydroxide sensitization,6-day OVA atomization excitation.Model+MSCs group:model rats,5×10~6BMMSCs were injected intravenously before nebulization challenge.Model+Exo group:model rats,50ugBMMSC-derived exosomes were injected intravenously before nebulization challenge.Model+miR-212-3p agomir group:model rats,100?mol miR-212-3p agomir were injected intravenously before nebulization challenge.Model+miR-212-3p anta group:model rats,100?mol miR-212-3p antagomir were injected intravenously before nebulization challenge.Model+MSCs+miR-212-3p anta group:model rats,5×10~6BMMSCs were injected intravenously before nebulization challenge,and then injected with 100?mol miR-212-3p antagomir.Model+Exo+miR-212-3p anta group:model rats,50ugBMMSC-derived exosomes were injected intravenously before nebulization challenge,and then injected with 100?mol miR-212-3p antagomir.3.5.2 Collect BALF of each group of rats,and then analysis the number of total cells,lymphocytes,eosinophils and neutrophils.3.5.3 Lung tissue sections were stained with HE and Masson,and analyzed the pathological conditions of each group.3.5.4 Realtome-PCR was used to detect the changes of the mRNA expression levels of E-cadherin?Vimentin?Shh?Ptch?smo?Gli1?Snail1?c-Myc and Cyclin D1 in the lung tissue of each group.3.5.5 WB was used to detect the changes of the protein expression levels of E-cadherin?Vimentin?Shh?Ptch?smo?Gli1?Snail1?c-Myc and Cyclin D1 in the lung tissue of each group.Results:1.1 Our isolated rat BMMSCs are typical fibroblast-like cells and increase rapidly.1.2 Flowcytometric analysis confirmed these cells are CD90,CD105 positive and CD34,CD45negative,which fits the phenotype of MSCs.1.3 The staining results of P3 BMMSCs after adipogenic,osteogenic and chondrogenic differentiation were all positive,indicating that the BMMSCs have good differentiation ability.1.4 TEM photos show that the diameter of exosomes is about 100nm,which is fit the typical size of exosomes.1.5 WB results showed that the levels of the exosome markers CD9,CD63 and CD81were higher in exosomes compared to BMMSCs.The expression of?-actin was negative,which shows that the purity of the extracted exosomes is good.2.1 MSCs and exosomes were successfully labeled with green fluorescence.2.2 The number of total cells,lymphocytes,eosinophils and neutrophils,in BALF of each group showed consistent trends.Compared with the Comtrol group,the number of cells in the Model group increased significantly(P<0.001).After model rats were reinfused with BMMSCs or BMMSC-derived exosome,the number of cells decreased significantly compared to the Model group(P<0.001).After intervention with Wnt agonist BML-284in treatment rats,whether in BMMSCs or BMMSC-derived exosome,the number of cells increased significantly(P<0.001).2.3 Pathological sections of HE staining and Masson staining showed that the control group rats showed normal alveolar tissue size,no obvious infiltration of inflammatory cells,and less collagen deposition under the trachea.In contrast,the lung tissue of the Model group showed a significant infiltration of inflammatory cells,alveolar expansion and damage,a large amount of collagen deposition.After BMMSCs or BMMSC-derived exosome infusion,the degree of inflammatory infiltration was significantly reduced,the damage of alveoli was reduced,and the degree of collagen deposition under the trachea was less.After intervention with Wnt agonist BML-284 in treatment rats,whether in BMMSCs or BMMSC-derived exosome,the inflammation infiltration,alveolar damage and Collagen deposition has increased significantly.2.4 Observe the frozen sections of the lung tissue of each group of rats with a fluorescent microscope,and only2-4 per field of viewof labeled MSCs and exosomes can be seen,indicating that the mesenchymal stem cells and exosomes can move to the lungs,but the number is less.2.5 The results of Realtime-PCR and WB showed that in the lung tissue,compared with the control group,the mRNA and protein level of E-cadherin and GSK-3?,the protein level of p-Ser-GSK-3?in the Model group were significantly reduced(P<0.001).Vimentin,Wnt,Frizzled,?-catenin,LEF1,Snail1,and c-Myc m RNA and protein,p-Tyr-GSK-3?protein expression increased(P<0.001)?After BMMSCs or BMMSC-derived exosome infusion,compared with the Model group,the mRNA and protein level of E-cadherin and GSK-3?,the protein level of p-Ser-GSK-3?were increased(P<0.001).Vimentin,Wnt,Frizzled,?-catenin,LEF1,Snail1 and c-Myc mRNA and protein,p-Tyr-GSK-3?protein expression levels were all reduced(P<0.001).It indicates the degree of EMT,and both the Wnt/?-catenin signaling pathway and its downstream transcription factors are suppressed.After intervention with Wnt agonist BML-284 in treatment rats,whether in BMMSCs or BMMSC-derived exosome,both showed a reactivation of EMT,Wnt/?-catenin signaling pathway and its downstream transcription factors.3.1 The results of Dual-LuciferaseReporter Assay System showed that in all 6co-transfecte groups,only the pmirGLO-Shh-3'UTR+miR-212-3p mimics co-transfection group had a significant decrease in fireflies luciferase activity/renilla luciferase activity(P<0.01).This shows that miR-212-3p can specifically recognize the complementary pairing site of 3'UTR of Shh gene and then interference the expression of its upstream protein.3.2 Cell experiments prove that in the model of TGF-?1 stimulation of rat bronchial epithelial cells EMT,co-cultured MSCs and exosomes can inhibit the activation of EMT and Shh pathway of epithelial cells.transfected miR-212-3p minics can also inhibit EMT and inhibit the Shh pathway,while the use of miR-212-3p inhibitors can reduce the effect of MSCs and exosomes on inhibiting EMT of epithelial cells.3.3 The number of total cells,lymphocytes,eosinophils and neutrophils,in BALF of each group showed consistent trends.Compared with the Comtrol group,the number of cells in the Model group increased significantly(P<0.001).After model rats were reinfused with BMMSCs or BMMSC-derived exosome,the number of cells decreased significantly compared to the Model group(P<0.001).After intervention with miR-212-3p antagomir in treatment rats,whether in BMMSCs or BMMSC-derived exosome,the number of cells increased significantly(P<0.001).The injection of miR-212-3p agomir in model rats can significantly reduce the total cell number and the number of three immune cells compared with the Model group(P<0.01).3.4 Pathological sections of HE staining and Masson staining showed that the control group rats showed normal alveolar tissue size,no obvious infiltration of inflammatory cells,and less collagen deposition under the trachea.In contrast,the lung tissue of the Model group showed a significant infiltration of inflammatory cells,alveolar expansion and damage,a large amount of collagen deposition.After BMMSCs or BMMSC-derived exosome infusion,the degree of inflammatory infiltration was significantly reduced,the damage of alveoli was reduced,and the degree of collagen deposition under the trachea was less.After intervention with miR-212-3p antagomir in treatment rats,whether in BMMSCs or BMMSC-derived exosome,the inflammation infiltration,alveolar damage and Collagen deposition has increased significantly.Model rats injected with mi R-212-3p agomir alone can reduce inflammation and collagen deposition in the lungs compared to the Model group.3.5 The results of Realtime-PCR and WB showed that in the lung tissue,compared with the control group,the mRNA and protein level of E-cadherin in the Model group were significantly reduced(P<0.001).Vimentin,Shh,smo,Gli1,Snail1,c-Myc and Cyclin D1mRNA and protein expression increased(P<0.001)?After BMMSCs or BMMSC-derived exosome infusion,compared with the Model group,the mRNA and protein level of E-cadherin,Ptch were increased(P<0.001).Vimentin,Shh,smo,Gli1,Snail1,c-Myc and Cyclin D1 mRNA and protein expression levels were all reduced(P<0.001).It indicates the degree of EMT,and both the Shh signaling pathway and its downstream transcription factors are suppressed.After intervention with miR-212-3p antagomir in treatment rats,whether in BMMSCs or BMMSC-derived exosomes,both showed a reactivation of EMT,Shh signaling pathway and its downstream transcription factors.Model rats injected with miR-212-3p agomir alone also significantly inhibited the activation of EMT and Shh signaling pathway compared to the Model group.Conclusion:1.In this experiment,we isolated high-purity,high-value-added and high differentiation ability rat BMMSCs.At the same time,we extracted high-quality BMMSC-derived exosomes.2.Animal experiments have proved that MSCs can inhibit the Wnt pathway through their exosomes,and then reducing EMT of the tracheal epithelium in asthma,reducing collagen deposition under the trachea,and improving tracheal remodeling.3.In vitro experiments,we proved mi R-212-3p can interference Shh gene.4.Cell experiments proved that co-culture of MSCs and exosomes can inhibit the activation of EMT and Shh pathways caused by TGF-?1 stimulation,and this effect can be blocked by miR-212-3p inhibitors.5.In vivo experiments,we proved that part of the mechanism of rat BMMSCsin the treatment of asthma is achieved by miR-212-3p degrading the mRNA of the key protein Shh in the shh pathway and then inhibiting the activation of the Shh pathway.