| Objective: Bronchial asthma is a common chronic respiratory disease in the world,especially in developed countries.According to WHO,the number of asthma patients worldwide is about 334 million.With the increasing incidence rate of asthma,it is estimated that the number of asthma patients will increase to 400 million by the end of 2025.Asthma is also a high incidence rate of chronic respiratory diseases in children,ranking among the top 20 conditions worldwide for disability-adjusted life years(DALY)in children.Therefore,asthma brings a heavy burden to global health and economy.Asthma is characterized by persistent chronic airway inflammation,airway hyperresponsiveness and irreversible airway remodeling.Irreversible airway remodeling aggravates airway hyperresponsiveness and eventually leads to progressive and irreversible persistent damage of lung function,which is closely related to the prognosis of the disease.The mechanism of airway remodeling is still unclear.Recent studies have shown that airway epithelium,as the first barrier for the body to contact with allergic or harmful stimulation,can participate in the process of airway remodeling in the form of epithelial mesenchymal transition(EMT).Therefore,further exploration and understanding of the pathogenesis of EMT in airway epithelial cells has become the focus of asthma prevention and treatment.MiRNAs are the main regulators of EMT in many diseases.More and more studies have proved that miR-21-5p is widely involved in EMT of many diseases.Our pre-experiment suggests that the expression of miR-21-5p in the lung tissue of asthmatic airway remodeling animal model increases with the progress of EMT in airway epithelial cells,and mainly exists in macrophages,suggesting that miR-21-5p is closely related to asthma EMT.Due to the existence of lipid bimolecular membrane structure in exosomes,the miRNAs in exosomes are not easy to degrade compared with free miRNAs and have better biological stability.MiRNAs are packaged in exosomes and act as paracrine messengers to further mediate intercellular communication between the same or different species.This process plays an important role in normal physiology,pathophysiology and pathological process.Based on the above research and views,this study explored whether miR-21-5p of macrophages could be transferred to bronchial epithelial cells through exosomes and affect the EMT process of epithelial cells,and further clarified the mechanism of miR-21-5p in macrophage-derived exosomes affecting EMT in bronchial epithelial cells.Methods: 1.The expression level and localization of miR-21-5p in airway remodeling model of asthma in rats.(1)Animal model preparation and specimen collection.Sixteen SD rats were divided into two groups: PBS-8 weeks group and OVA-8 weeks group.OVA and aluminum hydroxide were injected intraperitoneally to sensitize the rats on the 0,7 and 14 days of the experiment.OVA atomization stimulation was given to the rats from the 21 st day of the experiment,once every other day,and the stimulation lasted for 8 weeks.In PBS-8 weeks group,the rats were sensitized and challenged with PBS instead of OVA.The behavioral changes of rats in each group were observed.The total number of leukocytes in BALF and the classification of leukocytes were counted.Lung tissue samples were collected for HE staining and Masson staining to verify the preparation of asthma airway remodeling model,and to further evaluate the degree of asthma airway remodeling.Western blot was used to detect the protein expression of E-cadherin,a-SMA and vimentin in the lung tissue of asthmatic airway remodeling model,which further proved that EMT appeared in the airway epithelial cells of asthmatic airway remodeling model.(2)The expression and localization of miR-21-5p in the lung tissue of asthmatic airway remodeling model were detected.The expression of miR-21-5p was detected by PCR.FISH and IF were used to localize the expression of miR-21-5p in rat lung macrophage.2.Effects of miR-21-5p in rat alveolar macrophages-derived exosomes by LPS stimulated on EMT of rat bronchial epithelial cells.(1)Rat alveolar macrophage was cultured,LPS was used to stimulate rat alveolar macrophage to establish inflammatory model,and exosomes were isolated.The morphology and distribution of exosomes were verified by transmission electron microscopy(TEM)and nanoparticle tracking analysis(NTA).Western blot was used to detect the expression of exosome surface marker protein.The intervention techniques such as GW4869,miR-21-5p inhibitor were used to investigate the expression level of miR-21-5p in rat alveolar macrophage-derived exosomes by LPS stimulated.PCR was used to detect the expression of miR-21-5p in rat alveolar macrophage and their exosomes.The concentration of exosomes was analyzed by NTA.(2)Rat bronchial epithelial cells was cultured,the rat bronchial epithelial cells was treated with exosomes secreted by rat alveolar macrophage stimulated by LPS.PKH67 labeled exosomes were used to observe the uptake of exosomes by rat bronchial epithelial cells under fluorescence microscope.The intervention techniques such as GW4869 was used to investigate the expression level of miR-21-5p in rat bronchial epithelial cells treated with exosomes secreted by rat alveolar macrophage stimulated by LPS.Furthermore,the uptake of exosomes by rat bronchial epithelial cells and the transfer of miR-21-5p in rat alveolar macrophage-derived exosomes by LPS stimulated to rat bronchial epithelial cells were verified.The expression of miR-21-5p in rat bronchial epithelial cells was detected by PCR.(3)The intervention techniques such as GW4869,miR-21-5p inhibitor was used to investigate the effects of miR-21-5p in rat alveolar macrophage-derived exosomes by LPS stimulated on EMT of rat bronchial epithelial cells.Western blot was used to detect the protein expression levels of EMT related indicators(E-cadherin,a-SMA and vimentin)in rat bronchial epithelial cells.Immunofluorescence was used to detect the expression of a-SMA in rat bronchial epithelial cells.3.The mechanism of miR-21-5p in rat alveolar macrophages-derived exosomes by LPS stimulated promote EMT in rat bronchial epithelial cells.(1)Using independent online databases,targetscan,we predicted the target genes Smad7 and TGFβRII of miR-21-5p.(2)The expression of TGF-β1,TGFβRII and Smad7 in lung tissue of rats in PBS-8 weeks group and OVA-8 week group were detected by Western blot and PCR in protein and m RNA levels.(3)Rat bronchial epithelial cells were cultured.miR-21-5p mimics or inhibitor were used to investigate the regulation of TGF-β1/ Smad7 by miR-21-5p and its role in EMT in rat bronchial epithelial cells.The expression of miR-21-5p in rat bronchial epithelial cells was detected by PCR.Western blot was used to detect the protein expression levels of TGF-β1,TGFβRII,pSmad2,pSmad3,Smad7,E-cadherin,a-SMA and vimentin in rat bronchial epithelial cells,so as to verify the activation of TGF-β1/Smad7 signaling pathway and its correlation with EMT.Immunofluorescence was used to detect the expression of a-SMA protein in rat bronchial epithelial cells.The inhibitory effect of miR-21-5p on Smad7 in rat bronchial epithelial cells was further verified by dual luciferase reporter gene assay.(4)Rat bronchial epithelial cells was treated with exosomes secreted by rat alveolar macrophages stimulated by LPS.Objective to observe the effect of miR-21-5p in rat alveolar macrophages-derived exosomes by LPS stimulated on TGF-β1/Smad7 signaling pathway in rat bronchial epithelial cells.Western blot was used to detect the expression of TGF-β1 and Smad7 in rat bronchial epithelial cells.The binding of miR-21-5p secreted by LPS stimulated rat alveolar macrophages to Smad7 3’-UTR in rat bronchial epithelial cells was verified by dual luciferase reporter gene assay.(5)Rat bronchial epithelial cells was treated with exosomes secreted by rat alveolar macrophages stimulated by LPS.The intervention techniques such as miR-21-5p mimic or inhibitor,Smad7 siRNA、TGF-β1 siRNA was used to investigate the regulatory mechanism of miR-21-5p in rat alveolar macrophages-derived exosomes by LPS stimulated on TGF-β1/Smad7 signaling pathway and its role in EMT of rat bronchial epithelial cells.Western blot was used to detect the protein expression levels of TGF-β1,Smad7 、 E-cadherin 、 a-SMA and vimentin.Immunofluorescence was used to detect the expression of a-SMA protein in rat bronchial epithelial cells.Results:1.MiR-21-5p was localized in macrophages and increased in the airway remodeling model of asthma in rats.(1)Asthmatic airway remodeling model in rats,the number of leukocytes in BALF increased(P<0.05).He staining and Masson staining showed that compared with PBS-8 weeks group,bronchial epithelial cells swelled,some epithelial cells fell off,a large number of inflammatory cells infiltrated in the wall and around the blood vessels,the wall thickened,collagen deposition increased,and smooth muscle thickened.Compared with PBS-8 weeks group,the expression of E-cadherin protein in lung tissue of asthmatic airway remodeling model decreased(P<0.05),and the expression stromal cell markers,such as a-SMA and vimentin protein increased(P<0.05).These results indicate that EMT appears in the lung tissue of asthmatic airway remodeling model in rats.(3)PCR results showed that the expression level of miR-21-5p in the lung tissue of rat airway remodeling model was significantly higher than PBS-8 weeks group(P<0.05).FISH combined with IF showed that the expression of miR-21-5p increased in the lung tissue of rat airway remodeling model,and it mainly existed in macrophages.2.MiR-21-5p in rat alveolar macrophages-derived exosomes by LPS stimulate could promote EMT of rat bronchial epithelial cells.(1)LPS stimulated rat alveolar macrophages to establish inflammatory cell model.The results of PCR showed that the expression level of miR-21-5p in rat alveolar macrophages was significantly increased(P<0.05).The exosomes should be further identified after isolation and extraction.First,the morphology and size distribution of exosomes were verified by TEM and NTA.The expression of exosome specific endogenous proteins was detected by Western blot.PCR results showed that miR-21-5p was significantly increased in rat alveolar macrophages-derived exosomes by LPS stimulated(P<0.05).MiR-21-5p inhibitor significantly inhibited the expression of miR-21-5p in exosomes.GW4869 did not change the relative levels of miR-21-5p in the exosomes,but it caused reduction in the number of vesicles isolated from the same amount of medium(2)The green fluorescence value of PKH67 labeled exosomes was detected in rat bronchial epithelial cells,which indicated that rat bronchial epithelial cells could absorb the exosomes secreted by rat alveolar macrophages.PCR results showed that the expression level of miR-21-5p was higher in rat bronchial epithelial cells co cultured with the exosomes secreted by LPS stimulated rat alveolar macrophages(P<0.05),which could be inhibited by GW4869.(3)Western blot analysis showed that the expression of E-cadherin decreased and the expression of α-SMA and vimentin increased in rat bronchial epithelial cells co cultured with the exosomes secreted by LPS stimulated rat alveolar macrophages(P<0.05).MiR-21-5p inhibitor and GW4869 could inhibit this change.Immunofluorescence showed that the expression of α-SMA protein increased in rat bronchial epithelial cells co cultured with the exosomes secreted by LPS stimulated rat alveolar macrophages.MiR-21-5p inhibitor and GW4869 could inhibit this change.3.MiR-21-5p in rat alveolar macrophages-derived exosomes by LPS stimulated inhibited Smad7 in rat bronchial epithelial cells,activated TGF-β1/Smad7 signal transduction pathway,and promoted EMT in rat bronchial epithelial cells.(1)Bioinformatics predicted that the target genes of miR-21-5p were Smad7 and TGFβ RII.(2)Western blot analysis showed that compared with PBS-8 weeks group,the expression of TGF-β1and TGFβRII protein in lung tissue of asthmatic airway remodeling model increased,while Smad7 protein expression decreased(P<0.05);PCR analysis showed that compared with PBS-8 weeks group,the expression of TGF-β1and TGFβRII m RNA increased,and Smad7 m RNA decreased(P<0.05).(3)The expression of miR-21-5p in rat bronchial epithelial cells was detected by PCR.The results showed that the expression level of miR-21-5p in miR-21-5p mimics group was significantly higher than that in NC mimics group(P<0.05);The expression level of miR-21-5p in miR-21-5p inhibitor group was significantly lower than that in NC inhibitor group(P <0.05).Western blot analysis showed that Smad7 protein level was decreased,and TGF-β1,TGFβRII,pSmad2,pSmad3 protein levels were increased in rat bronchial epithelial cells transfected with miR-21-5p mimics(P<0.05).After transfection with miR-21-5p inhibitor,Smad7 protein level was increased and TGF-β1,TGFβRII,pSmad2,pSmad3 protein levels were decreased(P<0.05).Western blot analysis showed that the expression of E-cadherin protein was decreased and the expression of α-SMA and vimentin protein was increased in rat bronchial epithelial cells transfected with miR-21-5p mimics(P<0.05).After transfection with miR-21-5p inhibitor,the expression of E-cadherin protein was increased and the expression of α-SMA and vimentin protein was decreased(P<0.05).Immunofluorescence showed that the expression of α-SMA protein increased in rat bronchial epithelial cells transfected with miR-21-5p mimics.Luciferase reporter assay confirmed the inhibitory effect of miR-21-5p on Smad7.(4)Western blot analysis showed that Smad7 protein level was decreased and TGF-β1 protein level was increased in rat bronchial epithelial cells treated with the exosomes secreted by LPS stimulated rat alveolar macrophages(P<0.05).Luciferase reporter assay confirmed that miR-21-5p in rat alveolar macrophages-derived exosomes inhibited its expression in rat bronchial epithelial cells by binding to 3’UTR of Smad7 m RNA.(5)Si NC or Smad7 siRNA transfected rat bronchial epithelial cells were exposed to exosomes secreted by LPS stimulated rat alveolar macrophages pretreated with miR-21-5p inhibitor.Western blot analysis showed that compared with si NC rat bronchial epithelial cells co cultured with exosomes secreted by LPS stimulated rat alveolar macrophages pretreated with miR-21-5p inhibitors,the expression levels of TGF-β1,α-SMA and vimentin in rat bronchial epithelial cells were increased in combination with Smad7 siRNA treatment group(P<0.05).Immunofluorescence showed that the expression of α-SMA was increased in Smad7 siRNA rat bronchial epithelial cells co cultured with exosomes secreted by LPS stimulated rat alveolar macrophages pretreated with miR-21-5p inhibitors.By using TGF-β1 siRNA to down regulate the expression of TGF-β1 in rat bronchial epithelial cells,it could prevent the exosomes secreted by LPS stimulated rat alveolar macrophages induced protein levels of α-SMA(P<0.05).Immunofluorescence showed that the expression of α-SMA in rat bronchial epithelial cells was decreased in LPS stimulated rat alveolar macrophages exosomes combined with TGF-β1 siRNA treatment group.Conclusions: 1.MiR-21-5p is mainly located in macrophages in the airway remodeling model of asthma in rats.The increased expression of miR-21-5p was accompanied by the EMT in airway epithelium cells.2.LPS stimulated rat alveolar macrophages increased the expression of miR-21-5p,and also increased the expression level of miR-21-5p in exosomes.The exosomes secreted by rat alveolar macrophages stimulated by LPS could be taken up by rat bronchial epithelial cells,and miR-21-5p was transferred through exosomes,resulting in the increased expression of miR-21-5p in rat bronchial epithelial cells.The exosomes secreted by rat alveolar macrophages stimulated by LPS can promote EMT of rat bronchial epithelial cells and can play a role through miR-21-5p in exosomes.3.MiR-21-5p in rat alveolar macrophages-derived exosomes by LPS stimulated which inhibiting the expression of Smad7 in rat bronchial epithelial cells,can induce the expression of TGF-β1,and then promote the EMT of rat bronchial epithelial cells. |
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