Effect And Mechanism Of Circ-Eif3c Delivered By M2 Macrophage Exosomes On Airway Epithelial Injury In Asthma

Part Ⅰ: Protective effect of M2 macrophage derived exosomes on acute asthmatic miceObjective: To investigate the protective effect of M2 macrophage derived exosomes on acute asthmatic mice.Methods:1.C57BL/6 acute asthma mouse model was established by intraperitoneal injection of OVA(ovalbumin)and continuous atomization inhalation for 1 week.24 mice were divided into control group,OVA-induced model group,OVA+PBS group and OVA+M2(?)-Exo treatment group.Mice in OVA+M2(?)-Exo treatment group were injected with 100 μl M2macrophage-derived exosomes(M2(?)-Exo)into the tail vein for 4 consecutive days from day20 to day 23,and mice in OVA+PBS group were injected with 100 μl PBS into the tail vein for 4 consecutive days from day 20 to day 23.2.Evaluation of airway inflammation in asthmatic mouse models: The expression levels of inflammatory factors(TNF-α,IL-1β,IL-6)in peripheral blood of mice were detected by enzyme linked immunosorbent assay(ELISA),apoptosis was observed by immunohistochemical TUNEL(Td T-mediated d UTP nick end labeling)staining,fibrosis levels in lung tissues of mice were observed by Masson staining,CD31 expression levels in lung tissues were detected by immunofluorescence to assess endothelial cell injury,ROS levels in lung tissues were detected by fluorescence staining(dihydroethidium).Results:1.Compared with the control group,the expression of inflammatory factors(TNF-α,IL-1β,IL-6)in peripheral blood,the degree of apoptosis,pulmonary fibrosis and ROS levels in lung tissue were significantly increased in OVA-induced acute asthmatic mice(p<0.001),and the expression level of CD31 in lung tissue was significantly decreased compared with the control group(p<0.05).2.Compared with OVA+PBS model group,the expression of inflammatory factors(TNF-α,IL-1β,IL-6)in peripheral blood,the degree of apoptosis in lung tissue,pulmonary fibrosis and ROS levels in lung tissue were significantly alleviated in M2(?)-Exo-treated mice(p<0.01),while the expression level of CD31 in lung tissue was significantly increased(p<0.001).Conclusion:1.Mouse model of acute asthma could be successfully induced by OVA.On the mouse model of acute asthma,the level of inflammatory factors in peripheral blood,the cell apoptosis,fibrosis and ROS levels of lung tissue were significantly increased,and there was endothelial cell inflammatory injury.2.M2(?)-Exo tail vein injection can effectively reduce the expression of inflammatory factors in peripheral blood,inhibit cell apoptosis,lung fibrosis and ROS levels in lung tissue,and improve endothelial cell injury in acute asthmatic mice.Part Ⅱ: Role of circ RNA-Eif3 c in M2 macrophage-derived exosomes in improving acute asthma in miceObjective:1.To investigate differentially expressed circular RNAs(circ RNAs)in M2(?)-Exo and identify target circ RNA.2.To elucidate the therapeutic effect of target circ RNA in mice with acute asthma.Methods:1.High-throughput sequencing was used to detect the expression of cir RNA in M0(?)-exo and M2(?)-exo,among which the circ RNAs with high expression in M2(?)-exo were candidate differentially expressed circ RNAs.2.Quantitative PCR(q PCR)was used to detect the expression of candidate differential circ RNAs in M0(?)-Exo and M2(?)-Exo,and those with the highest relative expression in M2(?)-Exo and stable experimental results were screened as target circ RNA.3.IL-4 was used to induce M0 macrophages of wild-type and interfered groups to convert to M2 type.Target circ RNA interference vector was transfected into induced Raw264.7 cells and then we collect exosomes and made tail vein injection into asthmatic mice.4.ELISA was used to detect the expression levels of inflammatory factors(TNF-α,IL-1β,IL-6)in peripheral blood of mice;TUNEL staining was used to observe the apoptosis level in lung tissue of mice;Masson staining was used to observe the fibrosis level in lung tissue of mice;immunofluorescence was used to detect the CD31 level in lung tissue to assess endothelial cell injury;ROS levels in lung tissues were detected by fluorescence staining(dihydroethidium).Results:1.Nine circ RNAs differentially highly expressed in M2(?)-Exo were selected by highthroughput sequencing.Mmu＿circ＿0001628 was identified as the target circ RNA by q PCR,which had the highest relative expression level and stable results(p<0.001).2.By consulting the website circ Base(Fig.http://circbase.org/),mmu＿circ＿0001628(circ-Eif3c)was identified to be looped from reverse spliced RNA after transcription of the gene EIF3c(eukaryotic translation initiation factor 3,subunit C)and is 364 bp in size.3.Compared with the conventional M2(?)-Exo group,the levels of inflammatory factors in peripheral blood,apoptosis,fibrosis and ROS levels in lung tissue were significantly increased(p<0.001),and the expression level of CD31 was significantly decreased(p<0.001)in the M2(?)-Exo group after interfering with circ-Eif3 c expression by tail vein injection.Conclusion:1.circ-Eif3 c plays a critical and positive role in improving inflammatory factor secretion,pulmonary fibrosis,ROS levels in lung tissue and endothelial cell injury in asthmatic mice regulated by M2(?)-Exo.Part Ⅲ: circ RNA-Eif3 c regulates the effects of mi R-15a-5p/GSS and mi R-15a-5p/SOCS6 signaling pathways on oxidative stress in airway epithelial cellsObjective:1.To explore the downstream targets of circ RNA-Eif3 c and their interactions.2.To investigate the effect of circ RNA-Eif3 c in regulating mi R-15a-5p/GSS and mi R-15a-5p/SOCS6 on airway epithelial cells.Methods:1.Predict and confirm circ RNA-Eif3 c downstream target molecules(1)We predicted potential binding sites of circ RNA-Eif3 c downstream micro RNA(mi RNA)by searching multiple bioinformatics databases,made preliminarily screen by Arraystar mi RNA software,and then we screened target mi RNAs directionally through DIANA database.Bioinformatics data methods were used to screen downstream targets of target mi RNAs.(2)We explored the direct binding and targeted regulation between circ RNA-Eif3 c and target mi RNAs by dual-luciferase reporter assay.(3)We explored the direct binding and targeted regulation between target mi RNAs and downstream targets by dual-luciferase reporter assay.2.Effect of circ RNA-Eif3 c,mi RNA and downstream targets on airway epithelial cells(1)Inflammatory cell models were constructed by inducing human normal lung epithelial cells(BEAS-2B cells)with lipopolysaccharide(LPS).Circ RNA-Eif3 c overexpression group,circ-Eif3c+mi RNA mimic group,circ-Eif3 c overexpression+ mi RNA downstream target interference group were constructed by transfection of plasmids.(2)QPCR was used to detect circ RNA-Eif3 c,mi RNA and mi RNA downstream target expression levels in airway epithelial cells of each group.(3)Flow cytometry was used to detect the level of apoptosis in each group,immunofluorescence(dihydroethidium assay)was used to detect the level of reactive oxygen species(ROS)in airway epithelial cells,and Tubule assay was used to assess the angiogenic ability of airway epithelial cells.Results:1.Combined with multiple database prediction results,mi R-15a-5p may be a downstream target of circ RNA-Eif3 c,and GSS and SOCS6 may be downstream targets of mi R-15a-5p.2.The results of dual-luciferase reporter assay revealed that circ RNA-Eif3 c could bind to mi R-15a-5p,and mi R-15a-5p could bind to GSS and SOCS6.3.In a series of experiments using the BEAS-2B inflammatory cell model constructed with LPS,it was found that:(1)Compared with the control group,the expression levels of circ RNA-Eif3 c,GSS and SOCS6 in the circ RNA-Eif3 c overexpression group were significantly up-regulated(p<0.001),while the expression of mi R-15a-5p was significantly decreased(p<0.001).(2)Compared with the control group,the expression of circ RNA-Eif3 c,mi R-15a-5p,GSS and SOCS6 were significantly up-regulated in circ-Eif3 c overexpression +mi R-15a-5p mimic group(p < 0.01).Compared with circ RNA-Eif3 c overexpression group,the expression of circ RNA-Eif3 c in circ-Eif3 c overexpression +mi R-15a-5p mimic group was not significantly different(p>0.05),while mi R-15a-5p was significantly increased(p<0.01),and the expression levels of GSS and SOCS6 were decreased(p<0.01).(3)circ RNA-Eif3 c and GSS expression was significantly up-regulated(p<0.01),while mi R-15a-5p and SOCS6 expression was significantly decreased(p<0.001)in the circ-Eif3 c overexpression+SOCS6 interference group compared with the control group.Compared with the circ-Eif3 c overexpression group,there were no significant differences in circ-Eif3 c,mi R-15a-5p,and GSS expression in the circ-Eif3 c overexpression+SOCS6 interference group(p>0.05),while SOCS6 expression was significantly lower(p<0.001).(4)circ RNA-Eif3 c and SOCS6 expression were significantly up-regulated(p<0.01),while mi R-15a-5p and GSS expression was significantly decreased(p<0.001)in the circEif3 c overexpression +GSS interference group compared with the control group.Compared with circ-Eif3 c overexpression group,there were no significant differences in circ-Eif3 c,mi R-15a-5p,and SOCS6 expression in circ-Eif3 c overexpression+GSS interference group(p>0.05),while GSS expression was significantly lower(p<0.001).4.Flow cytometry indicated that the apoptosis levels of the other four groups were significantly decreased compared with the control group(p < 0.001),while compared with the circ RNA-Eif3 c overexpression group,the apoptosis levels of circ-Eif3 c overexpression+mi R-15a-5p mimic group,circ-Eif3 c overexpression+GSS interference group and circEif3 c overexpression +SOCS6 interference group were increased(p < 0.001).5.Intracellular ROS levels detected by immunofluorescence dihydroethidium showed that intracellular ROS levels were significantly lower in the circ RNA-Eif3 c overexpression group compared with the control group(p<0.001).The other three groups: circ-Eif3 c overexpression+mi R-15a-5p mimic group,circ-Eif3 c overexpression+GSS interference group,circ-Eif3 c overexpression+SOCS6 interference group had decreased intracellular ROS levels compared with the control group(p<0.05),but showed increased levels compared with circ RNA-Eif3 c overexpression group(p<0.001).6.Tubule experiment showed that angiogenesis ability was increased in circ RNA-Eif3 c overexpression group,circ-Eif3 c overexpression +mi R-15a-5p mimic group,circ-Eif3 c overexpression+GSS interference group and circ-Eif3 c overexpression+SOCS6 interference group compared with the control group(p<0.01).However,compared with the circ-Eif3 c overexpression group,the angiogenesis ability of the other three groups: circ-Eif3 c overexpression+mi R-15a-5p mimic group,circ-Eif3 c overexpression+GSS interference group,and circ-Eif3 c overexpression+SOCS6 interference group was decreased(p<0.01).Conclusion:1.circ-Eif3 c can bind to mi R-15a-5p,while mi R-15a-5p can bind to downstream GSS and SOCS6.2.Elevated expression of circ-Eif3 c can bind to mi R-15a-5p and further promote the up-regulation of downstream GSS and SOCS6 expression,thereby improving airway epithelial cell apoptosis,inhibiting the generation of ROS in airway epithelial cells and promoting angiogenesis,and repairing injury.