| Background:Bronchial asthma is a chronic inflammatory disease of the airways involving a variety of cells(eosinophils,mast cells,T lymphocytes,bronchial epithelial cells,bronchial smooth muscle cells)and cytokines,which is characterized by chronic airway inflammation,airway hyperreactivity and airway remodeling.Airway epithelial cells are an important part of the airway barrier structure.When airway epithelial cells show injury and apoptosis,it will lead to airway integrity damage,increased tracheal permeability,secretion of chemokines to induce inflammatory cell accumulation,and aggravate collagen deposition,etc.Studying the process of epithelial cell injury and finding key targets has become the key to treatment.In the process of epithelial cell damage,excessive oxidative stress promotes mitochondrial damage and activates the process of mitochondrial apoptosis may be the main pathway of apoptosis in epithelial cells.NOX4 is a key protein in endogenous reactive oxygen species(ROS)generation,and during exogenous stimulation NOX4 induces increased endogenous ROS production,produces oxidative stress and damages mitochondria,and damaged mitochondria cause decreased membrane potential,releases cytochrome c(Cytochrome C),and activates the mitochondrial apoptotic pathway,while damaged mitochondria can also activate NLRP3/IL-1β to aggravate the inflammatory response.Mitochondrial function is also affected by mitochondrial morphological changes,of which excessive mitochondrial fission is an important factor promoting impaired mitochondrial function,and mitochondrial fission is regulated by the mitochondrial fission protein Drp1.In epithelial cell injury,NOX4 may promote mitochondrial injury by regulating Drp1,activate the mitochondrial apoptotic pathway and NLRP3 inflammasome,and aggravate airway inflammation.MiRNAs are non-codingRNAs about 18 – 23 nucleotides in length,and miRNAs currently have great research potential for diagnosis,therapeutic targets,and efficacy judgment in bronchial asthma.Screening differentially expressed miRNAs and targeting key proteins that regulate and promote disease progression in asthma may be a new therapeutic approach to improve airway inflammation in asthma.Objective:To explore that IL-13 promotes epithelial cell inflammatory responses through NOX4/Drp1/NLRP3 and activates the mitochondrial apoptotic pathway to induce epithelial cell apoptosis.To explore that mi R 182-5P ameliorates the process of epithelial cell injury and ameliorates airway inflammation in asthma by targeting Nox4 expression.Methods:Part I: In vivo experiments: The model of chronic asthma induced by OVA was established,which were normal group(Con)and asthma group(OVA).Observation indicators:(1)airway hyperreactivity was measured using different concentrations of ACh inhalation challenge;(2)the percentage of eosinophils in BALF was measured by flow cytometry;(3)IL-4,IL-5,and IL-13 levels in BALF were measured by ELISA;(4)pathological changes in lung tissues were observed by HE,PAS,and Masson staining of lung tissue paraffin;(7)tissue immunochemical staining was used to observe NOX4 expression in lung tissues.In vitro experiments: BEAS-2B was stimulated with IL-13 to construct an epithelial cell injury model,and epithelial cells were pretreated with si-NOX4,Drp1 inhibitor Mdivi-1,respectively.Observation indicators:(1)intracellular ROS,MMP,mt ROS,and apoptosis levels were measured by flow cytometry;(2)intracellular ATP content was measured by ATP detection kit;(3)intracellular ROS,MMP,mt ROS,IL-1β,and mitochondrial morphological changes were observed by cytofluorescence;(4)NOX4,Drp1,p-Drp1(Ser616),NLRP3,ASC,Cleaved-Caspase-1,IL-1β,Bcl-2,BAX,Cytochrome C,Cleaved-Caspase-9,and Cleaved-Caspase-3 protein expression levels were measured by Western blot.Part II: In vivo experiments: The chronic asthma model induced by OVA was constructed,which were normal group(Con)and asthma group(OVA).Observation indicators:(1)Microarray analysis was used to analyze the different expressions of miRNA between the two groups,and performe cluster mapping and target gene prediction;(2)RT-q PCR was used to verify the expression changes of mi R-182-5P,NOX4 mRNA in tissues.In vitro experiments: BEAS-2B was stimulated by IL-13 to construct an epithelial cell injury model,and epithelial cells were pretreated with mi R-182-5Pmimic/NC.Observation indicators:(1)RT-q PCR was used to verify the changes in mi R-182-5P and NOX4 mRNA expression in the cells;(2)the targeting relationship between mi R-182-5P and NOX4 mRNA was clarified by dual luciferase reporter gene assay kit;(3)intracellular ROS,MMP,mt ROS,and apoptosis levels were measured by flow cytometry;(4)intracellular ATP content was measured by ATP detection kit;(5)intracellular ROS,MMP,mt ROS,IL-1β,and mitochondrial morphological changes were observed by cell fluorescence;(6)NOX4,Drp1,p-Drp1(Ser616),NLRP3,ASC,Cleaved-Caspase-1,IL-1β,Bcl-2,BAX,Cytochrome C,Cleaved-Caspase-9,and Cleaved-Caspase-3 protein expression levels were detected by Western blot.Part III: OVA-induced chronic asthma model was constructed and divided into four groups,which were normal group(CON),asthma group(OVA),asthma + mi R182-5P NC group(OVA + mi R 182-5P NC),and asthma + mi R 182-5P agomir group(OVA + mi R 182-5P agomir).Observation indicators:(1)Airway hyperreactivity was measured using different concentrations of ACh inhalation challenge;(2)The percentage of eosinophils in BALF was measured by flow cytometry;(3)IL-4,IL-5,and IL-13 levels in BALF of mice were measured by ELISA;(4)Tissue fluorescence was used to observe NOX4 expression in lung tissues;(5)RT-q PCR was used to verify NOX4 mRNA expression changes in tissues;(6)Paraffin lung tissues was stained with HE,PAS,Masson,and frozen tissue sections were stained with DHE to observe the pathological changes in lung tissues of mice;(7)Western blot was used to detect the protein expression of NOX4,NLRP3,ASC,Cleaved-caspase-1,IL-1β,Bcl-2,BAX,Cleaved-Caspase-9,and Cleaved-Caspase-3.Results:Part I:(1)OVA group showed significant airway hyperreactivity,increased the proportion of eosinophils in BALF,and increased the expression of Th2 cytokines IL-4,IL-5,and IL-13;histopathological staining showed significant inflammatory cell accumulation and infiltration around OVA airways,airway structure disorder,epithelial cell shedding,goblet cell proliferation,and collagen deposition significantly accompanied by airway wall thickening.(2)NOX4 protein expression was increased and intracellular ROS levels were significantly increased after stimulation with IL-13 in BEAS-2B epithelial cells.(3)IL-13 induced a significant increase in the levels of mitochondrial fission proteins Drp1 and p-Drp1(Ser616),which were transferred from the cytoplasm to mitochondria,with fragmented mitochondrial morphology,impaired mitochondrial function,decreased MMP levels,decreased ATP synthesis,and increased mt ROS expression.(4)IL-13 induced NLRP3 inflammasome activation and increased the expression of NLRP3,ASC,Cleaved-Caspase-1 and IL-1β in BEAS-2B epithelial cells,accompanied by a significant increase in the level of apoptosis and changes in apoptosis-related proteins,including a decrease in the level of the inhibitor of apoptosis protein Bcl-2,an increase in the level of the pro-apoptotic protein BAX,and an increase in the levels of Cytochrome C,Cleaved-Caspase-9,and Cleaved-Caspase-3 proteins.(5)After pretreatment with si-NOX4,the intracellular ROS level was decreased,while the levels of Drp1 and p-Drp1(Ser616)were inhibited,and the transfer of Drp1 from cytoplasm to mitochondria was inhibited,which restored some mitochondrial meshlike changes.(6)After administration of Drp1 inhibitor Mdivi-1,the protein levels of Drp1 and pDrp1(Ser616)were inhibited,the mitochondrial fragmentation was partially reversed,the mitochondrial function was restored,the decrease of MMP and ATP synthesis were improved,and the mt ROS expression was decreased.(7)Mdivi-1 inhibited NLRP3 inflammasome activation and reduced NLRP3,ASC,Cleaved-Caspase-1 and IL-1β expression;Mdivi-1 inhibited epithelial cell apoptosis,increased Bcl-2 levels,and reduced BAX,Cytochrome C,Cleaved-Caspase-9,and Cleaved-Caspase-3 protein levels.Part II:(1)The results of microarray analysis in OVA model showed that 63 miRNAs were changed in expression,50 miRNAs were increased and 13 miRNAs were decreased in OVA,of which miRNA182-5p expression was decreased.The target gene prediction results showed that miRNA182-5p targeted the NOX4 mRNA sequence,and the RTq PCR results showed that miRNA182-5p expression was decreased and NOX4 mRNA expression was increased,which were negatively correlated.(2)The dual-luciferase reporter results suggest that miRNA182-5p directly targets the NOX4 mRNA3 ′ UTR region and inhibits the transcription of NOX4.(3)After transfection with miRNA182-5pmimic,the mRNA and protein levels of NOX4 were decreased,the intracellular ROS level was decreased,the expression of Drp1 was inhibited,the excessive mitochondrial fission was inhibited and the mitochondrial function was restored,the MMP decrease and ATP synthesis were improved,and the mt ROS expression was inhibited.(4)After transfection with miRNA182-5pmimic,NLRP3 inflammasome activation was inhibited,NLRP3,ASC,Cleaved-Caspase-1 and IL-1β expression was reduced,epithelial cell apoptosis was inhibited,Bcl-2 levels were increased,and BAX,Cytochrome C,Cleaved-Caspase-9,and Cleaved-Caspase-3 protein levels were reduced.Part III:(1)OVA asthma model was established.After miRNA182-5pagomir intervention,the levels of NOX4 mRNA and protein in lung tissue were decreased.(2)miRNA182-5p agomir decreased NLRP3/IL-1β protein expression in the OVA model.(3)miRNA182-5p agomir decreased the expression of apoptosis-related proteins BAX,Cleaved-caspase-9,and Cleaved-caspase-3 in the OVA model.(4)miRNA182-5p agomir inhibits airway resistance and improves airway hyperresponsiveness in asthmatic mice.(5)miRNA182-5p agomir decreased the percentage of eosinophils in BALF of asthma model,and reduced the expression of Th2-type inflammatory cytokines IL-4,IL-5,and IL-13 in BALF.(6)miRNA182-5p agomir inhibited the increase of ROS,significantly improved the accumulation and infiltration of inflammatory cells,alleviated the thickening of airway wall,and delayed goblet cell proliferation and collagen deposition in asthma models.Conclusion:1.Increased NOX4 expression during IL-13 stimulation of BEAS-2B cells promotes ROS generation to mediate mitochondrial fission and induce impaired mitochondrial function.2.IL-13 promotes the release of IL-1β through the NOX4/DRP1/NLRP3 signaling axis to aggravate the inflammatory response,while inducing epithelial cell apoptosis,and its apoptotic pathway is the mitochondrial apoptotic pathway.3.There are differential miRNAs in OVA asthma model,in which miRNA182-5p targets NOX4 mRNA and miRNA182-5p mimic inhibits NOX4/DRP1/NLRP3 signaling axis to reduce the release of IL-1β and inhibit apoptosis in vitro model.4.In vivo models,miRNA182-5p agomir can inhibit OVA-induced chronic airway inflammation,reduce inflammatory cell infiltration,goblet cell proliferation and collagen deposition,and miRNA182-5p may be a new target for asthma treatment. |
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