| [Background]Acute Lung Injury and acute respiratory distress syndrome(ALI and ARDS)are hyperinflammatory response syndrome characterized by progressive dyspnea and refractory hypoxemia caused by various etiologies inside and outside the lung.ARDS is a serious form of ALI,with high morbidity and mortality worldwide.ARDS is one of the main causes of death for ICU patients.ALI/ARDS is characterized by severe pulmonary inflammation,which causes destruction of alveolar-vascular endothelial barrier,leading to influx of a large number of neutrophils and other inflammatory cells,release of inflammatory cytokines and inflammatory mediators,and protein-rich liquid causing alveolar hyperemia,surface active substance synthesis and metabolic disorders,and local hypercoagulable state.These pathological factors jointly impair the gas exchange and alveolar surface function of the lung,and the patient suffers from severe hypoxemia and elevated pulmonary artery wedge pressure.The etiology of ALI is complex.After half a century,great progress has been made in the research on the pathogenesis of ALI/ARDS,but the exact pathogenesis has not been fully clarified.At present,it is believed that excessive activation and recruitment of inflammatory cells in the lung lead to uncontrolled inflammatory reaction.A large number of inflammatory factors and effector cells are activated each other.Interaction is the main pathophysiological change of ALI.Therefore,inhibition of uncontrolled inflammatory reaction may be the key way to treat ALI.In the past few decades,researches on ALI/ARDS have mostly focused on the imbalance of cytokine network and observed the clinical efficacy of various cytokine antagonists in ALI/ARDS,but no major breakthrough and progress have been made In recent years,the molecular mechanism and gene regulation of cell signaling have graduALIy become a research hotspot.A large number of experiments show that the disorder of pro-inflammatory signaling pathways in inflammatory cells plays an important role in the pathogenesis of ALI/ARDS.Therefore,exploring the mechanism of regulating various pro-inflammatory signaling pathways in ALI acute lung injury has very important clinical significance for ALI to realize early diagnosis,early treatment and timely therapeutic effect judgment.Recent studies have found that Toll-like receptor 4(TLR4)is a pattern recognition receptor for LPS signal transduction in gram-negative bacteria,mediates the activation of NF-?B and the generation of inflammatory mediators,and is one of the earliest signal molecules in cell signal transduction.TLR4/NF-?B pathway is a classical pathway that initiates intracellular inflammatory signaling and plays an important role in LPS-induced acute lung injury.After TLR4 is combined with LPS,it mainly transmits signals step by step via myeloid differentiation factor 88(MyD88),phosphorylating NF-?B inhibitor of NF-?B,I?B,resulting in degradation of I?B and activation of NF-?B.The activated NF-?B rapidly shifts into the nucleus and upregulates IL-1?,TNF-?,IL-6,IL-8 and other pro-inflammatory cytokines.Rho kinase can regulate inflammatory reaction.Studies have shown that Rho kinase may activate NF-?B through the I?B kinase(I?K)or PKC pathway,while Rho kinase inhibitor can inhibit the activation of NF-? B.Therefore,Rh kinase may play an important role in inflammatory cascade amplification of sepsis.Nucleotide binding oligomerization domain-like receptor protein 3(NLRP3)inflammatory corpuscle is the most widely studied inflammatory corpuscle at present.It is a kind of multi-protein complex in cells,mainly distributed in intercellular substance and cell membrane,and can participate in a variety of host immune and inflammatory reactions.It is composed of receptor protein-NLRP3,connexin-apoptosis-related spotted protein(ASC)and precursor of effector egg-Caspase-1(caspase-1).NLRP3 inflammatory corpuscle can recognize a series of exogenous and endogenous stimulation stimuli,and convert caspase-1 into its activated form.The activated caspase-1 can promote the cleavage and maturation of pro-IL-1? and pro-IL-18,the cytokine precursors,while IL-1? and IL-18 are powerful inflammatory initiating factors,which can promote the occurrence of inflammatory reactions and aggravate tissue damage.Toll-like receptors(TLRs)are cytoplasmic pattern recognition receptors,which can quickly recognize various pathogen-associated molecular patterns(PAMPs)and various hazard-associated molecular patterns(DAMPs)at first,thus triggering inflammatory signaling pathways and generating non-specific immune responses.However,the pattern recognition receptor on the cell membrane surface cannot fully recognize ALI PAMP or DAMP.NLRP3 inflammatory corpuscles can recognize these PAMP or DAMP entering the cell in the cytoplasm of the cell,forming another defense barrier of the body.The signal pathway mediated by TLRs and NLRP3 inflammatory corpuscles can promote the release of a large number of IL-1? and IL-18 with activated forms,thus promoting the occurrence of inflammation and the development of diseases.Therefore,in LPS-mediated ALI,it is possible that multiple pathways including TLR4/NF-?B and NLRP3 inflammatory corpuscles jointly regulate the occurrence and development of inflammatory reactions.Acute lung injury(ALI)is the result of excessive inflammatory response.Regulating the occurrence and development of inflammatory response is of great clinical significance for preventing the occurrence of ALI and reducing mortality.Therefore,it is of great clinical significance to explore the biological markers of acute lung injury,and to carry out early diagnosis,early treatment,timely judgment of curative effect and correct evaluation of prognosis of diseases.MicroRNAs is a kind of endogenous smALI molecule non-coding RNA,which can regulate gene expression after transcription.Recent studies have shown that many miRNAs play an important role in the inflammatory response of sepsis.Some scholars have found that the expression of miR-125b in serum and lung tissue of ALI mice induced by LPS is obviously reduced,and up-regulating the expression of miR-125b in lung tissue of ALI mice can obviously inhibit ALI induced by LPS.Knocking down miR-7 or increasing miR-155 expression can also effectively reduce LPS-induced ALI inflammatory response in mice.However,some scholars found that the expression of miR-214 in lung tissue of LPS-induced ALI mouse model was significantly increased,suggesting that miR-214 may be closely related to LPS-induced ALI as an inflammatory factor.MiR-223 was initiALIy found to be dysregulated in tumor tissues and abnormal in peripheral blood of some solid tumor patients.The expression abundance of miR-223 in platelets is also very high.The expression level of miR-223 in platelets of patients with coronary heart disease can reflect the degree of platelet activation and is significantly positively correlated with the severity of coronary artery.Recent animal studies have found that miRNA-223 may play a role in regulating anti-inflammatory and cell repair during infection.Mice lacking miR-223 are observed to have spontaneous inflammatory pathological changes in the lungs,and a large number of tissues are destroyed during endotoxin action.Neudecker et al.also believe that miRNA-223 deficiency is related to severe lung inflammation,suggesting that miRNA-223 may participate in the regulation of immune function of lung inflammation,but the specific mechanism of action is still unclear.TLR4/NF-?B,NLRP3 inflammatory corpuscles two signALIng pathways may jointly participate in ali regulation of the occurrence and development of inflammatory reactions.Whether miR-223 has regulatory effect on the occurrence and development of LPS-induced acute lung injury and whether the mechanism of action is related to TLR4/NF-?B or NLRP3 inflammatory body signaling pathway remains to be discussed.LPS is the active component of bacterial endotoxin.Bacterial infection,especiALIy gram-negative bacteria,is one of the main pathogenic factors causing acute lung injury.In recent years,a series of animal models have been established to simulate acute lung injury caused by different pathogenesis,among which LPS-induced acute lung injury animal models are most commonly used.[Objective]The purpose of this study is to detect the changes of miR-223 and its correlation with inflammatory factor expression in LPS-induced acute lung injury models in vivo and in vitro.To investigate the effect of up-regulation or down-regulation of miR-223 on inflammatory response of LPS-induced acute lung injury model in vitro,and further explore the mechanism of miR-223 in regulating inflammatory response of acute lung injury,so as to find a new potential therapeutic target for ALI and a biological index for judging the prognosis of ALI.[Methods]1.Establishment of acute lung injury mouse modelSixteen C57BL/6 mice were selected and randomly divided into ALI group and control group,8 mice in each group.LPS was injected intraperitoneALIy into ALI group to establish septic mice model.The general conditions of the two groups of mice were observed,and lung tissue specimens were collected to observe the pathological changes of the surface color,volume,surface and section of lung tissue of mice,such as congestion and exudation.The pathological changes of lung tissue were analyzed,and the expressions of TNF-?,IL-1?,IL-6 and IL-18 in lung tissue of mice were detected by ELISA to judge whether ALI model was successfully constructed.2.Role of 2.miR-223 in LPS-induced ALI InflammationThe expression level of miRNAs in ALI mice was analyzed by gene chip technology,and miRNA with abnormal expression was screened.RT-qPCR method was used to detect miR-223 expression in lung tissues of LPS group and normal control group mice respectively to observe whether miR-223 has abnormal expression in LPS group mice.ALI model of A549 cells(adenocarcinoma human alveolar basal epithelial cells)was constructed by co-culture with LPS.One day later,miR-223 expression in A549 cells of LPS group and normal control group was detected by RT-qPCR method respectively,and IL-1? and IL-18 expression levels in A549 cells were detected by ELISA method,and were compared to observe whether miR-223 was abnormALIy expressed in ALI cell model.The expression of miR-223 in A549 cells was up-regulated by mimics transfected with miR-223 or down-regulated by mimics of anti-miR-223.The changes in the expression levels of IL-1? and IL-18 in A549 cells were detected and compared to explore whether miR-223 has regulatory effect on LPS-induced ALI inflammatory reaction.3.miR-223 Regulates ALI Target Genes and Validation3.1 In LPS-treated A549 cells,the expression level of miR-223 was knocked down by RNA interference technology,the downstream targets regulated by miR-223 were analyzed by gene chip technology,and the expression level changes of target gene mRNA and protein were detected by RT-qPCR,Western blot and immunofluorescence experiments respectively.Overexpression of miR-223 was detected by RT-qPCR and Western blot experiments to detect the changes of mRNA and protein expression levels of target genes respectively.Targetscan database was used to analyze the binding sites of miR-223 with NLPR3 and RHOB target proteins,and the binding sites were verified by double luciferase reporting experiment.the molecular mechanism of miR-223 regulating the expression of NLPR3 and RHOB was analyzed.3.2 After transfecting miR-223 mimics/inhibitor into LPS-treated A549 cells,the mRNA level of inflammatory factors was detected by RT-qPCR method,and the protein level of inflammatory factors was detected by ELISA method.In order to verify the upstream-downstream relationship between the target proteins regulated by miR-223,in LPS-induced cell injury model,the cells were divided into negative Control group,miR-223 inhibitor group and miR-223 inhibitor combined with one of the target protein overexpression groups in turn.Firstly,the changes of expression levels of ALI target proteins in different treatment groups were analyzed by Western blot experiment to explore the upstream-downstream relationship between the target proteins.Furthermore,the expression levels of IL-1? and IL-18 in the cells of the above-mentioned different treatment groups were detected by ELISA experiments to explore the role of each target protein in miR-223 regulation of ALI[Results]1.Establishment of acute lung injury mouse model Establishment of ALI mouse model and pathological changes of lung tissue.Normal control group mice showed no abnormal performance.Three hours after LPS injection,LPS group mice showed obvious symptoms of shortness of breath,poor mental response,temperature rise,slight cyanosis in limbs,lips and nose,anorexia,diarrhea and other endotoxemia manifestations.Some mice have pink secretions overflowing from their snouts.General appearance and pathological changes of mouse lung tissue:normal control group mice have normal lung tissue structure and morphology,LPS group mice have increased lung tissue volume,dark red color,punctate and flaky congestion or hemorrhage spots can be seen under the capsule,the section is loose and moist,and reddish bloody fluid exudes.HE staining of lung tissue paraffin sections showed that the normal control group mice had complete lung tissue structure and normal morphology.The lung tissue of LPS group mice showed some structural damage,alveolar insufficiency,alveolar fusion or collapse,alveolar wALI edema,exudate in alveolar cavity,obvious thickening of alveolar septum,and a large number of inflammatory cell infiltration.Pulmonary capillaries dilate and extravasate,and the lumen is filled with red blood cells.The expression of TNF-?,IL-1?,IL-6 and IL-18 in the lung tissue of mice detected by ELISA were significantly increased,suggesting that the establishment of acute lung injury mouse model was successful.2.Role of 2.miR-223 in LPS-induced acute lung injuryThe expression level of miRNAs in LPS group mice was analyzed by gene chip technology.it was found that miR-223 expression in lung tissue of LPS group mice was significantly lower than that of control group(P<0.01).RT-qPCR method was used to detect miR-223 expression in lung tissue of two groups of mice,and ALI group was significantly lower than that of control group(P<0.01).ALI model of A549 cells was constructed by co-culture with LPS.After 24 h,the expression of miR-223 in A549 cells detected by RT-qPCR method decreased significantly compared with that of the control group(P<0.01),and the expression levels of IL-1?and IL-18 in A549 cells detected by ELISA method increased significantly(P<0.01)Compared with the control group,overexpression of miR-223 significantly decreased the expression levels of IL-1? and IL-18 in A549 cells(P<0.01),downregulated the expression of miR-223,and significantly increased the expression of IL-1? and IL-18 in A549 cells(P<0.01)3.miR-223 Regulates ALI Target Genes and Validation3.1 miR-223 inhibits NLRP3,TLR4 and NF-?B signaling pathways in LPS-induced cell injuryIn order to analyze the mechanism of miR-223 regulating lung injury,we knocked down the expression level of miR-223 by RNA interference technology in LPS-treated A549 cells,and detected the expression level of candidate genes by gene chip.Compared with the control group,after inhibiting the expression level of miR-223,we can significantly up-regulate the expressions of TLR4,NF-?B and NLRP3.In order to further verify the results,RT-qPCR detection showed that,compared with the control group,inhibition of miR-223 significantly increased the expression levels of NLPR3,TLR4 and NF-?B mRNA(P<0.01).After detecting the expression level of miR-223 through Western blot experiment,we can significantly promote the expression of NLPR3,caspase-1,TLR4 and NF-?B protein levels(P<0.01).After overexpressing miR-223,it can significantly inhibit the expression of the above proteins(P<0.01).The above results show that miR-223 can regulate the expression levels of NLRP3,caspase-1,TLR4 and NF-?B in the acute lung injury model in vitro.3.2 miR-223 inhibits TLR4 signaling pathway and ALIeviates LPS-induced cell damageIn order to verify the upstream-downstream relationship of miR-223 regulating NLRP3,TLR4 and NF-?B expression,we divided cells into negative Control group,miR-223 inhibitor group and miR-223 inhibitor combined siTLR4(TLR4i)group in LPS-induced cell injury model.Firstly,the changes of expression levels of TLR4,NLRP3,NF-?B and Caspase-1 in different treatment groups were analyzed by Western Blot experiment.compared with Anti-223 group,the expression levels of TLR4 and NF-?B in TLR4i group were significantly decreased(P<0.01),indicating that siTLR4 can effectively inhibit the expression level of TLR4 and block the expression of nf-K b induced by miR-223 inhibitor.Moreover,the expression levels of NLRP3 and Caspase-1 in TLR4i group were not significantly different from those in the control group(P>0.05),indicating that miR-223 does not depend on TLR4 to regulate the expression of NLRP3 and Caspase-1.The expression levels of IL-1?and IL-18 in cells of the above different treatment groups were detected by ELISA experiments.compared with Control group,the expression levels of IL-1? and IL-18 in A549 cells transfected with miR-223 inhibitor increased significantly(P<0.01),while TLR4i group had no significant difference(P>0.05)with Control group.the results showed that miR-223 ALIeviated LPS-induced cell damage by inhibiting the expression of TLR4.3.3 miR-223 Inhibits NLRP3 and Reduces LPS-induced Cell Damage3.3.1 Mechanism 3.3.1 miR-223 Regulating NLRP3 ExpressionIn order to clarify the molecular mechanism of miR-223 regulating NLRP3 expression,we analyzed the binding sites of miR-223 and NLRP3 3'-UTR region using TargetScan database,showing that miR-223 and NLRP3 3'-UTR region have potential binding ability.The binding site was verified by double luciferase reporting experiment.The results showed that miR-223 targeted regulated NLRP3 expression by binding to NLRP3 3'-UTR region.In order to further clarify the regulatory effect of miR-223 on NLRP3 in A549 cells,negative Control and miR-223 inhibitor(Anti-223)were transfected into A549 cells respectively,and the effect on NLRP3 expression was detected by immunofluorescence 48 h later.Compared with the Control group,the expression level of NLRP3 in A549 cells transfected with miR-223 inhibitor increased significantly,indicating that downregulation of miR-223 level can promote the expression of NLRP3 in A549 cells.3.3.2 miR-223 inhibitor induces expression of inflammatory factors by up-regulating NLRP3 in LPS-induced cell injury model.In order to verify the role of NLRP3 in miR-223-regulated acute lung injury model,we divided the cells into negative Control group,miR-223 inhibitor(Anti-223)group or miR-223 inhibitor combined with siNLRP3(NLRP3i)group in LPS-induced cell injury model.First,we analyzed the changes of expression levels of NLRP3 and Caspase-1 in different treatment groups through Western Blot experiment.Compared with Anti-223 group,the expression levels of NLRP3 and Caspase-1 in NLRP3i group were significantly decreased(P<0.01),indicating that siNLRP3 can effectively inhibit the expression level of NLRP3 and block the expression of Caspase-1 induced by miR-223 inhibitor.The expression levels of IL-1? and IL-18 in cells of the above different treatment groups were detected by ELISA experiment.compared with Control group,the expression levels of IL-1? and IL-18 in A549 cells transfected with miR-223 inhibitor increased significantly(P<0.01),while NLRP3i group had no significant difference(P>0.05)with Control group.results miR-223 ALIeviated LPS-induced cell damage by inhibiting the expression of NLRP3.3.4miR-223 ALIeviates LPS-induced cell damage by regulating RHOB and inhibiting NF-?B signaling pathway3.4.1 Mechanism of miR-223 Regulating RHOB ExpressionIn order to verify the role of miR-223 in RHOB-activated inflammatory response,we analyzed the binding sites of miR-223 and RHOB 3'-UTR region using TargetScan database,showing that miR-223 and RHOB 3'-UTR region have potential binding ability.The binding site was verified by double luciferase reporting experiment.The results showed that miR-223 targeted regulated the expression of RHOB by binding to RHOB 3'-UTR region.In order to further clarify the regulatory effect of miR-223 on RHOB in A549 cells,we analyzed the effect of overexpression of miR-223 on RHOB expression level through Western blot experiment.compared with Negative group,the expression level of RHOB in A549 cells after overexpression of miR-223 was significantly decreased(P<0.01).The effect of miR-223 inhibitor on RHOB expression level was analyzed by Western Blot experiment.compared with Negative group,RHOB expression level in Anti-223 group was significantly increased(P<0.01).The above results show that miR-223 inhibits RHOB expression through targeted binding to the 3'-UTR region of RHOB.3.4.2 miR-223 inhibitor induces the expression of inflammatory factors by up-regulating RHOB to regulate NF-?B signaling pathway in LPS-induced cell injury modelIn order to verify the role of RHOB in miR-223-regulated acute lung injury model,cells were divided into Negative control group,miR-223 mimetic group and miR-223 mimetic combined RHOB group in LPS-induced cell injury model.Firstly,the Western blot experiment was used to analyze the changes in the expression levels of RHOB,TLR4 and NF-?B in different treatment groups.compared with miR-223 group,the expression levels of RHOB and NF-?B in RHOB group were significantly increased(P<0.01),while the expression levels of TLR4 were not significantly different,indicating that the RHOB plasmid was effectively expressed in A549 cells,and the expression level of NF-?B inhibited by overexpression of miR-223 could be restored,but the expression of TLR4 was not affected.The expression levels of IL-1? and IL-18 in the cells of the different treatment groups were detected by ELISA.Compared with Negative group,the expression levels of IL-1? and IL-18 in miR-223 group were significantly down-regulated(P<0.01),and there was significant difference between RHOB group and miR-223 group(P<0.01).the above results showed that miR-223 ALIeviated LPS-induced cell injury by inhibiting RHOB to down-regulate NF-?B expression.[Conclusions]1.In this study,we found that the expression of miR-223 was significantly down-regulated in LPS-induced acute lung injury,and was significantly negatively correlated with the expression levels of inflammatory factors IL-1? and IL-18 MiR-223 has protective effect on LPS-induced acute lung injury.2.miR-223 is a regulatory factor of ALI inflammatory response.In LPS-induced acute lung injury,negative feedback may be used to control RHOB-stimulated TLR4/NF-?B signals and inhibit NLRP3 inflammatory bodies to play a protective role.It is expected to become a new therapeutic target for ALI inflammation. |
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