| Background:Acute lung injury(ALI)/Acute respiratory distress syndrome(ARDS)has attracted much attention due to its high morbidity and mortality,and seriously threatens the life and health of patients.In the pathogenesis of ALI/ARDS,uncontrolled inflammation is currently considered to be the central link in the development of ALI/ARDS,in which macrophages play a key role and are very important for prevention and treatment ALI/ARDS.Further elucidating the mechanism of inflammatory regulation of macrophages during the occurrence of ALI/ARDS are hopful to effectively inhibit inflammation.Non-coding RNA,including Long non-coding RNA(LncRNA)and microRNA(miRNA),plays an important role in regulating a variety of physiological processes,including cell survival,differentiation,cell cycle and cell migration.Long non-coding RNA(LncRNA)is a class of RNA that is longer than 200 nucleotides and does not encode proteins.Recent studies have shown that LncRNA can work as an inhibitor of booster or inflammatory transcription,interacting with chromatin remodeling complexes as scaffold molecular RNA binding proteins,and regulating the dynamics and epigenetic control of inflammatory transcription in gene specificity and time-dependent.It has been reported that P50-associated COX-2 extragenic RNA(PACER)is a new long non-coding RNA,which has been found to activate COX-2 and be closely involved in inflammatory reactions,but its specific mechanism is not clear.MicroRNA(miRNA)is a class of 18? 25 nucleotide composed of endogenous non-encoded small molecule RNA,which regulates gene expression at the level after transcription by inhibiting the translation or degradation of the target gene.Studies have shown that miR-124 plays an important role in the regulation of inflammatory responses by inhibiting IL-6R,binding of signal transduction and transcriptional activator 3(STAT3),NF-?B p65 subunit and TNF-associated factor 6(TRAF6)to regulate LPS-induced cytokine production to reduce IL-6 production and TNF-? invertase(TACE)to reduce TNF-? release.However,it is not clear how miR-124 adjusts,and further research is needed.Peroxisome proliferators activated receptor ?(PPAR?),as a transcription factor,which is involved in regulating the development of glucose metabolism,lipid metabolism,inflammation and cancer.Relevant studies have reported that ligand-activated PPAR? can inhibit the activity of signal transduction kinase or transcription regulatory factors and associated signaling pathways,thereby inhibiting the expression of inflammatory factors or increasing anti-inflammatory factors and apoptosis-inducing inflammatory cells in inflammatory response.Therefore,some scholars believe that PPAR? can be used as a new therapeutic target to inhibit inflammatory response.However,how PPAR? is limited and the anti-inflammatory mechanism has not been fully explained.Therefore,a deeper understanding of the anti-inflammatory mechanisms of PPAR? will be beneficial in the treatment of some inflammatory diseases.Based on these studies,we speculate that PACER,PPAR? and miRNA may interact with each other in the occurrence and development of inflammation.Further studies on these three factors in inflammation are expected to provide new therapeutic strategies for ARDS.Objectives:To explore the important role of PACER in LPS-induced macrophage inflammatory response and the mechanisms involved in the regulation of inflammation by PPAR? and miR-124.Methods:1.The effect and mechanism of PACER in LPS-induced macrophage inflammation.1.1 THP-1 difference into macrophage(M?): THP-1 cells were cultred with 100?M Phorbol-12-myristate-13-acetate(PMA)for 48 h,It differentiates THP-1 into macrophages(M?).1.2 The expression of miR-124 and PACER was analyzed by Real-time PCR,after M? was treated with LPS.1.3 After transfection with PACER siRNA,THP-1 was induced into M? with PMA.The expressions of PACER,miR-124 were analyzed by Real-time PCR.Western blot was used to detect changes in the expression of PPAR? protein.After M? With LPS stimulation,the expressions of PACER and TNF-?,IL-6 and IL-10 were analyzed by Real-time PCR.2.The effect of PPAR? in LPS induced ALI2.1 Mice lung injury model was established by intraperitoneal injection of LPS.Lung histological examination(HE staining)was performed to observe the lung injury in mice.The expressions of TNF-? and IL-6 in the lung tissues of mice in each group were analyzed by real-time PCR;The expressions of TNF-? and IL-6 in serum of mice in each group were detected by flow multifactorial assay.Western blot was used to detect the changes of PPAR? expression in the lung tissues of mice.2.2 Mice pretreated rosiglitazone(Rosi),PPAR? agonists,and then copied the ALI model.Pulmonary histological examination(HEstaining)was performed to observe lung injury in mice in various groups.In the lung tissues of TNF-? and IL-6,and the level of expression in the serum of mice in each group was detected by real-time PCR and flow multifactorial assay.Western blot detects the changes of PPAR? expression in each group?3.Investigating the regulation and mechanism of PPAR? on miR-124 in LPS-inducedmacrophage inflammatory response.3.1 RAW264.7 cells were treated with different concentrations of PPAR? agonist,Ciglitazone(Cig),Pioglitazone(Pio),Troglitazone(Tro)or DMSO,respectively.The expressions of miR-124 and its target gene TRAF6 were detected by Real-time PCR.3.2 RAW264.7 cells were transfected with PPAR? siRNA or PPAR? NC and treated with Cig,Pio and Tro,respectively.The expressions of miR-124 and TRAF6 were observed by Real-time PCR.3.3 After transfection with antagomiR-124 or antagomir NC,RAW264.7 cells were treated with Cig,Pio and Tro,respectively,and the expression of TRAF6 was detected by Real-time PCR.3.4 RAW264.7 cells were transfected with empty vector(pGL3/Basic)or luciferase reporter vector containing the miR-124 promoter(pGL3/miR-124)then cells were treated with PPAR? agonists(Cig,Pio,Tro),the luciferase assays were performed.3.5 The promoter region of miR-124 predicted by bioinformatics analysis contains PPAR? reaction element(PPRE).RAW264.7 cells were transfected with luciferase reporter vectors containing the wild type or mutant PPRE,then cells were treated with PPAR? agonists(Cig,Pio,Tro),and the luciferase assays were performed.Firefly luciferase activity was normalized to that of renilla luciferase3.6 RAW264.7 cells were treated with Cig,Pio and Tro respectively,and the interaction between PPAR? and miR-124 was detected by ChIP assay.3.7 After transfection with antagomiR-124 or antagomir NC,RAW264.7 cells were treated with Cig,Pio,Tro,and then stimulated by LPS or PBS,cell supernatant was collected and the levels of inflammatory cytokines TNF-? and IL-6 were determined by ELISA.Results:1.After LPS stimulation of M? cells,the expression levels of PACER,TNF-? and IL-6 mRNA were significantly higher than the control group(P<0.05),while the expression levels of miR-124 and IL-10 mRNA were significantly reduced(P<0.05).2.Compared with NC,the expression level of PACER decreased significantly while the siRNA group of miR-124 mRNA and PPAR? protein were significantly increased(P<0.05).After LPS stimulation,the expression in the PACER siRNA Group of TNF-? and IL-6 mRNA was significantly lower than that of the NC group after LPS treatment,while the expression of IL-10 mRNA increased(P<0.05).3.Compared with the control group,the lung tissue of the ALI model group showed significant congestion,alveolar collapse and alveolar wall thickening,and inflammatory factor TNF-? and IL-6 the expression level in lung tissue and serum increased significantly(P<0.05).In addition,the expression level of PPAR? protein in lung tissue of the model group was significantly lower than that in the control group(P <0.05).4.Rosiglitazone pretreated mice can reduce lung tissue injury induced by LPS: lung tissue injury of mice pretreated by Rosi(LPS+Rosi group)is less than that of LPS group.Compared with the LPS group,the relative expression levels of inflammatory factors TNF-? and IL-6 in lung tissues and serum of the LPS+Rosi group were significantly decreased(P <0.05).5.Different PPAR? agonists(Cig,Pio,Tro)upregulated the expression of miR-124 in a concentration-dependent manner.Meanwhile,the expression of target gene TRAF6 mRNA of miR-124 was down-regulated(P<0.05).6.Compared with the control group,RAW264.7 cells used Cig,Pio and Tro,and the relative expression of miR-124 was significantly decreased after transfection with PPAR? siRNA,while the expression of TRAF6 mRNA was significantly increased(P <0.05).The antagomiR-124 transfected RAW264.7 cells were treated with Cig,Pio and Tro,respectively,and the relative expression of TRAF6 mRNA was significantly increased(P<0.05).7.Luciferase reporter gene assay showed that luciferase activity of luc-pGL3 / miR-124 in cells treated by Cig,Pio and Tro was significantly increased.After Cig,Pio and Tro treatment,luciferase activity of luc-pGL3 / miR-124-mut group was significantly lower than that of luc-pGL3 / miR-124-WT group(P<0.05).ChIP assay showed that PPAR? directly bound to PPRE in the promoter of miR-124.8.After transfection of antagomiR-124,RAW264.7 cells were treated with different agonists(Cig,Pio,Tro)and then stimulated by LPS,the levels of cytoinflammatory cytokines TNF-? and IL-6 were significantly increased(P<0.05).After transfection of antagomir NC,the levels of TNF-? and IL-6 in RAW264.7 cells treated with Cig,Pio,Tro and stimulated by LPS were significantly decreased(P<0.05).Conclusions:1.PACER is a key molecule regulating LPS-induced macrophage inflammatory response,which may play a role in regulating inflammatory response by regulating PPAR? and miR-124;2.PPAR? can enhance the transcriptional activity of miR-124 promoter and increased its expression by directly binding to PPRE in the promoter region of miR-124,thus inhibiting the production and release of inflammatory factors and exerting anti-inflammatory effects. |
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