| BackgroundIn the treatment of critical patients such as respiratory failure and acute respiratory distress syndrome(ARDS),mechanical ventilation is an indispensable treatment measure.However,mechanical ventilation can sometimes lead to acute lung injury,namely ventilator-induced lung injury(VILI),which worsens the prognosis of patients seriously.Explorating the mechanism of VILI and seeking prevention and treatment measures for VILI have important clinical significance.Studies have shown that biological injury caused by inflammatory reaction plays an important role in the occurrence of VILI.Inappropriate mechanical stretch acts on lung effector cells,induces a large release of inflammatory mediators and inflammatory cell infiltration,and finally causes diffuse alveolar damage and pulmonary edema.Regulating inflammatory response is the key to prevent acute lung biological injury and the main content of prevention and treatment of VILI.Alveolar macrophages(AMs)account for more than 90%of the total number of pulmonary macrophages.They are the first cells to activate and initiate inflammatory response after abnormal stimulation of the lung.Previous studies have shown that mechanical stretch can quickly activate AMs.AMs are the main source of inflammatory mediators in the lung.In the early stage of inflammation,AMs can synthesize and release inflammatory factors,adhesion factors,chemokines,danger signal molecules,complement and other inflammatory mediators;The pre-consumption of alveolar macrophages can reduce the synthesis and secretion of proinflammatory factors,reduce the recruitment of inflammatory cells such as neutrophils,and significantly alleviate VILI in rats.MicroRNA(miRNA)is a kind of powerful endogenous non-coding single stranded RNA with a length of about 18-25 bases.It is evolutionarily conserved and has a high degree of homology among different species.MiRNA can combine with the 3'noncoding region(3'UTR)of specific target gene mRNA,promote the degradation of target gene or prevent the translation of target gene,regulate gene expression at the post transcriptional level,and play an important role in cell differentiation,migration,apoptosis,disease occurrence and so on.At present,miRNA related research has gradually moved from laboratory to clinic,and more and more miRNAs have been used as biomarkers or therapeutic targets for disease diagnosis and treatment.MiR-145 is a tumor suppressor gene,which can target and negatively regulate a variety of oncogenes such as YES,FSCN1,STAT1 to inhibit the occurrence and development of tumors.In recent years,studies have shown that miR-145 also plays a certain anti-inflammatory role in some inflammatory diseases.For example,high expression of miR-145 can reduce the inflammatory response of vascular smooth muscle cells and thereby reduce the incidence of atherosclerosis,inhibit the hypoxia related inflammatory response of cardiomyocytes,reduce the skin inflammation of patients with psoriasis,and reduce sepsis caused by LPS and improve the overall survival rate of septic mice.Studies have found that the expression of miR-145 decreases when VILI occurs in mice,but there was no relevant report on whether miR-145 had an effect on VILI and its possible mechanism.This experiment will explore these issues on the mechanical stretch model of NR8383 cells(rat alveolar macrophages)and the rat VILI animal model.Programmed cell death 4(PDCD4)was first discovered in 1995,it can regulate target gene transcription through eIF4A dependent and non-eIF4A dependent pathways,and is involved in the pathogenesis of many diseases such as tumor and inflammation.In many inflammatory disease models,PDCD4 plays a pro-inflammatory role.For example,PDCD4 gene deletion can significantly reduce the inflammatory response of spinal cord in mouse autoimmune encephalomyelitis model;In the LPS-induced death model,the inflammatory injury of pdcd4-/-mice was significantly reduced,and the survival rate was much higher than that of wild-type mice;PDCD4 knockout can reduce vascular endothelial inflammatory response caused by high fat and reduce the area of atherosclerotic plaque.However,there are some contrary reports.For example,in the mouse acute liver injury model induced by LPS/D-galactosamine and the mouse acute colitis model induced by dextran sodium sulfate,the inflammatory response ofpdcd4/-mice is more serious than that of wild-type mice.PDCD4 is highly expressed in lung tissue,whether it can regulate the pulmonary inflammation induced by mechanical ventilation has not been reported.The prediction results of miRNA target gene information prediction software such as TargetScan and previous related research results suggest the targeted regulation of miR-145 on PDCD4,which is consistent with our pre-experiment results.We speculate that miR-145 may affect the pulmonary inflammatory response induced by mechanical stretch through targeted regulation of PDCD4 expression.To verify this hypothesis,we will take NR8383 cells(rat alveolar macrophages)and rat VILI model as the research object,adopt gene regulation technologies such as miRNA mimic transfection,miRNA inhibitor transfection and siRNA transfection,and use a variety of detection methods such as RT qPCR,ELISA,Western blot,dual luciferase reporter gene experiment;transcription factor enzyme-linked immunoassay and H?E staining,to explore the effects of miR-145 regulating PDCD4 on VILI from multiple levels.objective1.To explore the effect of mechanical stretch on the expression of miR-145 in alveolar macrophages and lung tissue;2.To explore the effect of miR-145 on the inflammatory response of alveolar macrophages induced by mechanical stretch and VILI;3.To explore the effect of PDCD4 on the inflammatory response of alveolar macrophages induced by mechanical stretch and its related mechanisms;4.To clarify the target regulation relationship of miR-145 to PDCD4,and to explore the mechanism of miR-145 reducing VILI.MethodsPart one:The expression of MiR-145 in NR8383 cell mechanical stretch model and its regulation of inflammation1.Effects of mechanical stretch on inflammatory response and miR-145 expression of NR8383 cells.1.1 NR8383 cell mechanical stretchAfter resuscitation,culture and passage of NR8383 cells,the cells in logarithmic growth stage were inoculated into the 6-well plate of Flex-cell stress loading system pre-coated with collagen,and the cell density was 1*105/cm2.Grouping as follows:(1)group C:the control group,without stretch;(2)group MS:the mechanical stretch group,loaded with mechanical stretch,with stretch strength of 20%,frequency of 0.5Hz,stretch relaxation ratio of 1:1 and stretch time of 4h.1.2 The content of IL-1?,IL-6 and TNF-? in the supernatant of each group was determined by ELISA.1.3 The expression of miR-145 was measured by RT-qPCR2.Regulation of miR-145 on inflammatory response of NR8383 cells induced by mechanical stretch2.1 Cells were transfected with miR-145 mimic,miR-145 inhibitor and their negative control(NC)NR8383 cells were grouped as follows:(1)group C,the control group,without transfection;(2)group Mimic,miR-145 mimic was transfected;(3)group Mimic-NC,miR-145 mimic-NC was transfected;(4)group Inhibitor,miR-145 inhibitor was transfected;(5)group Inhibitor-NC,miR-145 inhibitor-NC was transfected.Lipo2000 transfection reagent was used for transfection.After 6 hours of transfection,the medium was changed to complete medium and the cells were incubated for another 48 hours.2.2 RT-qPCR was used to determine the content of miR-145 in cells of each group to verify the transfection effect.2.3 Mechanical stretchAfter successful transfection,the cells were inoculated into a 6-well plate of Flexcell stress loading system precoated with collagen according to the grouping.The grouping and treatment were as follows:(1)group C,the control group,untransfected cells,without mechanical stretch;(2)group MS,perform mechanical stretch on untransfected cells,with the same parameters as before;(3)group Mimic+MS,perform mechanical stretch on the cells transfected with miR-145 mimic,with the same parameters as the MS group;(4)group Mimic-NC+MS,perform mechanical stretch on the cells transfected with miR-145 mimic-NC,with the same parameters as the MS group;(5)group Inhibitor+MS,perform mechanical stretch on the cells transfected with miR-145 inhibitor,with the same parameters as the MS group;(6)group InhibitorNC+MS,perform mechanical stretch on the cells transfected with miR-145 inhibitorNC,with the same parameters as the MS group.2.4 Inflammatory factors detectionThe content of inflammatory factors IL-1?,IL-6 and TNF-? in the supernatant of each group was determined by ELISA;The mRNA of inflammatory factor IL-1?,IL-6 and TNF-? was measured by RT qPCR.Part two:MiR-145 reduces the inflammatory response of NR8383 cells induced by mechanical stretch via targeting down-regulation of PDCD41.Dual luciferase reporter gene experiment1.1 Construction of wild-type(WT)and mutant type(MUT)PDCD4 3'UTR plasmid vectorsThe pmirGLO plasmid containing both the firefly luciferase gene and the Renilla luciferase gene was selected as the gene vector,insert the wild type(WT)and mutant type(MT)of PDCD4 3'UTR into the 3'UTR region of firefly luciferase gene in the pmirGLO plasmid respectively.The WT PDCD4 3'UTR sequence is the PDCD4 3'UTR fragment containing the miR-145 predicted binding site(AACUGGAA),and the MT PDCD4 3'UTR is a fragment of PDCD4 3'UTR after mutating the predicted binding site.1.2 Cell grouping and transfectionHEK293T cells were selected as tool cells.HEK 293T cells in the logarithmic growth phase were inoculated into 24-well plates and randomly divided into 4 groups:(1)group Mimic+WT,co-transfected with miR-145 mimic and wild-type plasmid;(2)group Mimic-NC+WT,co-transfected miR-145 mimic-NC and wild-type plasmid;(3)group Mimic+MUT,co-transfected miR-145 mimic and mutant type Plasmid;(4)group Mimic-NC+MUT,co-transfected miR-145 mimic-NC and mutant plasmid.Lipo2000 transfection reagent was used for transfection.After 6 hours of transfection,the medium was changed to complete medium and the cells were incubated for another 48 hours.1.3 Detection of dual luciferase activityThe reporter gene cell lysate was used to lyse the cells in each well.After centrifugation,the activities of firefly luciferase and renilla luciferase in the supernatant were determined.The renilla luciferase activity was used as the internal control,and the firefly luciferase activity was the main detection index.The result is expressed by the ratio of the two luminous values.2.The effect of up-regulation/down-regulation of miR-145 on PDCD4 mRNA and protein expression in NR8383 cellsThe RNA and total protein of group C,group Mimic,group Mimic-NC,group Inhibitor and group Inhibitor NC has been extracted in the first part of our experiment.The PDCD4 mRNA was detected by RT-qPCR,the PDCD4 protein was detected by western blot.3.Transfection of NR8383 cells with siRNA-PDCD43.1 Cell grouping and transfectionNR8383 cells in logarithmic growth stage were divided into groups and treated as follows:(1)group C was the control group without transfection;(2)group SiRNA,transfected with PDCD4 siRNA;(3)group SiRNA-NC,transfected with PDCD4 siRNA-NC.Lipo2000 transfection reagent was used for transfection.After 6 hours of transfection,the medium was changed to complete medium and the cells were incubated for another 48 hours.3.2 Verification of the transfection effectThe cells were collected after 48h,the total RNA and total protein were extracted.PDCD4 mRNA and PDCD4 protein were detected to verify the transfection effect.4.Effect of down-regulating PDCD4 on inflammatory response of NR8383 cells induced by mechanical stretch.4.1 Mechanical stretchAfter the cells were successfully transfected with PDCD4 siRNA,the cells were inoculated into the 6-well plate of Flex-cell stress loading system precoated with collagen according to the grouping,and the cell density was 1*105/cm2.The grouping and treatment were as follows:(1)group C,the control group,without transfection and mechanical stretch;(2)group MS,perform mechanical stretch on untransfected cells,with the same parameters as before;(3)group SiR+MS,perform mechanical stretch on cells transfected with PDCD4 siRNA,with the same parameters as the MS group;(4)group SiR-NC+MS,perform mechanical stretch on cells transfected with PDCD4 siRNA-NC,with the same parameters as the MS group.4.2 Inflammatory factors detectionThe content of inflammatory factor IL-1?,IL-6 and TNF-? in the supernatant of each group was determined by ELISA;the mRNA content of inflammatory factors in cells of each group was measured by RT-qPCR.5.The possible mechanism of down-regulation of PDCD4 reducing the inflammatory response of NR8383 cells induced by mechanical stretch.5.1 Effect of down-regulation of PDCD4 on NF-? B activationThe cytoplasmic protein and nuclear protein of cells were extracted,western blot and transcription factor enzyme-linked immunoassay were used to detect NF-?B activity.5.2 Effect of down-regulation PDCD4 on the expression of anti-inflammatory factor IL-10 during mechanical stretch of NR8383 cellsThe content of IL-10 in the supernatant was measured by ELISA,and the IL-10 mRNA content in cells was measured by RT-qPCR.Part three:Effects of miR-145 on pulmonary inflammatory response and lung injury induced by high tidal volume mechanical ventilation in rats1.MiR-145 transfection in vivo1.1 Rat grouping and transfection48 male clean grade Wistar rats were randomly divided into 3 groups:(1)group Control,24 rats,without transfection,injected with the same volume of 5%glucose as the transfection solution;(2)group MiR,12 rats,transfected with miR-145 mimic;(3)group MiR-NC,12 rats,transfected with miR-145 mimic-NC.The rats were transfected in vivo with EntransterTM in vivo transfection reagent.1.2 Verification of the transfection effect72 hours later,6 rats in each group were randomly selected,and lung tissues were taken after being killed,then total RNA and total protein were extracted.MiR-145 and PDCD4 protein in rat lung tissue were detected using RT-qPCR and Western blot to verify the transfection effect.2.Effect of up-regulation of miR-145 on VILI in rats2.1 Establishment of rat VILI modelAfter miR-145 was successfully transfected in vivo,VILI model was established by high tidal volume mechanical ventilation.The grouping and treatments of rats were as follows:(1)group SHAM,untransfected rats retain spontaneous breathing and serve as a control group;(2)group LMV,untransfected rats were ventilated with a low tidal volume of 6 ml/kg,this group is set up to exclude the effects of muscle relaxants;(3)group HMV,untransfected rats were ventilated with a high tidal volume of 20 ml/kg;(4)group MiR+HMV,rats transfected with miR-145 mimic,treated the same as group HMV;(5)group MiR-NC+HMV,rats transfected with miR-145 mimic-NC,treated the same as group HMV.The ventilation time was 4h.Rats undergoing mechanical ventilation were given the muscle relaxant Rocuronium during ventilation to prevent the confrontation between spontaneous breathing and mechanical ventilation,and the SHAM group was given an equal volume of normal saline.After ventilation,the rats were killed and the broncho alveolar lavage fluid(BALF)and lung tissue were collected.2.2 Detection of miR-145 content in lung tissueExtract RNA from lung tissue,and detect the content of miR-145 by RT-qPCR method.2.3 Detection of Lung injuryH?E staining,pathological examination,lung W/D ratio,MPO activity and total protein concentration in BALF.2.4 Detection of lung inflammatory factors and their mRNAsThe content of inflammatory factors(IL-1?,IL-6 and TNF-?)in BALF was determined by ELISA.The mRNA expression of inflammatory factors in lung tissues was detected by RT-qPCR.2.5 Detection of the NF-?B activationCytoplasmic protein and nuclear protein were extracted from lung tissue,then western blot and transcription factor enzyme-linked immunoassay were used to detect NF-?B activity.2.6 Detection of anti-inflammatory factor IL-10 and IL-10mRNAThe content of IL-10 in BALF was measured by ELISA,and the mRNA expression of IL-10 in lung tissue was detected by RT-qPCR.Statistical analysisWe use SPSS25.0 statistical software for analysis in this study.After normality test,the experimental data were presented as meanąStandard Deviation(SD).When analyzing the data,test for homogeneity of variance was performed first.The comparison between the two groups adopts independent sample t test(t' test is used when the variance is uneven);the statistical method of one way ANOVA is used for the comparison between multiple groups(the welch method is used when the variance is uneven),LSD method was used for multiple comparisons between groups.P<0.05 means statistical difference.ResultsPart one1.mechanical stretch can increase the level of inflammatory factors in NR8383 cells.The ELISA results showed that the concentrations of inflammatory factors IL-1?,IL-6,and TNF-? in the supernatant of the MS(mechanical stretch)group were significantly higher than the C group(P<0.01).2.mechanical stretch can decrease miR-145 level in NR8383 cells.The RT-qPCR results showed that the expression of miR-145 in the MS group was significantly lower than that in the C group(P<0.01).3.Transfection of miR-145 mimic and miR-145 inhibitor can successfully upregulate and down-regulate the miR-145 level in NR8383 cells.After transfection with miR-145 mimic,the expression of miR-145 was significantly up-regulated in NR8383 cells(P<0.01);After transfection with miR-145 inhibitor,the expression of miR-145 was down regulated(P<0.01).4.Up-regulation of miR-145 can reduce the inflammatory response of NR8383 cells induced by mechanical stretch.The results of ELISA and RT-qPCR showed:the supernatant concentrations of inflammatory factors(IL-1?,IL-6 and TNF-?)and mRNA levels of inflammatory factors in the Mimic+MS group were significantly lower than those in the MS group(P<0.01).5.Down-regulation of miR-145 can aggravate the inflammatory response of NR8383 cells induced by mechanical stretch.The results of ELISA and RT-qPCR showed:the supernatant concentrations of inflammatory factors(IL-1?,IL-6 and TNF-?)and mRNA levels of inflammatory factors in the Inhibitor+MS group were significantly higher than those in the MS group(P<0.01).Part two1.MiR-145 can target PDCD4 3'UTRWe co-transfected HEK293T cells with miR-145 mimic/NC and the wild-type(WT)or mutant(MUT)plasmid of PDCD4 3'UTR respectively for 48 hours,then detected the dual luciferase activity.The results showed:compared with the Mimic-NC+WT group,the firefly luciferase activity of the Mimic+WT group was significantly reduced(P<0.01);and after the predicted PDCD4 3'UTR binding site(AACUGGAA)was mutated,there was no significant difference of the firefly luciferase activity between the Mimic+MUT group and the Mimic-NC+MUT group.These results indicate that miR-145 can target the "AACUGGAA" base sequence of PDCD4 3'UTR.2.MiR-145 can negatively regulate the expression of PDCD4 in NR8383 cells at the post-transcriptional levelAfter up-regulating/down-regulating the content of miR-145 in NR8383 cells by miR-145 mimic and inhibitor transfection,we tested the effect of changes in miR-145 content on the expression of PDCD4 mRNA and PDCD4 protein in the cells.The results showed that:compared with the C group,the expression of PDCD4 mRNA in the Mimic group did not change significantly,but the expression of PDCD4 protein was significantly reduced(P<0.01);compared with group C,the expression of PDCD4 mRNA in the Inhibitor group did not change significantly,but the expression of PDCD4 protein increased significantly(P<0.01).These results and the results of the dual luciferase reporter gene experiment can jointly show that miR-145 can target PDCD43'UTR,inhibit the translation of PDCD4 mRNA,and negatively regulate the expression of PDCD4 protein at the post-transcriptional level.3.Transfection of PDCD4 siRNA can significantly down-regulate the expression of PDCD4 mRNA and PDCD4 protein48 hours after transfection of PDCD4 siRNA,we used RT-qPCR and Western blot to detect the transfection effect at the gene and protein levels.The results showed that:compared with the C group,the contents of PDCD4 mRNA and PDCD4 protein in the SiR group were significantly reduced(P<0.01).4.Down-regulation of PDCD4 can significantly reduce the inflammatory response of NR8383 cells induced by mechanical stretchELISA and RT-qPCR results showed:the supernatant concentrations of inflammatory factors(IL-1?,IL-6 and TNF-?)and mRNA levels of inflammatory factors in the SiR+MS group were significantly lower than the MS group(P<0.01).5.Down-regulation of PDCD4 can inhibit the activation of NF-?B induced by mechanical ventilation in NR8383 cellsThe detection of the distribution of NF-?B in the nucleus and cytoplasm can reflect the degree of activation.Western blot results showed:compared with the C group,the p65 content in the cytoplasm of the MS group was significantly reduced(P<0.01),and the content of p65 in the nucleus was significantly increased(P<0.01),indicating that more NF-?B was activated and transferred to the nucleus after mechanical stretch;compared with the MS group,the content of p65 in the cytoplasm of SiR+MS group increased(P<0.05),and the content of p65 in the nucleus was significantly reduced(P<0.01),indicating that down-regulation of PDCD4 expression can reduce the activation of NF-?B caused by mechanical stretch.Transcription factor enzyme-linked immunoassay showed a trend consistent with western blot.The above results indicate that mechanical stretch can induce the activation of NF-?B in NR8383 cells,and downregulation of PDCD4 can inhibit the activation of NF-?B induced by mechanical stretch.6.Down-regulation of PDCD4 can further increase IL-10 production at posttranscriptional levels during mechanical stretchELISA and RT-qPCR results showed:the concentration of IL-10 in the supernatant and IL-10 mRNA in cells of the MS group were significantly higher than the C group(P<0.01);the concentration of IL-10 in the supernatant of the SiR+MS group was significantly higher than the MS group(P<0.01),while there was no statistical difference in the IL-10 mRNA level between the SiR+MS group and the MS group.These results show that down-regulation of PDCD4 has no significant effect on the level of IL-10 mRNA itself,but it can increase the protein expression of IL-10 at the post-transcriptional level.Part three1.Transfection of miR-145 mimic can up-regulate the content of miR-145 in rat lung tissueRT-qPCR results showed:compared with the control group,the content of miR145 in the lung tissue of the MiR group was significantly increased(P<0.01).2.Transfection of miR-145 mimic can inhibit the expression of PDCD4 protein in lung tissueWestern blot results showed that the expression of PDCD4 in the lung tissue of the MiR group was significantly lower than the control group(P<0.01).These results prove that miR-145 mimic transfected in vivo has achieved the expected effect at the target protein level,and also prove the regulation effect of miR-145 on PDCD4 at the overall animal level.3.High tidal volume mechanical ventilation can reduce the content of miR-145 in the lung tissueRT-qPCR results showed:compared with the SHAM group,the miR-145 content in the lung tissue of rats in the HMV group decreased significantly;the miR-145 content of the MiR+HMV group was significantly higher than that of the SHAM group and the HMV group.These results show that high tidal volume mechanical ventilation can reduce the level of miR-145 in the lung tissue of rats;during mechanical ventilation,miR-145 content has always been at a high level in rats transfected with miR-145 mimic,which meets our experimental requirements.4.Up-regulation of miR-145 can alleviate the pathological injury of lung tissue induced by high tidal volume mechanical ventilation and reduce the pathological injury scoreCompared with the SHAM group,the lung tissue structure of the HMV group was severely damaged,some alveoli were broken,inflammatory cell infiltration increased,and alveolar and interstitial edema were obvious.The lung tissue microscopic pathological damage score of the HMV group was significantly higher than that of the SHAM group(P<0.01);The LMV group and the SHAM group were similar;the pathological changes of the rats in the MiR+HMV group were better than the HMV group,and the pathological damage score was significantly lower than the HMV group(P<0.01).5.Up-regulation of miR-145 can reduce pulmonary edema incduced by high tidal volume mechanical ventilationThe wet weight/dry weight(W/D)ratio of lung tissue can directly reflect the degree of pulmonary edema.The W/D ratio of the HMV group was significantly higher than that of the SHAM group(P<0.01);the W/D ratio of the MiR+HMV group was lower than that of the HMV group(P<0.01).6.Up-regulation of miR-145 can reduce neutrophil infiltration in lung tissue induced by high tidal volume mechanical ventilationMPO activity is proportional to the content of neutrophils.MPO activity in the HMV group was significantly higher than that in the SHAM group(P<0.01);MPO activity in the MiR+HMV group was significantly lower than that in the HMV group(P<0.01).7.Up-regulation of miR-145 can reduce protein exudation in the bronchoalveolar cavity induced by high tidal volume mechanical ventilationWe used the BCA method to detect the protein content in each group of BALF,and the results showed that the protein content of BALF in the HMV group was significantly higher than that in the SHAM group.The protein content of BALF in the MiR+HMV group was significantly lower than the HMV group(p<0.01).8.Up-regulation of miR-145 can reduce the secretion of inflammatory factors and the mRNA expression of inflammatory factors during high tidal volume mechanical ventilationThe results of ELISA and RT-qPCR showed:the concentrations of IL-1?,IL-6 and TNF-? in BALF of the HMV group and the mRNA expression of inflammatory factors in lung tissue were significantly higher than those in the SHAM group;after miR-145 mimic transfection,the BALF concentrations and miRNA levels of the three inflammatory factors were significantly reduced in the MiR+HMV group comparing with the HMV group.9.Up-regulation of miR-145 can reduce the activation of NF-?B induced by high tidal volume mechanical ventilationThe results of Western blot showed:compared with the SHAM group,the p65 in the cytoplasm of rats in the HMV group was significantly reduced(P<0.01),and the p65 in the nucleus was significantly increased(P<0.01),indicating that more NF-?B was activated and transferred to the nucleus after high tidal volume mechanical ventilation;compared with the HMV group,the p65 in the cytoplasm of the MiR+HMV group was significantly increased(P<0.01),and the p65 in the nucleus was significantly decreased(P<0.01),indicating that upregulation of miR-145 can inhibit NF-?B activation.Transcription factor enzyme-linked immunoassay showed a trend consistent with western blot.The above results indicate that high tidal volume mechanical ventilation can activate the NF-?B signaling pathway in lung tissue cells,and upregulation of miR-145 can inhibit the activation of NF-?B signaling pathway in lung tissue cells caused by high tidal volume mechanical ventilation.10.Up-regulation of miR-145 can increase IL-10 synthesis at post-transcriptional levelsThe results of ELISA and RT-qPCR showed:the IL-10 concentration of BALF and expression of IL-10 mRNA of lung tissues in the HMV group were higher than the SHAM group;after miR-145 mimic transfection,the IL-10 concentration of BALF in the MiR+HMV group was higher than that in the HMV group significantly(P<0.01),but the expression of IL-10 mRNA was not significantly different with the HMV group.ConclusionThe miR-145 can target down-regulate PDCD4 expression,and then inhibit the activation of NF-?B and increase the IL-10 production at post-transcriptional level,and finally reduce the inflammatory response of alveolar macrophages(NR8383 cells)induced by mechanical stretch and reduce ventilator-induced lung injury in rats. |
PDF Full Text Request