| BackgroundSepsis is one of the most common clinical critical illnesses and is a life-threatening organ dysfunction caused by uncontrolled host response to infection.There are many causes of sepsis,but the most common ones were severe diseases caused by surgery,multiple injuries,and severe burns.When sepsis occurs,it can cause acute damage to multiple organs,and the lungs are often the first to be affected.Acute lung injury(ALI)caused by sepsis,which has a high mortality rate in the patient population,can also have a serious adverse effect on the long-term quality of life of survivors.In recent years,there is much progress in anti-infective and organ function support therapies,and some progress have also been made in understanding the epidemiology and pathogenesis of sepsis-associated acute lung injury,however,clinical studies have shown that current treatment strategies have no significant effect in reducing mortality in patients with acute lung injury.Therefore,more basic research is urgently needed to explore the molecular mechanism of the development of acute lung injury,in order to obtain more effective treatment methods.Current research suggests that the prime cause of acute lung injury and acute respiratory distress syndrome is infection,systemic inflammatory response after trauma.Activation and regulation of the NF-?B(nuclear factor ?B)signaling pathway plays a key role in the inflammatory response.The NF-?B protein was first discovered by David Baltimore,which is a family of proteins that can selectively bind to the enhancer in kappa-light chain of B cell to regulate the expression of many genes.The signaling pathway of NF-?B is switched by the extracellular stimuli,and NF-?B is the first responder to harmful cell stimulation.There are many known NF-?B pathway activators,including lipopolysaccharide,TNF-a(tumor necrosis factor alpha),IL-1?(interleukin 1?),IL-6(interleukin 6),COX2(cyclooxygenase 2).chemokines,adhesion molecules,colony stimulating factors,etc.,which bind to receptors on the cell membrane and initiate a cascade of downstream reactions.The receptor protein activates I?B kinase(IKK)after stimulation,and IKK phosphorylates the serine of the I?B subunit of the NF-?B-I?B complex in the cell,so that the I?B subunit is ubiquitinated and further be degradated by protease,thereby releasing NF-?B dimer.Free NF-?B enters the nucleus and binds to a gene with a NF-?B binding site to initiate transcriptional processes,which in turn produces a large number of pro-inflammatory factors,particularly TNF-?,IL-1?,IL-6,which activate NF-?B signaling pathway again and leads to a cascade of inflammation,further more a continuous deterioration of lung damage.miRNAs are small,non-coding,single-stranded RNA molecules that play key regulatory roles in a variety of biological processes such as development,differentiation,and homeostasis by reducing mRNA stability or inhibiting mRNA translation involved in regulation of gene expression.In recent years,several studies have demonstrated that microRNAs(miRNAs)are effective regulators of systemic inflammation.Among them,miRNA-146a is the first miRNA that has been shown to play a regulatory role in immune and inflammatory responses.It has a reverse regulation ability in immune inflammatory response,and its normal expression plays a key role in the cascade of prevention and control of inflammation.It plays an important role in regulating the differentiation and function of innate immune and adaptive immune cells.One study indicated that miR-146a is highly expressed in LPS-treated rat lungs,but miR-146a mimetic treatment unexpectedly inhibits LPS-mediated TNF-?,IL-6 and IL-in rat alveolar macrophages.The mechanism of production may be achieved by inhibiting NF-KB-activated mediators interleukin-1 receptor-associated kinase 1(IRAK-1)and tumor necrosis factor receptor-associated factor 6(TRAF6).As a receptor protein,IRAKI is widely distributed in various cells and participates in signal transduction pathways such as TLR,which plays an important role in regulating inflammation.TRAF6 is indispensable in the activation of NF-?B and JNK induced by IL-1 and LPS.IRAK1 and TRAF6 are two key adaptor proteins in the LPS/TLR4/NF-?B signaling pathway and have been shown to be directly targeted by miR-146a,which promotes inflammation by inflammatory cytokines such as TNF-? and IL-1?.Dexmedetomidine(Dex)is a highly selective ?2-adrenergic receptor agonist widely used in sedation and clinical anesthesia in intensive care units for its sedative,analgesic and antisympathetic effects.At present,a number of studies had provided strong evidence for the beneficial effects of Dex in various inflammation-related diseases including sepsis,and pointed out that Dex has a role in alleviating acute lung injury,which has caused widespread concern.Meng et al.pointed out that Dex may regulate protective effects by the Toll-like receptor 4(TLR4)/NF-?B and PI3K/Akt/mTOR pathways mediated by high mobility group protein(HMGB1)in lipopolysaccharide induced acute lung injury in rats.Xu et al.established a mouse model of sepsis lung injury induced by LPS,indicating that Dex may alleviate acute lung injury by inhibiting the MAPK pathway.Jiang et al's research in human alveolar epithelial cells A549 showed that Dex pretreatment has an in vitro protective effect on ALI,and the possible mechanism is to inhibit cell death and improve cell antioxidant capacity.Despite many explorations,the mechanism by which Dex alleviates acute lung injury remains unclear and needs further study.A number of recent studies have indicated that Dex can exert its organ protection by regulating the expression of microRNAs.Wang et al.showed that dexmedetomidine inhibits proliferation and migration of osteosarcoma cells by regulating miR-520a-3p and promotes apoptosis.Li et al.established a rat COPD model and pointed out that Dex exerts lung protection by inhibiting apoptosis by regulating miR-146a level.Paeschke et al.established a rat model of central nervous system inflammation induced by LPS and found that Dex inhibits the expression of multiple miRNAs in rat brain induced by LPS and exerts brain protectionBased on the above studies,we propose the hypothesis that whether dexmedetomidine plays a protective role in sepsis related acute lung injury by regulating miR-146a and thereby affecting the expression of IRAK1 and TRAF6?In this study,a mouse model of acute lung injury associated with sepsis was established by intraperitoneal injection of lipopolysaccharide(LPS).The lung injury degree,lung histopathology,changes of inflammatory cytokines TNF-?,IL-1?,NF-?B p65-to-nuclear transportation and I-?B phosphorylation,miR-146a expression,IRAK1 and TRAF6 molecular and gene levels,were observed and determined in each group.The protective effect and its mechanism are intended to provide new ideas and theoretical basis for the treatment of acute lung injury in clinical practice.Objective1.To clarify the protective effect of dexmedetomidine on acute lung injury associated with sepsis;2.To explore the possible mechanism of dexmedetomidine in protecting against acute lung injury associated with sepsis.Methods54 healthy male BALB/c mice weighing 21 ±2g,purchased from Nanjing Kaidewensi Biotechnology Co.,Ltd.,kept in 21-25? clean animal room,free to eat,drink,maintain 12 hours of light and dark cycle.Prior to the experiment,the mice were adaptively bred for at least one week.In order to preliminarily explore the effect and mechanism of dexmedetomidine on reducing acute lung injury associated with sepsis,24 experimental mice were randomly divided into 4 groups,6 in each group,which were control group(Control group)and LPS group,Dex+LPS group,DXM(Dexamethasone)+LPS group.Control group:intraperitoneal injection of corresponding volume of normal saline;LPS group:intraperitoneal injection of LPS(10mg/kg)to establish a model of sepsis acute lung injury;Dex+LPS group:intraperitoneal injection of Dex(40?g/kg)15 minutes before the application of LPS;DXM+LPS group:DXM(50?g/kg)was intraperitoneally injected 15 minutes before the application of LPS as a positive control for reducing acute lung injury.To further verify the mechanism by which dexmedetomidine reduces acute lung injury by regulating miR-146a,we randomly divided 30 mice into 5 groups according to the above method,6 in each group,which were Control group,LPS group,Dex+LPS group,Dex+LPS+miR146a-ant(miR-146a antagonist)group and LPS+mi R146a-ant group,in which Dex+LPS+miR146a-ant group was intraperitoneally injected with mR146a-ant 15 minutes before LPS and Dex.The LPS+miR146a-ant group was intraperitoneally injected with miR146a-ant 15 minutes prior to the application of LPS.The experimental mice were treated and placed back in the cage,and fed freely into the water.After 6 hours,lung tissue,bronchoalveolar lavage fluid(BALF)and serum samples were collected.Partial lung tissue was cut to determine lung wet-to-dry ratio(W/D),reflecting lung water content,assessing the degree of lung tissue edema;Using Bicinchoninic acid(BCA)method to determine protein content in BALF;detecting inflammatory cells in BALF Number;pathological changes of lung tissue were evaluated by HE staining light microscope;Enzyme-Linked Immunosorbent Assay(ELISA)was used to detect the expression of proinflammatory cytokines TNF-? and IL-1? in lung tissue and serum;Quantitative Real-time-PCR was used to detect the expression of miRNA-146a,IRAK1 mRNA and TRAF6 mRNA in lung tissue.The expression of IRAKI,TRAF6,NF-?Bp65,p-I?B and I?B was detected by Western Blot.Results1.The model of acute lung injury in mouse sepsis was successfully establishedIn the LPS group,an acute lung injury model of sepsis was established by intraperitoneal injection of LPS(10 mg/kg).Mice in the LPS group showed chills,rapid respiration,and markedly reduced activity.Compared with the control group,LPS group had obvious pathological changes in lung tissue,including alveolar wall thickening,lung tissue destruction and inflammatory cell infiltration,lung injury score was significantly increased,W/D was significantly increased(P<0.01),the content of BALF protein and the number of inflammatory cells were significantly increased(P<0.01)2.Dexmedetomidine attenuates LPS-induced lung injury in mice with sepsisWe observed and compared the pathological changes of lung tissue in each group,the index of lung W/D reflecting the degree of pulmonary edema,the protein content in BALF reflecting permeability index of pulmonary capillary and the number of inflammatory cells.Compared with the control group,LPS group had obvious pathological changes in lung tissue,including alveolar wall thickening,lung tissue destruction and inflammatory cell infiltration,lung injury score was significantly increased,W/D was significantly increased(P<0.01),The content of BALF protein and inflammatory cells including macrophages,neutrophils and monocytes were significantly increased(P<0.01).Pathological changes of lung tissue:Compared with the control group,lung tissue of LPS group had obvious pathological changes,including alveolar wall thickening,lung tissue destruction and inflammatory cell infiltration,and lung injury score was significantly increased(P<0.01);Compared with the group,the pathological changes of lung tissue in the Dex+LPS group were significantly reduced,and the lung injury score was significantly lower(P<0.01).Pulmonary wet-to-dry ratio(W/D):Compared with the control group,the W/D of the LPS group was significantly increased,and the difference between the two groups was statistically significant(P<0.01).Compared with the LPS group,the Dex+LPS group was W/D was significantly lower,and the difference between the two groups was statistically significant(P<0.01).Protein content in BALF:Compared with the control group,the protein content in BALF of LPS group was significantly lower,and there was significant difference between the two groups(P<0.01).Compared with LPS group,the protein content in BALF of Dex+LPS group was significantly lower.The difference between the two groups was statistically significant(P<0.01).The number of inflammatory cells in BALF:Compared with the control group,the number of inflammatory cells in BALF of LPS group increased significantly(P<0.01).Compared with LPS group,the number of BALF inflammatory cells in Dex+LPS group decreased significantly(P<0.01).3.Dexmedetomidine significantly inhibits the secretion of inflammatory factors associated with acute lung injuryThe expressions of proinflammatory cytokines TNF-? and IL-1? in lung tissue and serum were detected by ELISA.The results showed that the expression of IL-1?and TNF-a in lung tissue and serum of LPS group was significantly increased compared with the control group,and there was significant difference(P<0.01).Compared with LPS group,the expression of IL-1? and TNF-? in lung tissue and serum of Dex+LPS group was significantly decreased(P<0.01).The results showed that Dex significantly reduced the expression of ALI-associated pro-inflammatory cytokines and inhibited the inflammatory response,suggesting that Dex can alleviate lung injury by inhibiting the production of inflammatory factors.4.Pretreatment with dexmedetomidine inhibits NF-?B activationWestern Blot detected the levels of NF-?B p65 nuclear transport and I?B phosphorylation,and the results showed:compared with the LPS group,NF-?B p65 was significantly reduced in nuclear transport and lung I?B phosphorylation in the Dex+LPS group(P<0.01).The above results indicate that the effect of Dex on ALI is related to the inhibition of NF-?B activation.5.Dexmedetomidine treatment reduces the levels of IRAKI and TRAF6 which are the upstream adaptor proteins in the NF-?B signaling pathwayWe detected the expression of IRAK1 and TRAF6 at the gene and protein levels,respectively.Compared with the control group,the IRAK1 TRAF6 protein,IRAK1 mRNA and TRAF6 mRNA levels in the LPS group were significantly increased(P<0.01).Compared with the LPS group,the levels of IRAK1 and TRAF6 proteins in the Dex+LPS group were significantly decreased(P<0.01).These results indicate that Dex exerts a lung protective effect by reducing the expression of the upstream adaptor proteins IRAK1 and TRAF6 in the NF-?B signaling pathway.6.Dexmedetomidine treatment significantly up-regulated the expression of miR-146aIn order to further reveal the mechanism by which Dex alleviates ALI,we examined the expression levels of miR-146a in mice lung tissue of each group.The results showed that the expression level of miR-146a in the LPS group was significantly higher than that in the control group(P<0.01).Compared with the LPS group,the expression level of miR-146a was significantly increased in the Dex+LPS group(P<0.01).The above results indicate that the lung protection of dexmedetomidine may be achieved by up-regulating the expression of miR-146a.7.miR146a-ant(miR-146a antagonist)significantly impaired the protective effect of Dex on sepsis-associated ALI7.1 Expression of miR-146a in each groupCompared with the control group,the expression of miR-146a was significantly increased in the LPS group(P<0.01);compared with the LPS group,the miR-146a level was significantly increased in the Dex+LPS group(P<0.01).Compared with the Dex+LPS group,the miR-146a level in the miR146a-ant+Dex+LPS group was significantly lower,and the difference between the two groups was statistically significant(P<0.01).Compared with the LPS group,the miR-146a level in the miR146a-ant+LPS group was significantly lower(P<0.01).7.2 The changes of IRAKI,TRAF6 protein levelCompared with the control group,the levels of IRAK1 and TRAF6 in the LPS group were significantly increased(P<0.01);Compared with the Dex+LPS group,the levels of IRAK1 and TRAF6 in the miR146a-ant+Dex+LPS group were significantly higher than those in the Dex+LPS group.The difference between the two groups has significant statistical significance(P<0.01);compared with LPS group,the levels of IRAK1 and TRAF6 in miR146a-ant+LPS group were significantly increased(P<0.05).7.3 NF-?B p65 nuclear transport and I-?B phosphorylation levels changeAfter LPS injection,the nuclear transport and I?B phosphorylation of NF-?B p65 in lung tissue of mice were significantly increased(P<0.01).Compared with LPS group,The transferation to nuclear of NF-?B p65 and the phosphorylation of I?B in lung tissue was significantly decreased in Dex+LPS group.(P<0.01).Compared with Dex+LPS group,NF-?B p65 activation and I-?B phosphorylation were significantly increased in lung tissue of miR146a-ant+Dex+LPS group(P<0.01).Compared with the LPS group,NF-?B p65 activity and I?B phosphorylation were significantly enhanced in the miR146a-ant+LPS group.7.4 Changes in expression of proinflammatory cytokines TNF-? and IL-1? in lung tissue and serumCompared with the control group,the expression of IL-1? and TNF-? in lung tissue and serum of LPS group were significantly increased,and there was significant difference between the two groups(P<0.01).Compared with LPS group,the expressions of IL-1? and TNF-? sourced from lung tissue and serum in Dex+LPS group were significantly decreased(P<0.01).Compared with the Dex+LPS group,the levels of IL-1? and TNF-? in the miR146a-ant+Dex+LPS group were significantly increased(P<0.01).In comparison of LPS group,the expression of IL-1? and TNF-?in lung tissue and serum of miR146a-ant+LPS group increased(P<0.05).7.5 Pathological changes in lung tissue and W/D changes:Compared with the control group,lung tissue thickening,lung tissue destruction and inflammatory cell infiltration,lung injury score and W/D were significantly increased in the LPS group(P<0.01).Compared with the LPS group,the pathological changes of lung tissue in the Dex+LPS group were significantly attenuated,and the lung injury score and W/D were significantly decreased(P<0.01).Compared with the Dex+LPS group,the lung tissue pathology of the miR146a-ant+Dex+LPS group has significant damage,lung injury score and W/D were significantly increased(P<0.01).Compared with LPS group,lung tissue pathological damage was significant in miR146a-ant+LPS group,lung injury score and W/D were significantly increased(P<0.05).The above results indicate that Dex exerts lung protection by significantly upregulating miR-146a,and this effect is markedly attenuated by its antagonist miR146a-ant.Conclusion1.This study can successfully simulate the pathological process of acute lung injury associated with sepsis by establishing the mouse model of acute lung injury induced by LPS;2.Dexmedetomidine can achieve lung protection by down-regulating the levels of IRAK1,TRAF6,inhibiting the activation of NF-?B,and the expression of proinflammatory cytokines TNF-?,IL-6 in lung tissue and serum,and improving the pathological damage of lung tissue,and the changes in IRAKI and TRAF6 protein levels are dependent on the up-regulation of miR-146a expression;3.Pretreatment with dexmedetomidine has a protective effect on acute lung injury associated with sepsis,suggesting that dexmedetomidine can be used as a target acting drug for lung protection,and providing new ideas for clinical treatment. |
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