The Mechanism Of LncRNA NEATf Knockdown Improves Inflammatory Response In Early Sepsis By Promoting M2 Macrophage Polarization

Sepsis,a syndrome of physiologic,pathologic,and biochemical abnormalities induced by infection,is a major critical clinical disease.Sepsis should be defined as life-threatening organ dysfunction caused by a dysregulated host response to infection.Sepsis can cause severe immune dysfunction,acute renal injury,catabolism and even death.The reported incidence of sepsis is increasing.The incidence of sepsis is rising with more than 19 million new cases each year and 6 million cases of death all over the world.Sepsis can be induced by the infected diseases.Other non-infectious diseases such as tissue ischemia,trauma,drug reactions and even cancers can also cause systemic inflammatory response syndrome or secondary infection,which further induces sepsis.However,the mechanisms underlying the pathogenesis of sepsis remains poorly understood.Macrophages are the key members of innate immune cells serving as the first line of host defenses against various pathogens.The functional alterations of macrophages were also closely associated with sepsis development.lncRNAs(Long non-coding RNAs)are a class of noncoding RNAs with>200 nucleotides in length,which could regulate the expression of functional genes as ceRNA(competing endogenous RNA)and play essential roles in multiple disorders,including cardiovascular diseases,cancers and sepsis.At present,there are few studies on the regulatory mechanism of lncRNA about sepsis.So in the development of sepsis,the regulatory mechanism of specific lncRNA remains to be further studied.ObjectiveThe aim of study was to explore the regulation of miR-125a-5p and TRAF6 signaling pathway on phagocyte polarization mediated by lncRNA NEAT1.And provide a scientific basis for the study of sepsis and new therapeutic targets.MethodMacrophage polarization was induced by LPS treatment,and flow cytometry was used to detect M1/M2 polarization state of macrophages.The relative expression level of macrophage M1 markers(TNF-α,IL-1β,IL-6 and iNOS),M2 markers(IL-4,IL-10 and Arg-1),TRAF6 and TAK1 genes were measured by RT-qPCR.WB(Western Blot,WB)was used to detect the expression of TRAF6,p-TAK1 and TAK1.The NEAT1 knockdown mice macrophage model was constructed by sh-NEAT1 to investigate the regulation of NEAT1 gene expression on macrophage polarization.The regulatory relationship between miR-125a-5p and TRAF6 was revealed by transfection or co-transfection with miR-125a-5p mimic and TRAF6 overexpression vectors,respectively.Bioinformatics technology find possible targets for lncRNA NEAT1 and miR-125a-5p.Dual luciferase reporter assay determine whether they and their target combines to each other directly to explore the molecular regulation mechanisms involved.A NEAT1 knockdown sepsis mice model was constructed by using CLP and tail vein injection of adenovirus-coated si-NEAT1.ELISA was used to detect the relative expression of pro-inflammatory factors(IL-1β,IL-6 and TNF-α)and anti-inflammatory factors(IL-4,IL-10 and TGF-β1)in mice serum.IHC staining assay was used to measure the expression of M2 marker(CD 163+)in lung,spleen and liver tissues of mice.RT-qPCR was used to detection relative expression of NEAT1,miR-125a-5p,TRAF6,TAK1,M1 type markers(IL-6,TNF-α and iNOS)and M2 markers(IL-10,IL-4 and Arg-1)in mice peritoneal macrophages to explore the regulation of lncRNA NEAT1 on sepsis in vivo.Results1.Knockdown of lncRNA NEAT1 promotes LPS-induced RAW264.7 cells from M1 to M2 polarization2.miR-125a-5p regulates macrophage M1/M2 polarization by TRAF6/TAK1 axis3.Knockdown of lncRNA NEAT1 helps to release the inflammatory response in mice with sepsis.ConclusionsThis study demonstrated the regulation of lncRNA NEAT1 in sepsis in vitro and in vivo.The study found that knockdown of lncRNA NEAT 1 promotes LPS-induced RAW264.7 cells from M1 to M2 polarization and lncRNA NEAT1 could combine with miR-125a-5p directly.The study futher demonstrated that TRAF6/TAK1 axis regulates macrophage M1/M2 type polarization was mediated by miR-125a-5p.The animal study indicated that knockdown of lncRNA NEAT 1 could increase the expression level of miR-125a-5p,enhanced its inhibitory effect on its target gene TRAF6 and thus relieves inflammatory response in sepsis mice.