Effects Of LncRNA-AC013472.3 On The LPS-induced Macrophage Inflammatory Response

1.Background Sepsis is one of the eternal topics of burn infection.Despite the improvement of diagnosis and treatment of burns,the mortality rate of sepsis patients is still far from satisfactory.Sepsis3.0 reveals the essence of sepsis is the organ dysfunction syndrome caused by severe uncontrolled infection reactions,indicating that the unbalanced inflammatory response would cause serious body damage.Accumulating studies show that the cell wall endotoxin of Gram-negative bacteria is the main cause of the out-of-control cascade of inflammation.TLR4 mainly recognizes Gram-negative bacteria lipopolysaccharide(LPS),while the TLR2 ligand is more extensive.After stimulation caused a series of intracellular signaling cascade such as MAPK pathway,NF-KB and other transcription factors,will eventually activate and raise all kinds of proinflammatory and anti-inflammatory cytokine expression,such as tumor necrosis factor alpha(TNF-?),interleukin 6(IL 6)and intercellular adhesion molecule-1(ICAM 1).Lnc RNAs are non-coding RNAs with transcripts of more than 200 nucleotides in length.Lnc RNAs serve not only as regulators,but also as combines with proteins,RNAs and DNAs;primarily function at the transcriptional and posttranscriptional levels.The regulation of epigenetic and gene transcription,and its relationship with the development of disease,especially the fields of Alzheimer's disease and cancer have long been the focus of lnc RNA research.The latest studies of lnc RNA's role in regulation of immune gene expression have shown that lnc RNA delicate controls the balance of the inflammatory response.In the previous study of our research group,lnc RNA-AC013472 was screened from sera of acute lung injury patients by using the expression profiling high-throughput chip technology.There was a significant expression difference of lnc RNA-AC013472 between the acute lung injury of sepsis and healthy controls.We studied the effect of lnc RNA-AC013472.3 on TNF-?,IL-6 and ICAM-1 m RNA,as well as on the MAPK(P38,JNK,ERK1 / 2)and NF-?B signaling pathway at the cellular level in inflammatory response.Research on the function and mechanism of lnc RNA in inflammatory reaction will provide new ideas and new methods for exploring the essence of sepsis and treating the uncontrolled inflammatory cascade in sepsis.2.ObjectivesIn this study,we took NR8383 rat alveolar macrophages as experimental subjects to explore the effect of lnc RNA-AC013472.3 in inflammatory response at cellular level:(1)The effect of lnc RNA-AC013472.3 on the release of TNF-?,IL-6 and ICAM-1 in NR8383 macrophages(2)Effects of lnc RNA-AC013472.3 on MAPK(JNK,ERK1 / 2)and NF-?B signaling pathways in NR8383 macrophages.3.Material and MethodsThe alveolar macrophages of NR8383 rats were cultured routinely,and the lnc RNA-AC013472.3 silent model was constructed by si RNA interference.The lnc RNA-AC013472.3 overexpression model was constructed by transfecting cells with designed plasmid.The stimulation group was stimulated with 10 ug / ml LPS for 24 hours.Part ? The effect of lnc RNA-AC013472.3 on the release of cytokines TNF-?,IL-6 and ICAM-1 in NR8383 macrophagesThe NR8383 alveolar macrophages were divided into 8 groups after routine culture.Both the control group and the experimental group included:(1)unstimulated and stimulated groups of silent model;(2)unstimulated and stimulated groups of overexpression model.The expression of TNF-?,IL-6 and ICAM-1 m RNA in the cell supernatant was detected by quantitative PCR(q PCR).Part ? Effect of lnc RNA-AC013472.3 on MAPK(p38,JNK,ERK1/2)and NF-?B signaling pathways in NR8383 macrophagesNR8383 Alveolar macrophages were divided into control group and experimental group after routine culture.The control group and experimental group included:(1)unstimulated group and stimulated group;(2)unstimulated group and stimulated group.The expression of ERK?,IKB?,p-IKB?,IRAK-1,JNK,MAPK,NF-?B and TRAF-6 were detected by Western Blot(WB).4.ResultsPart ?(1)The expression levels of TNF-?,IL-6 and ICAM-1 at the level of m RNA of macrophages at about 24 hours after stimulation with LPS(10ug / ml)all increased to about 2 times.(3)Under the stimulation of LPS,the expression level of TNF-?,IL-6 and ICAM-1 in lnc RNA-AC013472.3 silent group also increased at the m RNA level,and significantly increased by about 4 times compared with the level before stimulation.(4)The m RNA expression of TNF-?,IL-6 and ICAM-1 in lnc RNA-AC013472.3 overexpression group was not significantly increased at LPS stimulation.These results indicate that lnc RNA-AC013472.3 inhibits the expression of TNF-?,IL-6 and ICAM-1 at the m RNA level in LPS-stimulated macrophages.Part ?(1)lnc RNA-AC013472.3 in cells could inhibit the expression of ERK?,IRAK-1,JNK,P38,NF-KB and TRAF-6 at the protein level under LPS stimulation.(2)lnc RNA-AC013472.3 plays a regulatory role in inhibiting the phosphorylation of IKB?,and It may be necessary for cells to maintain the dynamic stability of I?B and p-I?B.5.Conclusions1.lnc RNA-AC013472.3 inhibits the release of cytokines TNF-?,IL-6 and ICAM-1 in NR8383 rat alveolar macrophages..2.lnc RNA-AC013472.3 can inhibit the activation of MAPK(P38,JNK,ERK1 / 2)and NF-?B signaling pathway in NR8383 rat alveolar macrophages.