| ObjectivesPneumonia is a common respiratory disease,severe community- acquired pneumonia(SCAP) has become a threat to human health because of its poor efficacy,the high rate which easy to trigger systemic inflammatory response syndrome(SIRS) or multiple organ dysfunction syndrome(MODS) and even death.The reasons which can cause SCAP are so much,with complicate pathogenesis,we general recognize that SCAP caused by bacteria/virus infection or same non-infection factors.When SCAP happened,neutrophils,lymphocytes,macrophages and other flammatory cells in lung will producte many endogenous or exogenous substances such as inflammatory cytokines,vasoactive peptides,adhesion molecules,and so on,to defense intruder.Previous view is that the main reason severe consequence caused by SCAP is pathogen or toxin.At present,more and more studies have shown that after the initial inflammatory activation signal,all kinds of inflammatory mediators, released out of control,then stimulated the chain "cascade effect",and lead to inflammation/anti-inflammatory response to the imbalance,immune dysfunction, eventually leading to SCAP and severe complications.The pro-inflammatory cytokines and anti-inflammatory cytokine balance is the key if patient develop to SCAP even sepsis.Pro-inflammatory cytokines main have TNF-α,IL-1,IL-6,IL-8 and so on,anti-inflammatory cytokine main include IL-4,IL-10,IL-13 and so on.TNF-αcan result in a wide range of biological effects.TNF-αcan activate endothelial cells and other cells,induce secondary inflammatory mediators release such as IL-1,IL-6,IL-8,lead to "inflammation cascade effect" in body.At the same time,TNF-αcan activate coagulation and complement system,further damage lung tissue.TNF-αwas considered the most important inflammation in SCAP pateins. Study found that TNF-αgeneral increase in early SCAP,and expression more in MODS,levels of TNF-αin blood to determine the progress of SCAP with earlywaming function.Interleukin-family have a number of members which closely related pneumonia, such as IL-1,IL-6 and IL-10.IL-1 is a acute phase immune response regulating protein,can cause pateins fever,in addition,IL-1 has a wide range of biological effects.IL-6 can secret obviously increase in the inflammatory response.At present,a large number of studies found that the increased levels of IL-6 can indicate a higher mortality in SCAP,and IL-6 is more sensitive than other indicatrix such as C-reactive protein.Monton etal found that the mortality rate will decrease when use glucocorticoid in SCAP to make IL-6 declined.IL-10 is anti-inflammatory cytokine, the main biological activity of IL-10 is can inhibite a lot of inflammatory cells activate,and can impact on pro-inflammatory cytokine mRNA stability,then reduce them production,it even can inhibite NF-κB activation.TNF-α,IL-1,IL-6,IL-10 correlate closely with SCAP,so how to contrl them expression too much to prevent inducing inflammatory/anti-inflammatory imbalance now is the issue we should to face.Treatment method are more,such as cytokines blockage,immune regulation,anti-oxidant treatment and so on,but clinical value is not very well.In recent years,with the development of molecular biology techniques, the use of siRNA gene silencing interference make the target in order to regulate target protein function,showing that the great potential applications.NF-κB is a important factor in the inflammatory response because of it participation in a variety of inflammatory mediators and regulation of gene transcription.NF-κB is usually exists in cells cytoplasm be form of homologous or heterodimer p50/p65,p65 can cause gene transcription regulation activation,p50 do not have this function.NF-κB plays a key role in the excessive release of inflammatory mediators.RNA interference is a normal phenomenon in creature.Its essence is the purpose of the adoption of double-stranded RNA to make mRNA degradation,thus specifically inhibiting the expression of the target protein. Compared with gene knockou,antisense oligonucleotides and small molecule blockers,siRNA has obviously advantages:effective,simple inexpensive,stability and accuracy.Although NF-κB is closely related with inflammatory responses,can enhance a variety of inflammatory mediators such as TNF-α,IL-1β,IL-8,using of silence in NF-κB to control SCAP has been reported rarely.Pneumonia treatment from the gene level of intervention in theory is a promising new approach.In this study,RNAi technology was been adopt to silence macrophage NF-κB p65 gene to explore the expression of cytokine which closely related to SCAP development.We hope that our finding can provide a theoretical basis to SCAP treatment.Methods1.Mouse macrophage Ana-1 cells were divided into control group and LPS-activated group.The siRNA directed to the mouse NF-kB p65 mRNA was transfected into both two groups of mouse marcophages.After transfected 24 hours, the transcriptin and translation of NF-kB p65 was detected by RT-PCR and Western Bloting 24 hour after transfecting.2.The Ana-1 cells which successful transfected NF-kB p65 siRNA as experimental group,and natural Ana-1 cells as control group, activated the two groups with 1ug/ml LPS,and the secretion of inflammatory mediator such as TNF-α,IL-1β,IL-6,IL-10 in the cells cultivate serum by ELISA at different times(0 hour,4 hours,12 hours,24hours).ResultsUse RNAi technique,it was found that the expression of the NF-kB p65 mRNA and proteins in Ana-1 cell were specifically and extensively suppressed after 24 hours transfection(P＜0.01),and the ELISA experiment indicate that the expression of inflammatory mediator such as TNF-α,IL-1β,IL-6 were cut down after NF-kB p65 siRNA transfection in the experiment group(P＜0.05),situation is opposition when IL-10 was measured,the expression was a little raised at 12 hours and 24 hours(P＜0.05).ConclusionRNAi technology can effectively silence murine macrophage NF-kB p65 gene, induced NF-kB p65 protein expression decreased,then caused decreased in pro-inflammatory cytokines expression and increased in IL-10 expression.It is a powerful theoretical support for more animal experiments and provide us a new idea on treatment the inflammatory diseases.
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