Effect On Macrophages Functions And Protection In Mouse Hepatic Ischemia Reperfusion By Glucocorticoid-Induced Leucine Zipper

Objective: GILZ have ability of anti-inflammation and regulation of epithelialsodium channels and many other effects.It's a new important mediator ofglucocorticoid action.SiRNA was looked as a revolutionary breakthrough in thehistory of genetic research, has the specificity and efficiency of the characteristics ininhibition of target mRNA . SiRNA technique is a powerful ways to inhibit geneexpression specifically. The present study, GILZ and siGILZ plasmid vector that hasbeen constructed transfect to the mouse peritoneal macrophages with sufficientquantity and purity in vitro , and detect expression of GILZ mRNA and proteinlevels.The model of high expression and silence of GILZ has been established forfurther study of the genetic characteristics of GILZ in macrophages. METHODS:Cultured sufficient quantity and purity of mouse peritoneal macrophages, siGILZ andGILZ transfected into mouse peritoneal macrophages by the French companyPolyplus-transfection's transfection reagents INTERFERin^TM and jetPEI^TMrespectively. The interference test is divided into a blank Control group, siC group andsiGILZ group and 1 hour, 3 hours, 6 hours, 12 hours and 24 hours were taken as theobservation point. GILZ gene transfer trials were divided into control group, emptyvector AAV group and GILZ group, were also taken after 1 hour, 3 hours, 6 hours, 12hours and 24 hours for observation. Realtime PCR detected the mRNA expressionlevels of GILZ in each treatment group, Western-blot detected expression of theprotein GILZ of cells in each treatment group. Results: Compared with the controlgroup and siC group, siGILZ significantly decreased the mRNA level and proteinlevels of GILZ after transfected for 6 hours (p <0.05); GILZ gene transferexperiments, compared with the control group and empty plasmid AAV group, GILZwas significantly increased the expression of GILZ in mRNA and protein levels (p<0.05), in which the highest expression of 6h group. Conclusion: Overexpression andsilence model of GILZ has been builded successfully. Objective: Macrophages are important innate immune cell, it's functions includingphagocytosis, antigen presentation and secretion of cytokines and other functions.In adifferent microenvironment, macrophages consists of M1 and M2-type model, M1Macrophage can directly engulf and destruct the pathogenic microorganisms andtumor cells, present antigen and secrete proinflammatory cytokines, involved in ppositive immune responserofessionally; M2 macrophages showed lowerantigen-presenting ability, ana secreted the inhibitory Cytokine such as IL-10 andTGF-βthat reduced immune response. This study attempts to explore whether GILZpromoting peritoneal macrophages changed to the M2-type macrophages and playinga role in the negative regulation. Methods: Mouse peritoneal macrophages was thestudyobject, the test divided into control group, DEX group, GILZ treatment groupand siGILZ treatment group. Realtime PCR Detected expression of symbol moleculeof M1 and M2 in each treatment group, ELISA method for detection of cytokines,flow cytometry for macrophage phagocytosis , Western-blot to detect the expression ofTLR4 signaling molecules. Results: Compared with the control group, Dex treatmentgroup and GILZ group express high level of Arginase-1, FIZZ-1 and Ym-1 of M2,low level of i-NOS of M1; inhibition of pro-inflammatory factor IL-1β, IL-6, TNF-αand IL-12 expression, and increased anti-inflammatory factor IL-10 expression,inhibition of macrophage phagocytic capacity, reduced the expression of TLR4signaling pathway molecules such as TLR4, IRAK1, IRAK2 and TRAF6; WhilesiGILZ has the low expression of Arginase-1, FIZZ-1 and Ym-1 of M2, highexpression of M1 molecules i-NOS; to promote pro-inflammatory factorαIL-1β, IL-6,TNF- and IL-12 Expression, and inhibition of anti-inflammatory factor IL-10expression, enhance macrophage phagocytosis , increased TLR4 signaling moleculesexpression such as TLR4, IRAK1, IRAK2 and TRAF6 (p <0.05). Conclusion: GILZ promote the production of M2 macrophages, inhibit the expression ofproinflammatory factors, the promot of anti-inflammatory factor IL-10 expression,and probably by inhibiting the mechanism of TLR4 signaling molecules. Objective: Hepatic ischemia reperfusion injury is a common pathophysiology of liversurgical diseases. Through the direct inhibition generation of neutrophil hydrogenperoxide and neutrophil swim out ,dexamethasone block the phospholipase A2 andarachidonic acid metabolism, thereby inhibiting neutrophil-mediatedischemia-reperfusion injury. This study investigated whether GILZ reduce the hepaticischemia-reperfusion injury by promoting peritoneal macrophages changes to theM2-type macrophages. Methods: Build GILZ transfection by transfection reagent invivo jetPEI^TM in mouse liver ischemia-reperfusion model in vivo.test divided intocontrol group, Dex group, AAV empty plasmid treated group, GILZ treatment group,taking 4 hours after reperfusion as observation point. ALT and AST test performed,HE staining for pathological changes, the level of cytokines by ELISA. Results:Compared with the control group and the AAV empty plasmid treatment group, Dextreatment group and GILZ group significantly reduced the expression of ALT andAST (p <0.05), significantly reduced liver ischemia reperfusion injury in pathology,inhibition expression of proinflammatory factors IL-12IL-1β, IL-6 and TNF-α, andincreased anti-inflammatory factor IL-10 expression (p <0.05). Conclusion: Thisstudy found that GILZ protecting the function of liver in liver ischemia /reperfusion ,reducing the expression of inflammatory cytokines by inducingproduction of M2-type macrophages, thereby protecting Structural integrity of liver inischemia-reperfusion.