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Role of Golgi Body and Inhibitory Effect of Npy in TLR4Hypersensitivity of Peripheral Blood Mononuclear Cell

Author: XiongJian
Tutor: ShenYue
School: Third Military Medical University
Course: Field Surgery
Keywords: TLR4hypersensitivity PBMC IL-6 IL-10 TNF–α Golgi body Fibronectin
CLC: R459.7
Type: Master's thesis
Year: 2012
Downloads: 11
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Abstract


ObjectiveClinical observation shows an increasing incidence of sepsis in severe trauma patients.Some research suggested that products of disruption of cell and tissue after severe traumacould initially activate the innate immune system. Furthermore, if the pathogens invade theinjured host, it will lead to occur excessive inflammatory cytokines release, and put forward anotion of TLR4hypersensitivity. Previous studies reported that there were a large number ofTLR4in Golgi body of inflammatory cells. However, the role of Golgi body in thedevelopment of sepsis and in the TLR4hypersensitivity in the progression of postinjurysepsis remains unclear. Previous studies confirmed the anti-inflammatory effect ofneuropeptide Y, and whether neuropeptide Y could inhibit the postinjury excessiveinflammatory response had not been verified. Hence, the authors selected the immune cellPBMC of healthy human, established a PBMC model of TLR4hypersensitivity, so as to studythe effect of Golgi body in TLR4hypersensitivity and the influence of neuropeptide Y in theexcessive inflammatory cytokines release, to explore the mechanism of TLR4hypersensitivity, and to provide a new way for treating excessive inflammatory response aftersevere trauma.Methods1. PBMC were obtained by density gradient centrifugation from concentrated leukocytesof healthy human.1x106cells/ml were incubated in24-well plates. and then divided into blankcontrol group, Fibronectin(Fn) group, lipopolysaccharide (LPS) group, and Fn plus LPSgroup respetively.5ug/ml Fn or (and)1ug/ml LPS were used to stimulate PBMC for0.5h,2h,4h,8h,12h, then cell-free supernatants were collected respectively. Concentrations of IL-6,TNF-α and IL-10in the supernatants were determined by enzyme-linked immunosorbentassay(ELISA); Western blotting was also used to estimate the expression of TLR4in PBMC.2. PBMC were isolated and cultured by the above procedures. PBMC were divided into blank control group, Fn group, NPY plus Fn group, LPS group, NPY plus LPS group, Fn plusLPS group and NPY plus Fn plus LPS group.5ug/ml NPY was applied to preincubate PBMCfor2hours, then5ug/ml Fn or (and)1ug/ml LPS was added in RPMI medium to continuouslystimulate PBMC for0.5h,2h,4h,8h,12h. Both the expressions of IL-6, IL-10, TNF-α and theexpression of TLR4were measured.3. PBMC were divided into Fn group, BFA plus Fn group, Fn plus LPS group and BFAplus Fn plus LPS group.5ug/ml BFA(brefeldin A, BFA)was used to preincubate PBMC for3hours, RPMI medium containing BFA was removed and cells were washed for3times inRPMI medium. Then5ug/ml Fn or (and)1ug/ml LPS was used to stimulate PBMC for0.5h,2h,4h,8h,12h. The expressions of IL-6, IL-10, TNF-α in the supernatants and the expressionof TLR4in PBMC were measured.Results1. Effect of Fn on the synthesis and release of inflammatory cytokines of PBMC inresponse to LPS stimulation(1)IL-6: The expressions of IL-6in blank control group were at low level, and nodifference was observed at all time points(p>0.05). IL-6expressions were not significantlyincreased in Fn group, LPS group and Fn plus LPS group at0.5h(p>0.05). The IL-6level wassignificantly higher in Fn plus LPS group than that in Fn group and LPS group at2h(p<0.05).IL-6expressions in Fn plus LPS group reached the peak at8h after stimulation, significantlyhigher those of the remaining three groups(p<0.05).(2)TNF-α: No difference was observed in expressions of TNF-α among groups at0.5hafter treatment(p>0.05). TNF-α expression was markedly higher in Fn plus LPS group thanthat in blank control group, Fn group and LPS group at2h,4h,8h,12h (p<0.05).(3)IL-10: IL-10expression was not significantly increased in Fn plus LPS group thanthat in blank control group, Fn group and LPS group at0.5h and2h after treatment(p>0.05).Notable increase of IL-10expression in Fn plus LPS group was observed at4h after treatment,significantly higher than that of Fn group and LPS group(p<0.05). An increase of IL-10expression was observed at8h and12h.(4)TLR4: The expression level of TLR4protein was not significantly increased in Fnplus LPS group than that in LPS group at0.5h(p>0.05). Notable increase of TLR4proteinlevel in Fn plus LPS group was observed at2h and4h after treatment, significantly higher than that of the remaining three groups(p<0.05).2. Effect of NPY on the release of inflammatory cytokines in hypersensitization of Fn toLPS stimulation to PBMC(1) IL-6: IL-6release level was not significantly decreased in NPY plus Fn group whilecompared with Fn group at0.5h. No difference was observed in the release level of IL-6between NPY plus LPS groups and LPS groups at all time points. IL-6release level wassignificantly lower in NPY plus Fn group than that in Fn group, and significantly lower inNPY plus Fn plus LPS group than that in Fn plus LPS group at2h,4h,8h(p<0.05).(2) TNF-α: At2h and4h after treatment, TNF-α release level was significantly lower inNPY plus Fn group than that in Fn group, and lower in NPY plus Fn plus LPS group than thatin Fn plus LPS group(p<0.05), and higher in NPY plus Fn plus LPS group than that in blankcontrol group and Fn group. IL-6release level was not significantly decreased in NPY plus Fngroup than that in Fn group at8h and12h(p>0.05).(3) IL-10: IL-10release level was not significantly lower in NPY plus Fn group than thatin Fn group at0.5h and2h after treatment(p>0.05). At4h and8h, IL-10release level wassignificantly lower in NPY plus Fn group than that in Fn group, and lower in NPY plus Fnplus LPS group than that in Fn plus LPS group(p<0.05).(4)TLR4: No difference was observed in TLR4protein level between Fn group and NPYplus Fn group at0.5h(p>0.05). At2h and4h after treatment, the level of TLR4wassignificantly lower in NPY plus Fn group than that in Fn group, and also lower in NPY plusFn plus LPS group than that in Fn plus LPS group(p<0.05).3. Effect of Golgi body protein transportion interfered by BFA on the synthesis andrelease of inflammatory cytokines in PBMC(1) At0.5h,2h and4h, the release level of IL-6, TNF-α and IL-10was significantlylower in BFA plus Fn group than that in Fn group(p<0.05), and significantly lower in BFAand Fn plus LPS group than that in Fn plus LPS group(p<0.05).(2)TLR4: The expression level of TLR4was significantly lower in BFA plus Fn groupthan that in Fn group at0.5h,2h and4h after treatment(p<0.05). At0.5h and2h, TLR4levelwas lower in BFA and Fn plus LPS group than that in Fn plus LPS group(p<0.05).Conclusions1. Simple Fn can stimulate PBMC to increase the release of inflammatory cytokines, while Fn in combination with LPS can further increase the release level of inflammatorycytokines and the expression level of TLR4, implying that Fn could enhance the sensitivity ofPBMC to LPS stimulatory response.2. NPY can inhibit the sensitivity of Fn in response to PBMC, resulting in the markedreduction of inflammatory cytokines by down-regulating the expressions of TLR4.3. BFA can inhibit protein transport in Golgi body and lead to reduction in the level ofTLR4and inflammatory cytokines, implying that Golgi body may participate in TLR4hypersensitivity.

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