| BackgroundSepsis is defined as the body’s overwhelming and life-threatening response to infection which can lead to organ failure.Sepsis is a big challenge for intensive care units because of the high mortality and morbidity. In the United States, there are about 751,000 cases of sepsis every year, in which the overall mortality of 100,000 people will be happen. Uncontrolled inflammation has been confirmed as one of the most important mechanisms of sepsis and that it is responsible for subsequent MODS. Large quantities of inflammatory mediators including TNF-a, IFN-y, IL-2 and so on are involved in the process of sepsis. It has been found that TNF-a increases in patients with sepsis, especially in early phase, is involved in a wide spectrum of cell responses and regulates the synthesis of several other critical cytokines and chemokines. TNF-a was also verified to play a vital role in the development of MODS in animal models of sepsis. Macrophage is the main source of pro-inflammatory cytokines including TNF-a.Recently, more studies focusing on extracellular histones which has been considered as a key mediator of death in sepsis and can lead to endothelial damage, cell apoptosis and organ failure because of its effect on inflammation and mitochondria. Extracellular histones can be derived from dying neutrophils in response to bacterial infections for host defense, which called neutrophil extracellular traps(NESTs) or from dying tissue cells. Extracellular histones play an inflammatory role in tissue injury. Histone H4 can induce the release of TNF-a,IL-1β,IL-6,IL-10 in vitro, while the histone H4 neutralization antibody preincubation showed a protective role. In addition, Peter Zhou reported that extracellular histones not only induce pro-inflammatory cytokines production, but also enhance LPS-mediated cytokines obviuosly.this further support the pro-inflammatory role of extracellular histones.ALA was first isolated from bovine live in 1950s and was considered as a potential vitamin.However, the present studies showed that ALA functions as a cofactor which exist in mitochondrial enzymes, Participating in Biological oxidation reactions Meanwhile,ALA which is a multifunctional antioxidant can improve symptoms in diseases through reducing the production of oxygen-free radicals.Zhang and colleagues found that ALA can reduce LPS-induced inflammatory responses in human monocytes via PI3k/AKT pathway.Moreover, Guo and colleagues have a similar result. Their research showed that ALA can prolong survival and attenuate the inflammatory responses through NF-κB pathway in rat model of sepsis. Documents have confirmed that extracellular histones are also invovlved in inflammation in sepsis and responsible for MODS and death. However the pathway by which extracellular histones mediate inflammation and the ALA’s effect on inflammation caused by extracellular histones are not clear. In this study, we used the cell line of RAW264.7 to conduct the investigation to explore the signal pathway by which extracellular histones mediate inflammation and the anti-inflammation effect of ALA.Objectives:1. To Determine whether the extracellular histones induce TNF-a release from RAW264.7 cells;2. To investigate the receptor and signaling pathway for the secretion of TNF-a by extracellular histone mediated RAW264.7 cells;3. To Determine the role and mechanism about ALA for extracellular histones induce TNF-α release from RAW264.7 cells;MethodsCell lineThe murine macrophage cell line RAW 264.7 (American Type Culture Collection ATCC) was cultured in DMEM containing 10% fetal bovine serum and 1% penicillin/streptomycine. Cells were grown at 37℃and with 5% CO2-humidified atmosphere. RAW264.7 were detached with 0.25% trypsinization, which plated in six-well plates at a density of 1×106 cells per well,and allowed to grow until reaching a confluence of 70-80%.TNF-α MeasurementRAW264.7cells were stimulated with various concentrations of extracellular histones (10,25,50μg/ml) for a set time (12h) or cultured with a set concentration (50μg/ml) of extracellular histones for various time durations (0,20,30,45,60,120 min). Then supernatant and RAW264.7cells were harvested for analysis of TNF-α and phosphorylation of ERK,p38 and NF-κB/p65. Inhibitors of activation of ERK (PD98059), p38 (SB203580) or NF-κB (PDTC) or dimethyl sulfoxide (DMSO) were incubated together with 50mg/ml extracellular histones for 1 h, and then the supernatants were harvested for TNF-α measurement 12h later. Supernatant was collected for ELISA test after treatment by ALA (50μg/ml) for 1h and followed by extracellular histones for 12h.TNF-α was quantified by TNF-α ELISA kits (ExCell Biology,shanghai, China) according to the manufacturer’s instruction.Determination of related protein expression by Western blot AnalysisA) detection of mitochondrial membrance protein ERKThe prepared RAW264.7 cells were washed 3 times with cold phosphate-buffered saline (PBS).The cell lysis buffer was supplemented with the protease inhibitor and a phosphatase inhibitor,incubating on ice for 30 min.The supernatant was collected with centrifμging at 10000 g for 10 min at 4℃.Equal quantity of each sample was fraction on SDS-PAGE with a current for 200v for approximately 90 min. Then the protein was transferred into PVDF membranes, after that the membrane was blocked with 5% BSA in TBST(20mM Tris-HCL,15mM Nacl,0.05%(v/v)Tween-20,[pH7.4]) for 1 hour at room temperature(RT), then washed 3 times with TBST for every 5min.The membranes were blotted with antibodies against phospho-ERK(1:2000diluted)and ERK(1:1000diluted)at 4℃overnight,then the membrane was washed 3 times with TBST for every 15 min.then,the membranes was incubated with about secondary antibodies at RT for one hour and washed 3 times with TBST for every 15min.The protein on the membranes were incubated with Immobilon Chemiluminescent HRP substrate for 1 minute,and exposed to X-ray film.B) detection of mitochondrial membrance protein p38The prepared RAW264.7 cells were washed 3 times with cold phosphate-buffered saline (PBS).The cell lysis buffer was supplemented with the protease inhibitor and a phosphatase inhibitor,incubating on ice for 30 min.The supernatant was collected with centrifuging at 10000 g for 10 min at 4 ℃.Equal quantity of each sample was fraction on SDS-PAGE with a current for 200v for approximately 90 min. Then the protein was transferred into PVDF membranes, after that the membrane was blocked with 5% BSA in TBST(20mM Tris-HCL,15mM Nacl,0.05%(v/v)Tween-20,[pH7.4]) for 1 hour at room temperature(RT), then washed 3 times with TBST for every 5min.The membranes were blotted with antibodies against phospho-p38(1:2000diluted)and p38(1:1000diluted)at 4℃overnight,then the membrane was washed 3 times with TBST for every 15 min.then,the membranes was incubated with about secondary antibodies at RT for one hour and washed 3 times with TBST for every 15min.The protein on the membranes were incubated with Immobilon Chemiluminescent HRP substrate for 1 minute,and exposed to X-ray film.C) detection of mitochondrial membrance protein p65The prepared RAW264.7 cells were washed 3 times with cold phosphate-buffered saline (PBS).The cell lysis buffer was supplemented with the protease inhibitor and a phosphatase inhibitor,incubating on ice for 30 min.The supernatant was collected with centrifuging at 10000 g for 10 min at 4 ℃.Equal quantity of each sample was fraction on SDS-PAGE with a current for 200v for approximately 90 min. Then the protein was transferred into PVDF membranes, after that the membrane was blocked with 5% BSA in TBST(20mM Tris-HCL,15mM Nacl,0.05%(v/v)Tween-20,[pH7.4]) for 1 hour at room temperature(RT), then washed 3 times with TBST for every 5min.The membranes were blotted with antibodies against phospho-p65(1:2000diluted)and p65(1:1000diluted)at 4℃overnight,then the membrane was washed 3 times with TBST for every 15 min.then,the membranes was incubated with about secondary antibodies at RT for one hour and washed 3 times with TBST for every 15min.The protein on the membranes were incubated with Immobilon Chemiluminescent HRP substrate for 1 minute,and exposed to X-ray film.Cell Viability Analysisthe 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT) assay that can be an indicator was determined to the cell viability.Briefly, RAW264.7 cells were placed in 96-well plates at a density of 1×10∧4 cells per well, which culture for 14h, and then treated with ALA. After removing the supernatant, these cells were incubated with MTT working solution (5mg/mL;) at 37℃ for 4 h. After treatment with 150μL DMSO to dissolve the crystals, absorbance was read at a wavelength of 490 nm using Absorbance Microplate Reader.Statistical AnalysisAll data were analyzed using SPSS software(version20.0). Results are expressed as the mean ± standard deviation(SD). Statistical analysis was carried out by one-way ANOVA analysis, and Bonferroni post test was used for multiple comparisons. A P＜0.05 was considered significant.Result1. extracellular histones induce TNF-α release from RAW264.7 cells via TLR2.ELISA was used to measure the TNF-α after treatment with the concentrations of 0,10,25,50 μg/ml of extracellular histones for 12h. Results indicated that extracellular histones can induce TNF-α release from RAW264.7 cells in a dose dependent way (269.19±124.07,1757.38±421.63,2132.69±578.54,2780.87±1032.61, *P<0.05and**p<0.001). The RAW264.7cells were treated with or without anti-TLR2 antibody that blocking the TLR2 signaling pathway significantly decrease the production of TNF-a (1081.02±755.09 vs 3754.49±551.43,#P<0.05)2.Extracellular histones induced activation of ERK1/2 and p38 pathwaysTo determine whether the ERK and p38 pathways are involved in the upregulation of TNF-a in extracellular histones-stimulated RAW264.7 cells, we detected the activation of ERK and p38 by western blotting. The results showed that the extracellular histones induced phosphorylation of ERK and p38 after 20 min and with the best effect at 30min. To decide whether extracellular histones activated the phosphorylation of ERK1/2 and p38through TLR2,RAW264.7 cells were pre-treated with or without anti-TLR2 antibody for lh prior to stimulation with extracellular histones.when blocking the TLR2 signal,it significantly reduced extracellular histones-induced phosphorylation of ERK and p38.3. activation of NF-κB pathways regulates extracellular histones-induced TNF-a via TLR2 signalTo determine whether NF-κB pathway is involved in the upregulation of TNF-a which extracellular histones-induced for RAW264.7 cell,we examined the activation of NF-κB pathway by western blotting with phospho-specific antibodies.our data showed that the extracellular histones induced phosphorylation of NF-κB after 30 min.To decide whether extracellular histones activated the phosphorylation of NF-κB through TLR2,RAW264.7 cells were pre-treated with or without anti-TLR2 antibody for 1h prior to stimulation with extracellular histones.when blocking the TLR2 signal,it significantly reduced extracellular histones-induced phosphorylation of NF-κB p65.4.inhibitors reduce extracellular histones-induced TNF-a release from RAW264.7cellskinate inhibitor was used to test the ERK1/2,p38 and NF-κB activity for the extracellular histones induced TNF-a production.we used PD98059,SB203580 or PDTC as an inhibitor for ERK1/2, p38 or NF-κB. RAW264.7 cells were pre-treated with or without PD98059,SB203580 or PDTC for 1h prior to stimulation with extracellular histones.TNF-α production was measured by ELISA.Result showed that pre-treatment with PD98059, SB203580 or PDTC significantly inhibited TNF-α production(2219.38±431.64,2030.61±306.26,2063.5±148.98vs4078.93±558. 02).these data suggested that the ERK1/2,P38 and NF-κB pathways are important in extracellular histones-induced production of TNF-αby TLR2 mediation.DMSO alone have no inhibitory effect.5.Effects of ALA on RAW264.7 cell viability and TNF-a expression.MTT was used to test the cell viability after treated with the concentration of 0,25 and 50 μg/ml of ALA.Results indicated,compared with those cultured in the normal medium,the RAW264.7 cell which were exposed to the different concentration of ALA maintain nearly same cell viability.RAW264.7 cell which treat with the extracellular histones can induce TNF-α release when compared with control group,Then treatment with ALA could markedly reduce TNF-α release from RAW264.7.6.ALA reduce extracellular histones-induced TNF-α release from RAW264.7 through ERK1/2,P38and NF-κB pathwayswe examined the effect of ALA on ERK1/2 and p38signal.30 min after extracellular histones treatment,western blotting was used to detect the expression of ERK1/2, p38 and NF-κB.The results indicated that,the phosphorylation of ERK1/2 and p38 were increase in RAW264.7cells after extracellular histones stimulation when compared with the control cells or cells which only were treated with ALA pretreatment of 50μg/ml ALA can significantly decrease the expression the phosphorylation of ERK1/2 and P38. our data showed that extracellular histones-induced activation of ERK and P38 pathway in RAW264.7 cells was partially inhibited by ALA pretreatment.Conclusion1. Extracellular histones can induce TNF-a production in macrophages via TLR2-MAPK and NF-κB pathway;2. ALA can reduce TNF-α production by inhibiting the ERK1/2, p38 and NF-κB pathways. |
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