Dual Effect of 3, 4-Dihydroxyacetophenone on LPS-induced Apoptosis in RAW264.7 Cells by Modulating the Production of TNF-α

To explore the pharmacological effect of 3,4-dihydroxyacetophenone (DHAP) on the apoptosis of RAW264. 7 macrophage cells and the mechanism, RAW264. 7 macrophage cells were treated with 100 or 500 mg/L lipopolysaccharide (LPS), with or without 10^-5 mol/L DHAP for 24 h. Trypan blue dye exclusion assay was used to assess cell viability. Cell apoptosis was morphological studied and flow cytometric assay was used. Tumor necrosis factor-α (TNF-α) level was measured by ELISA methods. IκB protein was determined by Western blotting. Our results showed that in 100 mg/L LPS stimulated macrophages, DHAP enhanced the cell apoptosis while in 500 mg/L LPS-stimulated macrophages, DHAP significantly inhibited the cell apoptosis. In both groups, DHAP increased the level of IκB but decreased the level of TNF-α. It is concluded that DHAP has dual effect on the apoptosis of RAW 264.7 cells treated with different concentrations of LPS. This effect may be due to the inhibition of activation of NF-κB and autocrine production of TNFα. Our study suggests that DHAP may have anti-inflammatory effect on LPS-activated macrophages.     

Caffeic acid ester fraction from Erigeron breviscapus inhibits microglial activation and provides neuroprotection

Objective:To investigate the effects of caffeic acid ester fraction(Caf)from Erigeron breviscapus, mainly composed of dicaffeoylquinic acids(diCQAs),on microglial activation in vitro and focal cerebral ischemia in vivo.Methods:The production of nitric oxide(NO),tumor necrosis factorα(TNF-α),and interleukin-1β(IL-1β)induced by lipopolysaccharide(LPS)treatment in rat primary cultured microglia were measured by Griess reaction or enzyme-linked immunosorbent assay.Cell viability of cortical neurons was measured using AlamarBlue reagent.The behavioral tests and the infarct area of brain were used to evaluate the damage to central nervous system in rat middle cerebral artery occlusion(MCAO)model of cerebral ischemia.Real time polymerase chain reaction was used to determine the expression of inducible nitric oxide synthase(iNOS), TNF-αand IL-1βmRNA in ischemic cerebral tissues.Results:Caf inhibited the production of NO,TNF-αand IL-1βinduced by LPS treatment in primary microglia in a dose-dependent manner.Exposure of cortical neurons to conditioned medium from Caf-treated microglia increased neuronal cell viability(P<0.01)compared with conditioned medium from LPS-treated alone.In MCAO rat model of cerebral ischemia,Caf could significantly improve neurobehavioural performance and reduce percentage infarct volume compared with the vehicle group (P<0.05).Caf could also significantly inhibit the up-regulation of iNOS,TNF-α,and IL-1βgene expressions in ischemic cerebral tissues.Conclusion:Caf could suppress microglial activation,which may be one mechanism of its neuroprotective effect against ischemia.     

In Vivo and In Vitro Anti-inflammatory Effects of Ethanol Fraction from Periploca forrestii Schltr.

Objective: To determine the anti-inflammatory effects of an ethanol fraction of Periploca forrestii Schltr. (EFPF) and to investigate the potential mechanisms underlying in vivo and in vitro models. Methods: The anti-inflammatory effects of EFPF were evaluated using the xylene-induced mouse ear edema and carrageenan-induced rat paw edema models in vivo. In vitro, RAW264.7 cells were exposed to 0–800 μg/mL EFPF and the cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Then cells were treated with different concentrations of EFPF (100–400 μg/mL) and stimulated with lipopolysaccharide (LPS, 1 μg/mL) for 24 h. The supernatant was analyzed for nitric oxide (NO) using the Griess reagent, and the levels of inflammatory mediators and cytokines were determined using enzyme-linked immunosorbent assays for prostaglandin E2 (PGE2), tumor necrosis factor α (TNF-α), interleukin (IL) 6, and IL-10. The protein expressions of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor κB (NF-κB), and mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK were examined by Western blot. Results: Compared with the control group, EFPF significantly reduced mouse ear edema and rat paw edema rate (P<0.05 or P<0.01). Compared with the LPS group, EFPF significantly inhibited the LPS-stimulated production of NO, PGE2, TNF-α and IL-6 (P<0.05 or P<0.01), and increased the IL-10 production (P<0.05). EFPF also significantly inhibited LPS-induced protein expressions of iNOS and COX-2, suppressed the phosphorylation and degradation of inhibitor of NF-κB-α, decreased p65 level, and inhibited the phosphorylation of p38, ERK1/2 and JNK (P<0.05 or P<0.01). Conclusion: EFPF exerted anti-inflammatory effect by reducing protein expressions of iNOS and COX-2 and the production of the inflammation factors, including TNF-α, IL-6, NO and PGE2, mainly through inhibition of LPS-mediated stimulation of NF-κB and MAPK signaling pathways.     

In Vivo and In Vitro Anti-inflammatory Effects of Ethanol Fraction from Periploca forrestii Schltr.

Objective:To determine the anti-inflammatory effects of an ethanol fraction of Periploca forrestii Schltr.(EFPF) and to investigate the potential mechanisms underlying in vivo and in vitro models. Methods:The antiinflammatory effects of EFPF were evaluated using the xylene-induced mouse ear edema and carrageenan-induced rat paw edema models in vivo. In vitro, RAW264.7 cells were exposed to 0–800 μg/m L EFPF and the cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay. Then cells were treated with different concentrations of EFPF(100–400 μg/m L) and stimulated with lipopolysaccharide(LPS, 1 μg/m L) for 24 h. The supernatant was analyzed for nitric oxide(NO) using the Griess reagent, and the levels of inflammatory mediators and cytokines were determined using enzyme-linked immunosorbent assays for prostaglandin E2(PGE2), tumor necrosis factor α(TNF-α), interleukin(IL) 6, and IL-10. The protein expressions of inducible NO synthase(i NOS), cyclooxygenase-2(COX-2), nuclear factor κB(NF-κB), and mitogen-activated protein kinases(MAPKs) including extracellular signal-regulated kinase(ERK), c-Jun N-terminal kinase(JNK), and p38 MAPK were examined by Western blot. Results:Compared with the control group, EFPF significantly reduced mouse ear edema and rat paw edema rate(P0.05 or P0.01). Compared with the LPS group, EFPF significantly inhibited the LPS-stimulated production of NO, PGE2, TNF-α and IL-6(P0.05 or P0.01), and increased the IL-10 production(P0.05). EFPF also significantly inhibited LPS-induced protein expressions of i NOS and COX-2, suppressed the phosphorylation and degradation of inhibitor of NF-κB-α, decreased p65 level, and inhibited the phosphorylation of p38, ERK1/2 and JNK(P0.05 or P0.01). Conclusion:EFPF exerted anti-inflammatory effect by reducing protein expressions of i NOS and COX-2 and the production of the inflammation factors, including TNF-α, IL-6, NO and PGE2, mainly through inhibition of LPS-mediated stimulation of NF-κB and MAPK signaling pathways.     

Short-term intensive atorvastatin therapy improves endothelial function partly via attenuating perivascular adipose tissue inflammation through 5-lipoxygenase pathway in hyperlipidemic rabbits

Background Atherosclerosis is a kind of disease with multiple risk factors,of which hyperlipidemia is a major classical risk factor resulting in its pathogenesis and development.The aim of this study was to determine the effects of short-term intensive atorvastatin （IA） therapy on vascular endothelial function and explore the possible mechanisms that may help to explain the clinical benefits from short-term intensive statin therapy.Methods After exposure to high-fat diet （HFD） for 8 weeks,the animals were,respectively,treated with IA or low-dose atorvastatin （LA） for 5 days.Blood lipids,C-reactive protein （CRP）,tumor necrosis factor-α （TNF-α）,interleukin-6 （IL-6）,nitric oxide （NO）,endothelin-1 （ET-1）,and endothelium-dependent vasorelaxation function were,respectively,measured.mRNA and protein expression of CRP,TNF-α,IL-6,macrophage chemoattractant protein-1 （MCP-1）,and 5-lipoxygenase （5-LO） were also evaluated in pericarotid adipose tissue （PCAT） and cultured adipocytes.Results HFD increased serum inflammatory factor levels; induced significant hyperlipidemia and endothelial dysfunction,including imbalance between NO and ET-1; enhanced inflammatory factors and 5-LO expression; and promoted macrophage infiltration into adipose tissue.Five-day IA therapy could significantly decrease serum inflammatory factor levels and their expression in PCAT; restore the balance between NO and ET-1; and improve endothelial function and macrophage infiltration without significant changes in blood lipids.However,all of the above were not observed in LA therapy.In vitro experiment found that lipopolysaccharide （LPS） enhanced the expression of inflammatory factors and 5-LO in cultured adipocytes,which could be attenuated by short-time （6 hours） treatment of high-dose （5 pmol/L） but not low-dose （0.5 μmol/L） atorvastatin.In addition,inhibiting 5-LO by Cinnamyl-3,4-dihydroxy-α-cyanocinnamate （CDC,a potent and direct 5-LO inhibitor） could significantly downregulate the     

Expression of IL-βα and TNF-α in MCMV Myocarditis and Its Role

In order to study the expression of IL-β and TNF-α in the myocardium of MCMV myocarditis and their role in the myocardial damages, 60 BALB/C mice of 4 weeks were randomly divided into two groups: 36 were injected intraperitoneally with MCMV and 24 served as control group. Immunohistochemistry was used to detect IL-β and TNF-α expression in the myocardium, and myocardial lesions were observed histopathologically. Histopathological study on the myocardium from infected mice revealed focal or diffuse lesions characterized by inflammatory cells and degeneration or necrosis of myocytes. The myocardial lesion score showed the degree of inflammatory cell infiltration was slight in MCMV myocarditis. The positive staining signals for IL-β and TNF-α proteins which mainly located in the infiltrating inflammatory cells and degenerative or necrotic myocytes were markedly detectable whereas there were no positive findings in the myocardium of control mice. IL-β and TNF-α was expressed in the myocardium of viral myocarditis murine model induced by MCMV. IL-β and TNF-α may play an important role in the pathogenesis of viral myocarditis.     

Interaction between Macrophage Colony—stimulating Factor and Inter—leukin—10onTNF—αand IL—8Generated by Human Monocytes

Objective To investigate the interaction between recombinant human macrophage colony-stimulating factor(rhM-CSF)and interleukin(IL)-10onTNF-αandIL-8generated by human peripheral bloodmonocytes.Methods Human monocytes,isolated from the blood of healthy donors,were cultured with M-CSForIL-10alone,or combinjed M-CSFwith Il-10.Seventy two hours later TNF-a in culture supernatans were determined by ELISA and the method of biological activity detection,andIL-8in culture supernatans were detected by ELISA48h after culture.Results(1)M-CSF was able to induce generation of TNF-αby monocytes and had a syn-ergetic effect with LPSin induction of TNF-α。WhileIL-10was capable to inhibit production of TNF-αand had an antagonism to in-duction of TNF-αbyLPSand M-CSF.(2)M-CSFhad a capability to induce secretion of IL-8by monocytes.While IL-10could sup-press secretion of IL-8and had an antagonism to i nduction of IL-8 by M-CSF.Conclusion M-CSF may promote monocytes to re-lease inflammatory cytokines and plays an important role in inflammatory reaction in vivo;whileIL-10,as a dramdtic inflammation-in-hibitor,may have an antagonism to in-flammatory effect of M-CSF.     

Organ-association phenomena during sepsis. TNF and IL-6 in different macrophages.

Sepsis was induced in rats by cecal ligation and perforation (CLP). Five and 15 hours post CLP, alveolar macrophage (AM), Kupffer cell (KC), and peritoneal macrophage (PM) were isolated and cultured for 18 hours. Culture supernatant was examined for bioactivity of tumor necrosis factor (TNF) and interleukin 6 (IL-6) and response to lipoplysaccharide (LPS) stimulation in vitro. Results showed that AM produced more TNF during sepsis as compared with KC and PM. Stimulation with LPS in vitro was responded only by AM at 15 hr after CLP. Pattern of IL-6 production was different from TNF while KC produced the highest level of IL-6 after induction of sepsis.

     

穿心莲内酯对巨噬细胞炎症因子表达的影响

目的研究穿心莲内酯对小鼠巨噬细胞氧化亚氮（NO）、肿瘤坏死因子（TNF-α）和白细胞介素-6（IL-6）生成的影响。方法培养小鼠巨噬细胞RAW264.7,分别加入脂多糖（lipopolysaccharide,LPS,终质量浓度1μg/mL）和不同浓度的穿心莲内酯（终浓度1、10、50μmol/L）进行干预,并设空白组和穿心莲内酯单独作用组作为对照。取培养24h细胞上清,用Griess法检测NO产量;用Elisa法检测TNF-α、IL-6浓度。结果与空白组比较,LPS明显促进了RAW264.7细胞NO、TNF-α、IL-6等炎症因子的生成;穿心莲内酯单独作用不影响炎症因子的表达,但穿心莲内酯能显著下调LPS诱导的炎症因子表达,与LPS组差异有显著性。结论穿心莲内酯可明显降低LPS诱导的巨噬细胞炎症因子表达,抑制炎症反应。     

人参二醇皂苷对失血-内毒素二次打击模型大鼠肿瘤坏死因子α和白细胞介素6含量的影响

目的：探讨失血-内毒素二次打击模型大鼠血清中肿瘤坏死因子α（TNF-α）和白细胞介素6（IL-6）含量的变化,以及人参二醇皂苷（PDS）对其影响。 方法：Wistar大鼠随机分为假手术对照组（S组）、失血性休克组（H组）、失血-内毒素双打击组（HL组）、地塞米松预治疗组（HLD组）及PDS预治疗组（HLP组）。在失血性休克首次打击的基础上,用内毒素作为第二次打击复制大鼠难治性休克模型,6 h后处死动物,用放射免疫分析法测定血清TNF-α和 IL-6的含量。结果：血清TNF-α和IL-6含量,H组、HL组 与S组比较均明显升高,但以HL组尤为明显 (P<0.01,P＜0.001),而HLP组和HLD组的TNF-α含量和IL-6含量则明显地低于HL组（均P<0.01)。 结论：在失血性休克基础上给予内毒素可显著地提高TNF-α和IL-6的血清水平;PDS和地塞米松有类似的抑制TNF-α和IL-6的释放,保护失血-内毒素二次打击大鼠的作用。     

TRPC对脂多糖诱导的星形胶质细胞TNF-α和NO分泌的影响

目的 探讨TRPC对脂多糖诱导的星形胶质细胞TNF-α和NO分泌的影响.方法 通过摇床筛选法纯化大鼠大脑皮层星形胶质细胞,用免疫荧光法鉴定其纯度.细胞培养至80%左右融合时加入0.5 μg/mL 脂多糖(LPS)后用维持液分别培养0、2、6、12、24和48 h,检测TNF-α和NO分泌情况,观察TRPC阻断剂 2-A...     

COX/5-LOX双重抑制剂ZLJ-6对脂多糖诱导下巨噬细胞炎症因子表达的影响

研究COX/5-LOX双重抑制剂ZLJ-6对脂多糖(LPS)诱导下RAW 264.7细胞炎症模型的抗炎作用。采用LPS (1μg/mL)刺激生长良好的RAW 264.7巨噬细胞建立细胞炎症模型,在不同浓度的ZLJ-6( 3,10,30 μmol/L)作用下,用Griess法检测细胞培养液中NO的含量,用ELISA法检测TNF-α、IL-6含量,并用Western blotting检测各组细胞中环加氧酶-2(COX-2)和诱导型一氧化氮激酶(iNOS) 的表达。ZLJ-6能剂量依赖性地抑制LPS诱导的RAW264.7细胞NO的释放以及TNF-α、IL-6等炎症因子的生成,同时抑制COX-2和iNOS的表达。结果表明,ZLJ-6具有抑制LPS诱导的RAW 264.7细胞炎症反应的作用,其抗炎机制可能通过抑制COX-2、iNOS的表达以及炎症介质NO以及TNF-α、IL-6的产生。     

瑞芬太尼和芬太尼对LPS诱导的细胞因子的影响

目的:观察瑞芬太尼与芬太尼对正常人离体外周血中白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)的影响。方法:采集10例正常自愿者外周血,每例血样分成14组。Ⅰ组,空白对照;Ⅱ组,脂多糖(LPS)100 mg/L;R1、R3、R5组分别加入瑞芬太尼1,10,100 mg/L;R2、R4、R6组分别加入瑞芬太尼1,10,100 mg/L和LPS100 mg/L;F1、F3、F5组分别加入芬太尼2,20,200 mg/L;F2、F4、F6组分别加入芬太尼2,20,200 mg/L和LPS100 mg/L。用ELISA检测处理后血样上清液IL-6和TNF-α的含量。结果:与Ⅰ组比较,所有加入LPS组外周血中IL-6和TNF-α浓度均增加(P<0.01),所有未加入LPS组外周血中IL-6和TNF-α浓度差异无显著性(P>0.05);而与Ⅱ组比较,加入LPS和瑞芬太尼或芬太尼组外周血中IL-6和TNF-α浓度均降低(P<0.05);与R2组比较,R4、R6组降低(P<0.05);与R4组比较,R6组降低(P<0.05);与F2组比较,F4、F6组降低(P<0.05);与F4组比较,F6组降低(P<0.05)。结论:①LPS可以促进正常人离体外周血中促炎性细胞因子IL-6和TNF-α的产生和含量的增加;②瑞芬太尼和芬太尼可抑制LPS诱导的正常人离体外周血中的IL-6和TNF-α的含量的增加,并呈浓度依赖性;③瑞芬太尼、芬太尼对正常人离体外周血的IL-6和TNF-α的含量均无影响。     

替米沙坦对脂多糖诱导3T3-L1脂肪细胞生成炎性因子的影响

目的探讨血管紧张素Ⅱ受体拮抗剂替米沙坦对小鼠3T3-L1脂肪细胞合成白细胞介素6（IL-6）和肿瘤坏死因子α（TNF-α）的影响。方法脂肪细胞诱导至90%成熟,随机分为5组：对照组,脂多糖（LPS）组,LPS＋替米沙坦组,其中替米沙坦设0.1μg/ml、1μg/ml和10μg/ml 3个浓度梯度。对照组中加DMSO,LPS＋替米沙坦组中加不同浓度替米沙坦,孵育2h后,再向LPS组和LPS＋替米沙坦组加入LPS（1μg/ml）,与替米沙坦共同孵育细胞1h。酶联免疫吸附法（ELISA）检测各组IL-6和TNF-α的蛋白分泌水平,Q-PCR检测各组IL-6和TNF-αmRNA基因表达。结果脂多糖处理后IL-6分泌较对照组增加约2.7倍,IL-6mRNA表达增加4倍。替米沙坦干预后各组IL-6浓度较脂多糖组均明显下降,10μg/ml组降低67%,IL-6mRNA表达随替米沙坦浓度升高而下降,10μg/ml组下降约63%。此外,与对照组相比,脂多糖处理组TNF-α分泌增加1.3倍,其mRNA表达增加3.5倍,替米沙坦干预后,各组TNF-α蛋白分泌及mRNA表达均较脂多糖组明显降低,大剂量替米沙坦干预后（10μg/ml）,TNF-α蛋白分泌下降约85%,mRNA表达下降约23%。结论替米沙坦通过影响脂肪细胞炎性因子的产生发挥抗炎作用,10μg/ml大剂量时作用尤为明显。     

冬凌草甲素对LPS诱导Raw264.7巨噬细胞炎症反应的抑制作用

目的 研究冬凌草甲素对脂多糖(LPS)诱导的Raw264.7巨噬细胞炎症反应的影响.方法 选用小鼠巨噬细胞Raw264.7,CCK-8法确定冬凌草甲素作用的最适浓度;设立正常对照组、模型对照组(LPS组)、实验组(冬凌草甲素预处理+LPS组)和阳性药组(地塞米松预处理+LPS组),实时定量PCR法检测TNF-α、IL-1β、IL-6、IL-10和TLR4 mRNA表达水平的改变;Western blot法检测NF-κB p65、磷酸化p65(p-p65)蛋白表达水平的改变.结果 冬凌草甲素作用于Raw264.7细胞的最佳浓度为10 μmol/L;与LPS组比较,冬凌草甲素预处理实验组Raw264.7细胞内TNF-α、IL-1β和IL-6 mRNA表达水平明显下降,IL-10 mRNA表达水平明显升高,TLR4基因表达降低,NF-κB活化入核减少.结论 冬凌草甲素能下调LPS诱导的Raw264.7细胞促炎因子表达,其抗炎免疫作用机制与抑制TLR4-NF-κB信号通路的活化有关.     

去甲斑蝥素对LPS 诱导的体外培养的肝细胞损伤的保护作用

目的:通过体外观察去甲斑蝥素(NCTD) 对LPS 所致肝细胞损伤和TNF-α 表达的影响,探讨NCTD 的作用及其机制。方法:胶原酶Ⅳ灌流分离培养大鼠肝细胞,将细胞分为对照组、LPS 组、NCTD 组。对照组用无血清DMEM 培养, LPS 组用LPS (40 mg/L) 诱导,NCTD 组用不同浓度NCTD(0.5,1.0,2.5 μg/mL) 与LPS (40 mg/L) 共同作用,各组作用时间均为24 h。MTT 法检测肝细胞的增殖情况、测定培养上清液乳酸脱氢酶(LDH) 含量,ELISA 法检测TNF-α 和IL-6 表达。结果:LPS (40 mg/L) 作用于原代培养大鼠肝细胞24 h 后,与对照组相比,细胞生长抑制率达27%,培养上清液LDH 含量增加20 倍,TNF-α 和IL-6 的表达以及NF-κB DNA 结合活性均明显增加(P<0.05)。给予不同浓度NCTD 后,培养上清液LDH,TNF-α 和IL-6 的含量及NF-κB DNA 结合活性均明显下降,且呈量效关系。结论:NCTD 拮抗LPS 所致的肝细胞损伤,其保护作用的机制可能与其抑制TNF-α 和IL-6 的表达有关。     

脐带间充质干细胞对小胶质细胞增殖及活化的影响

目的 观察人脐带来源的间充质干细胞(hUC-MSCs)在正常环境和炎症环境下对小胶质细胞活化的调节作用,探讨hUC-MSCs对中枢神经系统炎症反应的免疫调节作用。 方法 将BV2细胞与人脐带间充质干细胞来源的条件培养基(hUC-MSCs-CM)和(或)脂多糖(LPS)共培养24或48 h后利用MTT比色法分析BV2增殖活力的变化,ELISA试剂盒分析BV2细胞上清中TNF-α和IL-6的变化,免疫印迹法检测精氨酸酶1(Arg1)的表达, Real-time PCR分析TNF-α、IL-6、ARG1基因的表达水平。 结果 (1)BV2在LPS长程刺激下表现出明显的增殖效应,而hUC-MSCs-CM可以抑制这种增殖反应;(2)炎症刺激诱导BV2表达TNF-α和IL-6明显增多,而在hUC-MSCs-CM和LPS共刺激时TNF-α和IL-6表达明显低于LPS单刺激组;(3)hUC-MSCs-CM共培养组BV2细胞高表达M2型小胶质细胞标志物Arg1。 结论 脐带间充质干细胞抑制炎症所致的小胶质细胞增殖;脐带间充质干细胞通过旁分泌机制调节小胶质细胞向M2型活化,降低促炎因子表达,这可能是其最终发挥神经保护作用的机制之一。