Baicalin inhibits macrophage activation by lipopolysaccharide and protects mice from endotoxin shock

Baicalin (BA) exhibits anti-inflammatory effect in vivo and in vitro and is used to treat inflammatory diseases. Here, we report that BA inhibits the activation of macrophage and protects mice from macrophage-mediated endotoxin shock. The experiments in vitro showed BA suppressed the increased generation of nitric oxide (NO) and expression of inducible nitric oxide synthase (iNOS) induced by LPS or Interferon-gamma (IFN-gamma) without directly affecting iNOS activity in RAW264.7 cells and peritoneal macrophages. Similarly, BA inhibited the production of reactive oxidative species (ROS), whereas augmented the level of intracellular superoxide dismutase (SOD). Moreover, BA inhibited the production of inflammatory mediators including tumor necrosis factor (TNF)-alpha, endothelin (ET)-1 and thromboxane A2 (TXA2) induced by lipopolysaccharide (LPS) in RAW264.7 cells. In animal model, BA protected mice from endotoxin shock induced by d-galactosamine (D-GalN)/LPS possibly through inhibiting the production of cytokine and NO. Collectively, BA inhibited the production of inflammatory mediators by macrophage and may be a potential target for treatment of macrophage-mediated diseases.     

Toll-like receptor 4 signal transduction inhibitor, M62812, suppresses endothelial cell and leukocyte activation and prevents lethal septic shock in mice

Sepsis occurs when microbes activate toll-like receptors (TLRs) stimulating widespread inflammation and activating coagulation cascades. TLR4 signal transduction has been recognized as a key pathway for lipopolysaccharide (LPS)-induced activation of various cells and an attractive target for treatment of sepsis. We found a new benzisothiazole derivative, M62812 that inhibits TLR4 signal transduction. This compound suppressed LPS-induced upregulation of inflammatory cytokines, adhesion molecules and procoagulant activity in human vascular endothelial cells and peripheral mononuclear cells. The half maximal inhibitory concentrations in these assays ranged from 1 to 3 μg/ml. Single intravenous administration of M62812 (10-20 mg/kg) protected mice from lethality and reduced inflammatory and coagulatory parameters in a murine d-galactosamine-sensitized endotoxin shock model. M62812 (20 mg/kg) also prevented mice from lethality in a murine cecal ligation and puncture model. These results suggest that inhibition of TLR4 signal transduction can suppress coagulation as well as inflammation during sepsis and may be clinically beneficial in sepsis treatment.     

脓毒症小鼠树突状细胞B7-H1表达与免疫功能的改变

目的 观察脂多糖(Lipopoiysaccharide,LPS)所致脓毒症小鼠树突状细胞(DendriticCells,DCs)B7-H1表达及免疫功能的改变.方法 以10 ng/ml脂多糖刺激体外诱导小鼠树突状细胞建立脓毒症免疫麻痹体系,利用显微拍照技术、流式细胞术、Real-Time PCR和BrdU细胞增殖反应试剂盒分别检测树突状细胞形态、B7-H1表达及抗原特异性混合淋巴细胞增殖反应的变化.结果 LPS刺激引起DCs由未成熟转变为成熟,且B7-H1的RNA水平增加了11.07倍(P＜0.001),蛋白表达平均荧光密度值由12.39增加到43.81 (P＜0.001);然而抗原特异性混合淋巴细胞反应OD值由0.761减少为0.374,并且封闭B7-H1后OD值由0.374增加到0.595 (P＜0.001).结论 LPS所致脓毒症小鼠DCs表达高水平的免疫共抑制B7-H1分子,从而引起T细胞免疫反应低下,呈现免疫麻痹状态.     

Inhibitory effects of melittin on the production of lipopolysaccharide-induced matrix metalloproteinase 3 in human osteoarthritic chondrocytes

In continuation of our previous study which explored the effect of bee venom (BV) on the global gene expression profiles in lipopolysaccharide (LPS)-treated human chondrosarcoma cells, we investigated herein the effect of melittin, a major component of BV, on the productions of matrix metalloproteinases (MMPs) 1, 3, and 13 in primary cultured human arthritic chondrocytes. Increased generations of MMPs 1, 3, and 13 were observed by MMPs stimulating agents LPS, tumor necrosis factor α (TNF-α), and interleukin 1β (IL-1β). The generations of LPS (1 μg/ml)-induced MMPs 1 and 13 were not decreased by melittin, whereas that of LPS-stimulated MMP 3 was significantly inhibited by melittin. IL-1β (10 ng/ml) and TNF-α (10 ng/ml)-induced MMPs 1, 3 and 13, however, were not decreased by melittin. Immunoblot analysis revealed that melittin exerted no effect on the LPS-stimulated expression levels of MMPs 1 and 13 but attenuated the LPS-induced MMP 3, which is consistent with the enzyme-linked immunosorbent assay (ELISA) data. Taken together, these findings suggest melittin may exert its anti-arthritic effect, at least in part, by inhibiting LPS-stimulated MMP 3 production in human osteoarthritic chondrocytes.     

Inhibitory activity of quercetin and its metabolite on lipopolysaccharide-induced activation of macrophage U937 cells

The potential of quercetin and its metabolite 3-O-methyl quercetin in inhibiting lipopolysaccharide (LPS)-mediated activation of macrophage U937 cells was investigated. Cells were pre-incubated for different periods with 100 ng/mL phorbol myristate acetate (PMA), and later with LPS and quercetin or 3-O-methyl quercetin (30 μM). Later, the supernatant of each cell culture was assessed for catalase activity, nitric oxide, and the production of tumour necrosis factor-α (TNF-α), interleukin 6 (IL-6), and interleukin 1 (IL-1). The results showed that when the cells were incubated with LPS, there were elevations in the levels of all the markers over the cells not incubated with LPS (P < 0.05). For the cells that were incubated with LPS, there were significant differences between the various cells when they were pre-incubated with PMA for various periods (P < 0.05). However, greatest production of the markers was attained when the cells were pre-treated with PMA for 48 h. Both quercetin and 3-O-methyl quercetin (at 30 mM) reduced the levels of all the markers with 3-O-methyl quercetin possessing more inhibitory potential (P < 0.05). This suggests that the flavonoids possessed significant immunomodulatory activities which depend on methylation especially at position 3.     

Inhibitory effects of eicosapentaenoic acid on lipopolysaccharide-induced activation in BV2 microglia

Upon activation, microglia release proinflammatory mediators that play important roles in eliciting neuroinflammatory responses associated with neurodegenerative diseases. The anti-inflammatory properties of eicosapentaenoic acid (EPA) have been known, however, the effects responsible for lipopolysaccharide (LPS)-induced activation remain poorly understood in microglia. In the present study, we investigated the effects of EPA on the expression of proinflammatory mediators in LPS-stimulated BV2 microglia. EPA significantly inhibited the release of nitric oxide (NO), prostaglandin E(2) (PGE(2)) and proinflammatory cytokines such as interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNF)-alpha in a dose-dependent manner. EPA also attenuated the production of cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS) and proinflammatory cytokines at mRNA and/or protein levels. Moreover, EPA suppressed NF-kappaB activation by blocking IkappaB degradation, and also blocked the mitogen-activated protein kinases (MAPKs) such as ERK, p38 and JNK, and the Akt pathway. The anti-inflammatory properties of EPA may be useful for ameliorating neurodegenerative diseases as well as suppressing LPS-induced shock.     

Beneficial effects of magnolol in a rodent model of endotoxin shock

Magnolol is a compound extracted from the Chinese medicinal herb Magnolia officinalis. It has multiple pharmacological effects, notably as an anti-oxidant. The aim of this study was to evaluate the effects of magnolol on sepsis induced by intravenous (i.v.) administration of lipopolysaccharide (LPS; 10 mg/kg) in anaesthetized Wistar rats. Magnolol (4 μg/kg, i.v.) was administered at 30 min after LPS injection. Post-treatment with magnolol significantly attenuated the deleterious haemodynamic changes (e.g., hypotension and bradycardia) caused by LPS. Meanwhile, magnolol significantly inhibited the elevation of plasma levels of tumor necrosis factor alpha, glutamate-oxaloacetate transaminase, glutamate-pyruvate transaminase and blood urine nitrogen caused by LPS. The induction of inducible nitrous oxide (NO) synthase and the overproduction of NO and superoxide anions by LPS were also significantly reduced by post-treatment with magnolol. Moreover, the plasma level of the thrombin-antithrombin complex following administration of LPS was also reduced by post-treatment with magnolol. Thus, the beneficial effects of magnolol on LPS-induced sepsis result from its anti-inflammatory, anti-coagulatory, and anti-oxidant effects.     

Riboflavin protects mice against liposaccharide-induced shock through expression of heat shock protein 25

Riboflavin (vitamin B2) is a water-soluble vitamin essential for normal cellular functions, growth and development. This study aimed to investigate the effects of vitamin B2 on the survival rate, and expressions of tissue heat shock protein 25 (HSP25) and heat shock factor 1 (HSF1) in mice undergoing lipopolysaccharide (LPS) induced shock. Mice were assigned to four groups, saline vehicle, LPS, LPS plus low dose of vitamin B2 (LB2) and LPS plus high dose of vitamin B2 (HB2). Vitamin B2 (1 and 10 mg/kg BW) was administered intraperitoneally at 2 and 0 h before the i.p. administration of LPS. At the end of the experiment, the survival rate monitored was 10, 20, 60, and 100% for LPS, LB2, HB2, and saline mice, respectively. HSP25 expressions in the heart and lung were significantly enhanced in a time-dependent manner in the HB2 mice as compared to the saline mice (p < 0.05), but not altered in the LB2 mice. In the HB2 mice, plasma riboflavin concentrations reached 300 nM at 6 h post LPS and returned to the 0 h level at 72 h. The results showed that high dose of riboflavin could decrease LPS-induced mortality through an increased expression of HSP25.     

Anti-inflammatory effects of dimemorfan on inflammatory cells and LPS-induced endotoxin shock in mice

BACKGROUND AND PURPOSE: Dimemorfan (a sigma1 receptor agonist) showed neuroprotective properties in animal models of inflammation-mediated neurodegenerative conditions, but its effects on inflammatory cells and systemic inflammation remain unclear. EXPERIMENTAL APPROACH: The effects of dimemorfan on phorbol-12-myristate-13-acetate (PMA)- and N-formyl-methionyl-leucyl-phenylalanine (fMLP)- induced neutrophils and lipopolysaccharide (LPS)-activated microglial cells, as well as LPS-induced endotoxin shock in mice were elucidated. KEY RESULTS :Dimemorfan decreased PMA- and fMLP-induced production of reactive oxygen species (ROS) and CD11b expression in neutrophils, through mechanisms independent of sigma1 receptors, possibly by blocking ROS production and G-protein-mediated intracellular calcium increase. Dimemorfan also inhibited LPS-induced ROS and nitric oxide (NO) production, as well as that of monocyte chemoattractant protein-1 and tumour necrosis factor-alpha (TNF-alpha), by inhibition of NADPH oxidase (NOX) activity and suppression of iNOS up-regulation through interfering with nuclear factor kappa-B (NF-kappaB) signalling in microglial cells. Treatment in vivo with dimemorfan (1 and 5 mg kg(-1), i.p., at three successive times after LPS) decreased plasma TNF-alpha, and neutrophil infiltration and oxidative stress in the lung and liver. CONCLUSIONS AND IMPLICATIONS: Our results suggest that dimemorfan acts via sigma1 receptor-independent mechanisms to modulate intracellular calcium increase, NOX activity, and NF-kappaB signalling, resulting in inhibition of iNOS expression and NO production, and production of pro-inflammatory cytokines. These effects may contribute its anti-inflammatory action and protective effects against endotoxin shock in mice.     

Inhibitory effects of flavonoids extracted from licorice on lipopolysaccharide-induced acute pulmonary inflammation in mice

Airway inflammation plays important roles in the pathogenesis of acute respiratory distress syndrome (ARDS), asthma and chronic obstructive pulmonary disease (COPD), and anti-inflammatory treatment effectively improves the symptoms of these diseases. To develop the potentially therapeutic compounds for the treatment of pulmonary inflammation, we investigated the effects of licorice flavonoids (LF) extracted from the roots of Glycyrrhiza uralensis (licorice) on lipopolysaccharide (LPS)-induced acute pulmonary inflammation in mice. Acute pulmonary inflammation was induced by intracheal instillation with LPS, treatment with LF at dosages of 3, 10 and 30 mg/kg significantly reduced the LPS-induced inflammatory cells, including neutrophils, macrophages and lymphocytes accumulation in bronchoalveolar lavage fluids (BALF), among these inflammatory cells, LF predominately inhibited neutrophil infiltration, and the maximal effect (30 mg/kg) was as comparable as dexamethasone treatment at 1 mg/kg. Consistent with its effects on neutrophil infiltration, LF treatment significantly increased LPS-induced BALF superoxide dismutase activity, and significantly decreased lung myeloperoxidase activity as well. Furthermore, treatment with LF at 30 mg/kg significantly reduced LPS-induced lung TNFα and IL-1β mRNA expression at 6 h and 24 h after LPS instillation, respectively. Finally, LF at different dosages not only significantly decreased the elevation of lung water content, but also markedly attenuated LPS-induced histological alteration. Therefore, we suggest that LF effectively attenuates LPS-induced pulmonary inflammation through inhibition of inflammatory cells infiltration and inflammatory mediator release which subsequently reduces neutrophil recruitment into lung and neutrophil-mediated oxidative injury, and this study provides with the potential rationale for development of anti-inflammatory compounds from flavonoid extracts of licorice.     

氯胺酮对内毒素小鼠肺泡巨噬细胞核因子-κB活化的影响

目的观察不同浓度的氯胺酮对脂多糖(LPS)诱发的小鼠肺泡巨噬细胞核因子(NF)-κB的活化,进一步探讨氯胺酮对急性肺损伤(ALI)/急性呼吸窘迫综合征(ARDS)的应用前景。方法在小鼠肺泡巨噬细胞(PAM)培养液中加入脂多糖(LPS)10μg/ml得到内毒素肺损伤细胞模型。实验分为LPS刺激组(加LPS10μg/ml)、氯胺酮干预组1(同时加入LPS10μg/ml和氯胺酮10μmol/L)、氯胺酮干预组2(同时加入LPS10μg/ml和氯胺酮100μmol/L)、氯胺酮干预组3(同时加入LPS10μg/ml和氯胺酮1000μmol/L)4组。分别于刺激后0.5,1,4,6h留取细胞,用免疫组织化学染色图像分析法检测细胞核中NF-κB活性。结果①LPS+不同浓度的氯胺酮(10,100,1000μmol/L)后,NF-κBp50活性与LPS组相比在1h和4h处有明显的下降(P<0.05),且有剂量依赖性,氯胺酮浓度越大,NF-κBp50活性下降越明显,各组之间差异有统计学意义(P<0.05)。②NF-κBp65活性的检测与NF-κBp50的检测结果相似。结论氯胺酮通过对PAM内NF-κB活化的抑制,可阻止和延缓LPS诱发的ALI的发生,可能有一定的肺保护作用。     

Inhibitory effects of bacterial endotoxin and reserpine on the uptake of 3H-5-hydroxytryptamine by granule fractions of blood platelets

In rabbit platelets, bacterial endotoxin (lipopolysaccharide), at a concentration of 10⁻⁴ g/ml, inhibited the uptake of ³H-5HT by platelets, especially by the granule fraction. Reserpine, at a concentration of 10⁻⁷ g/ml, counteracted the uptake of ³H-5HT by platelets, especially by the granule fraction.     

Neuroendocrine, behavioral and macrophage activity changes induced by picrotoxin effects in mice

The relevance and property of studies related to stress effects on immune function are undisputable. All studies conducted on stress-immune relationships, however, provide from physical and/or psychological stressors. Indeed, as far as it is of our knowledge brain-innate immune responses were not analyzed after anxiogenic-like drugs use. The present experiment was then undertaken to analyze the effects of picrotoxin (0.3, 0.6 and 1.0mg/kg doses) on behavior, macrophage activity, serum corticosterone and noradrenaline (NE) levels and turnover in the brain of adult mice. Results showed that picrotoxin treatment in mice: (1) decreased motor and rearing activities in an open-field; (2) decreased the number of entries into the plus-maze open-arms and decreased the time spent in the exploration of the plus-maze open-arms; (3) decreased both motor activity and the level of holes exploration in the hole-board; (4) increased the levels of serum corticosterone in dose-dependent way; (5) increased noradrenaline (NE) and MHPG levels and NE turnover in the hypothalamus; and (6) increased Staphylococcus aureus and PMA-induced macrophage oxidative burst. However, and contrary to that reported after physical or psychological stress, this drug induced no effects on macrophage phagocytosis and NE levels and turnover in the frontal cortex. The present results are thus showing that picrotoxin induces some but not all neuro-innate immunity changes previously reported for inescapable foot-shock and psychological stressors in mice. These facts suggest that this chemical stressor triggers CNS pathways that might be somehow different from those fired by inescapable foot-shock and psychological stressors, leading to different neuro-innate immune responses.     

辛伐他汀对脓毒症小鼠肺损伤的保护作用

目的观察辛伐他丁对脓毒症小鼠急性肺损伤的保护作用,并探讨其可能机制。方法采用盲肠结扎穿孔术制备脓毒症小鼠模型,将72只雄性C57BL/6小鼠随机分成3组:假手术组、脓毒症急性肺损伤组(脓毒症组)、脓毒症+辛伐他汀治疗组(治疗组)。治疗组给予辛伐他汀0.2μg/g,q12h腹腔注射1周;假手术组、脓毒症组给予等量安慰剂腹腔注射1周。分别于造模后6、12、24 h留取肺脏标本。HE染色观察肺组织病理学变化,免疫组化检测肺组织toll样受体4(Toll-like receptor 4,TLR4)蛋白的表达,ELISA测定肺组织匀浆中IL-1β及TNF-α的表达水平。结果与假手术组比较,造模后6、12、24 h,脓毒症组肺组织病理学评分、肺组织TLR4蛋白的表达,以及TNF-α、IL-1β水平明显升高(P<0.05);与脓毒症组相比,治疗组上述指标明显降低(P<0.05)。结论辛伐他汀通过抑制TLR4信号转导通路,减少其下游炎症介质TNF-α、IL-1β的释放,对脓毒症导致的急性肺损伤具有一定保护作用。     

红景天苷对脂多糖诱导的小鼠心肌损伤保护作用的研究

目的:研究红景天苷对脂多糖诱导的小鼠心肌损伤的保护作用及其机制。方法:选取雄性Balb/c小鼠50只,随机分为对照组、模型组、地塞米松组、红景天苷低、高剂量组。地塞米松和红景天苷高、低剂量连续灌胃7 d,每天1次,最后一次灌胃后1 h,除对照组外,其余各组腹腔注射LPS,12 h后处死,取血和心脏。结果:红景天苷组小鼠心肌病理改变明显减轻于模型组;相比于模型组,红景天苷组能减少血清中TNF-α、IL-6的含量;减少心肌组织中TLR4、My D88和NF-κBp65蛋白的表达。结论:红景天苷减轻脂多糖诱导的小鼠心肌损伤,其机制可能通过调节TLR4/My D88/NF-κBp65信号通路,抑制炎症反应。     

地塞米松对早期脓毒症大鼠急性肺损伤的保护作用研究

目的 观察地塞米松对早期脓毒症大鼠急性肺损伤(acute lung injury,ALI)的保护作用.方法 将雄性大鼠随机分为3组:生理盐水组(N组)、脂多糖组(L组)、脂多糖+地塞米松组(LD组).各组大鼠于造模后4h麻醉处死,行动脉血气分析,测定肺组织湿/干质量比值(W/D),用RT-PCR法检测肺组织水通道蛋白1(aquaporin 1,AQP1 )mRNA表达水平.结果 与L组比较,LD组大鼠肺组织湿/千质量比明显降低,AQP1 mRNA表达水平升高,动脉血气改善.结论 地塞米松对早期脓毒症大鼠ALI有保护作用,该作用与上调肺AQP1 mRNA表达水平有关.     

Inhibitory effects of baicalein and wogonin on lipopolysaccharide-induced nitric oxide production in macrophages.

In order to elucidate the mechanism of the antiinflammatory action of baicalein and wogonin, flavonoids from the root of Scutellaria baicalensis, the effects of these compounds were investigated on lipopolysaccharide-induced nitric oxide production in a macrophage-derived cell line, RAW 264.7. Baicalein (5-25 microM) and wogonin (5-50 microM) inhibited lipopolysaccharide-induced nitric oxide generation in a concentration-dependent manner. The inhibitory effect of these compounds was observed only when they were added at the start of cell incubation soon after the stimulation with lipopolysaccharide. Baicalein (25 microM) and wogonin (25 microM) also inhibited protein expression of inducible nitric oxide synthase. This inhibitory effect of wogonin was stronger than that of baicalein, which agrees with the result that wogonin showed stronger inhibition of nitric oxide production than baicalein. These results suggest that baicalein and wogonin attenuate lipopolysaccharide-stimulated nitric oxide synthase induction in macrophages and thus may help to explain the antiinflammatory action of these flavonoid compounds.     

The protective effect of obeticholic acid on lipopolysaccharide-induced disorder of maternal bile acid metabolism in pregnant mice

The disorder of bile acid metabolism is a common feature during pregnancy, which leads to adverse birth outcomes and maternal damage effects. However, the cause and therapy about the disorder of bile acid metabolism are still poor. Microbial infection often occurs in pregnant women, which can induce the disorder of bile acid metabolism in adult mice. Here, this study observed the acute effect of lipopolysaccharide (LPS) on maternal bile acid of pregnant mice at gestational day 17 and the protective effect of obeticholic acid (OCA) pretreatment, a potent agonist of bile acid receptor farnesoid X receptor (FXR). The results showed LPS significantly increased the level of maternal serum and disordered bile acids components of maternal serum and liver, which were ameliorated by OCA pretreatment with obviously reducing the contents of CA, TCA, DCA, TCDCA, CDCA, GCA and TDCA in maternal serum and DCA, TCA, TDCA, TUDCA, CDCA and TCDCA in maternal liver. Furthermore, we investigated the effects of OCA on LPS-disrupted bile acid metabolism in maternal liver. LPS disrupted maternal bile acid profile by decreasing transport and metabolism with hepatic tight junctions of bile acid in pregnant mice. OCA obviously increased the protein level of nuclear FXR and regulated its target genes involving in the metabolism of bile acid, which was characterized by the lower expression of bile acid synthase CYP7A1, the higher expression of CYP3A and the higher mRNA level of transporter Mdr1a/b. This study provided the evidences that LPS disrupted bile acid metabolism in the late stage of pregnant mice and OCA pretreatment played the protective role on it by activating FXR.