人参皂苷Rg1对LPS诱导的BT325细胞株脑啡肽酶表达的影响

目的 :探讨人参皂苷Rg₁对LPS诱导的BT325细胞株脑啡肽酶表达的影响。方法 :应用MTT比色法检测一定剂量LPS和不同浓度人参皂苷Rg1对BT325细胞存活率的影响 ,应用RT PCR观察细胞中脑啡肽酶(neprilysin ,NEP)表达的变化。结果 :一定剂量LPS作用于BT325细胞株 ,BT32 5细胞存活率下降 ,细胞内NEP的表达降低。人参皂苷Rg₁能提高LPS诱导的BT325细胞的存活率 ,使细胞内NEP的表达增高。结论 :一定剂量的LPS能造成细胞损伤和细胞内NEP表达降低的模型 ,人参皂苷Rg₁对LPS造成的细胞毒性具有一定的保护作用 ,并可以增高细胞内NEP的表达。     

内毒素肝损伤中FADD mRNA表达及凉血化瘀方的影响

目的研究凋亡调节基因相关死亡区蛋白(FADD)mRNA在内毒素肝损伤中的表达与肝细胞凋亡的关系,以及凉血化瘀方对其的影响.方法腹腔注射内毒素脂多糖于D-氨基半乳糖致敏小鼠造成暴发性肝衰竭模型,用逆转录-聚合酶链反应半定量分析FADD mRNA表达情况,同时观察药物的影响作用.结果造模3h模型组和治疗组肝组织中FADD mRNA表达分别为110%、100%,至6h模型组的表达增加(124%),治疗组表达下调(81%).结论凉血化瘀方抑制肝细胞凋亡的作用,可能与其下调FADD mRNA表达有关.     

抗脂多糖单链噬菌体抗体在大肠埃希菌中的表达及筛选

目的研究小鼠鼠源性的抗脂多糖(lipopolysaccharide, LPS)单链可变区片段(single-chain fragment variable, ScFv),即在大肠埃希菌TG1中的表达,并筛选出对LPS具有较高亲和力的ScFv. 方法将预先构建好的有效库容量为4.75×106的噬菌体抗体库接种至SOBAG培养基上,隔夜培养后扩容至约1×1010;严格控制lac启动子的表达,仅当加入M13KO7辅助噬菌体后才使ScFv以附着型的形式表达,然后利用LPS淘筛特异性的ScFv,富集的噬菌体阳性克隆重新感染TG1;最后随机挑选出190个菌落在96孔培养板分别培养单个含特异性ScFv的TG1菌落,经间接ELISA检测抗LPS的ScFv. 结果淘筛一轮过后即有3×104阳性菌落长出,190个菌落经间接ELISA检测有两个阳性克隆.结论成功地使鼠抗LPS ScFv在大肠埃希菌中表达,并从中筛选出了两株抗LPS ScFv.     

CTRP4 acts as an anti-inflammatory factor in macrophages and protects against endotoxic shock

Despite the availability of antibiotics, current therapies to treat sepsis are still ineffective and many clinical trials aimed at neutralizing specific inflammatory cytokines have failed, suggesting the urgent need for new treatments. Using two models of LPS-induced endotoxemia and cecal ligation and puncture (CLP)–induced sepsis, we investigated the effects of C1q/TNF-related protein 4(CTRP4) on septic lethality and sepsis-induced inflammation. The effects of CTRP4 on survival, inflammation, organ damage, and bacterial clearance were assessed. Here, we found that CTRP4 decreased the mortalities of mice and alleviated pathological lung injury in mice model. In vivo depletion and adoptive transfer studies showed CTRP4-expressing macrophages as the key cell type inhibiting LPS-induced septic shock. The mechanism associated with the CTRP4 deficiency involved promoting of TLR4 internalization and activation of downstream pathways that resulted in a lethal, prolonged proinflammatory cytokine storm. Treatment of macrophages with exogenous CTRP4 abrogated proinflammatory cytokine production. Our results showed CTRP4 regulates inflammatory response and could be a promising strategy to treat septic shock.     

Nurr-1对小胶质细胞微环境的影响

目的:探讨Nurr-1基因对小胶质细胞活化状态及其微环境的影响。方法:分离培养新生SD大鼠小胶质细胞,纯化、鉴定并分别用脂多糖(LPS)刺激活化和过表达Nurr1基因。实验随机分为小胶质细胞组、Nurr1过表达组、LPS处理组、Nurr1过表达+LPS处理组。ELISA分析过表达Nurr1基因对不同状态小胶质细胞分泌TNF-α、IL-1等炎症相关因子和BDNF、GDNF和PDNF等神经营养因子的影响。结果:(1)小胶质细胞混合培养、纯化、CD11b/c免疫细胞化学鉴定为阳性,纯度在95%;(2)Nurr1基因过表达小胶质细胞转染阳性率90%;(3)ELISA法检测结果显示:过表达Nurr1基因的小胶质细胞显著降低了TNF-α、IL-1的分泌,并促使了BDNF、GDNF和PDNF等神经营养因子的分泌。结论:Nurr1基因可抑制小胶质细胞的活化和减少炎症相关因子的产生,同时可促进GDNF、BDNF、PDNF等与DA能神经元存活和成熟等相关的神经营养因子的分泌。     

Systemic inflammation promotes lung metastasis via E-selectin upregulation in mouse breast cancer model

Systemic inflammation might modulate the microenvironment in the lungs and promotes metastasis. E-selectin, an inflammation inducible endothelial cell adhesion molecule, has been reported to play an important role in homing metastatic cancer cells. To study the effects of E-selectin expression induced by systemic inflammation on breast cancer metastasis, we first treated BALB/c mice with lipopolysaccharide (LPS) to induce systemic inflammation. Pulmonary tissues were analyzed by wet/dry ratio, hematoxylin and eosin (H&E) staining and immunohistochemistry. Then 4T1 cells were injected via tail vein. Lung surface metastasis was counted and detected by histological analysis. LPS-induced E-selectin expression and tumor cells adhesion were assessed by western blotting and immunofluorescence. The circulating levels of proinflammatory cytokines in sera were evaluated by ELISA. Our results showed that a significant increase in breast cancer metastasis to lungs was observed in LPS-treated mice vs. the PBS-treated mice, accompanying with an increased E-selectin expression in pulmonary tissue of LPS-treated mice. In vitro studies showed a significant elevation of E-selectin production in MPVECs which enhanced the adhesion activity of 4T1 cells. Treatment with anti-E-selectin antibody significantly reduced the development of metastasis in vivo, and significantly reduced the adhesion of 4T1 cells to MPVECs in vitro. Our results suggest that systemic inflammation may increase the expression of E-selectin which mediated the lung metastasis of breast cancer in mouse model.     

Innate immune factors modulate ethanol interaction with GABAergic transmission in mouse central amygdala

Excessive ethanol drinking in rodent models may involve activation of the innate immune system, especially toll-like receptor 4 (TLR4) signaling pathways. We used intracellular recording of evoked GABAergic inhibitory postsynaptic potentials (eIPSPs) in central amygdala (CeA) neurons to examine the role of TLR4 activation by lipopolysaccharide (LPS) and deletion of its adapter protein CD14 in acute ethanol effects on the GABAergic system. Ethanol (44, 66 or 100 mM) and LPS (25 and 50 μg/ml) both augmented eIPSPs in CeA of wild type (WT) mice. Ethanol (44 mM) decreased paired-pulse facilitation (PPF), suggesting a presynaptic mechanism of action. Acute LPS (25 μg/ml) had no effect on PPF and significantly increased the mean miniature IPSC amplitude, indicating a postsynaptic mechanism of action. Acute LPS pre-treatment potentiated ethanol (44 mM) effects on eIPSPs in WT mice and restored ethanol’s augmenting effects on the eIPSP amplitude in CD14 knockout (CD14 KO) mice. Both the LPS and ethanol (44–66 mM) augmentation of eIPSPs was diminished significantly in most CeA neurons of CD14 KO mice; however, ethanol at the highest concentration tested (100 mM) still increased eIPSP amplitudes. By contrast, ethanol pre-treatment occluded LPS augmentation of eIPSPs in WT mice and had no significant effect in CD14 KO mice. Furthermore, (+)-naloxone, a TLR4-MD-2 complex inhibitor, blocked LPS effects on eIPSPs in WT mice and delayed the ethanol-induced potentiation of GABAergic transmission. In CeA neurons of CD14 KO mice, (+)-naloxone alone diminished eIPSPs, and subsequent co-application of 100 mM ethanol restored the eIPSPs to baseline levels. In summary, our results indicate that TLR4 and CD14 signaling play an important role in the acute ethanol effects on GABAergic transmission in the CeA and support the idea that CD14 and TLR4 may be therapeutic targets for treatment of alcohol abuse.     

Effects of Lipopolysaccharide on the Blood-Brain Barrier Permeability in Prolonged Nitric Oxide Blockade-Induced Hypertensive Rats

The authors investigated the effects of lipopolysaccharide (LPS) on the blood-brain barrier (BBB) integrity and the activity of astrocytes during the N^w-nitro-L-arginine methyl ester (L-NAME) hypertension followed by angiotensin (ANG) II in rats. They measured the changes in the BBB permeability using the Evans blue (EB) dye and concomitantly in the levels of TNF-a, IL-1b, and IL-6 in serum and nitric oxide in plasma. The authors performed two tight junction-specific proteins, zonula occludens-1 and occludin, and glial fibrillary acidic protein, by using immunohisto-chemical method. The serum levels of TNF-α, IL-1β, IL-6, and the plasma level of nitric oxide significantly increased in LPS-treated rats (p < .01). The EB dye extravasation increased in cerebellum (p < .001) and diencephalon (p < .05) of L-NAME plus ANG II-treated animals. However, LPS reduced the increased EB dye extravasation in the brain regions of L-NAME-induced hypertensive rats treated with ANG II (p < .001). In L-NAME, there was a considerable loss of staining in both zonula occludens-1 and occludin. Staining for zonula occludens-1 and occludin was highly intensive in animals treated with LPS. Glial fibrillary acidic protein staining was seen in a few astrocytes in brains of L-NAME-treated animals. However, this staining showed an increased intensity in the brain sections of animals treated with LPS. This study indicates that, in L-NAME hypertensive rats, ANG II leads to an increase in the extravasation of EB dye to brain as a result of decreased activity of tight junction proteins and astrocytes, and LPS could significantly attenuate the EB dye transport to the brain through the increased activity of tight junction proteins and astrocytes.     

Anti-inflammatory effects of benzenediamine derivate FC-98 on sepsis injury in mice via suppression of JNK,NF-κB and IRF3 signaling pathways

FC-98, a synthesized benzenediamine derivate, was reported to regulate Toll-like receptor 9-induced activation of dendritic cells in our previous study. In this study, we evaluated the anti-inflammatory properties of FC-98 both in macrophages and in septic mouse models. By using enzyme-linked immunosorbent assay and real-time quantitative PCR, we found that FC-98 (6.25, 25 and 100 μM) dose-dependently attenuated lipopolysaccharide (LPS)-induced tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and monocyte chemoattractant protein (MCP-1) productions in RAW264.7 and primary mouse peritoneal macrophages. These inhibitory effects were not due to inducing cell cytotoxicity or altering LPS binding or TLR4 expression. Subsequently, western blot, immunofluorescence and luciferase reporter assays were used to investigate the underlying mechanisms of its anti-inflammatory activities. Results showed that FC-98 blocked activation of the c-Jun N-terminal kinase (JNK), nuclear factor-κB (NF-κB) and interferon regulatory factor 3 (IRF3) signaling pathways. In vivo, FC-98 (30 or 100 mg/kg) was intraperitoneally administrated into LPS-induced or CLP-induced sepsis mice. It was observed to enhance the survival rate, inhibit pro-inflammatory mediator production, improve organ injuries and suppress bacterial propagation. In conclusion, FC-98 effectively inhibited macrophage inflammatory responses and ameliorated sepsis in mice through down-regulation of both MyD88 and TRIF-dependent pathways. These results suggest that FC-98 could be a promising therapeutic agent for inflammatory diseases.