云芝糖肽在ROS、p38信号通路中对巨噬细胞Raw264．7的免疫调节作用

目的：探究云芝糖hk（PSP）对静息和脂多糖（LPS）过度刺激两种不同状态的巨噬细胞Raw264．7产生活性氧簇（ROS）TLp38、p-p38的影响及其相关机制。方法：采用流式细胞术方法检测ROS的表达;Westernblot方法检测MAPK信号通路中p38磷酸化及非磷酸化的相对表达量。结果：100g／mlPSP下调1g／ml LPS单独作用于巨噬细胞时ROS及p-p38的表达量;100g／mlPSP上调正常巨噬细胞p-p38的表达量;p38表达量基本不变。结论：PSP通过ROS,p38两条通路参与巨噬细胞的免疫调节作用。     

云芝糖肽在ROS、p3 8信号通路中对巨噬细胞Raw264.7的免疫调节作用

目的:探究云芝糖肽(PSP)对静息和脂多糖(LPS)过度刺激两种不同状态的巨噬细胞Raw264.7产生活性氧簇(ROS)及p38、p-p38的影响及其相关机制。方法:采用流式细胞术方法检测ROS的表达;Western blot方法检测MAPK信号通路中p38磷酸化及非磷酸化的相对表达量。结果:100g/ml PSP下调1g/ml LPS单独作用于巨噬细胞时ROS及p-p38的表达量;100g/mlPSP上调正常巨噬细胞p-p38的表达量;p38表达量基本不变。结论:PSP通过ROS,p38两条通路参与巨噬细胞的免疫调节作用。     

Protective effect of 1,2,4-benzenetriol on LPS-induced NO production by BV2 microglial cells

Summary Hydroxyhydroquinone or 1,2,4-benzenetriol (BT) detected in the beverages has a structure that coincides with the water-soluble form of a sesame lignan, sesamol. We previously showed that sesame antioxidants had neuroprotective abilities due to their antioxidant properties and/or inducible nitric oxide synthase (iNOS) inhibition. However, studies show that BT can induce DNA damage through the generation of reactive oxygen species (ROS). Therefore, we were interested to investigate the neuroprotective effect of BT in vitro and in vivo. The results showed that instead of enhancing free radical generation, BT dose-dependently (10–100 μM) attenuated nitrite production, iNOS mRNA and protein expression in lipopolysaccharide (LPS)-stimulated murine BV-2 microglia. BT significantly reduced LPS-induced NF-κB and p38 MAPK activation. It also significantly reduced the generation of ROS in H₂O₂-induced BV-2 cells and in H₂O₂-cellfree conditions. The neuroprotective effect of BT was further demonstrated in the focal cerebral ischemia model of Sprague–Dawley rat. Taken together, the inhibition of LPS-induced nitrite production might be due to the suppression of NF-κB, p38 MAPK signal pathway and the ROS scavenging effect. These effects might help to protect neurons from the ischemic injury.     

Plant originated glycoprotein has anti-oxidative and anti-inflammatory effects on dextran sulfate sodium-induced colitis in mouse

Summary We investigated the preventive effect of glycoprotein (27 kDa) isolated from Gardenia jasminoides Ellis (GJE) fruits on colitis in dextran sulfate sodium (DSS, 3%)-induced A/J mice which were administrated orally for 7 days. Anti-inflammatory activity of GJE glycoprotein was assessed by neutrophil infiltration and colonic lipid peroxidation, and determined by myeloperoxidase (MPO) activity and levels of thiobarbituric acid reactive substances (TBARS), respectively in DSS treatment system. The activities of antioxidative enzymes [catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx)], activation of inflammation related mediators (iNOS, COX-2, and NF- B), and production of nitric oxide (NO) and reactive oxygen species (ROS) were also measured. The results of this study showed that GJE glycoprotein (g/ml) has a scavenging property to inhibit the intracellular ROS production in RAW 264.7 cells and that GJE glycoprotein (80 mg/kg BW) significantly suppressed the increase in the MPO activity, TBARS level, and NO production, inflammation related mediators [iNOS, COX-2, and NF-B (p50)] activity in DSS-induced mice. Interestingly, the activities of CAT, SOD, and GPx were gradually augmented after a supplement of GJE glycoprotein. Therefore, we suggest that GJE glycoprotein is preventive and therapeutic agent for the ulcerative colitis.     

黄芪甲苷对马兜铃酸诱导的巨噬细胞M1型极化的作用及机制研究

目的:探讨黄芪甲苷对马兜铃酸诱导的RAW264.7细胞向M1型极化的影响,并初步探索其可能的作用机制.方法:分别采用马兜铃酸和脂多糖(LPS)刺激RAW264.7细胞24h,伴或不伴黄芪甲苷进行药物干预处理.采用细胞计数检测试剂盒-8(CCK8)检测细胞活性变化,流式细胞仪检测巨噬细胞分型,酶联免疫吸附试验(ELISA...     

The Pseudomonas aeruginosa quorum-sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone stimulates phagocytic activity in human macrophages through the p38 MAPK pathway

Quorum-sensing is an important mechanism for the regulation of bacteria-to-bacteria communication. Recent advances have demonstrated that the Pseudomonas aeruginosa signaling molecule N-(3-oxododecanoyl)-L-homoserine lactone (3O-C(12)-HSL) is also a potent modulator of eukaryotic cells and may thus play an important role in the host response during P. aeruginosa infections. Little is known, however, about specific effects of 3O-C(12)-HSL molecules on human macrophages. To address this issue, we investigated the influence of 3O-C(12)-HSL on the phagocytic activity, production of reactive oxygen species, and activation of p38 and p42/44 MAPK signaling pathways in human macrophages. We show an effect of 3O-C(12)-HSL on the phagocytic capacity in human macrophages, which depends on concentration and time of exposure. When cells were exposed to 100 microM 3O-C(12)-HSL for 30 min or 1 h, the phagocytic activity increased 1.8 and 1.6 times, respectively. The 3O-C(12)-HSL treatments had no significant effect on the level of reactive oxygen species production. Furthermore, the p38 MAPK, but not the p42/44 MAPK, signaling pathway was activated in response to 3O-C(12)-HSL. In addition, specific blocking of p38 MAPK activation with 10 microM SB 203580 prevented the 3O-C(12)-HSL-induced increase in the phagocytic activity. These findings demonstrate that the bacterial quorum-sensing can play a significant role also in regulation of macrophage activity during infections caused by P. aeruginosa.     

In Vitro evaluation of the cytotoxic and anti-proliferative properties of resveratrol and several of its analogs

Resveratrol (RES), a component of red wine, possesses anti-inflammatory properties. The studies described in the present work were aimed at evaluating the potential for RES and related stilbene analogs (piceatannol, PIC; pterostilbene, TPS; trans-stilbene, TS; and trans-stilbene oxide, TSO) to exhibit toxicity towards RAW 264.7 mouse macrophages. The effect of TS, TSO, RES and TPS on RAW 264.7 macrophage viability was determined by two standard methods: (a) the MTT assay and (b) the trypan blue dye exclusion test. Whereas macrophages were more sensitive to PIC (LC_{50 trypan} ∼ 1.3 μM) and to TPS (LC_{50 trypan} ∼ 4.0 μM and LC_{50 MTT} ∼ 8.3 μM) than to RES (LC_{50 trypan} ∼ 8.9 μM and LC_{50 MTT} ∼ 29.0 μM), they were relatively resistant to TSO (LC_{50 trypan} ∼ 61.0 μM and LC_{50 MTT} > 100 μM) and to TS (LC_{50 trypan} ≥ 5.0 μM and LC_{50 MTT} ≥ 5.0 μM). The ability of selected stilbenes (RES, TPS and PIC) to exhibit growth inhibitory effects was also examined. Although RES and TPS were observed to inhibit cell proliferation in macrophages (IC₅₀ ≤ 25 μM), these cells were resistant to growth inhibition by PIC (IC₅₀ ≥ 50 μM). The data obtained in the present analysis demonstrate that substituted stilbene compounds such as RES have the capacity to exhibit cytotoxic and anti-proliferative activities in macrophages.     

NF-κB modulates activation of the <Emphasis Type="Italic">BMP-2</Emphasis> gene by trichostatin A

In this study, trichostatin A (TSA), a histone deacetylase inhibitor, increased the Bone morphogenetic protein-2 (BMP-2) mRNA level in a human osteoblasts line. Deletion analysis of the promoter region revealed that TSA-induced luciferase was regulated by the BMP-2 promoter spanning from −320 to −310. Electrophoresis mobility shift assay (EMSA) and Chromatin immunoprecipitation (CHIP) assay proved that this position was a nuclear factor (NF)-κB responsive element. The results above demonstrated that acetylation plays a crucial role in BMP-2 expression, and acetylation of NF-κB p65 and p50 subunits by TSA treatment may activate the BMP-2 promoter. Published in Russian in Molekulyarnaya Biologiya, 2008, Vol. 42, No. 6, pp. 984–989. The text was submitted by the authors in English.     

Anti-sense morpholino oligonucleotide assay shows critical involvement for NF-κB activation in the production of Wnt-1 protein by HepG2 cells: oncology implications

The link of proto-oncogenic protein Wnt-1 production with NF-κB activation has been functionally demonstrated in PC12 cells, a rat pheochromocytoma cell line of neural crest lineage, while it is not yet verified in human cells. The link can be indirectly supported in our previous report that functional proteomics identifies enhanced expression of NF-κB-associated Wnt-1 production in human hepatocellular carcinoma tissues. This study aimed to further validate this link in human cells using anti-sense strategy. The effects of sequence-specific anti-sense morpholino oligonucleotides (ONs) targeting against pre-mRNA sequences of human p50 and p65 subunits of NF-κB as well as Wnt-1 genes were investigated. It revealed that all the three morpholino ONs inhibited NF-κB activation in human hepatoblastoma cell line HepG2 cells along with decreased Wnt-1 production. Chromatin immunoprecipitation assay ascertained the direct binding of NF-κB-p50 to the Wnt-1 promoter. Additionally, anti-P50 and anti-P65 morpholino ONs also repressed the phosphorylation of Iκ Bα which temporarily correlated with the inhibition of NF-κB activation accompanied by decreased Wnt-1 production by HepG2 cells. In summary, NF-κB activation is critically involved in the production of Wnt-1 by HepG2 cells. These results may have important oncology implications in treating patients with NF-κB-associated Wnt-1-producing cancers. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Dual knockdown of p65 and p50 subunits of NF-kappaB by siRNA inhibits the induction of inflammatory cytokines and significantly enhance apoptosis in human primary synoviocytes treated with tumor necrosis factor-alpha

In order to develop an anti-NF-kappaB siRNA as a novel class of anti-inflammatory drug, we have isolated a highly efficient siRNA targeting the p65 (RelA) subunit of NF-kappaB, hereafter named REL1096. To determine whether down-regulation of p65 by REL1096 can block the induction of inflammatory cytokines after treatment with tumor necrosis factor-alpha (TNF-alpha), human primary fibroblast-like synoviocytes were isolated from patients with rheumatoid arthritis. When treated with REL1096, the TNF-mediated induction of downstream target genes such as inflammatory cytokines, chemokines, and anti-apoptosis genes was drastically inhibited. To enhance the inhibitory effect of REL1096, cells were treated with siRNA targeting the p50 subunit of NF-kappaB together with REL1096. In addition to effective downregulation of inflammatory cytokines, knockdown of both p65 and p50 resulted in much more extensive apoptosis when compared to cells treated with either REL1096 or p50-siRNA alone. Thus, our results provide evidence for the potential use of siRNA targeting NF-kappaB as an effective means to treat rheumatoid arthritis. In addition to effective amelioration of synovial inflammation by downregulation of inflammatory cytokines, increased apoptosis by dual knockdown of p65 and p50 may prove advantageous in preventing invasiveness and destructiveness of hyperplastic synoviocytes.     

Immunostimulatory Activities of Theobromine on Macrophages via the Activation of MAPK and NF-κB Signaling Pathways

Theobromine is mainly found in plant foods, such as tea; the primary source of theobromine is the seeds of the Theobroma cacao tree. Theobromine is an alkaloid belonging to the methylxanthine class of drugs, and it is similar to theophylline and caffeine. Theobromine is known for its efficacy and role in health and disorder prevention. We evaluated the effects of theobromine on macrophage function, including the phosphorylation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB). Theobromine significantly stimulated the production of nitric oxide (NO) and prostaglandin E2 through immune responses, which relate to the increased expression of inducible nitric oxide synthase and cyclooxygenase-2. Additionally, theobromine increased the production of inflammatory cytokines, including tumor necrosis factor-α and interleukin-6 in macrophages. Additionally, theobromine induced the translocation and activity of NF-κB in a concentration-dependent manner. Consistent with these results, the phosphorylation level of MAPKs was increased in theobromine-stimulated macrophages. Collectively, these data revealed that theobromine acts as an immune response stimulator via the NF-κB and MAPKs signaling pathways. Thus, theobromine might have protective effects against inflammatory disorders.     

Effects of marginal iron overload on iron homeostasis and immune function in alveolar macrophages isolated from pregnant and normal rats

The effects of changes in macrophage iron status, induced by single or multiple iron injections, iron depletion or pregnancy, on both immune function and mRNA expression of genes involved in iron influx and egress have been evaluated. Macrophages isolated from iron deficient rats, or pregnant rats at day 21 of gestation, either supplemented with a single dose of iron dextran, 10 mg, at the commencement of pregnancy, or not, showed significant increases of macrophage ferroportin mRNA expression, which was paralleled by significant decreases in hepatic Hamp mRNA expression. IRP activity in macrophages was not significantly altered by iron status or the inducement of pregnancy ± a single iron supplement. Macrophage immune function was significantly altered by iron supplementation and pregnancy. Iron supplementation, alone or combined with pregnancy, increased the activities of both NADPH oxidase and nuclear factor kappa B (NFκB). In contrast, the imposition of pregnancy reduced the ability of these parameters to respond to an inflammatory stimuli. Increasing iron status, if only marginally, will reduce the ability of macrophages to mount a sustained response to inflammation as well as altering iron homeostatic mechanisms.     

In Vitro Effects of SB202190 on Echinococcus granulosus

Spillage of cyst contents during surgical operation is the major cause of recurrence after hydatid cyst surgery. Instillation of a scolicidal agent into a hepatic hydatid cyst is the most commonly employed measure to prevent this complication. SB202190 is a pyridinyl imidazole derivative and is known to be a specific inhibitor of p38 MAPK. In the present study, the scolicidal effect of SB202190 was investigated. Freshly isolated Echinococcus granulosus protoscolices were subjected to SB202190 treatment (10, 20, 40, and 80 µM), and the effects on parasite viability were monitored by trypan blue staining. Corresponding effects were visualized by scanning and transmission electron microscopy. Dose-dependent protoscolex death within a few days of SB202190 treatment was observed. Although the in vitro scolicidal effect of SB202190 was satisfactory, the in vivo efficacy of this drug and also possible side effects remain to be further investigated.