ERK 1/2介导结缔组织生长因子刺激系膜细胞产生MCP-1

目的检测结缔组织生长因子（CTGF）是否刺激大鼠肾小球系膜细胞分泌单核细胞趋化蛋白-1（MCP-1）,并探讨其作用机制。方法应用CTGF刺激静息的培养的系膜细胞,在刺激后不同时间点应用RT-PCR方法测定MCP-1的mRNA表达,应用酶联免疫吸附试验（ELISA）测定上清液中MCP-1,应用趋化试验测定上清液对单核细胞（THP-1）的趋化作用。应用Western blot测定CTGF对细胞外信号调节激酶（ERK）1／2磷酸化的作用。应用ERK1／2抑制剂PD98059预处理,观察CTGF对上清液中MCP-1分泌的影响。结果应用CTGF刺激后,系膜细胞的MCP-1的mRNA表达上升,上清液中分泌量增加。MCP-1抗体可部分阻止上清液对单核细胞的趋化作用。CTGF诱导ERK1／2磷酸化,而PD98059可抑制这一作用,并部分抑制CTGF诱导的上清液中MCP-1的分泌。结论CTGF可引起系膜细胞分泌MCP-1,其作用机制部分依赖于ERK1／2的磷酸化。     

p42/44 MAPK介导结缔组织生长因子刺激系膜细胞合成分形素

目的检测结缔组织生长因子(CTGF)是否可诱导大鼠肾小球系膜细胞分泌趋化因子分形素(fractalkine,FLK)并探讨其作用机制。方法应用CTGF刺激培养的静息系膜细胞,在刺激后不同时间点,应用RT-PCR方法测定FLKmR-NA的表达,应用ELISA法测定系膜细胞培养上清液中FLK的水平。应用趋化试验测定系膜细胞的培养上清液对单核细胞THP-1的趋化作用。应用Western blot测定CTGF对系膜细胞中磷酸化的蛋白激酶(p42/44MAPK)表达的作用。应用p42/44MAPK抑制剂PD98059或UO126预处理,观察CTGF对该上清液中FLK分泌的影响。结果应用CTGF刺激后,系膜细胞中FLK mRNA的表达上升,其培养上清液中FLK的含量增加。抗FLK抗体可部分地阻止系膜细胞的培养上清液对单核细胞的趋化作用。CTGF可诱导p42/p44MAPK磷酸化,PD98059或UO126可抑制这一作用,并部分抑制CTGF诱导的系膜细胞培养上清液中FLK的分泌。结论CTGF可刺激系膜细胞分泌FLK,其作用机制与p42/p44MAPK的磷酸化有关。     

Membrane-bound form of fractalkine induces IFN-gamma production by NK cells

Natural killer (NK) cells participate in both innate and adaptive immunity, in part by their prompt secretion of cytokines including IFN-gamma, a pro-inflammatory cytokine with an important role in Th1 polarization. To assess the involvement of fractalkine in inflammatory processes, we examined the effect of fractalkine on IFN-gamma production by NK cells. Although soluble chemokines, including MCP-1 and RANTES as well as fractalkine, had a negligible effect on IFN-gamma production, immobilized fractalkine markedly induced IFN-gamma production by NK cells in a dose-dependent manner. Pretreatment of NK cells with the phosphatidylinositol 3-kinase (PI 3-K) inhibitor, wortmannin, completely inhibited the production of IFN-gamma induced by fractalkine, and pretreatment with the protein tyrosine kinase inhibitor, herbimycin A, partially suppressed the response, suggesting that augmentation of IFN-gamma production in response to fractalkine treatment of NK cells involves signaling through PI 3-K and protein tyrosine kinases. Furthermore, co-culture of NK cells with fractalkine-transfected 293E cells markedly enhanced IFN-gamma production by NK cells compared with co-culture with control 293E cells. These findings may indicate a paracrine feedback loop system in which endothelial cells may be activated to produce more fractalkine, and also suggest a role for fractalkine expressed on endothelial cells in Th1 polarization through the stimulation of IFN-gamma production by NK cells.     

结缔组织生长因子促进肾小球系膜细胞合成RANTES部分依赖于p42/44 MAPK

检测结缔组织生长因子(CTGF)是否诱导肾小球系膜细胞分泌正常T细胞表达分泌的活化调节因子(RANTES),并探讨其作用机制。应用CTGF刺激静息的培养大鼠肾小球系膜细胞,在刺激后不同时间点应用RT-PCR方法测定RANTES的mRNA表达,应用酶联免疫吸附试验(ELISA)测定上清液中RANTES。应用趋化试验测定上清液对单核细胞(THP-1)的趋化作用。应用Westernblot测定CTGF对丝裂原激活的蛋白激酶(p42/44MAPK)磷酸化的作用。应用磷酸化p42/44MAPK抑制剂PD98059预处理,观察CTGF对上清液中RANTES分泌的影响。结果显示,应用CTGF(100ng/ml)刺激后,系膜细胞的RANTES的mRNA表达上升,上清液中RANTES分泌量增加。RANTES抗体可部分阻止上清液对单核细胞的趋化作用。CTGF诱导p42/p44MAPK磷酸化,而PD98059可抑制这一作用,并部分抑制CTGF诱导的上清液中RANTES的分泌。研究表明,CTGF可引起系膜细胞分泌RANTES,其作用机制部分依赖于p42/p44MAPK的磷酸化。     

PKB在类胰蛋白酶诱导基因表达中的作用

目的：探讨蛋白激酶B(PKB)在类胰蛋白酶（tryptase）诱导基因表达中的作用。 方法： 采用RT-PCR和Western blotting 方法，检测tryptase对ECV304细胞PKB（Akt）的表达及其活性和对转录因子AP-1、NF-κB p65亚单位、JNK、p38MAPK、趋化因子IL-8表达的影响。 结果： 在ECV304中1 μg/L tryptase可使PKB蛋白质磷酸化水平增加并促进PKB、转录因子NF-κB P65亚单位、AP-1和趋化因子IL-8的表达，但对JNK、p38MAPK表达影响不大。PI3K特异性抑制剂LY294002可抑制PKB的表达增加，同时可抑制NF-κB P65亚单位和IL-8的表达增加；反义PKB质粒瞬时转染ECV304，可抑制PKB、AP1、NF-κB P65亚单位和IL-8的表达增加；PAR2的抗体可抑制PKB的磷酸化，但不能阻断PKB表达。 结论： 在ECV304细胞tryptase经其膜受体PAR2通过PI3K促进PKB的磷酸化而激活之，通过其下游途径促进趋化因子IL-8、转录因子AP-1、NF-κB P65亚单位和PKB本身的表达增加。     

Role of chemokines in promoting instability of coronary atherosclerotic plaques and the underlying molecular mechanism

Our aim was to investigate the role of chemokines in promoting instability of coronary atherosclerotic plaques and the underlying molecular mechanism. Coronary angiography and intravascular ultrasound (IVUS) were performed in 60 stable angina pectoris (SAP) patients and 60 unstable angina pectoris (UAP) patients. The chemotactic activity of monocytes in the 2 groups of patients was examined in Transwell chambers. High-sensitivity C-reactive protein (hs-CRP), monocyte chemoattractant protein-1 (MCP-1), regulated on activation in normal T-cell expressed and secreted (RANTES), and fractalkine in serum were examined with ELISA kits, and expression of MCP-1, RANTES, and fractalkine mRNA was examined with real-time PCR. In the SAP group, 92 plaques were detected with IVUS. In the UAP group, 96 plaques were detected with IVUS. The plaques in the UAP group were mainly lipid 51.04% (49/96) and the plaques in the SAP group were mainly fibrous 52.17% (48/92). Compared with the SAP group, the plaque burden and vascular remodeling index in the UAP group were significantly greater than in the SAP group (P<0.01). Chemotactic activity and the number of mobile monocytes in the UAP group were significantly greater than in the SAP group (P<0.01). Concentrations of hs-CRP, MCP-1, RANTES, and fractalkine in the serum of the UAP group were significantly higher than in the serum of the SAP group (P<0.05 or P<0.01), and expression of MCP-1, RANTES, and fractalkine mRNA was significantly higher than in the SAP group (P<0.05). MCP-1, RANTES, and fractalkine probably promote instability of coronary atherosclerotic plaque.     

Distinct compartmentalization of NF-κB activity in crypt and crypt-denuded lamina propria precedes and accompanies hyperplasia and/or colitis following bacterial infection

Citrobacter rodentium induces transmissible murine colonic hyperplasia (TMCH) and variable degrees of inflammation and necrosis depending upon the genetic background. Utilizing C. rodentium-induced TMCH in C3H/HeNHsd inbred mice, we observed significant crypt hyperplasia on days 3 and 7 preceding active colitis. NF-κB activity in the crypt-denuded lamina propria (CLP) increased within 24 h postinfection, followed by its activation in the crypts at day 3, which peaked by day 7. Increases in interleukin-α1 (IL-1α), IL-12(p40), and macrophage inflammatory protein 1α (MIP-1α) paralleled NF-κB activation, while increases in IL-1α/β, IL-6/IL-12(p40)/granulocyte colony-stimulating factor (G-CSF)/keratinocyte-derived chemokine (KC)/monocyte chemotactic protein 1 (MCP-1), and MIP-1α followed NF-κB activation leading to significant recruitment of neutrophils to the colonic mucosa and increased colonic myeloperoxidase (MPO) activity. Phosphorylation of the crypt cellular and nuclear p65 subunit at serines 276 and 536 led to functional NF-κB activation that facilitated expression of its downstream target, CXCL-1/KC, during TMCH. Distinct compartmentalization of phosphorylated extracellular signal-regulated kinase 1 and 2 ([ERK1/2] Thr(180)/Tyr(182)) and p38 (Thr(202)/Tyr(204)) in the CLP preceded increases in the crypts. Inhibition of ERK1/2 and p38 suppressed NF-κB activity in both crypts and the CLP. Dietary administration of 6% pectin or 4% curcumin in C. rodentium-infected mice also inhibited NF-κB activity and blocked CD3, F4/80, IL-1α/β, G-CSF/MCP-1/KC, and MPO activity in the CLP while not affecting NF-κB activity in the crypts. Thus, distinct compartmentalization of NF-κB activity in the crypts and the CLP regulates crypt hyperplasia and/or colitis, and dietary intervention may be a novel strategy to modulate NF-κB-dependent protective immunity to facilitate crypt regeneration following C. rodentium-induced pathogenesis.     

Aspirin Inhibits LPS-Induced Expression of PI3K/Akt,ERK, NF-κB,CX3CL1, and MMPs in Human Bronchial Epithelial Cells

This study focused on the effects of aspirin on lipopolysaccharide (LPS)-induced expression of phosphoinositide 3 kinase (PI3K)/protein kinase B (Akt), extracellular signal-regulated protein kinase (ERK), nuclear factor-κB (NF-κB), CX3CL1, and MMPs in human bronchial epithelial cells. Human bronchial epithelial cells were seeded in six-well plates. After 24 h, the cells were classified into six groups: control blank (CK) group; LPS group; PD98059 (ERK inhibitor) (PD) group, treated with LPS + ERK inhibitor; LY294002(PI3K/Akt inhibitor) (LY) group, treated with LPS + PI3K/Akt inhibitor; Aspirin (Asp) group, treated with LPS + aspirin; and Pyrrolidinedithiocarbamic acid (PDTC) group, treated with LPS + NF-κB inhibitor. After 4-h treatment, the cells were harvested. Western blot analysis was performed to detect the expression of PI3K/Akt, ERK, NF-κB, and CX3CL1. Real-time quantitative PCR (RT-qPCR) was used to determine the gene expression of MMP-7, MMP-9, and MMP-12. Compared to the CK group, expression of PI3K/Akt, ERK, NF-κB, and CX3CL1 was significantly increased in the LPS group (P?<?0.05). When compared to the LPS group, expression of PI3K/Akt, ERK, NF-κB, and CX3CL1 was significantly decreased in the PD group, PDTC group, and Asp group (P?<?0.05). In addition, expression of NF-κB in the LY group was significantly reduced by comparison with the LPS group (P?<?0.05). RT-qPCR: When compared to the LPS group, expression of MMP-7 and MMP-12 was significantly decreased in Asp group (P?<?0.05). Expression of MMP-12 was significantly reduced in LY group (P?<?0.05). LPS-ERK, NF-κB-PI3K/Akt, and CX3CL1 signal pathways exist in human bronchial epithelial cells. The PI3K/Akt inhibitor repressed expression of MMP-12. Aspirin inhibited LPS-induced expression of PI3K, Akt, ERK, NF-κB, CX3CL1, MMP-7, and MMP-12 in human bronchial epithelial cells.     

Chikusetsusaponin V attenuates lipopolysaccharide-induced liver injury in mice

Chikusetsusaponin V (CsV), a saponin from Panax japonicus, has been reported to inhibit inflammatory responses in lipopolysaccharide (LPS)-induced macrophage cells. However, whether CsV could alleviate LPS-induced liver injury in vivo and the potential mechanisms involved remain unclear. In the present study, we investigated the anti-inflammatory effects of CsV on LPS-induced acute liver injury in mice and further explored the potential mechanisms involved. Our results showed that CsV significantly attenuated elevation of alanine transaminase (ALT) and aspartate aminotransferase (AST) levels and improved liver histopathological changes in LPS-induced mice. In addition, CsV decreased serum tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) levels and inhibited mRNA expressions of inducible nitric oxide synthase (iNOS), TNF-α and IL-1β in LPS challenged mice. Furthermore, CsV inhibited nuclear factor kappa B (NF-κB) activation by downregulating phosphorylated NF-κB, IκB-α, ERK, c-Jun N-terminal kinase (JNK) and p38 levels in the liver tissue, which ultimately decreased nucleus NF-κB protein level. In conclusion, our data suggested that CsV could be a promising drug for preventing LPS challenged liver injury since it attenuated LPS-induced inflammatory responses, partly via inhibiting NF-κB and MAPK signaling pathways.     

Quercetin suppresses NF-κB and MCP-1 expression in a high glucose-induced human mesangial cell proliferation model

Diabetic nephropathy (DN), which is characterized by mesangial cell proliferation, is a common complication observed in diabetic patients. The protective effects of quercetin for DN have been reported; however, the mechanism has yet to be determined. We aimed to identify the underlying mechanism for quercetin protection against DN. High glucose (HG)-induced human mesangial cell (HMC) proliferation, a feature of the early stages of diabetic nephropathy, was employed as an in vitro model. Cells were grown in normal glucose (5.6 mM), high glucose (30 mM) or high glucose with various concentrations of quercetin. Cell proliferation, cell cycle progression, and expression of NF-κB and MCP-1 were examined by MTT assay, DNA staining, immunocytochemistry and western blot analysis, respectively. HMCs cultured in high glucose had signficantly greater proliferation, accumulation in the G1 phase, upregulated NF-κB and MCP-1 expression. Quercetin treatment reversed the effects of high glucose in a dose-dependent manner. Cotreatment of quercetin with pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB activation, suggest that the effects of quercetin are partially mediated by NF-κB signaling. Quercetin partially suppresses the effects of high glucose in HMC cultures, which are mediated at least in part through the suppression of NF-κB.     

髓样细胞激活受体2调控氧糖剥夺/复氧模型小鼠小胶质细胞向M2型极化

目的 探讨TREM2调控氧糖剥夺/复氧(OGD/R)模型小鼠小胶质细胞向M2型极化的机制.方法体外培养N9小胶质细胞系,采用TREM2过表达慢病毒(LV-TREM2)转染N9细胞,空载病毒LV-scramble作为对照组,并建立OGD/R模型.将OGD/R细胞随机分为OGD/R组、OGD/R+LV-scramble组和...     

Actions of the chemotactic cytokines MCP-1, MCP-2, MCP-3, RANTES,MIP-1α and MIP-1β on human monocytes

The activities of six synthetic CC chemokines, MCP-1, MCP-2, MCP-3, RANTES, MIP-1α and MIP-1β on human blood monocytes were studied. All CC chemokines elicited a bimodal migration response in vitro. Highest numbers of migrating cells were obtained with the monocyte chemotactic proteins (MCP) and RANTES, somewhat lower numbers with MIP-1α, and only weak migration with MIP-1β. The most potent attractants were MCP-1 and MIP-1α which reached maximum efficacy at 0.1 to 1 nM. All CC chemokines also induced the release of N-acetyl-β-D-glucosaminidase from cytochalasin B-pretreated monocytes. The MCP were most effective (MCP-1 > MCP-3 > MCP-2), RANTES and MIP-1α showed moderate (1/3 of MCP-1 activity), and MIP-1β only minimal activity. Cytosolic free Ca²⁺ changes and exocytosis were used to monitor receptor desensitization. Marked cross-desensitization was observed among MCP-1, MCP-2 and MCP-3 on the one hand, and RANTES, MIP-1α and MIP-1β on the other, indicating receptor sharing within these two subgroups of CC chemokines. The responses to RANTES, MIP-1α and MIP-1β were also moderately to markedly desensitized by pretreatment with MCP-1, MCP-2 or MCP-3, while the responses to the MCP were virtually unaffected by pretreatment with RANTES, MIP-1α and MIP-1β. These results suggest that the MCP also interact with receptors recognized by RANTES, MIP-1α and MIP-1β, but not vice versa. Binding studies were performed with radiolabeled MCP-1 or MIP-1α. All MCP competed readily for labeled MCP-1 yielding a concentration-dependent sigmoidal displacement curve. Displacement with RANTES, MIP-1α and MIP-1β was observed at higher concentrations, but was not complete. Radiolabeled MIP-1α was displaced efficiently by MIP-1α or MIP-1β, but only partially by RANTES. Of the MCP, only MC-3 completely displaced MIP-1α, while only partial displacement was observed with MCP-1 and MCP-2.     

核因子-κB活化参与Ox-LDL诱导人肾小球系膜细胞表达单核细胞趋化蛋白-1(英文)

目的：研究核因子-κB(NF-κB)在氧化低密度脂蛋白(Ox-LDL)诱导的体外培养的人肾小球系膜细胞表达单核/巨噬细胞趋化蛋白-1(MCP-1)中的作用。方法： 采用凝胶迁移率变动分析检测NF-κB的DNA结合活性变化，以免疫组化观测细胞内REL P65的核转位，用细胞ELISA法检测细胞内MCP-1及IκBα蛋白含量变化。结果： 不同浓度(10、25、50、100 mg/L)Ox-LDL刺激肾小球系膜细胞均可引起细胞NF-κB的DNA结合活性增强，50 mg/L Ox-LDL活化MCs效果最明显(8.50±1.14，P＜0.01 vs control； P＜0.05 vs 10, 25和100 mg/L Ox-LDL)。Ox-LDL刺激MCs 30-240 min均可以活化NF-κB，60 min时相点活性最强(11.0±2.11，P＜0.01 vs control； P＜0.05 vs 30 min or 240 min)。以50 mg/L Ox-LDL刺激MCs 1 h后，细胞内IκBα蛋白水平最低(0.050±0.006，n=5,P＜0.01 vs control)，作用24 h MCP-1表达水平最高(0.331±0.016， n=5,P＜0.01 vs control)。NF-κB活化的同时伴有REL P65核转位。上述效应可被NF-κB特异性抑制剂吡咯二硫氨基甲酸酯(PDTC)所抑制。结论： Ox-LDL刺激人肾小球系膜细胞产生MCP-1是由NF-κB调控，NF-κB参与了脂质肾损害的发病过程。     

卡维地洛对高血压大鼠主动脉的保护作用

目的 :研究卡维地洛对核因子 κB(NF κB)、单核细胞趋化蛋白 1(MCP 1)在自发性高血压大鼠 (SHR)大血管中表达的影响 ,探讨其对血管保护的机制。方法 :18只雄性 12周龄SHR随机分为阳性组 ,卡维地洛组 ( 30mg/kg·d) ,美托洛尔组 ( 50mg/kg·d) ,灌喂 8周 ;另选同龄雄性WistarKyoto大鼠为阴性组 (n =6 )。用免疫组化法测各组主动脉NF κB、MCP 1的表达 ,ELISA法测血清MCP 1含量。结果 :与阴性组比较 ,阳性组主动脉组织中NF κB、MCP 1表达增加 (P<0 .0 1) ,且两者正相关 (r=0 .72 8,P <0 .0 1) ;血清MCP 1含量升高 1.6 4(P <0 .0 1)。 8周后 ,治疗组之间血压无明显差异 (P >0 .0 5) ,但卡维地洛更显著抑制NF -κB、MCP 1表达 (P <0 .0 5)。结论 :卡维地洛可能独立于降压外抑制NF κB的活化来调控MCP 1表达。     

核转录因子-κB在实验性肝纤维化中的表达、分布及意义

目的:研究核转录因子-κB(NF-κB)在实验性肝纤维化过程中的表达、分布及意义。方法:雌性Wistar大鼠随机分为正常组、肝纤维化模型组和NF-κB抑制剂吡咯烷二硫代氨基甲酸盐(PDTC)组,PDTC组大鼠在给CCl4的同时给予PDTC灌胃。肝组织羟脯氨酸(Hyp)含量测定,鲎试剂法测定血浆内毒素水平,赖氏法测定丙氨酸氨基转移酶(ALT),TBA法检测丙二醛(MDA)的水平,免疫组化法测定NF-κB的表达,Western blot-ting检测结缔组织生长因子(CTGF)的表达。结果:免疫组化显示NF-κBp65在正常肝组织中少量表达于中央静脉周围的肝细胞胞浆内,肝纤维化模型组肝组织可见程度不等的细胞浆和(或)细胞核表达,主要分布于纤维化区、肝窦区、血管壁及部分胆管细胞、变性肝细胞也可见阳性染色,阳性细胞数从第1周末就显著高于正常对照组,PDTC组阳性细胞数明显低于同期肝纤维化模型组(P0.05);肝纤维化模型组内毒素含量、NF-κB以及CTGF表达明显升高,且两两均呈正相关;PDTC组内毒素含量呈先升高后降低趋势,NF-κBp65和CTGF表达显著低于同期肝纤维化模型组,且二者呈正相关(r=0.815,P0.01),内毒素与NF-κBp65和CTGF表达无相关性。结论:内毒素可能通过激活NF-κB通路上调CTGF的表达。