退银汤对寻常性银屑病患者外周血及皮损中CCL17的影响

目的：探讨退银汤对寻常性银屑病外周血及皮损中胸腺和调控活化的趋化因子（thymus and activation regulated chemokine,TRAC,又称CC chemokine ligand 17,CCL17）表达水平的影响。方法：采用双抗体夹心酶联免疫吸附法检测50例寻常性银屑病患者在使用退银汤治疗前后外周血血清CCL17水平,与40例正常对照组进行比较;另检测30例寻常性银屑病患者在使用退银汤治疗前后皮损组织液CCL17水平,与20例正常对照组进行比较。结果：治疗后,寻常性银屑病患者血清和皮损组织液CCL17表达水平分别与治疗前比较,差异均有统计学意义（P＜0.01）;治疗前、后进行期与稳定期银屑病患者外周血CCL17分别比较,差异也均有统计学意义（P＜0.01）,但治疗前进行期与稳定期银屑病患者皮损组织液CCL17表达水平比较,差异有统计学意义（P＜0.01）;而治疗后比较,差异无统计学意义（P＞0.05）。以上CCL17表达水平均与患者相应的PASI呈正相关。结论：退银汤可以降低寻常性银屑病血清及皮损组织液中CCL17的表达水平。     

Rosmarinic acid as a downstream inhibitor of IKK-beta in TNF-alpha-induced upregulation of CCL11 and CCR3

1. Tumor necrosis factor (TNF)-alpha is known to induce the expression of CCL11 and CCR3 via the activation of NF-kappaB. CCL11 (eotaxin), the C-C chemokine, is a potent chemoattractant for eosinophils and Th2 lymphocytes, and CCR3 is the receptor for CCL11. 2. In order to determine the effects of rosmarinic acid on the TNF-alpha-induced upregulation of CCL11 and CCR3 in human dermal fibroblasts, we performed an enzyme-linked immunosorbent assay for CCL11 and a Western blot assay for CCR3. The TNF-alpha-induced expression of CCL11 and CCR3 genes was attenuated by rosmarinic acid. 3. In our NF-kappaB luciferase reporter system, TNF-alpha-induced NF-kappaB activation was observed to be reduced by rosmarinic acid. In accordance with this result, rosmarinic acid also inhibited TNF-alpha-induced phosphorylation and degradation of IkappaB-alpha, as well as nuclear translocation of NF-kappaB heterodimer induced by TNF-alpha. This suggests that rosmarinic acid downregulates the expression of CCL11 and CCR3 via the inhibition of NF-kappaB activation signaling. 4. Using the NF-kappaB luciferase reporter system, Western blot analysis, and IKK-beta activity assay, we determined that rosmarinic acid inhibits IKK-beta activity in NF-kappaB signaling, which upregulates the expression of CCL11 and CCR3. Additionally, TNF-alpha-induced secretion of soluble intercellular adhesion molecule-1 and soluble vascular cell adhesion molecule-1 molecules was found to be attenuated by rosmarinic acid. 5. Our results show that rosmarinic acid inhibits the expression of CCL11 and CCR3 by suppressing the IKK-beta activity in NF-kappaB activation signaling. Further, these results suggest that rosmarinic acid might inhibit the expression of NF-kappaB promoter-related genes.     

Comparison of the anti-inflammatory activities of imidazole antimycotics in relation to molecular structure

The objective of this study was to compare the anti-inflammatory potencies of the imidazole antimycotics, fluconazole, itraconazole, ketoconazole and voriconazole (0.5 and 5 microM) in relation to their molecular structures. Anti-inflammatory activity was determined according to the magnitude of inhibition of production of leukotriene B4 and influx of Ca2+ following activation of the cells with the chemo-attractant platelet-activating factor (200 nM), using enzyme-linked immunosorbent assay and spectrofluorometric procedures, respectively. Treatment of platelet-activating factor-activated neutrophils with the imidazole antimycotics resulted in inhibition of production of leukotriene B4 and attenuation of Ca2+ influx, the order of potency being itraconazole > ketoconazole > fluconazole = voriconazole. These observations demonstrate the requirement for both the diazole/triazole moiety (all four agents), and the substituted phenylpiperazinyl ether side chain (itraconazole and ketoconazole only) for maximal anti-inflammatory activity of this class of pharmacological agents.     

Effect of cytochrome P-450 inhibitors econazole, bifonazole and clotrimazole on prostanoid formation

1. The present study was carried out to clarify the effect of the imidazole antimycotics econazole, bifonazole and clotrimazole on prostanoid biosynthesis. Osteoblast-like MC3T3-E1 cells stimulated by endothelin-1, melittin, ionomycin or arachidonic acid showed diminished prostaglandin E(2) (PGE(2)) production upon pretreatment with econazole. Following pretreatment with bifonazole, stimulation with ionomycin or arachidonic acid also resulted in decreased PGE(2) formation. Clotrimazole inhibited ionomycin but not arachidonic acid stimulated PGE(2) synthesis in MC3T3-E1 cells. 2. The results observed in osteoblast-like UMR-106 cells pretreated with econazole, bifonazole or clotrimazole and stimulated by arachidonic acid were similar with the exception of clotrimazole which was a more effective inhibitor of PGE(2) biosynthesis than in MC3T3-E1 cells. 3. Upon treatment with arachidonic acid thromboxane B(2) (TXB(2)) production in human platelets was abolished completely at concentrations of the three imidazole antimycotics higher than 5 microM (IC(50)<1 microM). 4. These data were confirmed by a direct assay using purified ram seminal vesicle prostaglandin H(2) synthase-1 (PGHS-1), which clearly showed inhibitory properties of econazole (IC(50) 4.7+/-2.3 microM), bifonazole (IC(50) 9.4+/-0.8 microM) and clotrimazole (IC(50) 4.4+/-0.6 microM). 5. Summarizing, these results indicate an inhibitory effect of econazole, bifonazole and clotrimazole on PGHS-1, varying in its potency dependent on the cell system used. In addition TXB(2) formation is affected at doses even lower than those needed to suppress PGE(2) biosynthesis.     

Functional analysis of chemically synthesized derivatives of the human CC chemokine CCL15/HCC‐2, a high affinity CCR1 ligand

Abstract: The CCL15 is a human CC chemokine that activates the receptors, CCR1 and CCR3. Unlike other chemokines, it contains an unusually long N‐terminal domain of 31 amino acids preceding the first cysteine residue and a third disulfide bond. To elucidate the functional role of distinct structural determinants, a series of sequential amino‐terminal truncated and point‐mutated CCL15 derivatives as well as mutants lacking the third disulfide bond and the carboxy‐terminal α‐helix were synthesized using 9‐fluorenylmethoxycarbonyl (Fmoc) chemistry. We demonstrate that a truncation of 24 amino acid residues (Δ24‐CCL15) converts the slightly active 92‐residue Δ0‐CCL15 into a potent agonist of CC chemokine receptor 1 (CCR1) and a weak agonist of CCR3 in cell‐based assays. The biological activity decreases from Δ24‐CCL15 to Δ29‐CCL15, and re‐increases from Δ29‐CCL15 to Δ30‐CCL15. Thus, an exocyclic N‐terminal region of only one amino acid residue is sufficient for efficient CCR1 activation. As none of the peptides investigated except for Δ24‐CCL15 activates CCR3, we suggest that CCR1 is the major receptor for CCL15 in vivo. Further we demonstrate that the third disulfide bond of CCL15 and an exchange of tyrosine in position 70 by a leucine residue, which is conserved in CXC chemokines, do not alter the interaction with CCR1. In contrast, a CCL15 derivative lacking the carboxy‐terminal α‐helix exhibits a complete loss of tertiary structure and hence loss of CCR1 agonistic and binding activity. This study demonstrates that specific protein residues in chemokines, which contribute to receptor–ligand interaction, vary significantly between chemokines and cannot be extrapolated using data from functionally related chemokines.     

Effect of ketoconazole and terbinafine on the pharmacokinetics of caffeine in healthy volunteers

The effects of single oral doses of ketoconazole 400 mg and terbinafine 500 mg on the hepatic microsomal system have been investigated in 8 healthy male volunteers. Microsomal activity caffeine was assessed by following the metabolism of 3 mg/kg bodyweight i.v. administered 1 h after the drug. The inhibitory effect of terbinafine was more pronounced than that of ketoconazole: clearance was decreased from 1.34 ml.kg-1.min-1 in controls to 1.06 and 1.21 ml.kg-1.min-1, respectively, and the corresponding half-life was increased from 5.8 h in controls to 7.6 and 6.7 h, respectively. The apparent volume of distribution remained unchanged. The serum levels of the antimycotics were within the therapeutic range in each subject. Although all three substances are metabolised by microsomes, the kinetic parameters (Cmax, half-life, elimination constant) of the antimycotics were poorly if at all correlated with the elimination of caffeine.     

TP receptor-mediated release of eosinophil chemotactic activity from human bronchial smooth muscle cells

There are reports indicating that thromboxane A₂ receptors (TP receptors) may stimulate the eosinophil accumulation in the lower airways of asthmatics, however, the mechanisms behind such an effect remain unknown. We quantified the synthesis of eosinophil chemotactic activity and eosinophilic CC chemokines, including CCL5, CCL7, CCL8, CCL11, CCL13, CCL24, and CCL26 in primary cultures of human bronchial smooth muscle cells (BSMC) stimulated with a prostanoid TP receptor agonist, IBOP (10^− 9–10^− 7 M). The activation of prostanoid TP receptors in BSMC induced the release of potent eosinophil chemoattractant(s) in the presence of interleukin (IL)-4. CCL11/eotaxin-1 was the only synthesis significantly increased by IBOP co-stimulated with IL-4, and pretreatment with an anti-CCL11 antibody abrogated the eosinophil chemotactic activity released from IBOP/IL-4-stimulated BSMC. The effect of IBOP was also completely blocked by pretreatment with a prostanoid TP receptor-specific antagonist, AA-2414. IBOP had no effect on the expression of IL-4 receptor-α, or on the IL-4-induced phosphorylation of STAT6 in BSMC. In conclusion, activation of prostanoid TP receptors in a Th2-dominant microenvironment might exacerbate the eosinophilic inflammation of the airways by synthesis and release of CCL11 from BSMC.     

柚皮苷改善CCL2所致大鼠学习记忆障碍及其机制

目的探究柚皮苷改善CCL2诱导大鼠学习记忆障碍及其机制。方法56只SD大鼠随机分为空白组、假手术组、模型组(CCL2)、阳性药(CCL2+memantine)组、柚皮苷低(CCL2+25 mg·kg^-1柚皮苷)、中(CCL2+50 mg·kg^-1柚皮苷)、高(CCL2+100 mg·kg^-1柚皮苷)剂量组。除空白、假手术组外,各组均通过脑部定位将CCL2注射至大鼠海马制作学习记忆障碍模型。Morris水迷宫检测各组大鼠的学习和记忆水平;HE染色观察大鼠海马CA1区神经元形态;试剂盒检测海马SOD、GSH-PX活力和MDA含量;qRT-PCR检测凋亡基因caspase-3、caspase-8、Bax、Bcl-2 mRNA的相对表达。结果与模型组相比,各柚皮苷给药组大鼠逃避潜伏期及游泳路程均明显减少,平台穿越次数增加;海马CA1区神经元排列紧密且形态良好;SOD、GSH-PX活力升高,MDA含量降低;凋亡基因caspase-8、caspase-3、Bax mRNA相对表达量减少;Bcl-2表达量增加。结论柚皮苷能明显改善CCL2所致大鼠学习记忆功能障碍,其机制与柚皮苷的抗氧化和抗凋亡效应有关。     

趋化因子CCL5表达与肾透明细胞癌病理特征及预后的相关性分析

目的:探讨趋化因子CCL5表达与肾透明细胞癌病理特征及预后的相关性分析。方法:回顾性分析2012年1月~2014年1月在某院肿瘤科治疗的100例肾透明细胞癌患者的临床资料,将其作为观察组,将同期100例健康体检者作为对照组,检测并比较两组的趋化因子CCL5水平,分析肾透明细胞癌组织及癌旁组织中CCL5表达的差异性、CCL5与肾透明细胞癌病理特征的关系、CCL5与肾透明细胞癌患者总体生存率的关系。结果:观察组CCL5水平明显高于对照组(P<0.05);肿瘤组织中CCL5表达阴性、弱阳性、中阳性、强阳性的比例与癌旁组织相比有明显差异(P<0.05);CCL5表达与肿瘤病理分期、肿瘤分级、肿瘤大小等密切相关,在分化低、级别高、直径大的肿瘤,CCL5表达越高(P<0.05),而与年龄、性别无显著关联(P>0.05);CCL5高表达的患者生存时间明显低于CCL5低表达的患者(P<0.05)。结论:趋化因子CCL5表达与肾透明细胞癌病理特征及预后有显著相关性,CCL5表达越高,病理恶性程度越高,预后越差,可作为临床评估病情严重程度的监测指标和潜在治疗靶点。     

Anti-Inflammatory Effect of Quercetagetin,an Active Component of Immature Citrus unshiu,in HaCaT Human Keratinocytes

Citrus fruit contain various flavonoids that have multiple biological activities. However, the content of these flavonoids are changed during maturation and immature Citrus is known to contain larger amounts than mature. Chemokines are significant mediators for cell migration, while thymus and activation-regulated chemokine (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22) are well known as the typical inflammatory chemokines in atopic dermatitis (AD), a pruritic and chronic inflammatory skin disease. We reported recently that the EtOH extract of immature Citrus unshiu inhibits TARC and MDC production. Therefore, we investigated the activity of flavonoids contained in immature Citrus on TARC and MDC levels. As a result, among the various flavonoids, quercetagetin has stronger inhibitory effects on the protein and mRNA expression of TARC and MDC than other flavonoids. Quercetagetin particularly has better activity on TARC and MDC level than quercetin. In HPLC analysis, the standard peak of quercetagetin matches the peaks of extract of immature C. unshiu. This suggests that quercetagetin is an anti-inflammatory component in immature C. unshiu.     

Downregulation of Angiotensin II-Induced 12-Lipoxygenase Expression and Cell Proliferation in Vascular Smooth Muscle Cells from Spontaneously Hypertensive Rats by CCL5

Angiotensin II (Ang II) plays an important role in vascular hypertension. The role of the chemokine CCL5 on Ang II-induced activities in vascular smooth muscle cells (VSMCs) has not been studied. In this study, we elucidated the effect of CCL5 on Ang II-induced 12-lipoxygenase (LO) expression and cell proliferation in spontaneously hypertensive rats (SHR) VSMCs. CCL5 decreased Ang II-induced 12-LO mRNA expression and protein production, and it increased Ang II type 2 (AT₂) receptor expression in SHR VSMCs. The inhibitory effect of CCL5 on Ang II-induced 12-LO mRNA expression was mediated through the AT₂ receptor. Although treatment of CCL5 alone induced SHR VSMCs proliferation, CCL5 inhibited Ang II-induced VSMCs proliferation and PD123,319, an AT₂ receptor antagonist, blocked the inhibitory effect of CCL5 on Ang II-induced VSMCs proliferation. Phosphorylation of p38 was detected in VSMCs treated with Ang II or CCL5 alone. But, decrease of p38 phosphorylation was detected in VSMCs treated with Ang II and CCL5 simultaneously (Ang II/CCL5) and PD123,319 increased p38 phosphorylation in VSMCs treated with Ang II/CCL5. Therefore, these results suggest that the inhibitory effect of CCL5 on Ang II-induced VSMCs proliferation is mediated by the AT₂ receptor via p38 inactivation, and CCL5 may play a beneficial role in Ang II-induced vascular hypertension.     

益康唑和克霉唑对血小板聚集及TXB_2和PGE_2生成的影响

益康唑,克霉唑,咪康唑和酮康唑体外抑制AA诱导兔血小板聚集比抑制ADP诱导大鼠血小板聚集的作用强。其中益康唑和克霉唑抑制AA诱聚兔血小板的IC_(50)分别是6.4及11.6μmol/L。放射免疫法测定大鼠血小板产生的TXB_2及PGE_2发现益康唑和克霉唑在5～50 nmol/L浓度下,能抑制TXB_2产生,同时增加PGE_2生成量,并呈良好的剂量效应相关。浓度为0.1～100μmol/L时,TXB_2的生成仍被抑制,而PGE_2的生成量反而逐渐降低。两药对TXB_2生成的抑制作用比Daz强。     

IL-10 enhances CCL2 release and chemotaxis induced by CCL16 in human monocytes

CCL16 is a CC chemokine originally identified as a liver-expressed chemokine. Its expression has been detected in activated monocytes where it is up-regulated by stimulation with IL-10. This is in contrast with IL-10's inhibition of the expression of most chemokines. CCL16 is chemotactic for monocytes, lymphocyte and dendritic cells. We investigated whether CCL16 displays biological activities other than chemotaxis and whether IL-10 affects monocyte response to CCL16. We show that CCL16 induces the expression of CCL2 at the mRNA and protein level, but does not affect that of CCL5, CCL18 and proinflammatory cytokines. This effect was prevented by treatment with pertussis toxin and may thus be mediated by G-protein-coupled receptors. IL-10 markedly increased CCL2 production induced by CCL16, but suppressed that of CXCL8. It also enhanced the chemotactic response to CCL16. Addition of antibodies blocking CCR1, but not CCR8, prevented this enhanced chemotactic response and suggested that CCR1 is primarily involved. We propose that IL-10 modulates the effects of CCL16 on monocytes by increasing their CCR1-dependent response. The coordinated secretion of CCL16 and IL-10 may thus enhance monocyte infiltration.     

Differential regulation of eotaxin-1/CCL11 and eotaxin-3/CCL26 production by the TNF-alpha and IL-4 stimulated human lung fibroblast

Allergic asthma and allergic dermatitis are chronic inflammatory diseases and are characterized by an accumulation of eosinophils at sites of inflammation. Eotaxin-1/CCL11 and eotaxin-3/CCL26 are members of the CC chemokine family, which are known to be potent chemoattractants for eosinophils. We observed that a human lung fibroblast, HFL-1 produces eotaxin-1 and -3 in response to TNF-alpha plus IL-4 stimulation, accompanied with NF-kappaB and STAT6 activation. We explored which signaling pathways are operative in the production of eotaxin-1 and -3 using several inhibitors. Eotaxin-1/CCL11 production was inhibited by a p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, but not by the MEK (MAPK/ERK kinase) inhibitors, PD98059 and U0126. In contrast, eotaxin-3/CCL26 production was inhibited similarly by PD98059 as well as U0126 and SB203580. In addition, two proteasome inhibitors, N-acetyl-leucyl-leucyl-norleucinal (ALLN) and bortezomib with significant inhibitory activity on NF-kappaB activation, inhibited eotaxin-1/CCL11 production with IC(50) 8 muM for ALLN and IC(50) 16 nM for bortezomib. In contrast, eotaxin-3/CCL26 production was not inhibited significantly up to 10 muM of ALLN (IC(50 )16 muM) and up to 10 nM of bortezomib (IC(50) 11 nM), giving inhibition of eotaxin-3/CCL26 less sensitive than eotaxin-1/CCL11 production by the proteasome inhibitors. Synergistic inhibition was observed among lower doses of SB203580 and proteasome inhibitors, particularly in the eotaxin-1/CCL11 production. No such prominent synergism was found on the eotaxin-3/CCL26 production. The suppression of eotaxin family production by these inhibitors may be efficacious against allergic diseases.     

CCL5/RANTES Gene Deletion Attenuates Opioid-Induced Increases in Glial CCL2/MCP-1 Immunoreactivity and Activation in HIV-1 Tat-Exposed Mice

To assess the role of CC-chemokine ligand 5 (CCL5)/RANTES in opiate drug abuse and human immunodeficiency virus type 1 (HIV-1) comorbidity, the effects of systemic morphine and intrastriatal HIV-1 Tat on macrophage/microglial and astroglial activation were assessed in wild-type and CCL5 knockout mice. Mice were injected intrastriatally with vehicle or Tat and assessed after 7 days. Morphine was administered to some Tat-injected mice via time-release implant (5 mg/day, s.c. for 5 days) starting at 2 days post injection. Glial activation was significantly reduced in CCL5(−/−) compared to wild-type mice at 7 days following combined Tat and morphine exposure. Moreover, the percentage of 3-nitrotyrosine immunopositive macrophages/microglia was markedly reduced in CCL5(−/−) mice injected with Tat ± morphine compared to wild-type counterparts, suggesting that CCL5 contributes to nitrosative stress in HIV-1 encephalitis. In CCL5(−/−) mice, the reductions in Tat ± morphine-induced gliosis coincided with significant declines in the proportion of CCL2/MCP-1-immunoreactive astrocytes and macrophages/microglia compared to wild-type counterparts. In knockout mice, neither Tat alone nor in combination with morphine increased the proportion of CCL2-immunoreactive astrocytes above percentages seen in vehicle-injected controls. Macrophages/microglia differed showing modest, albeit significant, increases in the proportion of CCL2-positive cells with combined Tat and morphine exposure, suggesting that CCL5 preferentially affects CCL2 expression by astroglia. Thus, CCL5 mediates glial activation caused by Tat and morphine, thereby aggravating HIV-1 neuropathogenesis in opiate abusers and non-abusers. CCL5 is implicated as mediating the cytokine-driven amplification of CCL2 production by astrocytes and resultant macrophage/microglial recruitment and activation.     

Involvement of matrix metalloproteinase-3 in CCL5/CCR5 pathway of chondrosarcomas metastasis

CCL5 (previously called RANTES) was originally recognized as a product of activated T cells, and plays a crucial role in the migration and metastasis of human cancer cells. It has been reported that the effect of CCL5 is mediated via CCR receptors. We found that human chondrosarcoma tissues had significant expression of the CCL5 and CCR5, which was higher than that in normal cartilage. We also found CCL5 increased the migration and matrix metalloproteinases-3 (MMP)-3 expression in human chondrosarcoma cells (JJ012 cells). In addition, MMP-3 small interfering RNA and inhibitor inhibited the CCL5-induced cell migration. Activations of phosphatidylinositol 3-kinase (PI3K), Akt and NF-κB pathways after CCL5 treatment was demonstrated, and CCL5-induced expression of MMP-3 and migration activity was inhibited by the specific inhibitor of PI3K, Akt and NF-κB cascades. Taken together, these results indicate that CCL5 and CCR5 interaction enhanced migration of chondrosarcoma cells through the increase of MMP-3 production.