Interleukin-17A induces cathepsin K and MMP-9 expression in osteoclasts via celecoxib-blocked prostaglandin E2 in osteoblasts

Interleukin-17 (IL-17) is a cytokine secreted primarily by T_H-17 cells that can stimulate the development of osteoclasts (osteoclastogenesis) in the presence of osteoblasts. IL-17, through osteoblasts, has indirect effects on the expression of bone resorption-related enzymes in osteoclasts, which have not been well clarified. Here, using MC3T3-E1 cells and RAW264.7 cells as osteoblasts and osteoclast precursors, we aimed to clarify these effects of IL-17A. MC3T3-E1 cells were cultured in the presence or absence of IL-17A for 72 h and the conditioned media collected (in the presence of soluble receptor activator of NF-кB ligand) and used to culture RAW264.7 cells. To assess osteoclast differentiation, adherent cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP). Our analyses demonstrated that the number of TRAP-positive multinucleated cells increases after 3 days of culture in conditioned medium from IL-17A-treated cells compared to untreated controls. In addition, we observed that the levels of cathepsin K and MMP-9 increase in the conditioned medium from IL-17A-treated cells, whereas CA II expression levels remain unaffected. PGE₂ production from MC3T3-E1 cells increased in the presence of IL-17A. Celecoxib, a specific inhibitor of cyclooxygenase-2 (COX-2), blocked both the IL-17A-stimulated increase in TRAP-positive multinucleated cells and the expression of cathepsin K and MMP-9. Furthermore, when MC3T3-E1 cells were transformed with small interfering RNA to silence COX-2 expression before IL-17A treatment, the resulting conditioned medium was less effective at inducing cathepsin K and MMP-9 expression in RAW264.7 cells. These results suggest that IL-17A induces the differentiation and function of osteoclasts via celecoxib-blocked prostaglandin, mainly PGE₂, in osteoblasts.     

Differential regulation of anti-inflammatory proteins in human rheumatoid synoviocyte MH7A cell by celecoxib and ibuprofen

Non-steroidal anti-inflammatory drugs are known to be the most widely used drugs to exert their anti-inflammatory activities. It was examined protein expression profiles of human rheumatoid fibroblast-like synoviocyte MH7A cells treated with celecoxib, a selective cyclooxygenase-2 inhibitor, or ibuprofen, a non-selective cyclooxygenase inhibitor, using two-dimensional gel electrophoresis for comparison the mechanism of the drugs. Altered expression pattern in response to celecoxib is significantly different from that of ibuprofen treated cells. When MH7A cells were treated with celecoxib, 28 proteins were affected at their expression levels. Among them, heat shock proteins (Hsp60 and 70), glucose regulated proteins (Hsp75 and 78) were observed to be up-regulated by 1 to 30 microM concentrations of celecoxib but those proteins were not affected in ibuprofen treated cells. On the other hand, the expression of 19 proteins was changed by ibuprofen and the expression of apolipoprotein E, RNA binding motif 4, CTP-phosphocholine cytidylyltransferase, and phospholipase A2 inhibitory protein was only altered by ibuprofen. The expressions of 15 proteins were affected by both celecoxib and ibuprofen. Our results showed that celecoxib and ibuprofen, though they are known to act as cyclooxygenase inhibitors, could exert a different mode of acting mechanisms in anti-inflammatory processes. The chemical proteomic approach will be useful for figuring out the mode of actions of drugs.     

Sulfotransferase 2A1 forms estradiol-17-sulfate and celecoxib switches the dominant product from estradiol-3-sulfate to estradiol-17-sulfate

Using recombinant sulfotransferases (SULTs) expressed in E. coli, β-estradiol (E2) sulfonation was examined to determine which SULT enzyme is responsible for producing E2-17-sulfate (E2-17-S). SULTs 1A1*1, 1A1*2, 1A3, 1E1 and 2A1 all sulfated E2 to varying extents. No activity was observed with SULT1B1. Among the SULTs studied, SULT2A1 produced primarily E2-3-sulfate (E2-3-S), but also some E2-17-S and trace amounts of E2 disulfate. SULT2A1 had a K_m value of 1.52 μM for formation of E2-3-S and 2.95 μM for formation of E2-17-S. SULT2A1 had the highest V_max of 493 pmol/min/mg protein for formation of E2-3-S, which was 8.8- and 47-fold higher than the maximal rates of formation of E2-17-S and E2 disulfate, respectively. SULT2A1 formed E2-3-S more efficiently. However, when celecoxib (0–160 μM) was included in the incubation with either SULT2A1 or human liver cytosol, sulfonation switched from E2-3-S to E2-17-S in a concentration-dependent manner. The ratio of E2-17-S/E2-3-S went up to 15 with SULT2A1, and was saturated at 1 with human liver cytosol. In both cases, more E2-17-S was formed, with the unreacted E2 remained unchanged, suggesting celecoxib probably bound to a separate effector site to cause a conformational change in SULT2A1, which favored production of E2-17-S. The ability of celecoxib to alter the position of sulfonation of E2 may in part explain its success in the experimental prevention and treatment of breast cancer.     

针刺疗法联合塞来昔布对老年类风湿性关节炎患者血清MMP-3, IL-6及自身抗体水平的影响

目的:研究针刺疗法联合塞来昔布对老年类风湿性关节炎患者的临床疗效及其对患者血清基质金属蛋白酶-3(MMP-3)、白细胞介素-6(IL-6)及自身抗体水平的影响。方法:选取2014年10月至2015年9月本院收治的86例类风湿性关节炎患者,随机分为观察组(43例)和对照组(43例)。对照组使用塞来昔布胶囊治疗,观察组在对照组基础上联合针刺疗法。分析和比较两组患者临床疗效、治疗前后20 m步行时间、晨僵时间、关节压痛数、治疗前后MMP-3、IL-6及自身抗体水平的变化。结果:观察组总有效率显著高于对照组[90.70%(39/43)比65.12%(28/43)](P0.05)。治疗后,两组患者20m步行时间、晨僵时间、关节压痛数均较治疗前显著降低或缩短,其中观察组明显低于或短于对照组(P0.05)。治疗后,两组患者血清MMP-3、IL-6水平较治疗前显著降低,其中观察组显著低于对照组(P0.05)。治疗后,两组患者AFA、抗-CCP抗体、抗Sa抗体、AKA、ANA自身抗体阳性率较治疗前显著降低,其中观察组显著低于对照组(P0.05)。结论:针刺疗法联合塞来昔布治疗老年类风湿性关节炎的临床疗效良好,能有效降低患者血清MMP-3、IL-6水平及自身抗体阳性率。     

二甲双胍联合塞来昔布对胰腺癌细胞增殖及Caspase-3活性的影响

目的:探讨二甲双胍和塞来昔布单独或联合应用对胰腺癌细胞增殖和Caspase-3活性的影响。方法:体外培养人胰腺癌BxPC-3和As PC-1细胞,用四甲基偶氮唑盐(MTT)法检测不同浓度二甲双胍和塞来细胞对胰腺癌细胞存活率的影响。联合实验分4组:对照组、二甲双胍组(MET,15 mmol/L)、塞来昔布组(CEL,100μmmol/L),二甲双胍和塞来昔布联合组(MET 15 mmol/L+CEL100μmmol/L),孵育48 h,用MTT检测细胞存活率,用Caspase-3比色测定试剂盒测定Caspase-3活性。结果:二甲双胍和塞来昔布单药均可以时间和剂量依赖性方式降低胰腺癌Bx PC-3和As PC-1细胞的生存率。两种细胞系的各加药组细胞存活率与对照组比较差异均存在统计学意义(P0.01),联合实验组的细胞存活率明显低于单独用药组(P0.01)。二甲双胍和塞来昔布单药处理的胰腺癌Bx PC-3和As PC-1细胞Caspase-3活性均显著高于对照组(P0.01),二甲双胍和塞来昔布联合实验组Caspase-3活性均明显高于单药处理组和对照组(P0.01),但二甲双胍组和塞来昔布组之间的Caspase-3活性比较差异无统计学意义(P0.05)。结论:二甲双胍和塞来昔布联合作用可协同抑制胰腺癌细胞的增殖,并通过激活Caspase-3活性促进胰腺癌细胞的凋亡,其联合应用可能成为胰腺癌药物治疗的有效策略。     

Structure–activity relationship of celecoxib and rofecoxib for the membrane permeabilizing activity

Non-steroidal anti-inflammatory drugs (NSAIDs) achieve their anti-inflammatory effect by inhibiting cyclooxygenase activity. We previously suggested that in addition to cyclooxygenase-inhibition at the gastric mucosa, NSAID-induced gastric mucosal cell death is required for the formation of NSAID-induced gastric lesions in vivo. We showed that celecoxib exhibited the most potent membrane permeabilizing activity among the NSAIDs tested. In contrast, we have found that the NSAID rofecoxib has very weak membrane permeabilizing activity. To understand the membrane permeabilizing activity of coxibs in terms of their structure–activity relationship, we separated the structures of celecoxib and rofecoxib into three parts, synthesized hybrid compounds by substitution of each of the parts, and examined the membrane permeabilizing activities of these hybrids. The results suggest that the sulfonamidophenyl subgroup of celecoxib or the methanesulfonylphenyl subgroup of rofecoxib is important for their potent or weak membrane permeabilizing activity, respectively. These findings provide important information for design and synthesis of new coxibs with lower membrane permeabilizing activity.     

Liquid chromatographic–mass spectrometric determination of celecoxib in plasma using single-ion monitoring and its use in clinical pharmacokinetics

Celecoxib is a cyclooxygenase-2 specific inhibitor, that has been recently and intensively prescribed as an anti-inflammatory drug in rheumatic osteoarthiritis. A robust, highly reliable and reproducible liquid chromatographic–mass spectrometric assay is developed for the determination of celecoxib in human plasma using sulindac as an internal standard. The run cycle-time is <4 min. The assay method involved extraction of the analytes from plasma samples at pH 5 with ethyl acetate and evaporation of the organic layer. The reconstituted solution of the residue was injected onto a Shim Pack GLC-CN, C₁₈ column and chromatographed with a mobile phase comprised of acetonitrile–1% acetic acid solution (4:1) at a flow-rate of 1 ml/min. The mass spectrometer (LCQ Finnigan Mat) was programmed in the positive single-ion monitoring mode to permit the detection and quantitation of the molecular ions of celecoxib and sulindac at m/z 382 and 357, respectively. The peak area ratio of celecoxib/sulindac and concentration are linear (r²>0.994) over the concentration range 50–1000 ng/ml with a lowest detection limit of 20 ng/ml of celecoxib. Within- and between-day precision are within 1.58–4.0% relative standard deviation and the accuracy is 99.4–107.3% deviation of the nominal concentrations. The relative recoveries of celecoxib from human plasma ranged from 102.4 to 103.3% indicating the suitability of the method for the extraction of celecoxib and I.S. from plasma samples. The validated LC–MS method has been utilized to establish various pharmacokinetic parameters of celecoxib following a single oral dose administration of celecoxib capsules in two selected volunteers.     

Determination of celecoxib in human plasma and rat microdialysis samples by liquid chromatography tandem mass spectrometry

Methods for the determination of celecoxib in human plasma and rat microdialysis samples using liquid chromatography tandem mass spectrometry are described. Celecoxib and an internal standard were extracted from plasma by solid-phase extraction with C₁₈ cartridges. Thereafter compounds were separated on a short narrow bore RP C₁₈ column (30×2 mm). Microdialysis samples did not require extraction and were injected directly using a narrow bore RP C₁₈ column (70×2 mm). The detection was by a PE Sciex API 3000 mass spectrometer equipped with a turbo ion spray interface. The compounds were detected in the negative ion mode using the mass transitions m/z 380→316 and m/z 366→302 for celecoxib and internal standard, respectively. The assay was validated for human plasma over a concentration range of 0.25–250 ng/ml using 0.2 ml of sample. The assay for microdialysis samples (50 μl) was validated over a concentration range of 0.5–20 ng/ml. The method was utilised to determine pharmacokinetics of celecoxib in human plasma and in rat spinal cord perfusate.     

脊柱三扳法联合塞来昔布对强直性脊柱炎患者胸腰椎活动和BMP/Smad信号通路的影响

摘要 目的：探讨脊柱三扳法联合塞来昔布对强直性脊柱炎（AS）患者胸腰椎活动和骨形态发生蛋白（BMP）/细胞信号转导分子(Smad)信号通路的影响。方法：选取2016年7月~2018年12月期间我院收治的AS患者150例,根据随机数字表法分为A组（n=50,给予脊柱三扳法治疗）、B组（n=50,给予塞来昔布治疗）和C组（n=50,给予脊柱三扳法联合塞来昔布治疗）,比较三组患者疗效、胸腰椎活动指标、BMP/Smad信号通路相关指标及不良反应发生率。结果：C组治疗6个月后的临床总有效率为94.00%（47/50）高于A组的66.00%（33/50）、B组的72.00%（36/50）（P<0.05）;C组治疗6个月后胸廓活动度、腰椎活动度均高于A组、B组（P<0.05）,指-地距离短于A组、B组（P<0.05）。C组治疗6个月后BMP-Ⅰ受体、BMP-Ⅱ受体、Smad1、Smad4表达水平均低于A组、B组（P<0.05）。三组不良反应发生率比较差异均无统计学意义（P>0.05）。结论：脊柱三扳法联合塞来昔布治疗AS患者,疗效显著,可有效改善胸腰椎活动,且不增加不良反应发生率,其具体作用机制可能是通过调节BMP/Smad信号通路实现。     

Influence of hydrophilic polymers on celecoxib complexation with hydroxypropyl β-cyclodextrin

Complexation of celecoxib with hydroxypropyl β-cyclodextrin (HPβCD) in the presence and absence of 3 hydrophilic polymers—polyvinyl pyrrolidone (PVP), hydroxypropyl methylcellulose (HPMC), and polyethylene glycol (PEG)—was investigated with an objective of evaluating the effect of hydrophilic polymers on the complexation and solubilizing efficiencies of HPβCD and on the dissolution rate of celecoxib from the HPβCD complexes. The phase solubility studies indicated the formation of celecoxib-HPβCD inclusion complexes at a 1∶1M ratio in solution in both the presence and the absence of hydrophilic polymers. The complexes formed were quite stable. Addition of hydrophilic polymers markedly enhanced the complexation and solubilizing efficiencies of HPβCD. Solid inclusion complexes of celecoxib-HPβCD were prepared in 1∶1 and 1∶2 ratios by the kneading method, with and without the addition of hydrophilic polymers. The solubility and dissolution rate of celecoxib were significantly improved by complexation with HPβCD. The celecoxib-HPβCD (1∶2) inclusion complex yielded a 36.57-fold increase in the dissolution rate of celecoxib. The addition of hydrophilic polymers also markedly enhanced the dissolution rate of celecoxib from HPβCD complexes: a 72.60-, 61.25-, and 39.15-fold increase was observed with PVP, HPMC, and PEG, respectively. Differential scanning calorimetry and X-ray diffractometry indicated stronger drug amorphization and entrapment in HPβCD because of the combined action of HPβCD and the hydrophilic polymers. Published: September 29, 2006