Development of a primary microglia screening assay and its use to characterize inhibition of system xc- by erastin and its analogs

The inflammatory response in the central nervous system involves activated microglia. Under normal conditions they remove damaged neurons by phagocytosis. On the other hand, neurodegenerative diseases are thought to involve chronic microglia activation resulting in release of excess glutamate, proinflammatory cytokines and reactive oxygen species, leading to neuronal death. System x_C^- cystine/glutamate antiporter (SXC), a sodium independent heterodimeric transporter found in microglia and astrocytes in the CNS, imports cystine into the cell and exports glutamate. SXC has been shown to be upregulated in neurodegenerative diseases including multiple sclerosis, ALS, neuroAIDS Parkinson's disease and Alzheimer's disease. Consequently, SXC inhibitors could be of use in the treatment of diseases characterized by neuroinflammation and glutamate excitotoxicity. We report on the optimization of a primary microglia-based assay to screen for SXC inhibitors. Rat primary microglia were activated using lipopolysaccharides (LPS) and glutamate release and cystine uptake were monitored by fluorescence and radioactivity respectively. LPS-induced glutamate release increased with increasing cell density, time of incubation and LPS concentration. Conditions to screen for SXC inhibitors were optimized in 96-well format and subsequently used to evaluate SXC inhibitors. Known SXC inhibitors sulfasalazine, S-4CPG and erastin blocked glutamate release and cystine uptake while R-4CPG, the inactive enantiomer of S-4CPG, failed to inhibit glutamate release or cystine transport. In addition, several erastin analogs were evaluated using primary microglia and found to have EC₅₀ values in agreement with previous studies using established cell lines.     

柳氮磺胺吡啶联合丽珠肠乐治疗溃疡性结肠炎的疗效及对患者炎症反应的影响

目的:观察丽珠肠乐联合柳氮磺胺吡啶治疗溃疡性结肠炎的疗效及对患者炎症反应的影响。方法:选取我院2014年6月-2017年6月期间收治的溃疡性结肠炎患者120例,根据数表法将患者分为观察组(n=60)和对照组(n=60)。对照组采用柳氮磺胺吡啶治疗,观察组在对照组的基础上增加丽珠肠乐治疗。连续治疗8周后观察两组患者的临床疗效以及结肠炎症状改善程度,并对两组患者治疗前后的血清炎症因子水平进行检测对比,同时观察两组患者不良反应发生情况。结果:观察组临床治疗的总有效率为93.33%(56/60),高于对照组的80.00%(48/60)(P0.05)。治疗后两组患者Sutherland指数评分、肠道菌群评分、肠镜评分均低于治疗前,且观察组低于对照组(P0.05)。治疗后两组患者CRP、IL-6、TNF-α水平均低于治疗前,且观察组低于对照组(P0.05)。观察组不良反应发生率为13.33%(8/60),与对照组的8.33%(5/60)比较差异无统计学意义(P0.05)。结论:柳氮磺胺吡啶联合丽珠肠乐治疗溃疡性结肠炎的临床疗效显著,能够改善患者的临床症状,降低患者体内的炎症因子水平,且无严重不良反应发生,临床应用价值高,值得进一步推广应用。     

Free radical scavenging reactions of sulfasalazine, 5-aminosalicylic acid and sulfapyridine: mechanistic aspects and antioxidant activity

Reactions of sulfasalazine (SAZ) and its metabolites, 5-aminosalicylic acid (5-ASA) and sulfapyridine (SP), with various oxidizing and reducing free radicals (hydroxyl, haloperoxyl, one-electron oxidizing, lipid peroxyl, glutathiyl, superoxide, tryptophanyl, etc.) have been studied to understand the mechanistic aspects of its action against free radicals produced during inflammation. Nanosecond pulse radiolysis technique coupled with transient spectrophotometry has been used for in situ generation of free radicals and to follow their reaction pathways. The transients produced in these reactions have been assigned and radical scavenging rate constants have been measured. In addition to scavenging of various primary and secondary free radicals by SAZ, 5-ASA and SP, 5-ASA has also been observed to efficiently scavenge radicals of biomolecules. 5-ASA has been found to be the active moiety of SAZ involved in the scavenging of oxidizing free radicals whereas reduction of SAZ produced molecular radical anion. The study suggests that free radical scavenging activity of 5-ASA may be a major path of pharmacological action of SAZ against inflammatory bowel diseases (IBD).     

川芎嗪联合益生菌及柳氮磺胺吡啶对溃疡性结肠炎的临床观察

目的：观察川芎嗪联合益生菌和柳氮磺胺吡啶对溃疡性结肠炎（Ulcerative Colitis,UC）的临床效果。方法：2013 年8 月至 2014 年8 月在我院接受治疗的UC 患者116 例根据其治疗分为观察组（n=58）和对照组（n=58）：对照组予以柳氮磺胺吡啶肠溶片 （1.0 g/次,4 次/d）、益生菌（2～4 粒/ 次,2 次/d）治疗,观察组增用川芎嗪（1～2 片/ 次,3 次/d）治疗,2 周后比较疗效、治疗前后 的炎症因子,以及药物不良反应。结果：观察组患者的总有效率高于对照组（96.55%vs 84.48%,P＜0.05）;观察组治疗前、后的白细 胞介素-4（Interleukin-4,IL-4; 6.84± 1.23 VS 18.24± 2.56 pg/mL）, IL-6 (168.90± 32.49 VS 16.24± 4.23 ng/L);IL-10 (15.17± 3.21 VS 16.77± 2.53 ng/mL);肿瘤坏死因子-alpha（Tumor necrosis factor alpha,TNF-alpha; 6.95± 7.25 VS 6.81± 1.84 ng/L); 与对照组治疗前、 后的IL-4（6.77± 1.52 VS 15.53± 2.75 pg/mL）;IL-6 (170.21± 25.68 VS 18.17± 2.25 ng/L）;IL-10（15.24± 2.83 VS 15.86± 2.24 ng/mL);TNF-alpha（17.01± 2.53 VS 7.63± 2.27 ng/L）相比差异有统计学意义（P＜0.05）。两组间药物不良反应总发生率无统计学意 义（13.79%vs 6.90%,P>0.05）。结论：川芎嗪联合益生菌及柳氮磺胺吡啶三联方案治疗UC 有较好的临床的疗效和安全性。     

美常安联合柳氮磺吡啶治疗溃疡性结肠炎的疗效及对血清TNF-alpha、IL-6、 IL-8水平的影响

目的：探索美常安联合柳氮磺吡啶治疗溃疡性结肠炎（UC）的疗效,及对血清TNF-alpha、IL-6、IL-8 水平的影响。方法：选择自 2012 年9月至2014 年12 月我院收治的100例UC 患者,按照随机数表法分成对照组和观察组,每组50 例。对照组患者口服给 予柳氮磺吡啶,观察组患者口服给予美常安联合柳氮磺吡啶治疗。统计分析两组患者的临床有效率、症状改善、不良反应发生情 况及治疗前后患者血清中TNF-alpha、IL-6、IL-8 水平的变化。结果：观察组中总有效率为92.00%显著高于对照组中总有效率为 48.00%（P<0.05）;两组患者治疗后的主要临床症状较治疗前均有明显改善（P<0.05）,且观察组患者治疗后主要症状缓解率明显高 于对照组（P<0.05）;两组患者治疗后血清中TNF-alpha、IL-6、IL-8 水平均显著低于治疗前,且观察组明显低于对照组（P<0.05）;两组患 者不良反应发生率之间的差异无统计学意义（P>0.05）。结论：美常安联合柳氮磺吡啶治疗UC具有良好的临床疗效,能显著改善 患者的临床症状和患者血清中炎症因子的水平,值得在临床上推广使用。