Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Box 6446, 14155 Tehran, Iran.
Environ Toxicol Pharmacol. 2007 Jul;24(1):60-6. doi: 10.1016/j.etap.2007.02.002. Epub 2007 Feb 16.
The present research introduces the method of Production of M2000 (β-d-mannuronic acid) and its therapeutic effect on experimental model of nephritis. M2000 was produced using enzymatic and chemical procedure on prepared alginate from Pseudomonas fluorescens. The experimental glomerulonephritis was induced in rats by a subcutaneous immunization and daily intravenous administration of bovine serum albumin (BSA). M2000 solution (30mg/kg) was administered intraperitoneally at regular 48-h intervals for 4 weeks. Onset of treatment was day 56. Urinary protein was measured weekly and serum anti-BSA antibody was assessed by ELISA method at different intervals. Animals were killed on day 84 and blood samples and kidney specimens were obtained. Serum (creatinine, BUN, cholesterol, and triglyceride) and urine (protein, urea, and creatinine) determinants were measured at the time of sacrifice. Kidney specimens were processed for light and immunofluorescent microscopic examination. The fibrosarcoma cell line was used for assaying tolerability and matrix metalloproteinase type 2 (MMP-2) activity. MMP-2 activity was assessed using zymography. Our data showed that M2000 therapy could significantly reduce the urinary protein excretion in treated rats versus non-treated controls. Anti-BSA antibody titer was lower in treated rats than in controls at the 12th experimental week. PMN infiltration and glomerular immune complex deposition was less intense in treated rats than in controls. Cytotoxicity analysis of M2000 showed a much higher tolerability compared with other tested drugs (diclofenac, piroxicam and dexamethasone). The inhibitory effect of M2000 in MMP-2 activity was significantly greater than that of dexsamethasone and of piroxicam at a concentration of 200μg/ml. Moreover, the toxicological study revealed that M2000 had no influence on serum (BUN, creatinine, triglyceride and cholesterol) determinants, urinary protein excretion and glomerular histology in healthy group receiving drug.
In this research, for the first time we introduced the procedure of production of M2000 (β-d-mannuronic acid) and our data suggest that treatment with M2000, as a novel anti-inflammatory drug can reduce proteinuria, diminish antibody production and suppress the progression of disease in experimental model of glomerulonephritis.
本研究介绍了 M2000（β-d-甘露糖醛酸）的生产方法及其在肾炎实验模型中的治疗效果。M2000 是通过酶法和化学法从荧光假单胞菌制备的海藻酸钠中制备的。通过皮下免疫和每日静脉给予牛血清白蛋白（BSA）在大鼠中诱导实验性肾小球肾炎。M2000 溶液（30mg/kg）每周腹膜内给予 4 周，间隔 48 小时。治疗开始于第 56 天。每周测量尿蛋白，并用 ELISA 法在不同时间间隔评估血清抗 BSA 抗体。第 84 天处死动物，采集血样和肾标本。在处死时测量血清（肌酐、BUN、胆固醇和甘油三酯）和尿液（蛋白质、尿素和肌酐）决定因素。对肾标本进行光镜和免疫荧光显微镜检查。使用纤维肉瘤细胞系评估耐受性和基质金属蛋白酶 2（MMP-2）活性。使用明胶酶谱法评估 MMP-2 活性。我们的数据表明，M2000 治疗可显著减少治疗大鼠的尿蛋白排泄，与未治疗对照组相比。在第 12 个实验周，治疗大鼠的抗 BSA 抗体滴度低于对照组。与对照组相比，治疗大鼠的PMN 浸润和肾小球免疫复合物沉积较轻。与其他测试药物（双氯芬酸、吡罗昔康和地塞米松）相比，M2000 的细胞毒性分析显示出更高的耐受性。M2000 对 MMP-2 活性的抑制作用明显大于地塞米松和吡罗昔康在 200μg/ml 浓度下的抑制作用。此外，毒理学研究表明，M2000 对接受药物的健康组的血清（BUN、肌酐、甘油三酯和胆固醇）决定因素、尿蛋白排泄和肾小球组织学没有影响。
在这项研究中，我们首次介绍了 M2000（β-d-甘露糖醛酸）的生产过程，我们的数据表明，作为一种新型抗炎药物，M2000 治疗可减少蛋白尿、减少抗体产生并抑制实验性肾小球肾炎模型中的疾病进展。
