对氨基水杨酸钠对锰致神经炎症的干预机制

Manganism patients not only had the clinical features of the extrapyramidal damage, but also had the signs and symptoms of the thalamus damage. Our findings from T1-weighted magnetic resonance imaging (MRI) and hydrogen proton magnetic resonance spectroscopy (1H-MRS) showed accumulated manganese level in globus pallidus, elevated gamma aminobutyric acid (GABA) and decreased myo-inositol (mI) levels in the thalamus and adjacent basal ganglia. The decreased myo-inositol (mI) level might be caused by manganese-induced damage of glial cells, which was associated with, nerve inflammation. The treatment of manganese poisoning patients with sodium para-aminosalicylic acid (PAS-Na) had firstly been applied by our department and this application had obtained a favourable effect. The research methods of in vivo and in vitro toxicology combined with case-control study, techniques of immunohistochemical staining, high performance liquid chromatography，single cell gel electrophoresis，enzyme-linked immunosorbent assay，real-time PCR and western blot will be used in the present project. The intervenient mechanism of PAS-Na on manganese neurotoxicity of the injuried neuron, glial cell in thalamus of the exposed rats will be explored. The intervenient mechanism of PAS-Na on manganese induced inflammation in thalamus of the exposed rats will be studied. The effect of nerve inflammation in occupational manganese-exposed workers and intervenient effect of PAS-Na on manganism patients will also be investigated. The present project will provide a scientific basis for prevention and treatment of manganese poisoning.

锰中毒病人不仅有锥体外系损伤的临床表现，也有丘脑损伤的症状和体征。我们的T1-加权磁共振成像（MRI）和氢质子磁共振波谱（1H-MRS）研究发现，锰冶炼工苍白球锰蓄积，丘脑及其邻近基底核γ-氨基丁酸(GABA)-感兴趣区 GABA水平明显增加，肌醇（mI）水平明显降低，后者可能是锰暴露引起神经胶质细胞损伤，且可能与神经炎症发生有关。我室率先应用对氨基水杨酸钠（PAS-Na）治疗锰中毒患者效果较好。本项目拟通过体内外毒理学研究和职业人群调查相结合的方法，采用免疫组化、高效液相色谱、单细胞凝胶电泳、酶联免疫吸附剂测定法、实时定量荧光PCR和蛋白质印迹法等技术探讨锰致大鼠丘脑神经元、神经胶质细胞损伤引起的神经毒性及PAS-Na的干预作用机制；探讨锰致大鼠丘脑炎症反应及PAS-Na的干预作用机制；探讨职业性锰暴露致工人神经炎症反应以及PAS-Na对中毒病人的治疗效果，为锰中毒防治工作提供科学依据。

锰中毒病人锥体外系和丘脑受损机制可能与神经炎症发生有关。本项目在对氨基水杨酸钠（PAS-Na）临床治疗锰中毒效果较好的基础上，探讨锰致大鼠丘脑神经细胞损伤、炎症反应及PAS-Na的干预作用机制；探讨锰暴露致工人神经炎症反应以及PAS-Na对中毒病人的治疗效果。结果显示，体外染锰使大鼠丘脑原代小胶质细胞、HAPI细胞和BV2小胶质细胞活性氧簇（ROS）增多，炎症因子白介素（IL-1β）、肿瘤坏死因子（TNF-α）分泌及其mRNA表达量增加，并通过NF-Kβ/ NLRP3/ Caspase-1通路介导炎症反应，PAS-Na干预使上述锰中毒性炎症反应减轻。短期染锰大鼠丘脑、海马GFAP、caspase-3阳性细胞表达量增高，丘脑IL-1β、COX-2、海马IL-1β、IL-6、COX-2、TNF-α、mRNA表达上调，丘脑p-ERK和p-JNK蛋白表达升高，血清IL-1β、IL-6、TNF-α、丘脑IL-1β、PGE2和海马PGE2含量增高，丘脑和海马Bcl-2阳性细胞表达量、Bcl-2/Bax比值降低。亚慢性染锰大鼠丘脑和海马GFAP、caspase-3阳性细胞表达量增加，血清、丘脑、海马、皮层IL-6、IL-1β、PGE2和TNF-α含量增高，丘脑IL-1β、COX-2和TNF-αmRNA、海马IL-1β、IL-6、COX-2、TNF-α表达升高。停止锰暴露6周，上述炎症指标仍然增高。锰可能通过MAPK通路引起大鼠皮层、海马和丘脑炎症反应，其中以激活ERK和p38磷酸化为主。PAS-Na预防或治疗使上述锰中毒性炎症反应降低，且通过抑制MAPK通路相关蛋白磷酸化来拮抗炎症反应。亚慢性染锰大鼠海马炎症小体NLRP3、caspase-1mRNA和蛋白表达、IL-1β、IL-18 mRNA表达增高。PAS-Na治疗使海马NLRP3、caspase-1蛋白表达、NLRP3、IL1-β、IL-18 mRNA 表达减少。本研究表明，PAS-Na对锰致大鼠神经炎症有拮抗作用，其机制可能与MAPK、NLRP3通路受阻有关。作业环境空气二氧化锰浓度几何均数为0.009 （0.005～0.041）mg/m3，锰暴露工人血清TNF-α、IL-1β含量增高，提示低水平锰暴露仍然对工人血清炎症因子产生一定的影响。该研究为锰中毒防治提供较好的科学依据，可望有较好的应用前景。