中药当归主要活性成分阿魏酸钠抑制淀粉样β蛋白激活炎症因子的量剂反应

Dose-dependent effect of sodium ferulate in inhibiting inflammatory factors activated by beta-amyloid protein

目的:观察中药当归提取的主要活性成分阿魏酸钠对淀粉样β蛋白激活的小鼠腹腔巨噬细胞α肿瘤坏死因子、诱导型一氧化氮合酶和一氧化氮水平的影响。方法:实验于2004-03/10在锦州医学院药理实验室和解剖实验室完成。选择8～10周龄的小鼠36只,随机分为6组,每组6只。正常对照组:巨噬细胞接种24h后用培养基继续培养。淀粉样β蛋白组:巨噬细胞接种24h后在培养基中加入经老化处理的终浓度为10μmol/L的淀粉样β蛋白。淀粉样β蛋白+10,100,500μmol/L,1mmol/L阿魏酸钠组:在加入淀粉样β蛋白的同时加入10,100,500μmol/L,1mmol/L的阿魏酸钠共同孵育。培养48h后采用酶联免疫吸附法、免疫组织化学方法和Griess反应分别检测肿瘤坏死因子α分泌量、诱导型一氧化氮合酶表达和一氧化氮的生成量。结果:36只小鼠均进入结果分析。①巨噬细胞的肿瘤坏死因子α分泌量:淀粉样β蛋白组明显高于正常对照组[(281.54±14.85),(17.55±4.84)ng/L,(P<0.01)]。淀粉样β蛋白+10,100,500μmol/L,1mmol/L阿魏酸钠组可以不同程度地减少由淀粉样β蛋白诱导产生的肿瘤坏死因子α的分泌量[(238.05±6.21),(186.90±7.44),(117.55±8.08),(71.77±10.50)ng/L,P<0.01],且有明显剂量依赖关系(P<0.05)。②巨噬细胞的一氧化氮生成量:淀粉样β蛋白组明显高于正常对照组[(85.04±6.16),(26.10±3.25)μmol/L,(P<0.01)],淀粉样β蛋白+10,100,500μmol/L,1mmol/L阿魏酸钠组可以不同程度地减少由淀粉样β蛋白诱导产生的一氧化氮的量[(69.80±4.96),(54.52±3.45),(43.88±4.04),(33.83±3.13)μmol/L,P<0.01],且有明显剂量依赖关系(P<0.05)。③诱导型一氧化氮合酶的表达:正常对照组只有零星细胞染色阳性;而淀粉样β蛋白组多数细胞胞浆被染成黄褐色。各剂量组的阿魏酸钠可以不同程度地抑制由淀粉样β蛋白激活巨噬细胞造成的诱导型一氧化氮合酶的表达,该抑制作用呈明显剂量依赖性。结论:①淀粉样β蛋白能激活巨噬细胞,使其分泌大量的肿瘤细胞坏死因子α和一氧化氮,诱导型一氧化氮合酶表达也相应增加。②阿魏酸钠通过其抗炎作用呈明显剂量依赖性抑制淀粉样β蛋白对巨噬细胞的激活作用,从而使巨噬细胞生成肿瘤细胞坏死因子α、诱导型一氧化氮合酶、一氧化氮水平降低。

AIM： To observe the effect of sodium ferulate, the main activity component extracted from Chinese angelica the traditional Chinese medicine, on the level of the tumor necrosis factor alpha （TNF-α）, induction nitricoxide synthase （iNOS） and nitrogen monoxide （NO） of abdominal cavity histocyte activated by amyloid beta protein in rats. METHODS： The experiment was completed in cooperation of the Department of Pharmacology and Department of Anatomy of Jinzhou Medical College from March to October 2004. Thirty-six mice aged from 8 to 10 weeks were selected and divided randomly into 6 groups with 6 in each group. Normal control group： Twenty-four hours after inoculation macrophages were cultured continuously by medium. Amyloid beta protein group： Twenty-four hours after inoculation macrophages were added in arnyloid beta protein with the 10μmol/L final concentration which was conducted ageing disposal in medium. The amyloid beta protein ±10, 100, 500μmol/L, 1 mmol/L sodium ferulate group： Sodium ferulate 10,100,500μmol/L, 1 mmol/L were added at the same time of adding amyloid beta protein to incubate together. The excretion of TNF-α, and the expression of iNOS and production of NO were detected by enzyme-linked immuno-sorbent assay, irnmuneohistochemistry technique and Greiss reaction respectively 48 hours after culture. RESULTS： Thirty-six mice were all involved in the result analysis. ① The excretion of TNF-α of histocyte： It was significantly higher in amyloid beta protein group than that in normal control group [（281.54±14.85）, （17.55±4.84） ng /L, （P 〈 0.01）]. The excretion of TNF-α induced by amyloid beta protein could decreased in different degree in amyloid beta protein ＋10, 100, 500 μmol/L, lmmol/L sodium ferulate group [（238.05±6.21）, （186.90±7.44）, （117.55±8.08）, （71.77±10.50）ng/L, P 〈 0.01] and had significant dosage dependence relation （P 〈 0.05）.② The production of NO of histocyte： It was significantly higher in amyloid beta protein group than that in normal control group [（85.04±6.16）, （26.10±3.25）μmol/L, （P 〈 0.01）]. The production of NO induced by amyloid beta protein could decreased in different degree in amyloid beta protein ＋10, 100, 500μmol/L, 1mmol/L sodium ferulate group [（69.80 ±4.96）, （54.52±3.45）, （43.88±4.04）, （33.83±3.13） μmol/L, P 〈 0.01] and had significant dosage dependence relation （P 〈 0.05）. ③ The expression of iNOS： Some cells in normal control group showed positive stain; While most of the cell plasm in amyloid beta protein group was stained with filemot. The sodium ferulate in every dosage group could inhibit the expression of iNOS induced by the activation of histocyte by amyloid beta protein in different degree. The inhibitive effect showed significant dosage dependence. CONCLUSION： ① The macrophages can be activated by amyloid beta protein and make it excrete a large number of TNF-α and NO, and the expression of iNOS also corresponding increases. ② The sodium ferulate shows significant dosage dependence through its anti-inflammation effect to inhibit the activation of amyloid beta protein on histocyte in order to reduce the level of TNF-α, iNOS and NO produced by histocyte.