通络方剂抗氧化作用及对大血管内皮细胞的保护

Antioxidation of Tongluofangji and its protective role in the great vessel endothelial cells

目的:观察通络方剂对1型糖尿病大鼠血清抗氧化指标和大血管内皮细胞损害的影响,并与抗氧化剂α-硫辛酸作用效果进行比较。方法:实验于2005-09/11在解放军第二军医大学动物实验中心及免疫研究所完成。①选用雄性SD大鼠23只,4～6周龄。随机抽取5只作为正常对照组大鼠(只注射等体积枸橼酸钠缓冲液),其余大鼠按60mg/kg剂量应用链脲佐菌素溶液,3d后,以空腹血糖持续≥16.7mmol/L,尿糖"者为造模成功,造模成功15只,将其按随机数字表法分为3组,每组5只:模型组,通络方剂组,α-硫辛酸组。通络方剂组:每天上午灌胃通络方剂水溶液1g/kg,通络方剂超微颗粒由人参、水蛭、全蝎、蜈蚣、土鳖虫、赤芍等药物组成,由以岭药业提供;α-硫辛酸组:每天上午灌胃α-硫辛酸(江苏常熟诚希化工有限公司)100mg/kg;其余2组均灌胃等体积的体积分数0.02的乙醇稀释液。②连续处理6周后,采用葡萄糖氧化酶法测血糖,采用可见光法检测过氧化氢酶活力,用TBA法测定丙二醛,用DTNB法定量测定谷胱甘肽过氧化物酶活力,比色法测超氧化物歧化酶活力与总抗氧化能力。透射电镜下观察胸主动脉内皮细胞的结构改变。结果:因造模失败脱失3只,其余均进入结果分析。①血清抗氧化指标:模型组血清超氧化物歧化酶、谷胱甘肽过氧化物酶、过氧化氢酶活力和总抗氧化能力明显低于正常对照组[(0.883±0.243)mkat/L,(2.056±0.219)mkat/L,(223.21±28.84)μkat/L,(5.25±0.84)kU/L;(2.826±0.199)mkat/L,(4.890±0.358)mkat/L,(387.91±13.00)μkat/L,(9.81±1.28)kU/L,P<0.05],丙二醛水平明显高于正常对照组[(9.99±1.7),(2.42±0.89)μmol/L,P<0.05]。通络方剂组和α-硫辛酸组血清超氧化物歧化酶和谷胱甘肽过氧化物酶活力明显高于模型组[通络方剂组:(1.665±0.140),(3.771±0.258)mkat/L;α-硫辛酸组:(1.851±0.132),(4.141±0.186)mkat/L,P<0.05],低于正常对照组(P<0.05)。②胸主动脉血管内皮结构透射电镜观察结果:模型组内皮细胞发生明显损害,通络方剂和α-硫辛酸组内皮细胞病变程度则明显改善。结论:通络方剂能提高糖尿病大鼠机体抗氧化能力,对糖尿病所致的大鼠血管内皮损害具有一定的保护作用。

AIM： To study the antioxidation of Tongluofangji and its protection on the great vessel endothelial cells of the Ⅰ type diabetic rats, and compare this effect with that of anti-oxidant α-lipoic acid. METHODS： The experiment was conducted in the Experimental Animal Center and Immunity Institute of the Second Military Medical University of Chinese PLA from September to November 2005. ①Twenty-three male SD rats of 4-6 weeks old were adopted in this study, and 5 of them were randomly selected as control group, which were injected with natrium citricum buffer solution only. And the others were given intraperitoneal injection of 60 mg/kg streptozocin to ensure a diabetic state, which fasting blood glucose levels were measured more than 16.7 mmol/L and urine glucose were ＋＋＋ three days after the injection. Fifteen diabetic rats were successful models and randomized into three groups： model group, Tongluofangji group and α-lipoic acid group, with 5 rats in each. Tongluofangji group： Tongluofangji water solution of 1 g/kg was given intragastrically in every morning. Tongluofangji uhrafine particle consisted of ginseng, leech, scorpion, centipede, ground beetle and red peony root, providing by Yiling Pharmaceutical Company; α-lipoic acid group： α-lipoic acid （product of Jiangsu Changshu Chengxi Chemical Engineering Limited Company） of 100 mg/kg was infused intragastrically into the rats in every morning; Other 2 groups were treated with equal ethanol dilution via intragastric injection in the volume fraction of 0.02. ②After six weeks of continuous management, the blood glucose was detected by glucose oxidase method, the activity of catalase （CAT） was measured by visible light, malonaldehyde （MDA） was assayed by TBA method, glutathione peroxidase （GSH-PX） was determined quantitatively by DTNB method, and superoxide dismutase （SOD） and total antioxidative capacity （T-AOC） on were estimated by chromometry. The pathologic changes of the aorta thoracic endothelial cells were observed with transmission electron microscope （TEM）. RESULTS： Twenty rats entered the result analysis because three failed models were lost.①Serum antioxidative index： Compared with the control group, the plasma activity of SOD, GSH-PX, CAT and T-AOC reduced significantly in model group [（0.883±0.243） mkat/L, （2.056±0.219） mkat/L, （223.21±28.84） μkat/L, （5.25±0.84） kU/L; （2.826±0.199） mkat/L, （4.890±0.358） mkat/L, （387.91±13.00） μkat/L, （9.81±1.28） kU/L, P 〈 0.05], while the plasma level of MDA increased significantly [（9.99±1.7）, （2.42±0.89） μmol/L, P 〈 0.05]. The plasma activities of SOD and GSH-PX were obviously higher in Tongluofangji group and α-lipoic acid group than in model group [Tongluofangji group： （1.665±0.140）, （3.771±0.258） mkat/L; α-lipoic acid group： （1.851±0.132）, （4.141±0.186） mkat/L, P 〈 0.05], but still lower than those of control group （P 〈 0.05）. ②TEM results of aorta thoracic endothelial cells： The endothelial cells were obviously damaged in model group, and the lesion was significantly relieved in Tongluofangji group and α-lipoic acid group. CONCLUSION： Tongtuofangji can raise the antioxidative ability and protect the diabetes-induced damage of aorta thoracic endothelia in rats.