背景:水飞蓟宾(silybin)具有抗自由基活性、抗脂质过氧化作用和抗脂氧酶作用、极低的毒性,但由于其人体生物利用度低,故影响其疗效.水飞蓟宾-磷脂酰胆碱复合物(Silybin-phosphatidylcholine compound,SPC)具有较好的生物利用度,并且SPC中所含磷脂酰胆碱也具有抗自由基活性、抗脂质过氧化作用,理论上可以认为SPC具有更好的药理作用.目的:了解水飞蓟宾-磷脂酰胆碱复合物(SPC)对大鼠脑缺血再灌注损伤的保护作用.设计:完全随机设计,对照实验研究.地点和材料:所有研究工作均在新乡医学院药物研究室进行.水飞蓟宾(Silybin,含量98%),辽宁盘锦第三制药厂提供;水飞蓟宾-磷脂酰胆碱复合物(SPC,含量＞95%),本室合成;丙二醛(MDA)试剂盒,南京建成生物工程研究所提供.仪器:SHIMADZU 2550紫外-可见光分光光度计,武汉科学仪器厂DL-6低温离心机.实验动物有新乡医学院动物中心提供.干预:各组受试大鼠给与供试样品灌胃;灌胃容积为0.5 mL/100 g.将SPC和Silybin分别悬浮于5 g/L羧甲基纤维素钠(CMC)溶液中;①伪手术组和②单纯脑缺血-再灌注模型组(单纯模型组):2次/d给与5 g/LCMC灌胃.③SPC-1组:每日2次给与SPC 5 mg/100g(5 g/L CMC悬浮液)灌胃.④SPC-2组:2次/d次给与SPC 10mg/100g(5 g/L CMC悬浮液)灌胃.⑤Silybin组:每日2次给与给与Silybin 10 mg/100g(5 g/LCMC悬浮液)灌胃.于第6次灌胃(第3 d)后2 h对各组大鼠进行手术.手术在100 g/L乌拉坦(125 mg/100g)腹腔注射麻醉下,沿颈部正中切口,小心钝性分离并暴露气管和双侧颈总动脉,用无损伤小动脉夹夹闭双侧颈总动脉和迷走神经,造成脑不完全缺血,60 min后放开动脉夹,再灌注6 h后,迅速取血、并断头处死取脑,迅速检测.伪手术组不夹闭颈总动脉.各项操作均由本研究室有经验实验师完成.主要观察指标:对比观察水飞蓟宾和两种剂量的水飞蓟宾-磷脂酰胆碱复合物(SPC)对大鼠脑缺血再灌注损伤模型脑组织含水量、病理学改变、血浆丙二醛(MDA)含量的影响.结果:①除伪手术组外,各组受试大鼠均出现脑水肿改变.单纯模型组大鼠脑组织表面肿胀,蛛网膜血管增粗,可见散在小出血点;光镜下可见细胞外间隙增大,毛细血管基底膜疏松,内皮细胞肿胀、饮泡扩大、胞浆疏松、核淡染;神经细胞和胶质细胞肿胀、胞浆出现少量空腔小泡,细胞器轻度扩张、染色变浅.SPC-1组、SPC-2组和Silybin组大鼠脑组织肉眼观察病变似单纯模型组,唯小出血点少见;光镜下细胞肿胀和细胞外间隙增大较轻,胞浆内空泡较少,SPC-2组细胞器未见扩张、核染色和伪手术组相似.②受试各组大鼠脑组织含水量存在差异:伪手术组(76.53±0.71)低于单纯模型组(80.90±0.62)、SPC-1组(79.35±0.32)、SPC-2组(77.63±0.68)和Silybin组78.81±0.57(t检验均P＜0.05).单纯模型组明显高于SPC-1组、SPC-2组和Silybin组(P＜0.01).SPC-1组类似于Silybin组(P＞0.05),而明显高于SPC-2组(P＜0.01).各组受试大鼠血浆MDA含量也存在明显差异:伪手术组低于单纯模型组、SPC-1组、SPC-2组和Silybin组;单纯模型组明显高于SPC-1组、SPC-2组和Silybin组(均P＜0.01);SPC-1组类似于Silybin组(P＞0.05),而明显高于SPC-2组(P＜0.01).结论:水飞蓟宾和水飞蓟宾-磷脂酰胆碱复合物对大鼠脑缺血-再灌注损伤均有保护作用,但不能完全阻断脑缺血-再灌注损伤;水飞蓟宾-磷脂酰胆碱复合物作用优于水飞蓟宾,并在一定范围内显示出量-效关系.%BACKGROUND: Silybin has the reactions of anti-free radicals, anti-lipoid peroxidation, and anti-lipoid oxidase with extremely low toxicity. However,the effectiveness is affected by its low biological utilization in organisms.Silybin-phosphatidylcholine compound(SPC) has preferably biological utility, and moreover, the phosphatidylcholine in SPC also has anti-free radical and anti-lipoid peroxidation reaction, and therefore, theoretically, SPC has better pharmacological reactions.OBJECTIVE: To understand the protective reaction of SPC in cerebral ischemic reperfusion injury in rats.DESIGN: A complete randomized controlled study.SETTING and PARTICIPANTS: Study was conducted in the Department of Pharmacy of Xinxiang Medical College. Silybin(98% ) was obtained from Panjin No. 3 Pharmaceutical Factory of Liaoning Province. SPC(＞ 95% ) was synthesized by our department. MDA reagent box was obtained from Nanjing Jiancheng Bioengineering Institute. Instruments: SHIMADZU 2550 model Ultraviolet-visible light spectrophotometer, DL-6 model low-temperature centrifuge(Wuhan Scientific Instrument Factory) . Experimental animals were obtained from the Experimental Animal Center of Xinxiang Medical College.INTERVENTIONS: Sample medications were given through stomach-perfusion to the rats in each group. The volume of stomach-perfusion was 0.5 mL/100 g. SPC and Silybin were suspended separately in 5 g/L of CMC group(model group): stomach-perfusion were conducted twice a day with 5 g/L ach-perfusion were conducted twice a day with 10 rg/100 g SPC(5 g/L of twice a day with 10 mg/100 g of Silybin(5 g/L of CMC suspension) . At 2hours after the sixth stomach-perfusion(on the third day), surgeries were operated in the rats of each group. 100 g/L of urathane was used for anesthesia through abdominal injection in a dose of 125 mg/100 g and a central incision was made along the neck. Trachea and common carotid arteries on both sides were carefully bluntly separated and exposed. Non-traumatic arterial clamps were used to clamp common carotid arteries and pneumogastric nerves of both sides to induce incomplete cerebral ischemia and the clamps were relieved after 60 minutes. Animals were executed after 6-hour of reperfusion and blood and brain were taken immediately for detection. The common carotid arties in sham-operation group were not clamped. All above operations were completed by experienced technicians of our department.MAIN OUTCOME MEASURES: A comparative observation of the impacts of Silyhin and SPC of two different doses on water content and pathological changes in cerebral tissues as well as serous MDA content in cerebral ischemic reperfusion model rats.occurred in all rats of other groups. In the rats of model group, the brain tissue surface swelled, arachnoid vessels became thicker; some scattered bleeding points were seen. Under microscope, extracellular space increased, basal membrane of capillary vessels loosened, endothelial cells swelled, vacuole enlarged, cytoplasm loosened and the staining in nuclei was light. Neurocyte and glial cells swelled and few vacuoles occurred in cytoplasm, and cell organelles mild enlarged and staining became light. The observatory results with naked eye of the pathological changes in cerebral tissues in SPC-1 group, SPC-2group and Silybin group were similar to that of model group but only with very few bleeding points. Under microscope, the swelling of cells and the increase of extracellular space were relatively mild and relatively fewer vacuoles in cytoplasm. There was no enlargement in cell organelles in SPC-2 group and the differences in water content in brain tissue in each group: the water content in sham-operation group(76.53 ±0.71) was lower than that of model group (80. 90 ±0.62), SPC-1 group(79. 35 ±0. 32), SPC-2 group(77.63 ±0. 68)and Silybin group(78.81 ±0.57) ( P ＜ 0.05). Water content of model group was significantly higher than that of SPC-1, SPC-2 and Silybin group( P ＜0.01), and water content of SPC-1 group was similar to Silybin group( P ＞0.05) but significantly higher than that of SPC-2 group(P ＜ 0.01) . The contents of serous MDA also had significant differences among groups: the content in sham-operation group was significantly lower than that of model group, SPC-1 group, SPC-2 group and Silybin group. Content in model group was significantly higher than that of SPC-1 group, SPC-2 group and Silybin group( P ＜ 0. 01). Content in SPC-i group was similar to that of Silybin group ( P ＞ 0. 05) but significantly higher than that of SPC-2 group ( P ＜ 0.01 ).CONCLUSION: Both Silybin and SPC have protective effects on cerebral ischemic-reperfusion injury in rats but can not completely block the injury of cerebral ischemic reperfusion. The reaction of SPC is better than that of Silybin, with displays a dose-effect relationship within certain range.
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