补阳还五汤及拆方影响脑缺血再灌注大鼠细胞凋亡与Bcl-2、Bax蛋白的表达

背景:补阳还五汤是一种临床上常用的治疗缺血性脑血管疾病的中药,其作用机制还有许多不明之处。目的:观察补阳还五汤及拆方对脑缺血再灌注SD大鼠模型细胞凋亡及Bcl-2、Bax蛋白表达的影响及其作用机制,并研究方剂配伍的意义。设计:随机对照的动物实验。单位:河南中医学院第一附属医院神经内科。材料:实验在河南省中医药研究院国家中医管理局三级实验室进行。选择上海西普尔-必凯实验动物有限公司提供的清洁级SD大鼠60只,月龄4～5个月;雌雄各半,雌性260～310g,雄性280～330g。活血组药物选用当归120g,桃仁60g,川芎60g,加水1440mL,提取挥发油,备用。药渣及提取液加入赤芍120g,地龙60g,红花60g,再加入1440mL水,煎煮1h,滤过,药渣再加入2880mL水,煎煮1h,滤过,合并两次煎液,浓缩至1200mL,加入挥发油及吐温-803mL,混匀,即得。黄芪组药物选用黄芪1200g,加水7200mL,煎煮两次,每次1h,滤过,合并煎液,浓缩至600mL,加入吐温-802mL,即得。补阳还五汤组选用当归60g,桃仁30g,川芎30g,加水720mL,提取挥发油,备用。药渣及提取液加入黄芪1200g,赤芍60g,地龙30g,红花30g,再加入920mL水,煎煮1h,滤过,药渣再加入8640mL水,煎煮1h,滤过,合并两次煎液,浓缩至600mL,加入挥发油及吐温-803mL,混匀,即得。(黄芪组和补血组药物为补阳还五汤的拆方。)活血组药物每毫升相当生药0.4g,黄芪组药物每毫升相当于生药2g,全方组药物相当于生药2.4g。方法:将60只清洁级SD大鼠按随机数字表法分为假手术组、模型组、活血组、黄芪组、总方组,每组分别为12只。每只SD大鼠分别通过灌胃给药:活血组8g/kg、黄芪组40g/kg、总方组48g/kg,假手术组及模型组分别给予生理盐水20mL/kg。通过四血管结扎法制作脑缺血再灌注模型。采用缺口末端标记法原位检测凋亡细胞(TUNEL),应用免疫组化法测定Bcl-2蛋白、Bax蛋白的表达。主要观察指标:①各组SD大鼠脑组织神经元细胞凋亡水平的比较;②各组SD大鼠脑组织神经元Bcl-2、Bax阳性细胞表达水平的比较。结果:①各组SD大鼠脑组织神经元细胞凋亡水平比较:与假手术组相比,其他各组细胞凋亡明显增加(t=6.07～11.70,P<0.01);与模型组相比,活血组、黄芪组细胞凋亡减少(t=2.71,2.06,P<0.05),总方组减少明显(t=5.34,P<0.01)。②各组SD大鼠脑组织神经元Bcl-2、Bax阳性细胞表达水平的比较:与假手术组相比,活血组、黄芪组、总方组Bcl-2染色阳性细胞的表达增多(t=4.59～8.82,P<0.01),Bax阳性细胞表达更加明显(t=4.59～8.55,P<0.01);与模型组相比,活血组、黄芪组、总方组Bcl-2染色阳性细胞的表达增多(t=3.48～7.75,P<0.01),Bax阳性细胞的表达降低(t=3.83～5.88,P<0.01)。活血组药物作用与黄芪组差异无显著性(P>0.05),总方组药物作用与黄芪组、活血组差异极具显著性(P<0.01)。结论:补阳还五汤及拆方通过抑制细胞凋亡的作用,达到抗脑缺血再灌注损伤的作用。黄芪组和活血组即拆方的协同作用,使总方组发挥最大的疗效。

Influences of Buyanghuanwu decoction and its decomposed formulas on cell apoptosis and expressions of Bcl-2 and Bax protein in rats with cerebral ischemia/reperfusion

BACKGROUND: Buyanghuanwu decoction is a kind of commonly used Chinese herb in clinic for ischemic cerebrovascular disease, but its mechanisms have been unknown.OBJECTIVE: To observe effect of Buyanghuanwu decoction and its decomposed formulas on the levels of nerve apoptotic cells and expressions of Bcl-2 and Bax of brain in the ischemia/reperfusion model of SD rats and its mechanisms, study the significance of formula compatibility.DESIGN: Randomized controlled animal experiment.SETTING: Department of Neurology, First Affiliated Hospital, Henan College of Traditional Chinese Medicine.MATERIALS: The experiment was performed in 3rd Grade Laboratory of State Administration of Traditional Chinese Medicine, Henan Academy of Chinese Medicine. Totally 60 clean-grade SD rats aged 4-5 months, male and female (half and half), with the body mass of 260-310 g in females and 280-330 g in males were employed in the experiment, provided by Shanghai SIPPR/BK Experimental Animal Co., Ltd. In blood activation group, 120 g danggui (Radix Angelicae Sinensis), 60 g taoren (Semen Persicae), 60 g chuanxiong (Szechwan Lovage Rhizome) were selected, and then water 1 440 mL was added and volatile oil was extracted for reserving. 120 g chishao (Radix Paeoniae Rubra), 60 g dilong (Lumbricus), 60 g honghua (Flos Carthami) and 1 440 mL water were added to the gruffs and extract, and then filtered after 1 hour decoction, added 2 880 mL water to the gruffs again, decocted for 1 hour and filtered. The decocted liquid was merged and condensed to 1 200 mL, and then we get it by adding volatile oil and tween -80 3 mL and misce bene. In huangqi (Radix Astragaliseu Hedysary) group, 1 200 g huangqi was selected and 7 200 mL water was added, decocted twice, every time for one hour, and then filtered and the decoction was merged, concentrated to 600 mL, obtained by addition of tween-80 2 mL. In Buyanghuanwu decoction group, 60 g danggui (Radix Angelicae Sinensis), 30 g taoren (Semen Persicae), 30 g chuanxiong (Szechwan Lovage Rhizome) were selected, 720 mL water was added, and volatile oil was extracted for reserving. 1 200 g huangqi (Radix Astragali),60 g chishao (Radix Paeoniae Rubra), 30 g dilong (Lumbricus), 30 ghonghua (Flos Carthami) and 920 mL water were added in the gruffs and extract, and then filtered after 1-hour decoction. 8 640 mL water was added in the gruffs again, decocted for 1 hour and filtered, and then the decocted liquid was merged and condensed to 600 mL, then we get it by adding volatile oil and tween -80 3 mL and misce bene (huangqi group and blood activation group was the decomposed formulas of Buyanghuanwu decoction group). The 1 mL drug in blood activation group was equal to 0.4g raw herbs, 1 mL huangqi (Radix Astragali) in the huangqi group to 2 g raw herbs, and the drug in the general formula group to 2.4 g raw herbs.METHODS: A total of 60 SD rats of clean grade were randomly assigned into sham operation group, model group, blood activation group, huangqi group and general formula group with 12 in eachgroup. The medicine was applied by gastric perfusion at the dosages of 8 g/kg, 40 g/kg and 48 g/kg in blood activation group, huangqi group and general formula group successively; and 20 mL/kg saline was applied in both sham operation group and model group. The models of cerebral ischemia/reperfusion were prepared by four vessel ligature method. Apoptotic cells were determined with TDT-mediated dUTP biotin nick ending labeling (TUNEL). Expressions of Bcl-2 protein and Bax protein were measured with immunohistochenical method.MAIN OUTCOME MEASURES: ①Comparison of apoptotic levels of neurons in brain tissues of SD rats in each group; ②Comparison of expressions of Bcl-2 and Bax positive cells in neurons of brain tissue of SD rats in each group.RESULTS: ①Comparison of neuron apoptotic level of brain tissues in SD rats of each group: Compared with the sham operation group, the apoptosis increased in other groups (t=6.07-11.70,P＜0.01). Compared with the model group, the apoptosis decreased in the blood activation group and huangqi group (t=2.71, 2.06, P＜0.05), and decreased obviously in the general formula group (t=5.34, P＜0.01 ). ②Comparison of Bcl-2 and Bax positive cells in the neurons of brain tissues in SD rats of each group:Compared with the sham operation group, the expression of Bcl-2 staining positive cells increased in the blood activation group, huangqi group and general formula group (t=4.59-8.82,P＜0.01 ), and the expression of Bax positive cells became very significant (t=4.59-8.55,P＜0.01 ). Compared with the model group, the expression of Bcl-2 staining positive cells increased in the blood activation group, huangqi group and general formula group (t=3.48-7.75,P＜0.01 ), and the expression of Bax positive cells decreased (t=3.83-5.88,P＜0.01 ). There was no significant difference between the blood activation group and huangqi group (P＞0.05). There were significant differences in the drug effect of general formula group with huangqi group and blood activation group (P＜0.01).CONCLUSION: Buyanghuanwu decoction and its decomposed formulas act on being against the cerebral ischemia/reperfusion injury achieved by inhibiting cell apoptosis. Synergism of huangqi group and blood activation group leads to the strongest curative effect of general formula group.