补阳还五汤及其拆方对脑缺血大鼠神经功能及血管生成的影响

Effects of Buyang Huanwu decoction and its decomposed recipes on neural function and angiogenesis after focal cerebral ischemia in rats

目的探讨补阳还五汤及其拆方对局灶性脑缺血后大鼠神经功能和血管生成的影响及机制。方法采用随机数字表法,将清洁级SD雄性大鼠(52只)随机分为假手术组、模型组、全方组、补气组、活血组,每组各8只。除假手术组外,其余各组大鼠均采用线栓法建立大脑中动脉闭塞模型,以首次Longa神经功能评分1~3分者为造模成功。补阳还五汤全方由黄芪120.0 g、当归6.0 g、赤芍4.5 g、川芎3.0 g、地龙3.0 g、红花3.0 g、桃仁3.0 g组成,补气方由黄芪120.0 g组成,活血方由当归6.0 g、赤芍4.5 g、川芎3.0 g、地龙3.0 g、红花3.0 g、桃仁3.0 g组成。术后第1天开始灌胃给药,全方组、补气组、活血组灌胃剂量分别为13.1、10.8、2.2 g/kg,假手术组和模型组分别灌服等容量的等渗盐水,1次/d,连续14 d。腹腔注射5-溴脱氧尿嘧啶核苷(Brd U,50 mg/kg),1次/d,连续14 d。在术后第1、7、14天,采用改良的神经症状严重程度评分(m NSS)和角实验评价感觉运动功能。术后第14天,采用Brd U和大鼠血管性血友病因子(v WF)免疫荧光双标检测缺血周边区血管生成情况;Western Blot检测血管内皮生长因子(VEGF)的蛋白表达。结果 (1)与模型组比较,术后第7、14天,全方组大鼠m NSS评分较低[(6.8±1.0)分比(8.5±1.1)分、(6.1±0.8)分比(8.0±1.4)分,均P<0.01],全方组右转次数减少[(7.1±0.6)次比(8.6±1.2)次、(6.1±0.8)次比(7.9±1.1)次,均P<0.01],补气组右转次数减少[(7.5±0.5)次比(8.6±1.2)次、(6.2±1.0)次比(7.9±1.1)次,均P<0.01];术后第14天,全方组缺血周边区Brd U/v WF标记免疫阳性细胞数量明显增多,组间差异有统计学意义[(30±8)个/mm2比(24±7)个/mm2,P<0.01],VEGF蛋白表达增加[(0.33±0.01)比(0.30±0.01),P<0.01]。(2)与补气组比较,术后第7、14天,全方组大鼠m NSS评分较低[补气组分别为(8.2±1.3)、(7.5±0.9)分,均P<0.05],术后第14天,全方组缺血周边区Brd U/v WF免疫阳性细胞增多[补气组为(26±5)个/mm2,P<0.05],VEGF蛋白表达增加[补气组为(0.31±0.01),P<0.01]。(3)与活血组比较,术后第7、14天,全方组大鼠m NSS评分较低[活血组分别为(8.5±0.9)、(7.6±0.7)分,均P<0.05],右转次数减少[活血组分别为(8.5±0.8)、(7.6±0.9)次,均P<0.05],术后第14天,全方组缺血周边区Brd U/v WF免疫阳性细胞增多[活血组为(26±6)个/mm2,P<0.05],VEGF相对表达水平升高[活血组为(0.31±0.01),P<0.05]。结论补阳还五汤能促进脑缺血后血管生成和神经功能恢复,其机制可能与上调VEGF蛋白有关,方中补气药和活血药具有协同作用。

Objective To investigate the effects of Buyang Huanwu decoction and its decomposed recipes on neurological function and angiogenesis after focal cerebral ischemia in rats and its mechanism.Methods Fifty-two clean grade SD male rats were randomly divided into sham-operation, model, whole prescription, invigorating qi, and promoting blood circulation groups （n = 8 in each group） according to the random number table. In addition to the sham-operation group, the middle cerebral artery occlusion models of the rats in other groups were induced by the suture method. The patients with the first Longa nerve function scores 1 to 3 were used as successful modeling. The whole Buyang Huanwu decoction included dried root of Astragalus membratmceus 120.0 g ,dried root of angelica sinensis 6.0 g ,dried root of Paeonia lactiflora 4.5 g, dried rhizome Ligusticum chuanxiong 3.0 g, dried body of Pheretima aspergillum 3.0 g, dried flowers of Carthamus tinctorius 3.0 g, and seed of Prunus persica 3.0 g ; the invigorating qi prescription included dried root of Astragalus membranaceus 120.0 g;the promoting blood circulation prescription included dried root of angelica sinensis 6.0 g, dried root of Paeonia lactifiora 4.5 g, dried rhizome Ligusticum chuanxiong 3.0 g, dried body of Pheretima aspergillum 3.0 g, dried flowers of Carthamus tinctorius 3.0 g, and seed of Prunus persica 3.0 g. On the first day after procedure,the rats began to be administered intragastrically. The intragastric doses of the whole prescription group, invigorating qi group, and promoting blood circulation group were 13.1,10.8, and 2.2 g/kg, respectively. The sham-operation group and the model group were given equal volume of isotonic saline,once a day for 14 days. 5-bromodeoxyuridine （BrdU,50 mg/kg） were injected intraperitoneally, once a day for 14 days. The modified neurological severity score （mNSS） and the corner test were used to evaluate sensorimotor function at day 1,7 and 14 after procedure. BrdU and rat von Willebrand factor （vWF） double immunofluorescent staining were used to detect the angiogenesis in ischemic peripheral region;Western Blot was used to detect the protein expression of vascular endothelial growth factor （VEGF）. Results （ 1 ） Compared with the model group,the mNSS score in rats in the whole prescription group was lower at day 7 and 14 after procedure （6.8 ± 1.0 vs. 8.5 ± 1.1,6.1 ± 0.8 vs. 8.0 ± 1.4 ; all P 〈 0.01 ）. The number of turning right in the whole prescription group was reduced （7.1 ± 0.6 vs. 8.6 ± 1.2 and 6.1 ± O. 8 vs. 7.9 ± 1.1 ;all P 〈0.01 ）. The number of turning right in the invigorating qi group was reduced （7.5± 0.5 vs. 8.6± 1.2 and 6.2 ± 1.0 vs. 7.9 ± 1. 1 ; all P 〈 O. 01 ）. At day 14 after procedure, the number of BrdU/vWF co-labeled immunopositive cells in ischemic peripheral zone of the whole prescription group was increased significantly. There was significant difference between the groups （30± 8/mm^2 vs. 24 ± 7/mm^2 ; P 〈 0.0 l ）. The VEGF protein expression was increased （0.33±0.01 vs. 0.30 s0.01 ;P 〈0.01 ）. （2） Compared with the invigorating qi group,the rat mNSS scores of the whole prescription group were lower at day 7 and 14 after procedure （ the invigorating qi group 8.2 ± 1.3 and 7.5 ± 0.9 respectively ; all P 〈 0.05 ）. The number of BrdU/vWF immunopositive cells in the whole prescription group was increased at day 14 after procedure （26±5/mm^2 in the invigorating qi group;P 〈 0. 05）. The VEGF protein expression was increased （0. 31 ± 0.01 in the invigorating qi group ;P 〈 0.01 ）. （3） Compared with the promoting blood circulation group, the mNSS scores of the whole prescription group were lower at day 7 and 14 after procedure （ the promoting blood circulation group 8.5± 0. 9 and 7.6 ± 0.7 respectively; all P 〈 0. 05 ）. The number of turning right was reduced （8.5 ± 0. 9and 7.6 ± 0.9 respectively in the promoting blood circulation group ; all P 〈 O. 05 ）. The number of BrdU/vWF immunopositive cells in isehemie peripheral zone of the whole prescription group at day 14 after procedure was increased （ 26± 6/mm^2 , P 〈 0.05 ）. The relative expression level of VEGF was increased （0.31 ± 0.01 in the promoting blood circulation group ,P 〈 0.05 ）. Conclusion Buyang Huaawu decoction can promote angiogenesis and recovery of neurological function after cerebral isehemia. Its mechanism may be associated with the up-regulation of the VEGF protein. The traditional Chinese medicines for invigorating qi and invigorating the circulation of blood in the prescription have synergistic effect.