针刺预处理对卒中后血管性认知障碍大鼠血管新生的影响

Effect of Acupuncture Pretreatment on Angiogenesis in Rats with Vascular Cognitive Impairment After Stroke

目的 探讨针刺预处理方法对卒中后血管性认知障碍(Post-stroke vascular cognitive impairment, PSVCI)大鼠血管新生的影响。方法 将48只5~6周龄SPF级健康雄性Sprague-Dawley(SD)大鼠按照随机数字表法分为空白对照组、模型组、针刺组和药物组,每组各12只,每组分7 d、14 d两个亚组,每个亚组各6只。针刺组针刺“百会”“水沟”、双侧“风池”“内关”“神门”,药物组予盐酸氟桂利嗪混悬液腹腔注射,针刺组和药物组1次/d干预,连续7 d;模型组正常条件下饲养7 d,空白对照组不作处理。7 d后,针刺组、药物组和模型组同时采用双侧颈总动脉永久结扎法制备VCI大鼠模型。模型制备后正常饲养,1周后对各7 d亚组大鼠采用免疫组化法检测血管内皮内皮生长因子(Vascular Endothelial Growth Factor, VEGF)蛋白平均光密度值(Average Option Disenty, AOD)、Western blot法检测VEGF蛋白表达量、荧光定量PCR法检测Notch 1、Dll 4相对表达量;2周后对各14 d亚组大鼠除上述检测外再行Morris水迷宫实验观察其学习记忆能力。结果 与针刺组14 d亚组比较,药物组14 d亚组、模型组14 d亚组平均逃避潜伏期明显延长,差异有统计学意义(P<0.05,P<0.01),平台所在象限时间明显降低,差异有统计学意义(P<0.05)。与空白对照组比较,针刺组14 d亚组平均逃避潜伏期明显延长,差异有统计学意义(P<0.05),平台所在象限时间明显延长,差异有统计学意义(P<0.05)。免疫组化实验结果显示,与针刺组两个亚组比较,药物组两个亚组VEGF蛋白表达AOD值均明显降低,差异有统计学意义(P<0.05);针刺组两个亚组VEGF蛋白表达的AOD值与模型组两个亚组均明显升高,差异有统计学意义(P<0.05)。WB实验结果显示,与针刺组两个亚组比较,药物组同时相亚组VEGF蛋白表达量明显降低,差异有统计学意义(P<0.01,P<0.05);与针刺组比较,模型组同时相两个亚组VEGF蛋白表达表达量均明显降低,差异有统计学意义(P<0.05)。qPCR实验结果显示,与药物组同时相两个亚组比较,7 d针刺组、14 d针刺组Notch 1 mRNA的相对表达量均明显升高,差异有统计学意义(P<0.01),针刺组两个时相Notch 1 mRNA的相对表达量与同时相模型组明显升高,差异有统计学意义(P<0.05);7 d针刺组、14 d针刺组与药物组同时相亚组Dll 4 mRNA的相对表达量明显升高,差异有统计学意义(P<0.01),针刺组两个时相亚组与模型组同时相亚组之间,差异有统计学意义(P<0.05)。结论 通过通督调神法针刺提前干预,能够有效提高VCI大鼠模型大脑组织内VEGF蛋白含量、Notch 1、Dll 4 mRNA相对表达量,能够促进VCI大鼠海马组织及大脑组织血管新生,进而预保护VCI模型大鼠认知功能。

Objective To investigate the effect of acupuncture pretreatment on angiogenesis in rats with post-stroke vascular cognitive impairment(PSVCI).Methods Forty-eight SPF male Sprague-Dawley(SD)rats(5 to 6 weeks)were divided into blank control,model,acupuncture,and medication groups by random number table,with 12 in each group.The groups were further divided into 7-day and 14-day subgroups each,with 6 in each group.The acupuncture group received needling at Baihui(GV 20),Shuigou(GV 26),bilateral Fengchi(GB 20),Neiguan(PC 6),and Shenmen(HT 7).The medication group was intraperitoneally injected with flunarizine hydrochloride suspension.The intervention for the acupuncture and medication groups was once daily for 7 consecutive days.The model group was raised under normal conditions for 7 days,and the blank control group received no treatment.After 7 days,PSVCI rat model was induced in the acupuncture,medication,and model groups by permanently ligating the bilateral common carotid arteries.After 1 week of normal feeding,immunohistochemistry was used in each 7-day subgroup to determine the average optical density(AOD)of vascular endothelial growth factor(VEGF)protein;Western blot to measure the expressions of VEGF protein;fluorescence quantitative PCR to detect the relative expressions of Notch1 and Dll4.Additionally,after 2 weeks,Morris water maze test was performed in each 14-day subgroup to evaluate the learning and memory abilities of the rats.Results The 14-day subgroups of the medication and model groups exhibited prolonged average escape latency and reduced time spent in the platform quadrant compared with the 14-day subgroup of the acupuncture group(P<0.05,P<0.01).The 14-day subgroup of the acupuncture group showed extended average escape latency and increased time spent in the platform quadrant compared with the blank control group(P<0.05,P<0.05).Immunohistochemical results indicated that both subgroups of the acupuncture group had higher AOD values of VEGF protein than those in the subgroups of the medication group(P<0.05),and these values were also higher compared with the model group(P<0.05).Western blot revealed that the the subgroups of the acupuncture group had higher expressions of VEGF protein than those of the medication group(P<0.01,P<0.05)and the model group(P<0.05).The qPCR results showed increased relative expression of Notch1 mRNA in the 7-day and 14-day subgroups of the acupuncture group compared with the condition in the subgroups of the medication group(P<0.01)and the model group(P<0.05).The relative expression of Dll4 mRNA were also elevated in the two subgroups of the acupuncture group compared with the condition in the subgroups of the medication group(P<0.01)and model group(P<0.05).Conclusion Early acupuncture intervention using the Tongdu Tiaoshen method could effectively increase VEGF protein and the relative expression of Notch1 and Dll4 mRNA in the brain tissues of PSVCI rats,promoting angiogenesis in hippocampal and brain tissues and thereby proactively protecting the cognitive function of PSVCI rats.