摘要
目的观察柚皮苷联合头穴针刺对脑缺血大鼠脑组织血管新生及Notch1/丝氨酸/苏氨酸蛋白激酶(Akt)/内皮型一氧化氮合酶(eNOS)信号通路的影响。方法将120只SD大鼠随机分组为假手术组、模型组、尼莫地平组、柚皮苷组、头穴针刺组和柚皮苷+头穴针刺组,每组20只。除假手术组外,其余组大鼠采用线栓法复制大鼠局灶性脑缺血模型。造模成功后,尼莫地平组给予尼莫地平混悬液12 mg/kg灌胃,柚皮苷组给予柚皮苷溶液80 mg/kg灌胃,头穴针刺组给予头穴针刺干预,柚皮苷+头穴针刺组给予柚皮苷溶液80 mg/kg灌胃和头穴针刺干预,假手术组及模型组给予等量生理盐水灌胃,各组均每天干预1次,连续干预7 d。干预结束后,记录各组大鼠神经功能评分,干/湿重法和TTC染色观察大鼠脑组织含水量及脑梗死体积,ELISA法检测大鼠血清中血管内皮生长因子(VEGF)、血管内皮生长因子受体2(VEGFR-2)水平,免疫组化法检测海马组织中VEGF阳性表达情况,Western blot法检测海马组织中Notch1、Akt、磷酸化Akt(p-Akt)、eNOS、磷酸化eNOS(p-eNOS)蛋白表达情况,RT-PCR法检测海马组织中Notch1、p-Akt、p-eNOS mRNA表达情况。结果模型组大鼠存在明显神经功能损伤及脑梗死,脑组织含水量、血清VEGF和VEGFR-2水平、海马组织中VEGF阳性表达数和Notch1蛋白及mRNA相对表达量均明显高于假手术组(P均<0.05),海马组织中p-Akt/Akt和p-eNOS/eNOS比值及Notch1、p-Akt、p-eNOS mRNA相对表达量均明显低于假手术组(P均<0.05)。与模型组比较,尼莫地平组、柚皮苷组、头穴针刺组和柚皮苷+头穴针刺组大鼠神经功能评分、脑含水量和梗死体积均明显降低(P均<0.05),且柚皮苷+头穴针刺组和尼莫地平组均明显低于柚皮苷组、头穴针刺组(P均<0.05);血清VEGF和VEGFR-2水平,海马组织中VEGF阳性表达数、p-Akt/Akt和p-eNOS/eNOS比值、Notch1蛋白及Notch1、p-Akt、p-eNOS mRNA相对表达量均明显增高(P均<0.05),且柚皮苷+头穴针刺组和尼莫地平组均明显高于柚皮苷组、头穴针刺组(P均<0.05);柚皮苷+头穴针刺组和尼莫地平组各指标比较差异均无统计学意义(P均>0.05)。结论柚皮苷联合头穴针刺治疗可明显改善脑缺血大鼠神经功能,促进大鼠脑组织血管新生,其作用可能与激活Notch1/Akt/eNOS通路相关。
Objective It is to observe the effect of naringin combined with scalp acupuncture on angiogenesis and Notch1/serine/threonine kinase(Akt)/endothelial nitric oxide synthase(eNOS)signaling pathway in cerebral ischemia rats.Methods One hundred and twenty SD rats were randomly divided into sham operation group,model group,nimodipine group,naringin group,scalp acupuncture group and naringin+scalp acupuncture group,20 rats in each group.The rats in all groups except for the sham operation group were used to replicate focal cerebral ischemia models by the suture-occluded method.After successful modeling,the nimodipine group was given 12 mg nimodipine suspension by gavage,the naringin group was given 80 mg naringin solution by gavage,the scalp acupuncture group was treated with scalp acupuncture,the naringin+scalp acupuncture group was treated with 80 mg naringin solution by gavage and scalp acupuncture,while the sham operation group and the model group were given the same amount of normal saline by gavage.Each group was intervened once a day for 7 consecutive days.After the intervention,the neurological function scores of the rats in each group were recorded,the water content and cerebral infarction volume of the rat brain tissue were observed by the dry/wet weight method and TTC staining,the levels of serum vascular endothelial growth factor(VEGF),vascular endothelial growth factor receptor 2(VEGFR-2)were detected by ELISA method,the positive expression of VEGF in hippocampus tissue was detected by immunohistochemical method,the protein expression of Notch1,Akt,phosphorylated Akt(p-Akt),eNOS,phosphorylated eNOS in hippocampus tissue(p-eNOS)was detected by Western blot method,the mRNA expression of Notch1,p-Akt,p-eNOS mRNA in hippocampus was detected by RT-PCR method.Results The rats in the model group had obvious neurological damage and cerebral infarction,and the water content in the brain tissue,the levels of VEGF and VEGFR-2 in serum,the positive expression of VEGF in the hippocampus and the relative expression of Notch1 protein and mRNA were significantly higher than those in the sham operation group(all P<0.05),the ratios of p-Akt/Akt and p-eNOS/eNOS and the relative expressions of Notch1,p-Akt and p-eNOS mRNA in hippocampus were significantly lower than those in the sham operation group(all P<0.05).Compared with the model group,the neurological function score,brain water content and infarct volume in the nimodipine group,naringin group,scalp acupuncture group and naringin+scalp acupuncture group were significantly decreased(all P<0.05),and the naringin+scalp acupuncture group and nimodipine group were significantly lower than the naringin group and scalp acupuncture group(all P<0.05);the levels of serum VEGF and VEGFR-2,the quantity of positive expression of VEGF in hippocampus,p-Akt/Akt and p-eNOS/eNOS ratios,relative expression of Notch1 protein and Notch1,p-Akt and p-eNOS mRNA were significantly increased(all P<0.05),and the naringin+scalp acupuncture group and the nimodipine group were significantly higher than the naringin group and scalp acupuncture group(all P<0.05);the differences in each index between the naringin+scalp acupuncture group and the nimodipine group were not significant(P>0.05).Conclusion Naringin combined with scalp acupuncture can significantly improve the neurological function and promote angiogenesis in brain tissue in the rats with cerebral ischemia,the mechanism be related to the activation of Notch1/Akt/eNOS pathway.
作者
陈悦
王珍珍
王文生·王月
李喆
CHEN Yue;WANG Zhenzhen;WANG Wensheng·WANG Yue;LI Zhe(Urumqi Hospital of Traditional Chinese Medicine,Urumqi 830000,Xinjiang,China;Naval Medical Center of Naval Military Medical University,Shanghai 200052,China)
出处
《现代中西医结合杂志》
CAS
2022年第16期2218-2224,共7页
Modern Journal of Integrated Traditional Chinese and Western Medicine
基金
新疆维吾尔自治区自然科学基金资助项目(2021D01C225)。
