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Title: [Effect of electroacupuncture at "Baihui" (GV20) and "Zusanli" (ST36) on angiogenesis in the brain of middle cerebral artery occlusion rats based on HIF/VEGF/Notch signaling pathway].
Author: Yang YY, Wu S, Ma SN, Guan MY, Wang JY, Ren BB.
Journal: Zhen Ci Yan Jiu; 2024 Oct 25; 49(10):1030-1039. PubMed ID: 39433363.
Abstract:
OBJECTIVES: To observe the effect of electroacupuncture (EA) on hypoxia-inducible factor (HIF)/vascular endothelial growth factor (VEGF)/Notch signaling pathway and related factors in rats with cerebral ischemia (CI), so as to explore its regulatory mechanism underlying improvement of CI by promoting cerebral microangiogenesis. METHODS: Fifteen male SD rats were randomly selected as the sham surgery (sham) group, while other 85 rats were used to prepare CI model according to the modified Zea-Longa method. The successful CI model rats (n=60) were randomly allocated to model, EA, EA+inhibitor, and inhibitor groups, with 15 rats in each group. EA stimulation (1 Hz/20 Hz, 1-2 mA) was applied to "Baihui"(GV20) and right "Zusanli"(ST36) for 30 min, once a day for 7 days. Rats of the 2 inhibitor groups received intraperitoneal injection of YC-1 (HIF-1α inhibitor, 2.5 mg/kg), once a day for 7 days. The modified neurological severity scale (mNSS, including the motor ［muscular state, abnormal movement］, sensory ［visual, tactile and proprioceptive］, balance and reflex functions, 0-18 points) was used to assess the rats' neurological deficit state. The percentage of cerebral infarct volume was measured after 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining, and pathological changes of the ischemic penumbra cortex were observed after H.E. staining. The immunoactivity of CD34 was determined using immunohistochemistry, followed by calculating the microvascular density (MVD) in the ischemic penumbra cortex, and the expression levels of HIF-1α, VEGF, vascular endothelial growth factor receptor 2 (VEGFR2), Notch1, and Delta like 4 ligand (DLL4) mRNAs and proteins in the ischemic penumbra of brain tissue were detected using real-time PCR and Western blot, respectively. RESULTS: In contrast to the sham group, the mNSS score, percentage of cerebral infarct volume, MVD, and expression levels of HIF-1α, VEGF, VEGFR2, Notch1 and DLL4 proteins and mRNAs were all significantly increased in the model group (P<0.01). In comparison with the model group, the mNSS score and percentage of cerebral infarct volume were significantly decreased in the EA group (P<0.01), and increased in the inhibitor group (P<0.01), and the MVD, and expression levels of HIF-1α, VEGF, VEGFR2, Notch1 and DLL4 proteins and mRNAs were further strikingly increased in the EA group (P<0.01), but obviously decreased in the inhibitor group (P<0.05, P<0.01). Comparison between EA and EA+inhibitor groups showed that the effects of EA were basically eliminated in lowering mNSS score and percentage of cerebral infarct volume (P<0.01), and in up-regulating MVD, and expression levels of HIF-1α, VEGF, VEGFR2, Notch1 and DLL4 mRNAs and proteins (P<0.01). The levels of mNSS score and percentage of cerebral infarct volume were markedly higher (P<0.01) in the inhibitor group than those in the EA+inhibitor group, while the MVD, expression levels of HIF-1α, VEGF, VEGFR2, Notch1 and DLL4 mRNAs and proteins were significantly lower in the inhibitor group than in the EA+inhibitor group (P<0.01), suggesting an elimination of EA after administration of HIF-1α inhibitor. H.E. staining showed loosened structure and disordered arrangement of neurons, swollen and vacuolized cells with ruptured nuclei, swollen and deformed microvessels in the ischemic penumbra of the brain tissue in the model group, which was improved in the EA group, including reduction of cellular swelling degree and vacuol-like changes, relatively intact of nuclei, and increase of new capillaries. CONCLUSIONS: EA of GV20 and ST36 can improve neurological deficit and reduce cerebral infarct volume in CI rats, which may be associated with its functions in promoting angiogenesis in ischemic penumbra area and in up-regulating the activities of HIF/VEGF/Notch signaling pathway and related factors. 目的: 观察电针对脑缺血大鼠缺氧诱导因子（HIF）/血管内皮生长因子（VEGF）/神经源性位点缺口同源蛋白（Notch）信号通路及相关因子的影响，探讨电针对大鼠脑缺血后大脑微血管新生的调控机制。方法: 从100只雄性SD大鼠中随机选取15只为假手术组，其余85只大鼠采用改良Zea-Longa法制备左侧大脑中动脉缺血模型，将造模成功的大鼠随机分为模型组、电针组、电针＋抑制剂组、抑制剂组，每组15只。电针组大鼠予“百会”、右侧“足三里”电针治疗，疏密波，30 min/次，1次/d；电针+抑制剂组大鼠在电针治疗后，腹腔注射HIF-1α抑制剂YC-1 （2.5 mg/kg），1次/d；抑制剂组大鼠腹腔注射HIF-1α抑制剂YC-1 （2.5 mg/kg），1次/d。以上各组均连续治疗7 d。采用改良神经功能损伤严重程度量表（mNSS）评分评估大鼠神经功能缺损情况；TTC染色法观察大鼠脑梗死体积百分比；HE染色法观察大鼠缺血半暗带皮层病理学变化；免疫荧光染色法检测CD34表达以计算缺血半暗带皮层微血管密度（MVD）；Western blot法和荧光定量PCR法分别检测缺血半暗带皮层脑组织HIF-1α、VEGF、血管内皮生长因子受体2（VEGFR2）、Notch1、Delta样4配体（DLL4）蛋白及mRNA表达水平。结果: 与假手术组比较，模型组大鼠mNSS评分、脑梗死体积百分比及脑组织缺血半暗带MVD均升高（P<0.01），缺血侧脑组织HIF-1α、VEGF、VEGFR2、Notch1、DLL4的蛋白及mRNA表达水平升高（P<0.01）。与模型组比较，电针组大鼠mNSS评分、脑梗死体积百分比均降低（P<0.01），脑组织缺血半暗带MVD和缺血侧脑组织HIF-1α、VEGF、VEGFR2、Notch1、DLL4的蛋白及mRNA表达水平均升高（P<0.01）；抑制剂组大鼠mNSS评分及脑梗死体积百分比均升高（P<0.01），脑组织缺血半暗带MVD和HIF-1α、VEGF、VEGFR2、Notch1、DLL4的蛋白及mRNA表达水平降低（P<0.05，P<0.01）。与电针＋抑制剂组比较，电针组大鼠mNSS评分、脑梗死体积百分比均降低（P<0.01），脑组织缺血半暗带MVD和HIF-1α、VEGF、VEGFR2、Notch1、DLL4的蛋白及mRNA表达水平均升高（P<0.01）；抑制剂组大鼠mNSS评分、脑梗死体积百分比均升高（P<0.01），大鼠脑组织缺血半暗带MVD和HIF-1α、VEGF、VEGFR2、Notch1、DLL4的蛋白及mRNA表达水平均降低（P<0.01）。HE染色显示模型组大鼠缺血半暗带脑组织神经元结构疏松，排列紊乱，细胞肿胀；与模型组比较，电针组大鼠缺血半暗带脑组织排列紊乱程度有所改善，肿胀程度减轻，而抑制剂组缺血半暗带脑组织上述损伤程度更重；与电针+抑制剂组比较，抑制剂组大鼠缺血半暗带脑组织上述损伤程度更重，而电针组缺血半暗带脑组织上述损伤程度较轻。结论: 电针“百会”“足三里”可促进脑缺血大鼠缺血半暗带血管新生，减轻缺血后脑损伤，其作用机制可能与提高HIF/VEGF/Notch信号通路及相关因子的表达有关。.

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