基于miR-126-3p调控mTOR/HIF-1α信号通路探讨电针促脑缺血大鼠血管新生的机制

Involvement of miR-126-3p via mTOR/HIF-1α signaling pathway in effect of electroacupuncture on angiogenesis in rats with cerebral ischemia

目的:观察电针对脑缺血模型大鼠miR-126-3p及哺乳动物雷帕霉素靶蛋白(mTOR)/缺氧诱导因子-1α(HIF-1α)信号通路的影响,探讨电针疗法对脑缺血后血管新生的调控机制。方法:SD大鼠随机分为对照组、模型组、电针组和抑制剂组,每组按治疗时程分为3、7、14 d共3个亚组,各亚组12只。采用Zea Longa法建立右侧大脑中动脉缺血(MCAO)大鼠模型。电针组予“大椎”“百会”电针治疗,疏密波,频率2 Hz/20 Hz,强度0.5 mA,20 min/次,术后4 h开始电针治疗,1次/d,分别治疗3、7、14 d。抑制剂组在电针治疗的基础上予腹腔注射mTOR特异性抑制剂雷帕霉素(0.1 mg/mL),每次0.3 mg/kg, 1次/d,分别注射3、7、14 d。改良神经功能缺损评分(mNSS)评估大鼠神经功能缺损情况,透射电镜观察大鼠缺血半暗区皮层神经元及微血管内皮超微结构,免疫组织化学法检测大鼠缺血半暗区皮层内皮微血管密度(MVD),Western blot法和荧光定量PCR法分别检测大鼠缺血半暗区皮层mTOR、HIF-1α蛋白及其mRNA和miR-126-3p的表达。结果:与同时点对照组比较,模型组大鼠mNSS升高(P<0.01),缺血半暗区皮层神经元及微血管内皮细胞水肿,细胞结构损坏明显,MVD值与缺血半暗区皮层mTOR、HIF-1α蛋白及mRNA表达均升高(P<0.01), miR-126-3p表达降低(P<0.01)。与同时点模型组比较,两干预组mNSS显著降低(P<0.01,P<0.05),缺血半暗区皮层神经元与微血管内皮细胞超微结构有不同程度改善且电针组更佳,两治疗组3、14 d时MVD值升高(P<0.01),电针组7 d时MVD值升高(P<0.05),两干预组缺血半暗区皮层mTOR、HIF-1α蛋白及mRNA及miR-126-3p表达均升高(P<0.01,P<0.05)。与同时点电针组比较,抑制剂组MVD值降低(P<0.05,P<0.01),缺血半暗区皮层mTOR、HIF-1α蛋白及mRNA及miR-126-3p表达均降低(P<0.01)。与本组4 h和3、7 d时比较,模型组、电针组、抑制剂组mNSS评分均在14 d时最低(P<0.01);与本组3 d时比较,模型组、电针组、抑制剂组7、14 d时MVD值和缺血半暗区皮层mTOR蛋白表达均升高(P<0.01,P<0.05);与本组3、7 d时比较,模型组和电针组14 d时缺血半暗区皮层mTOR mRNA、miR-126-3p表达均升高(P<0.01,P<0.05);与本组3 d时比较,抑制剂组7、14 d时缺血半暗区皮层mTOR、HIF-1αmRNA表达升高(P<0.01)。结论:电针“大椎”“百会”穴可显著提高MCAO大鼠缺血半暗区皮层mTOR、HIF-1α表达,其作用机制可能是通过激活miR-126-3p表达进而靶向调控mTOR/HIF-1α信号通路,最终促进血管新生,从而发挥其脑保护效应。

Objective To observe the effect of electroacupuncture(EA) on miRNA-126-3 p and mammalian target of rapamycin(mTOR)/hypoxia-inducible factor-1α(HIF-1α) signaling pathway in rats with cerebral ischemia(CI), so as to explore the underlying mechanism of EA on angiogenesis. Methods Male SD rats were randomly divided into control group, model group, EA group and EA+inhibitor group(inhibitor group), which were further divided into 3, 7 and 14 d subgroups, with 12 rats in each sub-group. The CI model was established by occlusion of the middle cerebral artery. EA(2 Hz/20 Hz, 0.5 mA) was applied to “Dazhui”(GV14), “Baihui”(GV20) for 20 min, once daily for 14 days at most. Rats of the inhibitor group were given an intraperitoneally injection of mTOR inhibitor(0.1 mg/mL, 0.3 mg/kg) before daily EA. The neurological function was evaluated by modified neurological severity score(mNSS). The ultrastructure of cortical neurons and microvascular endothelial cells in ischemic penumbra was observed by transmission electron microscope, and the microvessel density(MVD) of cortical endothelium in ischemic penumbra was detected by immunohistochemistry. Western blot and quantitative real-time PCR were used to detect the protein and mRNA expression of mTOR, HIF-1α and the expression of miR-126-3 p in the cortex of ischemic penumbra, respectively. Results After modeling, compared with the control group at the same time point, the mNSS of the model group was increased(P<0.01), and decreased over time(P<0.01). The cortical neurons and brain microvascular endothelial cells in the ischemic penumbra were edema, and the cell structure was damaged obviously in the model group.The MVD value and the expressions of mTOR、HIF-1α proteins and mRNAs were increased(P<0.01), while the expression of miR-126-3 p decreased(P<0.01) in the model group relative to the control group. Compared with the model group at the same time point, the mNSS of both intervention groups was significantly reduced(P<0.01, P<0.05), the neuron and cerebral microvascular structure improved to varying degrees, and the MVD value, the expressions of mTOR and HIF-1α protein and mRNA, and the expression of miR-126-3 p of the two treatment groups were increased(P<0.01, P<0.05) at all time points(excep MVD at day 7 in the inhibitor group). Compared with the EA group at the same time point, MVD, the expressions of mTOR, HIF-1α proteins and mRNAs and miR-126-3 p in the inhibitor group were all decreased(P<0.05,P<0.01). Compared with the group itself at 4 hours after modeling and day 3 and day 7, the mNSS was decreased at day 14(P<0.01) in the model, EA and inhibitor groups. Compared with the group itself at day 3, the MVD value and the expression of mTOR protein were increased at day 7 and day 14 in the model, EA and inhibitor groups(P<0.01, P<0.05). Compared with the group itself at day 3 and day 7, the expression of mTOR mRNA and miR-126-3 p were up-regulated at day 14 in the model and EA groups(P<0.01, P<0.05).Compared with the group itself at day 3, the mRNA expressions of mTOR and HIF-1α were increased at day 7 and day 14(P<0.01, P<0.05) in the inhibitor group. Conclusion EA at GV14 and GV20 can alleviate neurological deficit and improve angiogenesis in rats with CI, which may be related with its effect in up-regulating the expression of mTOR and HIF-1α, improving activation of miR-126-3 p in the cortex of ischemic penumbra.