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

Objective: To observe the effect of electroacupuncture (EA) on miRNA-126-3p 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-126-3p 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 (<0.01), and decreased over time (<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 (<0.01), while the expression of miR-126-3p decreased (<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 (<0.01, <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-126-3p of the two treatment groups were increased (<0.01, <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-126-3p in the inhibitor group were all decreased (<0.05,<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 （<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 (<0.01, <0.05). Compared with the group itself at day 3 and day 7, the expression of mTOR mRNA and miR-126-3p were up-regulated at day 14 in the model and EA groups (<0.01, <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 (<0.01, <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-126-3p in the cortex of ischemic penumbra.