Objective To investigate whether electroacupuncture (EA) at Quchi (LI11) and Zusanli (ST36) acupoints may regulate microRNA-34a (miR-34a) to promote neural stem cells differentiation in ischemic peripheral areas in rats with cerebral ischemia-reperfusion injury or not.
Methods A total of 108 rats were randomly assigned into sham group, model group and EA group, and each group was divided into three subgroups (three days, seven days and 14 days), with twelve rats in each subgroup. Besides, 16 rats were randomly divided into EA+dimethyl sulfoxide (DMSO) group and EA+miR-34a inhibitor group, with eight rats in each group. The middle cerebral artery occlusion (MCAO) model was induced for focal cerebral ischemia in rats. EA group was electroacupunctured at the ipsilateral Quchi and Zusanli acupoints on the second day. The dilatational wave was 1/20 Hz, 30 minutes every time, once a day for seven days, totally. At the same time, 5-Bromo-2′-Deoxyuridine (BrdU) was intraperitoneally injected twice a day, with an 8-hours interval. The DMSO and miR-34a inhibitor were injected into the lateral ventricle before modeling. The co-location condition was evaluated by immunofluorescence. The expression of miR-34a in ischemic peripheral areas was detected by reverse transcription real-time quantitative polymerase chain reaction.
Results The Longa's score was lower in EA group than in the model group (t > 2.084, P < 0.05). At the same time points, the paw print areas (right forepaw, right hind paw) and maximum pressures (right forepaw, right hind paw) of the affected limbs decreased in the model group than in the sham group (P < 0.05), and the paw print area of right hind paw gradually increased in the model group (P < 0.05); the paw print areas (right forepaw and right hind paw) of the affected limbs improved in EA group, compared with the model group (P < 0.05); and there was no significant difference in the maximum pressure of the affected limbs three days and seven days after electroacupuncture (P > 0.05); however, it was higher in EA group than in the model group 14 days after electroacupuncture (P < 0.05). And the paw print area of the right hind paw and the maximum pressure of the right forepaw gradually increased in EA group three days and seven days after electroacupuncture, which was in time-dependent manner (P < 0.05). The Nestin+/GFAP+ and BrdU+/GFAP+ cells expressed in ischemic peripheral areas both in the model group and EA group. And the Nestin+/GFAP+ and BrdU+/GFAP+ double positive cells increased in EA group compared to the model group three days, seven days and 14 days after electroacupuncture (t > 3.292, P < 0.05), and they reached peak seven days after electroacupuncture. The expression of miR-34a in ischemic peripheral areas was higher in the model group than in the sham group seven days after modeling (P < 0.01), however, the expression of miR-34a further increased in EA+DMSO group after electroacupuncture (P < 0.05). After injection of miR-34a inhibitor, the expression of miR-34a and BrdU+/GFAP+ cells was lower in EA+miR-34a inhibitor group than in EA group (P < 0.05).
Conclusion Electroacupuncture at Quchi and Zusanli acupoints could promote the neural stem cells differentiation in ischemic peripheral areas by regulation of miR-34a expression.
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