| Objective:1. To observe the effect of electroacupuncture on the neurological recovery in rats with MCAO/R;2. To investigate the effect of electroacupuncture on the number of CD34+EPCs in the bone marrow and peripheral blood in rats with MCAO/R;3. To investigate the effect of electroacupuncture on the expression of p-AKT in the bone marrow in rats with MCAO/R;4. To investigate the effect of electroacupuncture on the expression of p-AKT, e NOS, MMP-9 in the ischemic cortex in rats with MCAO/R;5. To investigate the effect of electroacupuncture on the microvascular density and the cerebral infarction area in rats with MCAO/R;6. To investigate the effect of electroacupuncture on the angiogenesis in ischemic cortex via PI3K/AKT by promoting the mobilization of EPCs in bone marrow into peripheral blood in rats with MCAO/R.Methods:A total of 120 healthy male adult Sprague Dawley(SD) rats were randomly assigned to Sham group, model group(I/R), electroacupuncture group(I/RE) and I/RE plus LY-294002(A specific antagonist of PI3K) group(I/REL). The SD rat model of MCAO/R was prepared by the thread embolism method, all groups were divided into 24 h, 48 h and 7d subgroups after reperfusion following 90 min-middle cerebral artery occlusion except for the Sham group. Electroacupuncture was given at “Baihui”(GV20)/“Siguan” on the left side(Hegu LI 4/Taichong LR 3) acupoints for 20 min per day. The percentage of CD34+EPCs in rat bone marrow and peripheral blood were quantified by flow cytometer. Western Blot was applied to detect the expression of p-AKT in bone marrow and ischemic cortex. Immunohistochemical method was used to detect the expression of e NOS, MMP-9 and CD34+microvessels. The m RNA expression of e NOS was measured by fluorogenic quantitative PCR. TTC staining was used to detect the infarction area of each group at the seventh day after ischemia.Results:1. The neurological function deficit scores in each groupEach group operated with MCAO/R surgery to some degrees showed neurological function deficit behavior. Compared with I/R group, the I/RE group improved neurological function deficit behavior with the lasting of reperfusion time gradually and got the lower neurological function deficit scores significantly(P<0.05) at the 7th day after reperfusion. With the intervening of LY-294002, a specific antagonist of PI3 K, electroacupuncture failed to significantly improve neurological function after reperfusion following 90 min-middle cerebral artery occlusion.2. The effect of electroacupuncture on the number of CD34+EPCs in the bone marrow of rat with MCAO/RThe count of bone marrow derived CD34+EPCs in I/R group increased significantly compared with the Sham group(P < 0.01) at 24 h and 48 h after 90 min-middle cerebral artery occlusion, and reached a peak at 24 h, but slightly higher at 7th d than the Sham group with no significant difference(P>0.05). With electroacupuncture, the number of CD34+EPCs in bone marrow significantly increased compared with the I/R group at 24 h and 48 h after reperfusion respectively(P<0.05), and then gradually decreased with prolonged reperfusion time. After blocking the role of PI3 K, electroacupuncture failed to significantly increase the count of CD34+EPCs in bone marrow compared with I/RE group(P < 0.05).3. The effect of electroacupuncture on the number of CD34+EPCs in the peripheral blood of rat with MCAO/RCompared with the Sham group, the number of CD34+EPCs in peripheral blood of I/R group significantly increased at 24 h and 48 h after reperfusion(P<0.01), reached a peak at 48 h, and then gradually decreased with prolonged reperfusion time till 7th day with statistic difference(P<0.05) than the Sham group. The number of CD34+EPCs in I/RE group at each time point increased more significantly than I/R group(P < 0.05). With the intervention of intraperitoneal injection of LY – 294002, the count decreased significantly than the I/RE group(P < 0.05).4. The effect of electroacupuncture on the expression of p-AKT protein in bone marrow of rat with MCAO/RSham group expressed a small amount of p-AKT protein in the bone marrow. The expression of p-AKT protein in I/R group increased gradually with the prolonged reperfusion time and significantly higher than Sham group at 24 h, 48 h, 7d after 90 min-middle cerebral artery occlusion(p<0.01). Compared with I/R group, expression of p-AKT in I/RE group at each time were significantly upregulated(p<0.05). With the blocking of LY294002, electroacupuncture failed to improve the expression of p-AKT, and the expression was obviously lower than I/RE group(p <0.05).5. The effect of electroacupuncture on the expression of p-AKT protein in the ischemic cortex of rat with MCAO/RSham group showed a little expression of p-AKT protein in ischemic cortex. I/R group displayed a gradually increased expression of p-AKT protein in the ischemic cortex with the extension of reperfusion time, and the expression was significantly higher than Sham group(p<0.01). The expression in I/RE group was significantly higher than I/R(p<0.05) at each time point. With LY294002 blocking the effect of PI3 K, electroacupuncture can not improve the expression of p-AKT, and the expression was lower than the I/RE group(p<0.05).6. The effect of electroacupuncture on the expression of e NOS protein and m RNA in the ischemic cortex of rat with MCAO/RSham group expressed a little e NOS protein and m RNA on the right side of the cerebral cortex. I/R and I/RE group showed obviously higher expression of e NOS at each detected time point after reperfusion than Sham group(P<0.01), and I/RE expressed significantly more than the I/R group(P<0.05) and the I/REL group which was processed by LY294002(P<0.05).7. The effect of electroacupuncture on the expression of MMP-9 protein in the ischemic cortex of rat with MCAO/RSham group expressed a little MMP-9 protein. The expression of MMP-9 in I/R group began to increase(P<0.01) at 24 h after reperfusion, and reached a peak at 48h(P<0.01), began to decline(P<0.01) at 7th day than Sham group with a little MMP-9 expression in cerebral cortex. The protein expression of MMP-9 in I/RE group was lower than that of I/R group at 24 h and 48 h, but higher in I/RE group compared with I/R group(P<0.05) at 7th day after reperfusion. With the effect of LY-294002, the expression of MMP-9 in I/REL group was of no significant difference compared with I/RE group(P>0.05).8. The effect of electroacupuncture on the CD34+microvessel density(MVD) in the ischemic cortex of rat with MCAO/RRats with focal cerebral ischemia/reperfusion injury is given priority to CD34+ individual cells at 24 h and new blood vessels of small diameter. At 7th day, microvessel density increased and vessels with different diameters coexisted in the ischemic cortex. The amount of CD34+ microvessels in I/RE group was higher than that of I/R group(P<0.05) and I/REL group which was followed by the intraperitoneal injection of LY-294002(P<0.05).9. TTC staining reveals the infarction area in each group at 7th day after focal cerebral ischemia/reperfusionAt 7th day after cerebral ischemia/reperfusion, electroacupuncture treatment can decreased cerebral infarction area obviously than the I/R group significantly(P<0.05), while LY294002 blocking PI3 K specifically, electroacupuncture stimulation failed to decrease the cerebral infarction area.Conclusions:1. Electroacupuncture can promote the neurological function recovery in rat with MCAO/R;2. Electroacupuncture can promote the mobilization of EPCs in bone marrow into peripheral blood, and the effect can be weakened by blocking the PI3K/AKT signal transduction pathway, thus speculating that PI3K/AKT signal transduction pathway is closely related to the mobilization of EPCs in bone marrow after cerebral ischemia injury;3. Electroacupuncture can effectively promote CD34+microvascular density and reduce the infarction area by regulating the expression of p-AKT, e NOS, MMP-9 in ischemic cortex. Electroacupuncture play a role in brain protection via the regulation of the PI3K/AKT, e NOS/MMP-9 signal pathway, which may contribute to the homing of EPCs to the ischemic cerebral cortex after focal cerebral ischemia/reperfusion. |
PDF Full Text Request