| BackgroundCerebral ischemia has been one of the common diseases leading to bothhigh deformity and mortality. Researches focused on the cure and preventionof cerebral ischemia has never been stopped. Electroacupuncture, as atradition non-medicine therapy, plays a definite role in improving cerebralischemia recovery and is widely used in clinic. However, the exactmechanism is unclear. Recent research has indicated that endothelialprogenitor cells （EPCs）participate in not only vasculogenesis of embryostage but also vascularization through self-mobilization, migration anddifferentiation after birth. EPCs can also promote neuranagenesis andangiogenesis after cerebral ischemia.In recent years, there has been a large number of experimentsdemonstrated that PI3K/Akt involved in proliferation, migration and homingof EPCs. But for the research of its downstream signal eNOS and MMP-9are relatively rare. After the activation of PI3K/AKt can make the phos- phorylation of the eNOS Ser1177, and increase the generation of NO. Thenthe matrix metalloproteinase-9（MMP-9）can be activated by NO, completethe EPCs mobilization, migration and homing. It has confirmed that eNOS isone of the markers of EPCs, participates in the mobilization, migration andhoming of EPCs.Stem cell factor （SCF）is a multifunctional cytokines, which has beenfound in recent years. Through binding to its receptor c-kit，SCF can lead tomobilization, migration and homing of EPCs. Many reports have held thatSCF/c-kit pathway could promote neuranagenesis after cerebral ischemia;however, whether EA improves recovery after ischemia through thispathway remains unclear. In our research, we will make a rat model of focalcerebral ischemia/reperfusion, use point acupuncture as a way of treatment,observe the protein and mRNA expression of eNOS, MMP-9, SCF and c-kit,probe the effect of the SCF/c-kit after focal cerebral ischemia/reperfusionand discuss the underlying mechanism of EA.Objective1. To research the function of EA on the protein and mRNA expressionof eNOS, MMP-9, SCF and c-kit of rat after focal cerebral ischemia/reperfusion.2. To research the mechanism of eNOS, MMP-9on revascularizationafter focal cerebral ischemia/reperfusion.3. To research the mechanism of SCF/c-kit on revascularization after focal cerebral ischemia/reperfusion.MethodsOne hundred and twenty-five male Sprague-Dawley male rats wererandomly assigned to three groups: the sham group, the model group and EAgroup. The model group and EA group were further randomly divided intofive sub-groups receiving reperfusion1,3,7,14and21days after2hischemia（n=5in each sub-group）. At2h after reperfusion, in the EA group,it was the time given the EA at bilateral “Hegu” point (LI4).Neurobehavioral evaluation was used to detect whether EA treatment canimprove behavioral recovery after ischemia/reperfusion. We selected theImmunohistochemical method to detect the expression of eNOS, MMP-9,SCF and its receptor c-kit protein in the cortical ischemic region. RT-PCRmethod was used to detect the expression of eNOS, MMP-9, SCF and itsreceptor c-kit mRNA and Western Blot was selected to examed theexpression of eNOS, MMP-9and SCF protein. The SCF and c-kit mRNAand protein quatitative were detected at3d,7d,14d after reperfuion. TheeNOS and MMP-9protein and mRNA were detected at1d,3d,7d afterreperfusion.Results1. Neurobehavioral evaluationAfter ischemia the rats showed neurological deficit behavior at everytime point. At1d,3d after reperfusion, the neurological deficit score of model group and EA group gradually decreased, but no significantdifference was found（p>0.05）. At7d,14d after reperfusion, the ratsneurological function had slightly recovered, the neurological deficit scoreof EA group was lower compared with model group (p<0.05), suggestingthat EA treatment improves behavioral recovery after ischemia/reperfusion.2. The mRNA and protein expression of eNOS after focal cerebralischemia/reperfusion and the intervention of EAThe results of the Immunohistochemistry, Western blot and RT-PCRindicated that in sham group the expression of eNOS protein and mRNA wasbarely detectable. In model group, the expression of eNOS protein andmRNA started to upregulate at1d, reached the peak at3d and began todecrease at7d after reperfusion. In EA group the changes of eNOS proteinand mRNA expression were the same as that in model group, and thedifference between EA and model groups had statistical significance(P<0.05). The expression of eNOS protein and mRNA in model and EAgroups was higher than that in sham group (P<0.01).3. The mRNA and protein expression of MMP-9after focal cerebralischemia/reperfusion and the intervention of EAThe results of the Immunohistochemistry, Western blot and RT-PCRshowed that the expression of MMP-9protein and mRNA kept on a lowlevel in control group. In model group, the expression of MMP-9proteinand mRNA started to upregulate at1d and reached the peak at3d but decreased at7d after reperfusion. In EA group the MMP-9protein andmRNA expression were lower than that in model group at1d (P<0.01), whilehigher at3d and7d after reperfusion (P<0.01). Compared the sham group,the expression of eNOS protein and mRNA in model and EA groups werehigher (P<0.01).4. The mRNA and protein expression of SCF after focal cerebralischemia/reperfusion and the intervention of EAImmunohistochemistry result demonstrated that in normal group theexpression of SCF protein was barely detectable. The expression of SCFprotein in model and EA groups was upregulated compared with that incontrol group (P<0.01). In model group the SCF protein expression wasdetected as early as1d and continuously increased for up to7d, and at7dreached the peak. After then, it descended from14d to21d after reperfusion.In EA group the SCF protein expression were the same as that in modelgroup, the expression were higher than that in model group at the same timepoint, and the difference between EA and model groups had a statisticdifference (P<0.01).The results of Western blot and RT-PCR showed that in normal braintissue the expression of SCF protein and mRNA kept on a low level. Theexpression of SCF protein and mRNA in model and EA groups wasupregulated compared with that of in sham group (P<0.01). In model group,the expression of SCF protein and mRNA begun to increase at3d, reached the peak at7d after reperfusion and begun to decrease at14d afterreperfusion. In EA group the changes of SCF protein and mRNA expressionwere the same as that in model group, but the expression were higher thanthat in model group at the same time point, and the difference between EAand model groups had statistical significance (P<0.01).5. The mRNA and protein expression of c-kit after focal cerebralischemia/reperfusion and the intervention of EAImmunohistochemistry result showed that in sham group theexpression of c-kit protein kept on a low level. The expression of c-kitprotein in model and EA groups was upregulated compared with that in shamgroup (P<0.01). In model group, the c-kit protein expression started toupregulate at1d, reached the peak at7d. After then, it descended from14d to21d after reperfusion. In EA group the changes of c-kit protein expressionwere the same as that in model group, but the expression were higher thanthat in model group at the same time point, and the difference between EAand model groups had statistical significance (P<0.01) except1d afterreperfusion (P>0.05).The result of RT-PCR showed that in sham group the expression of c-kitmRNA was barely detectable. The expression of c-kit mRNA in model andEA groups was higher than that in sham group (P<0.01). In model group,c-kit mRNA expression began to increase at3d after reperfusion, reached thepeak at7d after reperfusion and began to decrease at14d after reperfusion. In EA group the changes of c-kit mRNA expression were the same as that inmodel group, but the expression were higher than that in model group at thesame time point, and the difference between EA and model groups hadstatistical significance (P<0.01)Conclusions1. EA can availably facilitate defective behavioral recovery and thenerve recovery after focal cerebral ischemia/reperfusion rats by improvingrevascularization.2. EA can up-regulate the eNOS, MMP-9, SCF, c-kit protein andmRNA expression in ischemic cortex after focal cerebral ischemia/reperfusion.3. EA may increase expression of eNOS, MMP-9and the binding ofSCF to its receptor c-kit, results in the promotion of the EPCs mobilization,migration and homing, which facilitate the vasculogenesis and recoveryafter cerebral ischemia.4. SCF/c-kit signal pathway may participate in the revascularization inischemic cortex after focal cerebral ischemia/reperfusion and improve thenerve functional recovery. |
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