Electroacupuncture At GV20 And ST36 Exerts Neuroprotective Effects Via EPO-mediated JAK2/STAT3 Pathway In Cerebral Ischemic Rats

[Objective]While electroacupuncture(EA)in cerebral ischemia has been used to promote functional recovery clinically,the underlying mechanism of its protective effect remains poorly understood.We investigated the effects of electroacupuncture(EA)at GV20 and ST36 observing the change of EPO-mediated Janus family tyrosine kinases-2(JAK2)/signal transducers and activators of transcription-3(STAT3)cell pathway.And the electroacupuncture effect on cell apoptosis and brain emema was also investigated.[Method]Thirty-six specific pathogen free Sprague-Dawley(SD)male rats(12 for each group)were randomly assigned into 3 groups:sham-operated group(S group),middle cerebral artery occlusion group(MCAO,M group)and electroacupuncture group(EA group).Rats in S group received the same surgical procedures as those in M group,but the suture was not advanced beyond the internal carotid bifurcation.Middle cerebral artery occlusion surgery was produced for rats in M group and EA group.Two hours later the thread was removed.There were no special intervention for rats in group S and group M.The rats in group EA were given electroacupuncture therapy at GV20 and left ST36 with disposable,sterile acupuncture needles once a day for 2 times.Two electrodes were attached for acupuncture and continuous-wave stimulation for 20min at a frequency of 2Hz(intensity 2mA).The first EA was given immediately after the withdrawl of the thread and the second was 2h before euthanasia.Finally,rats in the group M and group EA were euthanized after 24h reperfusion.Ludmila Belayev 12 score test was measured to quantify the damage of sensorimotor function.Forelimb placing test was performed to evaluate sensorimotor integration in forelimbs responses on rats undergoing CIRI.Screen test was used to examine the grasping capability and muscle strength of the rats before euthanized.TTC staining proceeded to show the ischemic volume and HE staining was detected to show the morphological changes.Immunohistochemistry staining for the protein expression of EPO,EpoR,JAK2,STAT3 and AQP9.Immunofluorescence staining for colocalization of JAK2 with cellular markers(NeuN and GFAP)was performed to detect the cellular localization of the cell pathway.The protein and mRNA expression of EPO,EPO receptor(EpoR),p-JAK2,JAK2,p-STAT3,STAT3 and AQP9 were examined to explore EA effect on rats of cerebral ischemia by Western Blot and qPCR(Quantitative Real-Time Polymerase Chain Reaction).TUNEL(terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick end labeling)staining was also performed to detect the neuron apoptosis in rats’ brain.The colocalization of EPO+cells and TUNEL+cells was showed by immunofluorescence staining in cerebral ischemic penumbra area.[Results]1.The effect of electroacupuncture at GV20 and ST36 on morphological changes in ischemic brains of rats.TTC staining showed that the ischemic area was white compared with the normal brain tissues,which was red.No white area in S group of the rats’ brain indicated that no injury was happening.The white area of rats’ brain in EA group was less than the model group.HE staining showed that the ischemic area belonged to blood-supply area of the middle cerebral artery(most in the area of cortex and corpus striatum).HE staining indicated that the brain structure of the rats was normal while the structure in ischemic brains of M group was loose.Karyopyknosis and disappearance of the nucleus could be observed in ischemic area of model group.The animals in EA group had less damage compared to the M group.2.The effect of electroacupuncture at GV20 and ST36 on the neurologic function and behavior changes in ischemic ratsLudmila Belayev12 score test displayed that rats in S group had the normal scores.The M group rats had higher scores 2h and 24h after reperfusion compared with the rats in EA group.But the difference between M group and EA group was only significant in 24h(P<0.05).The results showed that there was no difference between M group and EA group for the test in timepoint 2h.Forelimb placing test revealed that the rats in M group had a higher score compared with the S group rats.Electroacupuncture at GV20 and ST36 could downregulate the score of the test(P<0.05).Screen test proved that the rats in M group had a higher score compared with the S group rats.Electroacupuncture at GV20 and ST36 could reduce the score of the test(P<0.05).3.The effect of electroacupuncture at GV20 and ST36 on EPO mediated JAK2/STAT3 cell pathway in cerebral ischemic rats Immunohistochemistry staining,Western Blot and qPCR results showed that:compared with S group,the EPO,EpoR mRNA and protein expression level in M group was increased significantly(P<0.05).And acupuncture therapy could upregulate both the mRNA and protein expression level(P<0.05).Western Blot results showed that compared with S group,the ratios of phosphorylation level of JAK2,STAT3 to total JAK2,STAT3 in EA group were remarkably increased(P<0.05).4.The cellular localization of the expression of JAK2Double-labelling immunofluorescent showed that neuronal origin of mostJAK2-labelingcells was evident by strong colocalization between JAK2 and NeuN immunoreactive cells.And JAK2 immunofluorescence was most often dissociated from GFAP immunofluorescence.5.The effect of electroacupuncture at GV20 and ST36 on cell apoptosisTUNEL staining showed that a lot of TUNEL positive cells were expressed in ischemic rat brains.Compared with M group,Electroacupuncture at GV20 and ST36 remarkably reduced the numbers of apoptosis cells.Merged pictures with arrows showed only in rare occasions the TUNEL-labelled apoptotic cells also co-expressed EPO.The dissociation between EPO-positive and TUNEL-labelled apoptotic cells further indicated that the inhibition of cell apoptosis was the possible mechanism of the electroacupuncture-mediated protective effectsagainst cerebral ischemic reperfusion injury.6.The effect of electroacupuncture at GV20 and ST36 on expression of the brain edema relatied AQP9 mRNA and proteinImmunohistochemistry staining,Western Blot and qPCR results showed that:compared with S group,the AQP9 mRNA and protein expression level in M group was decreased significantly(P<0.05).And acupuncture therapy could downregulate both the mRNA and protein expression level(P<0.05).[Conclusion]1.Electroacupunctureatacupoints GV20 and ST3 6 could function as a therapy for:1)reducing cerebral infarction area and karyopyknosis.2)attenuating neurofunction deficits and behavior changes in ischemic rats.3)regulating the protein expression of p-JAK2,p-STAT3 and the mRNA and protein expression level of EPO,EpoR,JAK2,STAT3 to reduce cell apoptosis.The protein and mRNA expression of AQP9 was also downregulated to relieve brain edema.2.The cellular localization of the expression of JAK2 was mainly in neurons but not in astrocytes.[Innovation]Our results found that electroacupuncture at GV20 and ST36 had the function of reducing brain edema and cell apoptosis by regulating EPO-mediated JAK2/STAT3 cell pathway.And the expression of EPO,EpoR,p-JAK2,p-STAT3,JAK2,STAT3 and AQP9 were regulated.The results provide the evidence that electroacupuncture therapy could influence the activation of JAK2/STAT3 cell pathway after CIRI.Our results also found that the cellular localization of the expression of JAK2 was mainly in neurons but not in astrocytes.We haven’t seen any report on the effect of electroacupuncture at GV20 and ST36 observing the change of EPO-mediated JAK2/STAT3 cell pathway in rats undergoing CIRI.