| Objective To explore the content and expression of some cytokines and proteins in PI3K/AKt/mTOR signaling pathway of the rats with ischemic stroke treated by Tujia musk acupuncture or not,in order to reveal the mechanism of Tujia musk acupuncture in the treatment of ischemic stroke,especially the action li-nks and targets in motor function and cognitive function.Methods The rat model of ischemic stroke was induced by modified suture method,the rats scored 1 to 3 by Zea-longa method were randomly divided into model group,Tujia musk acupuncture group and acupuncture group,30 in each group.In addition,the sham operation group were induced as the same method but with no embolization.Rats in Tujia musk acupuncture group were treated by the homemade acupuncture needle contained musk to stimulate the muscular layer of the lateral scalp by twist the needle above 200r/min until the acupuncture zone has a slight fever.After 5min of twist the needle and 5min of retaining the needle,the operation was repeated for 3 times.Repeatedly twisting the needles and leaving them on 3times,once a day for 5 days,with 2 days rest between treatments,for 3 courses of continuous treatment.The acupuncture control group was simulated in the same way and at the same position with ordinary needles,the model group and sham operation group were not given any treatment.The motor function by balance beam test before and after 1,2 and 3 courses of treatment;Morris water maze behavior test after 2courses of treatment was used to evaluate the memory function;HE staining and pathological sections were performed on the brain tissue of each group of rats to observe the pathological changes of brain tissue after 3 courses of treatment;ELISA method was used to detect the contents of IL-6 and IL-1βin each group of rats before and after courses of treatment;Rat brain tissue of each group were fixed and made pathological section after 3 courses of treatment.Images of microglial processes in the cortical region were captured by two-photon laser confocal microscopy system.Image J was used for skeleton analysis.The total number and the total length of Microglia process were collected.After 3 treatments,the plasma and brain tissues of the rats in each group were taken,and the plasma VEGF and HIF-1αcontents were detected by ELISA,and the PI3K,AKt and mTOR protein expressions in the brain tissues were detected by Western-Blot.Results The balance beam test results show that comparing with sham operated group,the Balance Beam test scores of other group decreased significantly(P<0.05)and there was no significant difference among the group(P>0.05)before treatment;With the increasing of treatment course,the balance beam test score of each model rats increased significantly and in the third course of assessment,the score of Tujia musk acupuncture group was higher than that of acupuncture group,meanwhile,the score of acupuncture group was higher than that the model group(P<0.05).The result of Morris water maze behavior test showed that comparing with the model group(P<0.05),memory function of each treatment group has obvious difference after treatment,in which memory function of the Tujia musk acupuncture group was more similar to that of the sham operation group.The results of the neurons in the hippocampus which were observed under microscope showed that the neurons in the hippocampus were disordered,the arrangement of neuronal cells is disordered,the number is obviously reduced,and the structure is incomplete,a large number of neuronal cells are necrotic and vacuolized in the brain of the rats with ischemic stroke.After the Tujia musk needle treatment,the neuronal cells of the brain tissue were basically neatly arranged,the cytomembrane structure was intact,and the morphology was basically close to that of the sham operation group.According to the observation results of Microglia protuberances,the total number and length of Microglia in the model control group were significantly lower than the sham operated group after treatment(~aP<0.05);the total number and length of protuberances of Tujia musk acupuncture group increased significantly after acupuncture treatment(~bP<0.05),and acupuncture treatment can significantly increase the total length of protuberances(~bP<0.05)but it was no significant effect on the total number of protuberances(~bP>0.05).The determination results of IL-6 and IL-βin plasma of rats showed the contents of IL-6 and IL-1βin plasma of each model group were significantly increased(~aP<0.05)and there was no significant difference(~bP>0.05,~cP>0.05)in comparison with the sham group before treatment.After 3 courses treatment,compared with sham operation group,the levels of IL-1βand IL-6 in plasma of each group were decreased significantly(~aP<0.05,~△P<0.05);comparing with the model control group,the levels of IL-6 and IL-1βin the plasma of Tujia musk acupuncture group were significantly lower(P<0.05),the level of IL-6 in plasma of acupuncture group was significantly lower(~bP<0.05),but the level of IL-1βshowed no significant difference(~b P>0.05).The determination results showed that the levels of HIF-1αand VEGF in plasma of rats in each model groups were significantly higher than those in the sham operated group(~aP<0.05)before treatment.After 3 treatment courses,the contents of HIF-1αand VEGF in plasma of model control group were increased(~△P<0.05);the contents of HIF-1αand VEGF in plasma of Tujia musk acupuncture group and acupuncture group were significantly increased(~△P<0.05,~bP<0.05).The expression of PI3K、AKt、mTOR protein in brain tissue showed that before treatment the expression of PI3K and AKt in hippocampus of other groups decreased significantly(~aP<0.05)compared with Sham Operation Group,AKt expression decreased significantly(~aP<0.05),the expression of mTOR was significantly increased(~aP<0.05);as the treatment course increases,the expression of PI3K and AKt in the hippocampus of the Tujia musk acupuncture group increased gradually(~bP<0.05,~▲P<0.05,~△P<0.05,~●P<0.05),and the expression of mTOR had no obvious change after a course or two treatment course(~bP<0.05,~▲P>0.05,~△P>0.05),however,after three treatment courses,the expression of mTOR in hippocampus was decreased in Tujia musk acupuncture group(~●P<0.05).Conclusion After ischemic stroke,the motor function and memory and cognitive abilities of the rats were obviously decreased,which may be related to the neuronal necrosis caused by the increased expressions of IL-6,IL-1β,VEGF and HIF-1αin the plasma,the decreased expressions of PI3K and AKt in hippocampus and the increased expression of mTOR.The acupuncture therapy of Tujia musk significantly improved the motor function,memory and cognitive abilities of rats with ischemic stroke,the mechanism of"promoting blood circulation and removing blood stasis"may be related to(1)decreasing the level of IL-6 and IL-1βin Proinflammatory Cytokine;(2)activating PI3K/AKt/mTOR signal pathway,which promotes the increase of HIF-1αand VEGF in plasma,then promotes the formation of new blood vessels,(3)regulating intracellular molecular switches in MCAO rats and decreasing the transformation of Microglia to M1,which reduces neurotoxicity,then improving inflammation of brain tissue,thereby promoting the recovery of ischemic stroke. |
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