| Objective:Based on the TCM theory of "Zhi-shen Tiao-xing",to explore the biological mechanism of scalp-acupuncture therapy to improve the ischemic PSS symptoms through the regulation of BDNF/TrkB/CREB and PI3K/Akt/GSK-3? signaling pathway to promote the repair and cell protection of damaged brain tissue cells in ischemic PSS rats.Methods:1.Experiment 1: Establishment and validation of ischemic PSS rat modelBibliometrics was used to search CNKI,Wanfang,CQVIP,Pub Med and other major Chinese and English databases.The frequency of modeling methods in animal experiments on PSS and related diseases after stroke was counted,and the appropriate modeling methods of ischemic PSS model were determined according to the conditions of the intervention of scalp-acupuncture in the experiment.Sixty Sprague Dawley rats(SD rats)were randomly divided into blank group(n = 20),sham operation group(n = 20)and model group(n = 20).The rats in the model group were modeled according to the established modeling scheme.The number of death,spasticity and improved Ashworth(MAS)score of rats in each group were recorded at 1d,3d,7d and 14 d after modeling.2.Experiment 2:Verification of the frequency and time of scalp-acupuncture on ischemic PSS120 SD rats were assigned to blank group(n=10),sham group(n=10)and experimental group(n=100),and the PSS model of experimental group was established.The rats were randomly divided into model group,SA group A(200r/min,3min),SA group B(200r/min,1min),SA group C(100r/min,3min)and SA group D(100r/min,1min),and given corresponding treatment for 7 days.The Zealonga score and MAS was recorded on the 7th day.3.Experiment 3: The effect of scalp-acupuncture on behavioral changes in ischemic PSS ratsA total of 60 SD rats were randomly divided into sham operation group(n = 20)and experimental group(n = 40).Ischemic PSS model was established in the experimental group.The Ischemic PSS model rats were randomly divided into the model group and the acupuncture group.The rats were treated for 7 days.Zealonga score,MAS score,BWT score were observed and recorded every 2 days.4.Experiment 4: The effect of scalp-acupuncture on the recovery of brain tissue in ischemic PSS ratsTo extract in cortical tissue of rats after 1 week,with 2,3,5-triphenyl four azole nitrogen chloride staining method to observe ischemic PSS rats infarction volume change,using HE staining and Nissl staining observe the pathological changes of neurons and the change of nissl bodies,FITC-glucan cerebral perfusion method are used to observe state change brain blood vessels.5.Experiment 5: The effect of BDNF/TrkB/CREB Signaling Pathway in Ischemic PSS ratsThe protein expression and mRNA expression of BDNF,TrkB,CREB,p-CREB in the cerebral cortex were detected by Western Blotting and PCR in the injured cerebral cortex of Ischemic PSS rats.6.Experiment 6: Effect of PI3K/Akt/GSK-3? Signaling Pathway in Ischemic PSS RatsThe protein expression and mRNA expression of PI3 K,Akt,p-Akt,GSK-3?,p-GSK-3? in the cerebral cortex were detected by Western Blotting and PCR in the injured cerebral cortex of Ischemic PSS rats.Results:1.Experiment 1: Establishment and validation of ischemic PSS rat modelThe number of related animal experimental research papers was 25 cases,among which 18 papers were published in Chinese and 7 in English.The modeling methods mainly included MCAO,MCAO+NMDA with internal capsule injection and photochemical method.Among them,the application probability of MCAO was the highest(60%),followed by MCAO + NMDA with internal capsule injection(32%),and the photochemical method(8%).Considering that both of the latter could cause cranial lesions,permanent middle cerebral artery occlusion(MCAO)was preliminary determined as the modeling method.The rats in the model group were treated with MCAO by thread bolting method.After modeling,15 rats in the model group survived,while 5 rats died during modeling.There was no death or spasticity in the blank group and the sham group within 14 days,and the MAS level was 0.Compared with blank control group,1 case died and 3 cases developed spasticity on day 1 in model group,and there was no significant difference in the average level of MAS score(P>0.05).At 3d,3 rats in the model group died and 8 rats in spasmodic state,and there was a significant difference in the average MAS score(P<0.05).At 7d,4rats in model group died and 10 rats in spasmodic state,and there was significant difference in the average level of MAS score(P<0.05).On day 14,7 rats in the model group died and6 rats in spasticity,and there was no significant difference in the average level of MAS score(P > 0.05).Therefore,MCAO can be used as the animal modeling method in this study.2.Experiment 2:Verification of the frequency and time of scalp-acupuncture on ischemic PSSAfter 7 days of scalp-acupuncture intervention,Zealonga score and improved Ashworth score of acupuncture SA group B were significantly lower than those of SA group A,C and D(P<0.05),the Zealonga score and improved Ashworth score of acupuncture group B were significantly higher than those of acupuncture group 1d(P<0.05).Therefore,the final selection of 200r/min,1min as the hand-manipulating of scalp-acupuncture.3.Experiment 3: The effect of scalp-acupuncture on behavioral changes in ischemic PSS ratsCompared with sham group,Zealonga score in model group was increased,while BWT score was decreased,indicating that motor nerve function was decreased,while MAS score was increased,indicating that limb spasm degree was increased(P<0.05).Compared with model group,Zealonga score decreased and balance beam walking score increased in scalp-acupuncture group,indicating that motor nerve function improved after scalp-acupuncture treatment,and MAS score decreased,indicating that limb spasm degree decreased(P<0.05).4.Experiment 4: The effect of scalp-acupuncture on the recovery of brain tissue in ischemic PSS ratsCompared with sham group,the proportion of ischemic infarcts in PSS model rats was significantly higher in model group(P<0.05),there was obvious liquefaction necrosis area,the number of cells decreased,the number of neurons decreased and irregular arrangement,compared with the model group,the proportion of ischemic infarcts in the brain tissue of PSS model rats in the scalp-acupuncture group was significantly reduced(P<0.05),there was no obvious pathological change,the number of cells increased,the number and morphology of neurons were normal.5.Experiment 5: Effect of BDNF/TrkB/CREB Signaling Pathway in Ischemic PSS ratsCompared with the sham group,the expression of BDNF,TrkB,CREB,p-CREB protein in the motor cortex of the affected side of rats was decreased in the model group(P<0.05),the expression of BDNF,TrkB,CREB mRNA was decreased(P < 0.05);Compared with model group,BDNF,TrkB,CREB,p-CREB protein expression and BDNF TrkB CREB related mRNA expression increased in scalp-acupuncture group(P<0.05).6.Experiment 6: Effect of PI3K/Akt/GSK-3? Signaling Pathway in Ischemic PSS RatsCompared with the sham group,the expression of PI3 K,Akt,p-Akt and p-GSK-3? in the motor cortex of the affected side of rats in model group was decreased(P<0.05),the expression of GSK-3? was increased(P<0.05),and the mRNA expression of PI3 K and Akt was decreased(P<0.05).Compared with model group,the expressions of PI3 K,Akt,p-Akt and p-GSK-3? in scalp-acupuncture group were increased(P < 0.05),the expression of GSK-3? was decreased(P < 0.05),and the mRNA expressions of PI3 K and Akt were increased(P<0.05).Conclusion:This experiment based on "the BDNF/TrkB/CREB signaling pathways and PI3K/Akt/GSK-3? signaling pathways of nerve and vascular tissue cells in brain tissue repair and protection,promote the improvement of the damage of nerve and vascular network,and then improve the spasticity of ischemic rats for PSS,have proposed rehabilitation of traditional Chinese medicine “Zhi-shen Tiao-xing” in the theory of scientific hypothesis,observed from ethology,histopathology,three aspects carries on the analysis of molecular biology,come to a conclusion as follows:1.From the perspective of animal behavior,behavioral experiment demonstrated that scalp-acupuncture can improve the neurological function,the degree of muscle spasm and the motor function of PSS model rats,and proved that scalp-acupuncture therapy has the therapeutic effect of "Tiao-xing" by regulating the head treatment area.2.Experiment 4 demonstrated from the histological level that acupuncture can effectively reduce the volume of ischemic infarction in PSS model rats,improve the pathological state of brain tissue,promote the recovery of the morphology and number of upper motor neurons,as well as the recovery of the cerebrovascular state.Combined with experiment 1,it is proved that the head acupuncture can play a role of "Tiao-xing",that is,promoting the improvement of behavior,through the treatment of "brain damage" proposed in the pathogenesis.3.Experiment 5 confirmed scalp-acupuncture can effectively improve the expression of BDNF,TrkB,after CREB protein,increase of BDNF/TrkB/after CREB signaling pathway,so as to promote the virtuous cycle of BNDF autocrine,which promote the BDNF on repair of tissue cells and cell protection,combined with the experiment 1 and 2,scalp-acupuncture for the influence of the signal path proves that the "Tiao-shen" of scalp-cupuncture can improve "Sui-xu",and improve the effect of "Tiao-xing".4.Experiment 6 proves that the scalp-acupuncture can activate the PI3K/Akt/GSK-3?signaling pathways,and possibly by activating the signaling pathways promote GSK-3?phosphorylation,inhibition of GSK-3? effect on promoting apoptosis,thus to protect brain cells,combined with the above,the conclusion scalp-acupuncture may through increase the expression of BDNF and TrkB,further activate the downstream of PI3K/Akt/GSK-3?signaling pathways,play a therapeutic effect of "Nao-sun" organization,namely the "Zhi-shen",that is,promoting the recovery of the behavior,namely "Tiao-xing",From this point of view,the relationship between "Zhi-shen" and "Tiao-xing" is explained. |
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