| ObjectiveVascular dementia(VaD)refers to a clinical syndrome with severe cognitive impairment as the main symptom caused by cerebrovascular diseases and/or cardiovascular diseases,which is widely regarded as the type of dementia second only to Alzheimer disease(AD).Western medicine can partially relieve the symptoms of VaD,but it can’t cure it completely.In recent years,it has been found that scalp acupuncture is effective in increasing blood supply to cerebral cortex,increasing metabolism level of brain cells,activating potential neurons and promoting synapse formation of neurons.Acupuncture at Baihui point and Shenting point has been clinically proved to be effective in treating vascular dementia.Up to now,the understanding of nerve plasticity by acupuncture is not comprehensive,and the potential intervention mechanism of electroacupuncture on synaptic formation of neurons is not clear.The purpose of this study is to explore the mechanism of electroacupuncture improving learning and memory ability from the perspectives of behavior,morphology and molecular biology by electroacupuncture at Baihui and Shenting points of vascular dementia rats,and to study the effects of electroacupuncture on miR-81,its target gene IL-16 and downstream protein PSD-95 in frontal cortex and hippocampus of vascular dementia rats.Methods1.In Chapter 2,in order to study the mechanism of electroacupuncture at Baihui Point and shenting point to improve learning and memory ability,we made the rat VaD models.(1)VaD model was prepared by 2-VO two vessel occlusion method,which was divided into sham-group,model-group,non-acupoint group and electroacupuncture-group.The electroacupuncture-group was treated by acupuncture at Baihui Point and shenting point(Shumi wave,2-100Hz,3.0mA,30min/time/day)×14d),the non-acupoint-group was replaced by 1cm above the bilateral iliac nodules on the back,and the model-group and sham-group were only grasping and fixing at the same time without electroacupuncture treatment;(2)The learning and memory abilities of rats were tested through the positioning navigation and space exploration experiment of Morris water maze(MWM).The morphological changes of neurons in frontal cortex and hippocampal CA1 and CA3 areas were observed by HE staining.The changes of postsynaptic dense protein(PSD-95)in frontal cortex and hippocampal CA1 and CA3 areas were detected by immunohistochemical method,and the changes of CA1 and CA3 in frontal cortex and hippocampus were detected by TUNEL method Apoptosis of neurons in CA3 area.2.In Chapter 3,in order to study the effects of electroacupuncture at Baihui and shenting on microRNA(miRNAs)in frontal cortex and hippocampus of vascular dementia rats,we used PC12 cells.(1)The total RNA of frontal cortex and hippocampus was extracted by real-time polymerase chain reaction(RT-PCR),and the miR-81 with significant difference among sham-group,model-group,non-acupoint-group and electroacupuncture-group was screened;(2)The target of miR-81 was predicted through the biological information website,and the double luciferase reporter gene experiment was used to explore whether miR-81 and target gene IL-16 were directly targeted.3.In Chapter 4,it was found that:(1)Compared with the control-group,the RNA expression of IL-16 in PC12 cells transf-ected with miR-81 mimic decreased significantly,which verified the experimental results that miR-81 targeted and regulated the expression of IL-16.4.In chapter 5,taking oxygen glucose deprivation/reperfusion PC12 cells as the research object,IL-16 interference fragment was transferred into PC12 cells,and then transferred into miR-81 mic and miR-81 inhibitor respectively.Then oxygen glucose deprivation/reperfusion(OGD/R)experiment was carried out to observe the expression of postsynaptic dense protein PSD-95,and explore the effect of Electroacupuncture on miR-81,its target gene IL-16 and downstream protein PSD-95 in frontal cortex of vascular dementia rats.Results1.Through the second chapter of exper-imental research,it is found that(1)The results of MWM positioning navigation experiment showed that the escape latency of rats in the electroacupuncture-group on the fourth and fifth days was shorter than that in the modelgroup(P<0.05).The results of space exploration experiment showed that the electroacupuncture-group had the shortest time and swimming distance when there was no significant difference in the number of crossing the platform between the electroacupuncture-group and other groups(P<0.05);(2)HE staining showed that compared with the model-group,the neurons in the frontal cortex and hippocampal CA1 and CA3 areas of the electroacupuncture-group had complete structure,clear edges and layers,round and centered nuclei,compact and orderly arrangement of cells,plump and round cells,sufficient and transparent cytoplasm and rare nuclear pyknosis;(3)The results of immunohistochemistry showed that the average optical density of PSD-95 protein in frontal cortex in electroacupuncture-group was higher than that in model-group(P<0.05),but there was no significant difference in the average optical density of PSD-95 protein in hippocampus between groups(P>0.05);(4)TUNEL method showed that the apoptosis rate of neurons in frontal cortex in electroacupuncture-group was lower than that in model-group(P<0.05),but there was no significant difference in the apoptosis rate of neurons in hippocampus between groups(P>0.05).2.Through the experimental study in Chapter 3,it is found that:(1)RNA was extracted from the frontal cortex and hippocampus of rats,and miR-81 with significant differences among groups was screened.The content of miR-81 in the frontal cortex of rats in electroacupuncture-group was significantly less than that in model-group(P<0.05),but this difference was not found in hippocampus.(2)The target gene of miR-81 was predicted to be IL-16 through bioinformatics website,and it was verified by double luciferase reporter gene that miR-81 directly targeted the 3’UTR of IL-16 mRNA.3.Through the in vitro experimental study in Chapter 4,it was found that:compared with the normal control-group,the RNA expression of IL-16 in PC 12 cells transfected with mir-81 mimic decreased significantly(P<0.05),which verified the experimental results that mir-81 directly targeted down regulated the expression of IL-16.4.The experimental study of oxygen-glucose deprivation/reperfusion(OGD/R)in chapter 5 found that:(1)PC 12 cells in normal culture could express more PSD-95 protein without IL-16,P<0.05;(2)In OGD/R environment,PC12 cells expressed less PSD-95 protein(P<0.05);(3)In OGD/R environment,PC 12 cells could express more PSD-95 protein without IL-16,P<0.05;(4)In OGD/R environment,PC 12 cells could express more PSD-95 protein under the stimulation of miR-81 mic,P<0.05;(5)In OGD/R environment,PC12 cells expressed less PSD-95 protein in the presence of miR-81 and IL-16,P<0.05.In conclusion,compared with NC-IL-16 group,PSD-95 protein level in si-IL-16 group increased under OGD/R condition or not,but the overall level of both decreased under OGD/R condition.However,this was alleviated by mir-81 mimic,but in the absence of IL-16,the expression of PSD-95 protein was down regulated in cells treated with miR-81 mimic.This suggests that IL-16 is necessary for mir-81 to regulate the role of PSD-95 in OGD/R injury.Conclusions1.Acupuncture at Baihui and shenting points of vascular dementia rats can reduce the apoptosis of frontal cortical neurons and increase the formation of postsynaptic dense protein.2.Acupuncture at Baihui and shenting can stimulate the frontal cortex of vascular dementia rats to produce miR-81,and the expression of its target gene IL-16 can be directly down regulated.3.Through the ODG/R model of PC 12 cells,it was verified that miR-81 increased the formation of PSD-95 protein by down regulating the expression of IL-16. |
PDF Full Text Request