Clinical Study On Navel Acupuncture Combined With Conventional Acupuncture Therapy For Insomnia In Perimenopausal Period

Objective:According to the previous studies on the mechanism of acupuncture in the treatment of stroke,the role of IL-1 β-p38MAPK-TRPV1 pathway initiated by microglia-derived interleukin 1-β(IL-1β)in the right primary somatosensory cortex(cortex S1)brain region in the recovery of swallowing sensory dysfunction after electroacupuncture at Yifeng on the left side of stroke,and the effect of polarization after microglia activation on synaptic remodeling in the S1 brain region of mice were discussed,which provided a theoretical basis for the mechanism of electroacupuncture at Yifeng to improve swallowing sensory dysfunction after stroke.Methods:Experiment 1:Electroacupuncture improves post-stroke dysphagia(PSD)sensory dysfunction and cerebral blood perfusion at the stroke site,and activates Transient receptor potential vanillic acid 1(TRPV1)ion channels in the throat mucosa to promote superior laryngeal nerve conduction.This experiment is divided into three parts:(1)The first part is to verify the stability of the swallowing sensory dysfunction model of ischemic cerebral infarction caused by photochemical injury through behavioral experiments.The experiment was divided into three groups:control group(sham)model group(model)and EA group(EA).The model group and the EA group were subjected to photochemical damage to the right S1 brain region for modeling.The mice in the sham group were intraperitoneally injected with the same reagent and dose,and no photochemical damage was performed.Before and after modeling,water supply-induced electromyography,brain tissue blood flow perfusion,and 24-hour water intake were evaluated.(2)To verify the therapeutic effect of electroacupuncture on swallowing sensory dysfunction after cerebral infarction.EA group will start acupuncture at the left Yifeng point after 24 hours of modeling,continuous treatment for 7 days(current 1mA,frequency 2Hz,20 minutes),model group and sham group without acupuncture.Subsequently,the three groups of mice were evaluated after 7 days of electroacupuncture treatment using water-induced swallowing electromyography and brain tissue blood flow laser speckle.(3)To verify that electroacupuncture improves the expression of TRPV1 in pharyngeal mucosa and is related to the recovery of swallowing sensory function.In this part of the experiment,the mice in the sham group,model group and EA group were treated with electroacupuncture for 7 days.The posterior pharyngeal wall and vocal cord mucosa were taken,and the TRPV1 was detected by q-PCR and WB techniques.TRPV1 is involved in the process of electroacupuncture improving the recovery of swallowing sensory function after stroke.Experiment 2:The molecular mechanism of electroacupuncture in improving PSD dysphagia is related to the IL-1β-p38MAPK inflammatory pathway in the right S1 brain region.This experiment is divided into two parts:(1)In the first part,through the omics gene sequencing of the brain tissue and pharyngeal mucosa of the right S1 brain region,to understand the gene changes and pathway enrichment.In this part of the experiment,the mRNA gene sequencing of the right S1 brain region and pharyngeal mucosa of each group of mice was carried out,and then the top 50 genes with large differences were screened.According to the KEGG pathway enrichment of the differential genes,the target pathway in the process of acupuncture treatment of swallowing sensory dysfunction was screened:IL-1β-p38MAPK-TRPV1 pathway.(2)In the second part,q-PCR,WB,ELISA and other experimental techniques were used to further verify the molecular expression differences of the pathway after electroacupuncture at the molecular level.This part of the experiment was divided into 3 groups:sham group,model group and EA group.The protein expression of IL-1β,p38 MAPK and TRPV1 in the right S1 brain region of each group was detected by q-PCR,WB and ELISA.Image.J,SPSS25.0 software for statistics.Experiment 3:Electroacupuncture at the left Yifeng point promotes the polarization of microglia in the right S1 brain region,which is the key to the recovery of swallowing sensory function.This experiment is divided into two parts:(1)In the first part,immunofluorescence staining was performed on microglia in brain tissue to understand the polarization changes of microglia.In this part of the experiment,immunofluorescence staining was performed on the microglia phenotype IBA-1 in the right S1 brain region of each group of mice,and Image.J software was used to analyze the branches and fluorescence intensity of microglia.(2)In the second part,the morphological changes of synapses in the target brain area were observed to understand the effects of electroacupuncture on the morphological changes of synapses and the expression of related proteins.In this part of the experiment,ELISA and WB techniques were used to detect the expression of IL-10,Brain-derived neurotrophic factor(BDNF),Glial cell line-derived neurotrophic factor(GDNF)and Postsynaptic density protein-95(PSD95)in the right S1 brain region of each group of mice to determine the correlation between synaptic remodeling and microglia polarization anti-inflammatory.Experiment 4:The down-regulation of IL-1β-p38MAPK expression by microglia polarization is related to the treatment of dysphagia by electroacupuncture at left Yifeng acupoint.This experiment is divided into two parts:(1)In the first part,the expression of IL-1β,p38MAPK and TRPV1 was detected by q-PCR through targeted inhibition of p38MAPK.This part of the experiment was divided into 4 groups:model group,EA group,SB inhibitor group,and SB inhibitor combined with electroacupuncture group(EA+SB group).The p38MAPK was targeted by intraperitoneal injection of SB203580 in C57BL/6 mice.The expression of IL-1β,p38MAPK and TRPV1 mRNA in the right S1 brain region of each group was detected by q-PCR.(2)In the second part,the protein expression of IL-1β,p38MAPK and TRPV1 after p38MAPK targeted inhibition was detected by WB technology to understand the action site of electroacupuncture.Results:Experiment 1:After photothrombosis,the blood flow in the right S1 brain region of the model group decreased significantly,and the water consumption decreased.Electromyographic detection showed delayed swallowing reflex.There was no statistical difference between the model group and the EA group before treatment.After electroacupuncture,the results showed that the cerebral blood flow in the right S1 brain region of the model group was lower than that of the EA group,and the cerebral blood flow of the model group was lower than that of the sham group.The comparison between the three groups was statistically significant(P<0.05).The swallowing reflex initiation time of the model group was longer than that of the EA group,which was statistically significant(P<0.05).After electroacupuncture treatment,the expression of TRPV1 in the throat mucosa of the three groups showed that the expression of TRPV1 in the model group was lower than that in the sham group,and the expression of TRPV1 in the EA group was higher than that in the model group,the difference was statistically significant(P<0.05).In experiment 2:In experiment 2,after electroacupuncture treatment,mRNA gene sequencing was performed on the three groups of photothrombotic area and pharyngeal mucosa,and the KEGG pathway of differential genes was enriched.the IL-1β-p38MAPK-TRPV1 pathway was screened out.The results of q-PCR showed that the p38MAPK value of the model group was significantly higher than that of the sham group,and the difference was statistically significant(P<0.05),while the p38MAPK value of the EA group decreased,and the difference was statistically significant(P<0.05).The change trend of WB test results was consistent with this.The q-PCR results of TRPV1 showed that the model group was significantly lower than the sham group,and the difference was statistically significant(P<0.05),while the TRPV1 value of the EA group was significantly higher than model group,and the difference was statistically significant(P<0.05).The ELISA test results showed that the IL-1β value of the model group was significantly higher than that of the sham group,and the EA group was significantly lower than that of the model group,and the difference was statistically significant(P<0.05).In experiment3:Immunofluorescence results of microglial surface antigen IBA-1 showed that compared with the EA group,the microglial cells in the model group had fewer branches,lower branch length,lower fluorescence intensity,and lower IL-10 expression level.The difference was statistically significant(P<0.05).The above indicators in the EA group increased,and the difference was statistically significant(P<0.05).Electroacupuncture reversed the above trend.In terms of neuronal function remodeling:Compared with sham,BDNF,GDNF,and PSD95 in the model group were all reduced,with statistical differences(P<0.05),and the EA group was higher than the model group,with statistical differences(P<0.05).In experiment 4:after using p38MAPK inhibitor SB203580,the results of q-PCR and WB showed that there was no significant difference in the expression of p38MAPK and TRPV1 between the EA group,the inhibitor SB group and the EA+SB group compared with the model group.This proves that electroacupuncture at Yifeng acupoint plays a role through the IL-1β-p38MAPK-TRPV1 pathway.In the determination of IL-1β,the expression of IL-1β in the EA group was lower than that in the SB group,and the difference was statistically significant(P<0.05),while the expression in the EA+SB group was lower than that in the EA group.There was a statistically significant difference between the two groups(P<0.05).Considering that the effect of electroacupuncture on the expression of IL-1β was more obvious,combined with the results of the third part of the experiment,it was considered that electroacupuncture may act on microglia.Conclusion:The results of this study show that the right S1 brain region is involved in the regulation of swallowing sensory function,and the dysfunction of swallowing sensation after stroke is related to the inactivation of TRPV1 ion channel in pharyngeal mucosa.electroacupuncture at Yifeng on the left side can reduce the expression of IL-1β and p38MAPK in the right S1 brain region of mice and increase the expression of TRPV1.The inflammatory polarization process of microglia in the right S1 brain region is the key to the treatment of swallowing sensory dysfunction by electroacupuncture.It may be related to the activation of microglia-controlled inflammatory pathways that promote the release of nerve growth factor,thereby regulating synaptic remodeling,thereby improving swallowing sensory function.