Exploring the Mechanism of Electroacupuncture's Anxiolytic Based on Hippocampal Metabolomics and 16S rRNA Sequencing
Objective Exploring the mechanism of electroacupuncture's anti anxiety effect through methods such as hippocampal metabolomics and 16S rRNA sequencing.Methods Rats were randomly divided into normal group,model group,electroacupuncture group,and diazepam group.A rat model of anxiety disorder was prepared using chronic restraint stress method.During the modeling period of the electroacupuncture group,simultaneous"Zusanli"acupoint electroacupuncture intervention was performed for 30 minutes each time,once a day,for 21 days.The diazepam group received daily gavage of diazepam for a total of 21 days.After the completion of modeling,open field experiments and elevated cross maze experiments were conducted to observe the behavior of rats.Subsequently,pathological staining was performed to observe the pathological changes in the hippocampus and colon of rats.Metabolomics detection of changes in metabolites in the hippocampus of rats.16S rRNA and short chain fatty acid analysis were used to detect changes in gut microbiota and short chain fatty acids in rats.Western blot detection of Occludin in the colon and TLR4,NF-κB P65 and NLRP3 in the hippocampus of rats.Immunofluorescence detection of IBA-1,NLRP3,and IL-1β in the hippocampus Protein expression.Result The results of the open field experiment showed that compared with the normal group,the total activity distance(P<0.001),central area activity distance(P<0.01),and average velocity(P<0.01)of the model group decreased.Compared with the model group,the total activity distance(P<0.05,P<0.001),central area activity distance(P<0.05,P<0.01),and average velocity(P<0.05,P<0.01)of the electroacupuncture group and diazepam group all increased.The results of the elevated cross maze experiment showed that compared with the normal group,the model group had a decrease in OE%(P<0.001)and OT%(P<0.001).Compared with the model group,both the electroacupuncture group and the diazepam group could increase OE%(P<0.05,P<0.001)and OT%(P<0.01,P<0.001).The HE staining results of the hippocampus showed that the CA1 area of the hippocampus in the normal group rats showed clear and hierarchical structures in various regions of the hippocampus.In the hippocampus of the model group,a small amount of neural cell nuclei in the CA1 area were observed to be wrinkled and deeply stained,with unclear cell boundaries and irregular arrangement,and the cytoplasm was vacuolate.The intervention of electroacupuncture and diazepam can alleviate the above pathological phenomena to varying degrees,respectively;The results of hippocampal metabolomics showed that electroacupuncture can improve hippocampal metabolic disorders caused by modeling,mainly involving taurine and low taurine metabolism,cysteine and methionine metabolism,and regulation of glycine,serine,and threonine metabolic pathways;The results of 16S rRNA sequencing and short chain fatty acid detection showed that electroacupuncture can improve the disturbance of intestinal microbiota caused by modeling,and can regulate serum LPS and levels of butyric acid,caproic acid,and valeric acid.HE staining and Western blot results showed that the model group had pathological damage to the intestine,and compared with the normal group,the expression of Occludin in the colon of the model group decreased(P<0.05).However,the intervention of electroacupuncture and diazepam could improve the pathological damage of the colon and upregulate the expression of Occludin(P<0.05,P<0.05).The immunofluorescence results showed that compared with the normal group,the expression levels of IBA-1,NLRP3,and IL-1β in the hippocampus of the model group increased(P<0.05,P<0.01,P<0.01),while compared with the model group,the electroacupuncture group and diazepam group could reduce the expression levels of IBA-1(P<0.05,P<0.05),NLRP3(P<0.05,P<0.05),and IL-1β(P<0.05,P<0.05).Western blot results showed that compared with the normal group,the expression levels of TLR4,NF-κB P65,and NLRP3 proteins in the hippocampus of the model group increased(P<0.05,P<0.001,P<0.05).Compared with the model group,the electroacupuncture group and diazepam group were able to downregulate the expression levels of NF-κB P65(P<0.01,P<0.001)and NLRP3(P<0.05,P<0.05)proteins.Conclusion The anxiolytic of electroacupuncture involves the regulation of the"microbiota-gut-brain"axis.
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