基于海马代谢组学与16S rRNA测序探讨电针抗焦虑作用机制

Exploring the Mechanism of Electroacupuncture's Anxiolytic Based on Hippocampal Metabolomics and 16S rRNA Sequencing

目的通过海马代谢组学与16S rRNA测序等方法,探索电针抗焦虑作用机制。方法将大鼠随机分为正常组、模型组、电针组以及地西泮组。采用慢性束缚应激方法制备焦虑症大鼠模型。电针组造模期间同时进行“足三里”穴位电针干预,每次30 min,每天1次,持续21天。地西泮组每天灌胃地西泮,持续21天。造模结束后进行旷场实验以及高架十字迷宫实验观察大鼠行为学。随后进行病理染色观察大鼠海马脑区与结肠病理改变。代谢组学检测大鼠海马脑区代谢物改变。16S rRNA与短链脂肪酸分析检测大鼠肠道菌群与短链脂肪酸变化。Western blot检测大鼠结肠Occludin,海马TLR4、NF-κB P65和NLRP3蛋白含量表达变化。免疫荧光检测海马IBA-1、NLRP3和IL-1β蛋白表达。结果旷场实验结果显示,与正常组相比,模型组总活动距离(P<0.001)、中央区活动距离(P<0.01)和平均速度(P<0.01)下降,与模型组相比,电针组和地西泮组的总活动距离(P<0.05,P<0.001)、中央区活动距离(P<0.05,P<0.01)以及平均速度(P<0.05,P<0.01)均升高。高架十字迷宫实验结果显示,与正常组相比,模型组OE%(P<0.001)和OT%(P<0.001)降低,与模型组相比,电针组和地西泮组皆能升高OE%(P<0.05,P<0.001)和OT%(P<0.01,P<0.001)。海马HE染色结果显示正常组大鼠海马CA1区可见海马体各区域结构清楚,层次分明。模型组海马体可见CA1区有少量的神经细胞胞核皱缩深染,细胞边界不清晰,排列不规则,胞质呈空泡状。而给予电针和地西泮的干预分别能不同程度缓解上述病理现象;海马代谢组学结果显示电针能改善造模引起的海马代谢紊乱,其中主要涉及对牛磺酸和亚牛磺酸代谢调节;16S rRNA测序与短链脂肪酸检测结果显示电针能改善造模引起的肠道菌群紊乱,并且能调节血清LPS以及丁酸、己酸和戊酸水平。HE染色与Western blot结果发现模型组肠道出现病理损伤,并且与正常组相比,模型组结肠Occludin表达下降(P<0.05),而电针和地西泮的干预能改善结肠病理损伤,上调Occludin表达(P<0.05,P<0.05);免疫荧光结果显示,与正常组相比,模型组海马IBA-1、NLRP3和IL-1β表达水平上升(P<0.05,P<0.01,P<0.01),而与模型组相比,电针组和地西泮组能降低IBA-1(P<0.05,P<0.05)、NLRP3(P<0.05,P<0.05)和IL-1β(P<0.05,P<0.05)表达水平;Western blot结果显示,与正常组相比,模型组海马TLR4、NF-κB P65和NLRP3蛋白表达水平上升(P<0.05,P<0.001,P<0.05),与模型组相比,电针组和地西泮组能下调NF-κB P65(P<0.01,P<0.001)和NLRP3(P<0.05,P<0.05)蛋白表达水平。结论电针抗焦虑作用涉及对“微生物-肠-脑”轴的调节。

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-1βin 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-1βin 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.