| Objective:This study aims to explore the mechanism of electroacupuncture treatment of ERS-mediated diabetic cognitive impairment(DCI)from the perspectives of endoplasmic reticulum stress,hippocampal neuronal apoptosis,abnormal insulin signaling pathway transduction and differential metabolites,and to provide high-quality experimental evidence for acupuncture intervention in DCI.At the same time,further deepening the research on the effect mechanism of acupuncture intervention on DCI is beneficial to reveal the scientific connotation of electroacupuncture effect of"Adjust Zang and Arouse Spirit"electroacupuncture,and provide basis for its treatment of DCI and optimization of clinical diagnosis and treatment plan.Methods:1.In the experiment,9-week-old male db/db mice with spontaneous type 2 diabetes were selected as the research model.The cognitive function of mice were evaluated by Morris water maze behavior test,so as to screen db/db mice with cognitive impairment.diabetic cognitive impairment mice model(n=30)were randomly divided into"Adjust Zang and Arouse Spirit"electroacupuncture treatment group(Acup)(n=15),model group(Mod)(n=15),and db/m mice fed under the same conditions(n=15)were used as normal control group(Con).Acup mice were restrained with a homemade mouse sleeve and then treated with electroacupuncture for 20 min/time,1 time/day,6 times as a course of treatment,with 1day of rest between courses,for 4 consecutive courses of treatment.Con and Mod mice were raised under the same conditions,to keep in line with Acup,use the same mouse sleeve for 20 minutes.The mice in each group were tested by Morris water maze on the 22th day of the experiment.The behavioral test was synchronized with the electroacupuncture intervention of"Adjust Zang and Arouse Spirit".The hidden platform experiment and spatial exploration experiment were carried out successively for 6 days.The escape latency,the time and distance of crossing the target quadrant,the time and distance of crossing the water quadrant and the number of crossing the platform were recorded.2.General indexes were detected,including body weight,blood glucose,food intake and water intake of mice in each group.Elisa method was used to detect the contents of INS,TC and TG in serum of mice in each group.To observe the effect of"Adjust Zang and Arouse Spirit"electroacupuncture on general behavior and physiological indexes of db/db mice with cognitive impairment.3.HE staining was used to observe the pathological morphology of hippocampal CA1region in each group.Nissl staining was used to observe the number and distribution of nissl bodies in hippocampal neurons.TUNEL fluorescence staining was used to observe the apoptosis ratio of hippocampal neurons.4.Western blot and real-time quantitative PCR were used to detect the expression of ERS related molecules(GRP78,IRE1α,TRAF2,ASK1),apoptosis related molecules(JNK,p-JNK,Bcl-2,Bax,caspase 3),insulin signaling pathway related molecules(IRS1,p-IRS1,PI3K,p-PI3K,Akt,p-Akt,GSK-3β,p-GSK-3β)and p-Tau,Aβin hippocampus of mice.5.The ~1H NMR metabolomics technique combined with multivariate statistical analysis was applied to determine different metabolites,metabolic profiles and the correlation between metabolites with ERS and cognitive impairment in the hippocampus of db/db mice before and after the electroacupuncture intervention of"Adjust Zang and Arouse Spirit".Results:1.Physiological indicators:(1)Food intake,water intake:Compared with Con,the food intake and water intake of Mod mice increased significantly(p<0.05,p<0.01).After treatment,compared with Mod,the intake and water consumption of Acup were significantly decreased,with significant differences(p<0.05,p<0.01).(2)Body weight,blood glucose:Compared with Con,the body weight and blood glucose of Mod mice were significantly increased(p<0.05).Compared with Mod,the body weight and blood glucose of Acup mice were significantly decreased in the second week and the third week of the intervention,and there were significant differences compared with Mod(p<0.05).(3)INS、TC、TG:Compared with Con,the serum INS,TC and TG levels of Mod mice were significantly increased(p<0.05,p<0.01).Compared with Mod,the contents of INS,TC and TG in Acup decreased significantly(p<0.05,p<0.01).2.Morris water maze test:Compared with Con,the average escape latency of Mod mice was significantly prolonged(p<0.05).Compared with Mod,the average escape latency of Acup mice was shortened,with statistical difference(p<0.05).The results of the number of crossing platforms were consistent with those of the escape latency.After electroacupuncture treatment,the number of Acup crossing the platform increased significantly(p<0.05);Compared with Con and Acup mice,the number of mice crossing the platform within 60 seconds,swimming time and distance in the water entry and target quadrant were significantly increased in Mod mice,showing a certain tendency,and the swimming time and distance in the target quadrant were longer in Con and Acup mice(p<0.05).It shows that"Adjust Zang and Arouse Spirit"electroacupuncture can improve the learning and spatial memory of db db mice.3.HE,Nissl and TUNEL staining:(1)HE staining:Compared with Con,the neurons in the hippocampal CA1 region of Mod mice were widely lost,with nuclear rupture,nuclear pyknosis and disordered arrangement.However,the neurons in the hippocampal CA1 region of Acup mice were lost,with complete nuclear morphology and neat arrangement;(2)Nissl staining:Compared with Con,a large number of Nissl bodies in hippocampal CA1 region of Mod mice were lost,and the staining was weak.After treatment,the number of Nissl bodies in hippocampal CA1 region of Acup mice was significantly increased;(3)TUNEL staining:Compared with Con,neuron cells in hippocampus of Mod mice showed obvious apoptosis characteristics,and the ratio of apoptosis-related protein caspase 3 and Bax/Bcl-2 increased significantly.Electroacupuncture treatment of"Adjust Zang and Arouse Spirit"has a certain recovery effect on the morphological changes of neuronal apoptosis,and can reduce the ratio of caspase 3 and Bax/Bcl-2.It is indicated that"Adjust Zang and Arouse Spirit"electroacupuncture has a certain protective effect on the neurodegenerative changes in db/db mice.4.ERS exists in the hippocampus of db/db mice and activates ERS by activating the proapoptotic complex formed by the upstream molecule IRE1α-TRAF2-ASK1,which increases the expression of GRP78;After the initiation of ERS,the downstream JNK molecule was activated and phosphorylated,and p-JNK led to the phosphorylation of IRS1serine in the insulin signal transduction pathway,causing the insulin signal transduction disorder,reducing the phosphorylation of PI3K and Akt,and increasing the phosphorylation expression of GSK3β,thereby causing the phosphorylation of Tau and the deposition of Aβ,the marker of cognitive impairment;"Adjust Zang and Arouse Spirit"electroacupuncture can improve cognitive impairment in db/db mice by interfering with the above signal transduction.Pearson correlation analysis showed that the apoptosis level of hippocampal neurons was significantly correlated with the expression of key proteins of ERS and insulin signaling pathway(p<0.05).5.~1H NMR Metabolomics:Compared with Con,the hippocampal metabolites of db/db mice had significant indigenous changes,including 22 differential metabolites related to diabetic cognitive impairment,and 15 differential metabolites showed a tendency of retuning after"Adjust Zang and Arouse Spirit"electroacupuncture intervention.5 major metabolic pathways were involved,including amino acid metabolism,neurotransmitter metabolism,energy metabolism,membrane metabolism and osmotic regulation.Pearson correlation analysis was performed between differential expression metabolites in hippocampus and water maze-related behavioral parameters,and the results showed that the change level of metabolites was significantly correlated with the behavioral changes of db/db mice(p<0.05),indicating that the change of differential metabolites in hippocampus of db/db mice was closely related to cognitive function.Pearson correlation analysis was performed between differential metabolites in hippocampus and ERS marker molecules.The results also showed that the change of differential metabolites in hippocampus was significantly correlated with ERS(p<0.05),indicating that when ERS appeared in the hippocampus of db/db mice,the metabolites in the brain also changed,and this change was closely related to cognitive function.Conclusion:1."Adjust Zang and Arouse Spirit"electroacupuncture can not only effectively improve the changes of general indexes such as food intake,water intake,body weight and blood glucose in db/db mice with cognitive impairment,regulate the levels of TC,TG and INS,but also significantly improve the learning and memory ability of mice and improve cognitive impairmen.2."Adjust Zang and Arouse Spirit"electroacupuncture can delay the damage degree of hippocampal neurons in db/db mice and improve the morphology of neurons,so as to protect neurons,so as to protect neurons and restore the normal function of neurons.3."Adjust Zang and Arouse Spirit"electroacupuncture can improve cognitive impairment by regulating the level of ERS in the hippocampus of db/db mice with cognitive impairment,inhibiting the activation of JNK,reducing the expression of neuronal apoptosis protein,thereby inhibiting the serine phosphorylation of IRS1,returning to normal insulin signal transduction,thereby reducing the expression of p-Tau and Aβ.4."Adjust Zang and Arouse Spirit"electroacupuncture may improve cognitive dysfunction by interfering with the changes of multiple amino acid metabolites in the hippocampus of db/db mice,thereby regulating the level of ERS.5."Adjust Zang and Arouse Spirit"electroacupuncture can improve cognitive impairment by inhibiting ERS,restoring insulin signalling pathway transduction,reducing insulin resistance and regulating the level of amino acid metabolites in the brain,which may be one of the mechanisms of its treatment of diabetic cognitive dysfunction. |
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