Mechanism Of Dietary Restriction On Cognitive Neurological Dysfunction Associated With Copper Homeostasis Imbalance

Objective So far,dietary restriction(DR)has been regarded as the most powerful non-drug intervention measure to prolong life and delay age-related diseases.Studies have shown that DR can not only reshape intestinal flora,but also reduce inflammatory response and improve cognitive ability.Copper ion is an essential trace element for human body.Excessive copper may lead to intestinal barrier damage,increased inflammatory response and decreased cognitive function.Copper homeostasis imbalance is considered to be related to the pathogenesis of Alzheimer’s disease(AD),but it is still controversial whether copper ion can improve or promote the pathological characteristics of AD.At present,it seems that all the unfavorable factors caused by copper steady-state imbalance can be improved by DR,but there is very little direct research on the impact of DR on copper steady-state imbalance.Therefore,it is necessary to explore the impact of DR or copper steady-state imbalance on cognitive function and whether DR can improve the impact of copper steady-state imbalance.Methods This study consists of three parts:(1)the first part studies the effects of copper ions on intestinal flora and behavioral electrophysiology of mice.Ordinary young mice were intervened with copper ions of 10ppm(low copper)and 20ppm(high copper).After two months of intervention,stool samples were collected and sequenced,and finally behavioral and electrophysiological experiments were carried out.The experimental groups were divided into control group(Ctrl),low copper group(LCu)and high copper group(HCu).(2)the second part is to study the effect of DR on copper ion intervention,using ordinary young mice,using 30% DR and 10ppm/20 ppm copper ion intervention.After two months of intervention,stool samples were collected and sequenced,and finally behavioral and electrophysiological experiments were carried out.The experimental groups were divided into four groups: free diet group(AL),diet restriction group(DR),low copper diet restriction group(LCu DR)and high copper diet restriction group(HCu DR).(3)the third part was to study the effects of DR and copper ion intervention on intestinal flora and behavioral cognition of mice.AD young mice were intervened with copper ion concentration of 20 ppm and 30% DR.Fecal samples were collected and sequenced after four months of intervention,and then behavioral experiments were carried out.The experimental groups were free diet group(AL),diet restriction group(DR)and copper group(Cu).In the intestinal flora test,the V3-V4 region of 16 S r DNA in mouse feces was sequenced,and then the differences of intestinal microflora in different treatment groups were analyzed.In the behavior experiment,Barnes maze,Y maze and elevated maze experiments were mainly carried out.In the electrophysiological experiment,the mice need to be operated on,and then the levels of spontaneous basal EEG and sensory gating in the medial prefrontal lobe(m PFC),the right hippocampus(Hip)and the left posterior cortex(l PCtx)are measured.In the data analysis,the study of intestinal flora mainly analyzed the diversity of Alpha and Beta and the significant differences between groups(LEf Se analysis).Behavior mainly analyzed the escape time of mice in Barnes,the time and times of exploring new different arms in Y maze,and the time and times of exploring open arms in elevated cross maze.EEG data are mainly analyzed in five basic frequency bands(Delta:2-4Hz;Theta:4-8Hz;Alpha:8-12Hz;Beta:12-20Hz;Gamma:20-100Hz)and the inhibition ratio(T/C)of amplitude values of P50 and N100 induced by test sound and conditioned sound in sensory gating.Results The first part of the research results: Copper ion intervention did not affect the weight of young mice.Intestinal flora analysis showed that copper ion intervention had no effect on the Alpha diversity index of mice.LEf Se analysis screened out 7 kinds of differential bacteria between Ctrl group and LCu group,among which Akkermansiaceae and Verrucomicrobia in LCu group had high relative abundance.The behavioral results showed that the escape latency of HCu group in Barnes maze was significantly higher than that of Ctrl group and LCu group,and there was no significant difference in the number of times LCu group explored new arms in Y maze.The results of EEG showed that the relative power of Delta wave in r Hip in LCu group was significantly lower than that in Ctrl group,while the Alpha wave was significantly higher than that in HCu group.Sensory gating showed that the latency of P50 in r Hip and m PFC regions of LCu group was prolonged,while the T/C(N100)in l PCtx region of HCu group was significantly higher than that of Ctrl group.The results of the second part: 30% DR intervention significantly reduced the weight of young mice,but copper ion intervention had no effect on the weight.The results of intestinal flora showed that the Alpha diversity of intestinal flora was reduced by DR and the intervention of DR plus copper ions.LEf Se analysis screened out 56 kinds of differential bacteria among the four groups.The relative abundance of Lactobacillus and Bifidobacteriaceae in DR group was higher,Bacteroidetes in LCu DR group was higher,and Helicobacteraceae in HCu DR group showed higher relative abundance.The behavioral results showed that the escape latency of LCu DR group in Barnes maze was significantly higher than that of the other three groups,and the escape latency of DR group was the shortest but not significant.There was no significant difference in the time,frequency and distance of exploring the new arm in Y maze in LCu DR group,while the behaviors of DR group and HCu DR group were the best.The probe times and total distance of DR group in the elevated cross maze were significantly lower than those of AL group.The results of EEG showed that the relative power of Alpha waves in r Hip region of DR group,LCu DR group and HCu DR group decreased significantly compared with AL group.The relative power of Alpha wave in m PFC of LCu DR group was significantly lower than that of AL group and DR group.Compared with AL group,the relative power of Alpha wave in l PCtx region of HCu DR group decreased significantly,the relative power of Beta wave in DR group and LCu DR group increased significantly,and the relative power of Gamma wave in LCu DR group and HCu DR group increased significantly.Sensory gating results showed that T/C(N100)in r Hip and l PCtx regions of DR group was significantly higher than that of AL group.The amplitude difference of N100 in LCu DR group was significantly lower than that in AL group.In the first stimulation,the latency of N100 in LCu DR group was significantly lower in m PFC area than that in AL and DR groups,and significantly lower in l PCtx area than that in AL group.The latency of N100 in HCu DR group was significantly lower than that in AL group in l PCtx region.The results of the third part: 30% DR intervention significantly reduced the weight of young AD mice,and copper ion intervention had no effect on the weight.The results of intestinal flora showed that the ALpha diversity of DR group was significantly lower than that of Al group and Cu group.LEf Se analysis selected 15 kinds of differential bacteria between DR and Cu groups.The difference bacteria in the DR group are mainly Lactobacillaceae,Lactobacillales,Dubosiella,etc.in the phylum Sclerochaeta,while the bacteria groups such as Turicibacte and Clostridium＿bacteria＿CIEAF＿012 in the Cu group have high relative abundance.The behavioral results showed that the escape latency of Cu group in the first day of training in Barnes maze was significantly shorter than that of AL group,and the escape latency of DR group in the detection period was the shortest but not significant.In group AL,the exploration time and distance of the new arm in Y maze were significant,but the exploration times were not significant.The exploration time,times and distance of the new arm in DR group were significant,while those in Cu group were not significant.Conclusion The intervention of low and high copper ions will lead to the increase of inflammation-related flora in common mice and AD mice,and will also lead to the decline of cognitive function and the change of EEG.DR can reshape intestinal flora,increase the relative abundance of beneficial bacteria such as Lactobacillus and Bifidobacterium,and improve cognitive ability to a certain extent.However,whether DR causes anxiety and sensory gating damage needs further study.DR can improve the effect of high copper ion on flora and cognition of common mice,but it has no significant effect on low copper ion.This study has confirmed some beneficial effects of DR on the body,and it is also expected that we can continue to study the effects of DR on prolonging life and delaying aging in the future.