| Objective To investigate the effect of nicotinamide riboside(NR)on the depression of alcoholexposed mice.Methods 1.Production of the mouse model of alcohol-induced depression and intervention strategy: Twenty-one mice were housed in the same cage and adaptively fed for 3 weeks.Thereafter,the mice were randomly divided into the following three groups(seven mice per group): Control,Model,and NR groups.Each mouse was housed individually in a cage.From Monday to Thursday,the mice in the Model and NR groups were provided with fresh tap water from 8:00 a.m.to 4:00 p.m.,followed by 15% alcohol solution(v/v)until 8:00 a.m.the next day.The mice in the Control group drank tap water during the 4 days.On Friday and Saturday,the mice did not receive any liquid.On Sunday,the mice were given fresh tap water.During the experiment,all the mice were allowed ad libitum access to food.The 15% alcohol solution(v/v)was prepared immediately prior to use(supplementary Fig.1A).Mice in the Control and Model groups received normal saline(0.2 mL/d)via an intragastric tube at 12:00 p.m.each day.The NR group received 400 mg/kg of NR by gavage daily.The alcohol exposure process lasted for 10 weeks.2.Faecal microbiota transplantation(FMT): After 8 weeks of alcohol exposure,fresh faeces were collected daily from the Control,Model and NR groups for a period of 2 weeks.Eighteen C57 BL/6J recipient mice were randomly divided into 3 groups(6 mice per group)for FMT as follows: FMT Control,FMT Model,and FMT NR groups.The mice were provided drinking water containing broad-spectrum antibiotics(ampicillin,1g/L;neomycin sulfate,1g/L;metronidazole,1g/L;vancomycin,500mg/L)for 3 weeks before receiving FMT.Each day,after the donor mice received intragastric of normal saline or NR,fresh faecal pellets were collected to make supernatants.Supernatants from the Control,Model and NR groups were administered by gavage to the FMT Control,FMT Model and FMT NR groups,respectively(0.2 mL per mouse per day).3.Behavioral tests: Depression-like behaviors in mice were assessed using open field test,forced swim test,sucrose preference tests,elevated plus maze,and Y-maze task.4.Hematoxylin-eosin(HE)stainings was used to observe the pathological changes of CA1 region in hippocampus of mice.5.The morphological changes in the microglial cells in the hippocampus were evaluated by Immunofluorescence.6.The levels of Brain-derived neurotrophic factor(BDNF)in serum were measured by Enzyme-linked immunosorbent assay(ELISA).7.Western blot was used to detect the expression of TMEM119 and CD68,BDNF and its receptors TrkB,p-Akt(ser473),p-Akt(thr308),p-GSK 3?(ser9),GSK 3? and ?-catenin in hippocampus.8.16 S rDNA gene sequencing technology was used to analyze the changes of intestinal microbiota in mice.Results 1.During the 10 weeks of alcohol exposure,the growth rate,body weight,and daily food consumption in the Model group were generally lower than those in the Control and NR groups.2.In the open field test,there was no significant difference in the total distance among the three groups of mice,but the time spent in the central area of the Model group were decreased compared with the Control and NR group.The sucrose preference at 12 h of the mice in the Model group was significantly reduced by 15.1% compared with the Control group(P < 0.05).Similarly,compared with the Control group,the immobility time in the forced swim test of the mice in the Model group was significantly increased by 56.3%(P < 0.05).In the elevated plus maze test,the number of entries and the time spent in the open arms of the mice in the Model group were significantly reduced by 63.6% and 62.7% respectively compared with the Control group(P < 0.05).In the Y-maze test,compared with the Control group,the time spent by the mice in the novel arm in the Model group was significantly reduced by 43.5%,while the time spent in the start arm was significantly increased by 109.4%(P < 0.05).After NR intervention,the depression-like behavior of alcohol exposed mice was significantly improved compared with the model group(P < 0.05).3.The results of HE stainings showed that NR intervention improved neuronal damage induced by alcohol exposure to some extent.4.This result shows that while alcohol exposure did not change the number of microglia in the hippocampus,it significantly increased their activation(P < 0.05).NR treatment effectively protects against microglial hyperactivation.5.The levels of serum BDNF of mice in the Model group was(438.9 ± 46.7)pg/ml,which was significantly reduced by 31.2% compared with the Control group(638.1 ± 77.3)pg/ml(P < 0.05).Compared with the Control group,the levels of BDNF and TrkB in the hippocampus of mice in the Model group were also significantly reduced by 40.3% and 58.1% respectively(P < 0.05).The Akt/GSK 3?/?-catenin signalling pathway is significantly suppressed after alcohol intake,and activated following treatment with NR.6.16 S rDNA sequencing revealed that,compared with control and NR-treated mice,the gut microbiome richness and composition were significantly altered in the depressed mice.7.After faecal microbiota transplantation,cognitive behaviours,microglial activity,levels of cytokines and BDNF,and activation state of the AKT/GSK 3?/?-catenin signaling pathway in recipient mice were similar to those in donor mice.Conclusion 1.Chronic intermittent alcohol intake can cause depressive disorders in mice,showing typical depression like behaviours and pathophysiological changes.2.Nicotinamide ribose,as an important nutritional supplement,can effectively prevent and alleviate depression-like behaviours and related pathophysiological damage in alcoholexposed mice.NR protects against alcohol-induced depression might by improving the intestinal microbiota.The changes in the diversity and structure of intestinal microbiota may affect the occurrence and development of alcohol-induced depressive disorder through various pathways on the microbiota-gut-brain axis,including microglia activation,expression of BDNF and its receptor TrkB,and regulation of the AKT/GSK 3?/?-catenin signal transduction pathway. |
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