| Objective:To observe the antidepressant effect of metformin(Met)on mice and explore its main mechanism.Methods:This study used 20-25g ICR mice and were randomly divided into 4 groups:control group,metformin group,spatial restraint stress group and spatial restraint stress+Met group.The antidepressant effect of metformin on spatial restraint stress mice was verified by behavioral experiments(such as tail-suspension test,TST and forced swimming test,FST).To further confirm the antidepressant effect of metformin,treatment of Ahi1 KO mice was also used because Ahi1 KO mice had a stable depression-like phenotype.Meanwhile,Western Blot was used to determine the expression of brain-derived neurotropic factor(BDNF)in mice of each group after treatment with metformin.To further explore the mechanism of action of metformin,C6 cells were used to study the mechanism of dexamethasone and metformin treated cells,the level of BDNF in cells and the ratio of pAMPK to AMPK were measured by Western Blot.To further confirm whether metformin affects BDNF by activating AMPK,Compound C(a specific AMPK inhibitor)were used in cells,BDNF levels and the ratio of pAMPK to AMPK in each group of cells were also analyzed by Western Blot.In a subsequent experiment,Compound C and/or metformin were used in mice respectively to validate our hypothesis through Western Blot and behavioral tests,Finally,α-KG levels in ICR mice were detected by using the Mouse α-KG ELISA Kit and used EpiQuik Hydroxymethylated DNA immunoprecipitation(hMeDIP)Kit to enrich DNA containing 5hmC fragments of prefrontal cortex in mice,then detected the expression level of Tet enzyme and 5hmC modification level of the BDNF gene in the promoter region by Q-PCR.Results:The immobility time of Stress group mice was significantly higher than that of Con group(P<0.01)by the tail-suspension test and forced swimming test.After treatment with metformin,metformin did not change the immobility time of Control group in TST,but significantly reduced the immobility time of stress group(P<0.01).Also in FST,metformin also significantly reduced the immobility time in stress group(P<0.01)at 1 week and 2 weeks after treatment.Behavioral experiments showed that immobility time of Ahil KO mice also decreased in both TST and FST(P<0.01).The levels of BDNF in prefrontal cortex were detected by Western Blot and the level in stress group were decreased(P<0.05),the treatment of metformin increased BDNF levels in Stress group(P<0.01).In C6 cells,metformin reversed the decrease of BDNF(P<0.05)and the ratio of pAMPK to AMPK(P<0.05)caused by dexamethasone.After compound C inhibits the activity of AMPK,Western Blot results showed that BDNF levels in cells were significantly decreased(P<0.05).Treatment with Compound C and/or metformin in mice showed that Compound C decreased the ratio of pAMPK to AMPK(P<0.05)and BDNF levels(P<0.01).Behavioral experiments also showed that inhibition of AMPK activity blocked the antidepressant effect of metformin in mice(P<0.01),The a-KG content of ICR mice after metformin treatment was increased detected by ELISA(P<0.05);Q-PCR results showed that metformin increased the expression level of Tet enzyme(P<0.05)and 5hmC modification level of BDNF gene in the promoter region of prefrontal cortex in mice(P<0.05).Conclusion:1,Metformin can improve the depression-like behaviors of mice and increase the level of BDNF.2.Metformin can increase the level of BDNF by activating AMPK.3.Inhibition of AMPK activity decreases the expression of BDNF and blocks the antidepressant effect of metformin in mice.4.Metformin Leads to Increased 5hmC Modification of BDNF Gene Promoter in Mouse Prefrontal Cortex... |
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