Tet1 Inhibitor Eletriptan Has An Antidepressant Effect In Mice

Background:Depression is currently one of the most common mental disorders,and poses a serious threat to human health.It is predicted to become the main cause of the global disease burden by 2030.Researches have showed that environmental stress can affect the onset of depression through epigenetic regulatory mechanism,with DNA methylation modification playing a key role.Recent studies have revealed the significance of DNA hydroxymethylation modification in depression.Previous studies by our team have found that the deletion of Ten-eleven translation 1(Tet1)can render mice less vulnerable to chronic restraint stress(CRS),indicating an antidepressant effect.Considering that most antidepressants currently cause unwanted side effects and are slow to act,with limited efficacy for some individuals with depression,identifying new antidepressants based on epigenetic mechanisms may be an important strategy for discovering treatments for depression.This study primarily seeks to screen for small molecule inhibitors of Tetl and examines their antidepressant effects on mice with depression-like symptoms.Methods:The Tet1 chemiluminescent assay kit was utilized for the in vitro screening of small molecule compounds that inhibit Tet1 enzyme activity;Dot Blot was used to detect 5hmC modification level in genomic DNA in cells and mice brain tissues;The mRNA expression levels of Tet1,Tet2 and Tet3 in HT22 cells and selected target genes in prefrontal cortex of mice were detected by real-time fluorescence quantitative PCR(qPCR);Western blot analysis was used to determine the protein expression levels of Tet1,Tet2,and Tet3 in HT22 cells and prefrontal cortex of mice;To establish a chronic mild stress(CMS)mouse model of depression,two different stimuli were randomly administered to mice daily for five weeks;Depression-like behaviors of mice were evaluated by the forced swimming test(FST)and tail suspension test(TST);Anxiety-like behaviors of mice were evaluated by the elevated plus maze(EPM);The potential molecular mechanism of antidepressant effect of Tet1 inhibitor was analyzed by sequencing DNA hydroxymethylation in mouse prefrontal cortex.Results:In order to screen for small molecule compounds that inhibit Tet1 enzyme activity,cell-free Tet1 chemiluminescent assay kit were used to detect small molecule compounds that affect Tet1 enzyme activity in the small molecule compound library.The research results found that 10 small molecule compounds significantly inhibit Tet1 enzyme activity.Then these compounds were used to treat the neural cell line HT22 respectively.Dot Blot results showed that Eletriptan,Aripiprazole,BRL-54443,Trazodone and Azasetron HCl could significantly reduce the level of DNA 5hmC modification,further suggesting that these compounds might reduced the level of DNA 5hmC modification by inhibiting Tet1 enzyme activity in cells.To further investigate whether these compounds affect the intracellular the level of 5hmC modification by inhibiting Tet1 enzyme activity in cells,Eletriptan was selected for the study after literature review.In order to investigate whether Eletriptan specifically inhibits activity of Tet1 enzyme,Tet1-CD,Tet1,Tet2,and Tet3 were overexpressed in 293T cells and treated with Eletriptan.Dot Blot results showed that after overexpressing Tet1-CD and full-length Tet1,the Eletriptan treatment significantly reduced the intracellular DNA 5hmC modification level compared to the untreated group,while after overexpressing Tet2 and Tet3,the Eletriptan treatment did not change the level of 5hmC modification.This suggests that Eletriptan can specifically inhibit activity of Tet1 enzyme.Western blot and qPCR results showed that Eletriptan did not affect the protein and mRNA expression of Tet1,Tet2 and Tet3 in cells.In order to investigate whether the Tet1 inhibitor Eletriptan has antidepressant effects,treatment with Eletriptan was administered to depression-like mice induced by chronic mild stress.The behavioral results showed that treatment with Eletriptan significantly reduced the immobility time of mice in forced swimming test and tail suspension test,indicating that Eletriptan improved depression-like behaviors in mice.The results of the elevated plus maze experiment showed that the treatment with Eletriptan increased the time and frequency of mice entering the open arm and decreased the frequency of entering the closed arm,indicating that Eletriptan treatment can improved anxiety-like behaviors of mice.In order to explore the potential mechanism of antidepressant effect of Eletriptan,whole genome DNA hydroxymethylation modification sequencing was performed on the prefrontal cortex of mice.The sequencing results showed that Eletriptan altered the whole genome 5hmC distribution of the prefrontal cortex of stressed mice;GO analysis shows that the genes causing 5hmC modification changes caused by Eletriptan are related to neural related biological processes,such as neuronal migration,synaptic and presynaptic assembly,dendrite development,and neuron recognition,etc;KEGG analysis showed that these genes were mainly involved in serotonergic synapse,GABAergic synapse,glutamatergic synapse and other signaling pathways;IGV visualization analysis shows that Eletriptan affects the 5hmC modification of the synaptic related genes Gabrb3;qPCR results showed that CMS modeling increased the level of Gabrb3 mRNA expression,but treatment with Eletriptan reduced the level of Gabrb3 mRNA expression.This suggests that the antidepressant mechanism of Eletriptan may be altering the 5hmC modification of Gabrb3 to regulate gene expression through Tet1 enzyme,thereby restoring synaptic structure or function.Conclusion:In this study,medicine screening was conducted using a cell model and it was found that Eletriptan is an efficient Tet1 enzyme inhibitor,and Eletriptan has a good antidepressant effect in depressed mice;As Eletriptan is already clinically applicable drug,it may prove to be an effective treatment for depression in clinical practice.Therefore,the research results indicate that targeting epigenetic enzymes such as Tet1 may be an important strategy for searching for novel antidepressants.As such,these results provide new insights for the development of future antidepressant medications.