Wang et al. Translational Psychiatry (2020) 10:49 https://doi.org/10.1038/s41398-020-0731-z Translational Psychiatry ARTICLE Open Access Dl-3-n-butylphthalide attenuates mouse behavioral deficits to chronic social defeat stress by regulating energy metabolism via AKT/CREB signaling pathway Wei Wang1,2,3,TingWang1,2,4, Shunjie Bai1,2,5, Zhi Chen1,2, Xunzhong Qi1,2 andPengXie1,2,3,4,6,7 Abstract Major depressive disorder (MDD) is a severe mental disorder associated with high rates of morbidity and mortality. Current first-line pharmacotherapies for MDD are based on enhancement of monoaminergic neurotransmission, but these antidepressants are still insufficient and produce significant side-effects. Consequently, the development of novel antidepressants and therapeutic targets is desired. Dl-3-n-butylphthalide (NBP) is a compound with proven efficacy in treating ischemic stroke, yet its therapeutic effects and mechanisms for depression remain unexplored. The aim of this study was to investigate the effect of NBP in a chronic social defeat stress model of depression and its underlying molecular mechanisms. Here, we examined depression-related behavior and performed a targeted metabolomics analysis. Real-time quantitative polymerase chain reaction and western blotting were used to examine key genes and proteins involved in energy metabolism and the AKT/cAMP response element-binding protein (CREB) signaling pathway. Our results reveal NBP attenuates stress-induced social deficits, anxiety-like behavior and despair 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; behavior, and alters metabolite levels of glycolysis and tricarboxylic acid (TCA) cycle components. NBP affected gene expression of key enzymes of the TCA cycle, as well as protein expression of p-AKT and p-CREB. Our findings provide the first evidence showing that NBP can attenuate stress-induced behavioral deficits by modulating energy metabolism by regulating activation of the AKT/CREB signaling pathway. Introduction takes weeks to produce a measurable benefit. Further, Major depressive disorder (MDD) is an increasingly some drugs produce significant side-effects including common psychiatric disorder that globally affects > 300 fatigue, weight gain, nausea, headache, and sexual dys- – million people of all ages, and is also one of the leading function3 5. Hence, the search for new treatments of causes of disability worldwide1,2. Pharmacotherapy is the MDD has always been a focus in biomedical research, primary choice for medical management of MDD. Classes with investigation of alternative treatments urgently nee- of first-line antidepressant medications have been ded to reduce the heavy burden of MDD on patients and approved to reduce depressive symptoms, but it often society. Increasing evidence suggests the involvement of energy metabolism pathways in MDD. We have previously Correspondence: Peng Xie ([email protected]) shown dysfunction in energy metabolism in most brain 1NHC Key Laboratory of Diagnosis and Treatment on Brain Functional Diseases, The First Affiliated Hospital of Chongqing Medical University, Chongqing, regions and plasma in a rodent model of chronic mild – China stress6 9. Corroboratively, our recent study demonstrated 2 Institute of Neuroscience and the Collaborative Innovation Center for Brain the disruption of glucose energy metabolism in naturally Science, Chongqing Medical University, Chongqing, China Full list of author information is available at the end of the article occurring depression in cynomolgus monkeys and These authors contributed equally: Wei Wang, Ting Wang, Shunjie Bai © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Wang et al. Translational Psychiatry (2020) 10:49 Page 2 of 14 patients with MDD10. At present, energy metabolism increases its binding to CREB-binding protein (CBP) and abnormalities in depression mainly include: (1) dysfunc- enhances CREB-mediated transcription of genes that are tion of nutrient utilization in glycolysis/gluconeogen- critical for survival36. CREB is a nuclear transcription esis11,12; (2) mitochondrial damage in constituents of the factor that has an important role in direct transcriptional – tricarboxylic acid (TCA) cycle9,13 16; and (3) functional activation of gluconeogenic genes. Thus, our study damage in the mitochondrial respiratory chain17,18. investigated whether the effect of NBP administration on Minimizing perturbations in energy metabolism may energy metabolism was regulated by the AKT/CREB sig- prevent development of depression, although anti- naling pathway. depressants with the relevant properties are not yet Our primary aim was to determine whether NBP clinically available. Thus, targeting energy metabolism administration can modulate or prevent stress-induced pathways may be important for identifying novel behavioral deficits, and in addition, investigate candidate antidepressant drugs. signaling pathways to determine the potential mechanism Dl-3-n-butylphthalide (NBP) represents a family of in the hippocampus (HP) and prefrontal cortex (PFC). compounds initially isolated from the seeds of Apium Our study may lead to identification of potential ther- graveolens Linn (celery), which are approved for the apeutic targets for MDD and be important to anti- treatment of acute ischemic stroke. Previous studies have depressant drug studies. shown that NBP improves stroke outcome by protecting mitochondrial function and improving energy metabo- Materials and methods – lism19 21. NBP exerts improvements on cerebral energy Animals metabolism by protecting the integrity of mitochondrial Healthy male C57BL/6 J mice (aged 7–8 weeks and structure22, increasing activity of mitochondrial complex weighing 20–23 g, N = 40) and male CD1 mice (aged enzymes23, improving activity of mitochondrial ATPase24, 18–20 weeks and weighing 35–40 g, N = 90) were pur- and maintaining stability of cell membrane potential25. chased from the Experimental Animal Centre of Moreover, recent studies have shown that by promoting Chongqing Medical University (Chongqing, China). energy metabolism, NBP may be effective in treating During the entire study, the mice were maintained in a neurological disorders beyond the management of stroke. temperature and humidity controlled room (21–22°C, NBP exhibits protective effects against mitochondrial 55 ± 5%) under a 12 h light/dark cycle with access to food damage by inhibiting amyloid β (Aβ)-induced mitochon- and water freely. The study were approved by the Ethics drial dysfunction, whereas Aβ induces active caspase-3, Committee of Chongqing Medical University. caspase-9, and cytochrome c expression in Alzheimer’s disease26. NBP ameliorates SH-SY5Y cell survival against Drug treatment rotenone, and improves mitochondrial membrane All mice were randomly divided into control with pla- potential reductions and reactive oxygen species genera- cebo group (CON + PLA group) and CSDS with placebo tion and apoptosis in Parkinson’s disease27. MDD is the group (CSDS + PLA group) and CSDS with NBP group most frequent psychiatric disorder involving mitochon- (CSDS + NBP group) after 1 week of adaptive feeding. drial dysfunction and altered energy metabolism28, yet no The CON + PLA group (N = 12) and CSDS + PLA group studies investigating whether NBP exerts antidepressant (N = 15) were treated with soybean oil, and the CSDS + effects have been reported. Accordingly, we administered NBP group (N = 13) was treated with NBP (60 mg/kg, NBP to mice subjected to chronic social defeat stress diluted in soybean oil)37. Mice received intragastric (CSDS), a well-validated model of depression, to investi- administration daily in a volume of 200 μl between 14:00 gate the antidepressant effects of NBP. PM and 14:30 PM for 24 days. Many studies have focused on signaling pathways involved in regulation of energy metabolism. One of the CSDS most studied mediators of these pathways is AKT (also An established CSDS protocol was used to induce – known as protein kinase B)29 31. AKT is a serine/threo- depressive-like behavior in mice38,39. The CSDS procedure – nine kinase and signaling molecule of cell growth and in the study was performed as previously reported40 42. differentiation, which acts as a central node of many The protocol is beginning with the selection of aggressive signaling pathways. AKT may regulate glucose metabo- CD1. Forty-one CD1 mice with appropriate character- lism by trafficking cellular uptake of glucose and altering istics as described below were selected from 90 CD1. gene expression32 and the mitochondrial membrane gra- There are two criteria for screening the aggressive beha- – dient33 35. AKT is a phosphoprotein that is capable of vior of CD1 mice as below: first, CD1 mice must have