Treatment with BDPP promotes resilience to social stress

To test the efficacy of BDPP in promoting resilience in stress-mediated depression, we treated C57BL/6 male mice with BDPP or vehicle for 2 weeks prior to and throughout RSDS and then performed social avoidance/interaction (SI) testing (Fig. 1a). Treatment with BDPP significantly increased the proportion of mice resilient to stress as indicated by increased social interactions compared to the vehicle-treated animals (Fig. 1b, c). Overall, over 70% of mice receiving BDPP showed a resilient behavioral phenotype, whereas <40% were resilient in the vehicle control group.

Fig. 1 Oral administration of BDPP promotes resilience to RSDS. a Schematic design of the experiment. b Treatment with BDPP increases the proportion of mice showing a resilient phenotype, as measured by social interaction ratio (two-tailed unpaired t-test, t 42 = 2.786, P = 0.008). c Representative heatmaps of social avoidance behavioral test. Graphs represents mean ± s.e.m., **P < 0.01 Full size image

IL-6 and Rac1 modulate synaptic plasticity

Evidence garnered from the RSDS model indicates the novel roles of IL-6 and Rac1 as contributing factors to depression phenotypes11, 30. Recent studies demonstrate that increased glutamatergic transmission on ventral striatum medium spiny neurons (MSNs) mediates stress-induced susceptibility following RSDS31, 32. We therefore investigated the biological roles of IL-6 and Rac1 on glutamatergic synapses. To test the effect of peripheral IL-6 on synaptic plasticity in the NAc, we generated bone marrow (BM) transplanted chimeras using BM from wild type (WT) or IL-6 knockout mice (IL-6−/−). Following transplantation and recovery, chimeras were exposed to RSDS and the density of PSD-95, a postsynaptic marker of excitatory synapses, was measured in the NAc. RSDS induced a robust increase of PSD-95 puncta in WT BM chimeras compared to unstressed WT or IL-6−/− BM chimeras, while no increase of PSD-95 puncta was observed in stressed IL-6−/− BM chimeras (Supplementary Fig. 1a). Whether stress-induced IL-6-mediated up-regulation of PSD95 in the NAc is specific to select cell types (e.g., Drd1 or Drd2 medium spiny neurons, or inter-neurons) needs further characterization. Measurements of circulating IL-6 revealed that IL-6 was significantly higher in WT chimeras compared to IL-6−/− BM chimeras following RSDS (Supplementary Fig. 1b). The level in stressed IL-6−/− BM chimeras was comparable to that of non-stressed WT chimera, suggesting leukocytes are the main source of circulating IL-6 and alternative sources may only make minor contributions to stress-induced IL-6 increase. Previous studies showed that Rac1 decreases excitatory spine density on MSNs and attenuates RSDS-induced susceptibility11. We found that exogenous expression of Rac1 in MSN-enriched primary cultures significantly reduced the expression of PSD-95 and vesicular glutamate transporter 2 (vGlut2) while having no effects on GABAergic vesicular GABA transporter (VGAT) (Supplementary Fig. 1c). Collectively, these data demonstrate that IL-6 and Rac1 can each modulate synaptic plasticity in neurons from NAc, supporting these mechanisms as targets for stress-induced depression (Supplementary Fig. 1d).

In vitro screening for IL-6 inhibition and Rac1 promotion

BDPP contains a variety of polyphenols. Orally consumed polyphenols are typically bioavailable in various organs and tissues as metabolites (phase II polyphenol conjugates and phenolic acids) following xenobiotic metabolism and gastrointestinal microbiome fermentation. It is possible that some metabolites may counteract the positive effect of the others. Therefore, if supplied as BDPP, the overall benefits might be reduced due to potential ‘cancellation’ effects. By identifying individual metabolites that selectively target key pathological mechanisms (e.g., Rac1 and IL-6) we can greatly improve the efficacy. We and others recently identified 21 BDPP-derived phenolic metabolites that accumulate in blood and/or in the CNS (14 polyphenolic metabolites and 7 phenolic acids, Supplementary Table 1)25, 28, 33, among which 14 are currently accessible28, 34. We initiated a high-throughput screen of these metabolites for their ability to modulate IL-6 and Rac1. Peripheral blood mononuclear cells (PBMCs) isolated from C57BL/6 mice were pretreated with plasma bioavailable metabolites (Supplementary Table 1) for 16 h followed by stimulation with lipopolysaccharide (LPS) for 16 h. We found that 3(3,4-dihydroxy-phenyl) propionic acid (DHCA) was the most effective in reducing LPS-induced IL-6 production (Fig. 2a) with a calculated IC50 of 17.69 µM (Fig. 2b). In parallel studies, we screened 9 brain bioavailable metabolites using E18 MSN-enriched primary culture. We found that malvidin-3′-O-glucoside (Mal-gluc) significantly increased Rac1 expression (Fig. 2c) with a calculated EC50 of 3.52 nM (Fig. 2d). Among all the metabolites currently available for screening, we did not find any compounds that can simultaneously reduce IL-6 in PBMCs and increase Rac1 in primary MSN-enriched cultures.

Fig. 2 In vitro screening of phenolic metabolites to modulate IL-6 and Rac1 and mechanistic investigation of the role of DHCA on IL-6 and Mal-gluc on Rac1. a, b Screening of the effect of plasma bioavailable phenolic metabolites in inhibition of IL-6 in PBMCs following LPS stimulation. a Primary screening of 14 plasma bioavailable phenolic metabolites (one-way ANOVA, F 15,63 = 16.30, P < 0.0001, n = 3–8 per culture condition). b Dose response and EC50 calculations of the effects of DHCA on IL-6. c, d Screening of the effects of brain bioavailable phenolic metabolites in promotion of Rac1 expression in primary MSN-enriched cultures. c Primary screening of 9 brain bioavailable phenolic metabolites (one-way ANOVA, F 9,39 = 5.32, P = 0.0002, n = 4 per culture condition). d Dose response and EC50 calculations of the effects of Mal-gluc on Rac1 expression. e The expression of de novo methylation/demethylation genes in PBMCs following DHCA treatment (two-tailed unpaired t-test, t 12 = 3.220, P = 0.0074 for DNMT1, n = 7 per culture condition). f Assessment of methylation-mediated promoter-like activity of CpG-rich sequences in the regions of IL-6 promoter in transfected N2A cells in the presence or absence of methylation inhibitor AZA-DC or DHCA (one-way ANOVA, F 5,17 = 1.278, P = 0.335). g Assessment of methylation-mediated enhancer-like activities of CpG-rich sequences in the regions of IL-6 introns in transfected N2A cells in the presence or absence of methylation inhibitor AZA-DC or DHCA (one-way ANOVA, F 5,16 = 161.7, P < 0.0001 for intron 1; F 5,17 = 25.54, P < 0.0001 for intron 3; F 5,16 = 0.825, P = 0.558 for intron 4). h HDAC genes expression in MSN-enriched primary culture following Mal-gluc treatment (two-tailed unpaired t-test, t 6 = 2.781, P = 0.0017 for HDAC2, n = 4 per culture condition). i, j Quantitative CHIP assessment of permissive H3 acetylation (i) and repressive trimethylation on H3K27 (j) along the mouse Rac1 promoter and upstream in MSN-enriched primary neurons following Mal-gluc treatment (two-tailed unpaired t-test, t 8 = 8.284, P = 0.0071 for ~500 bp upstream; t 8 = 13.5, P = 0.0017 for ~−50 bp in the promoter; n = 5 per culture condition for acetylation). All graphs represent mean ± s.e.m., **P < 0.01, ***P < 0.001 Full size image

DHCA modulates intronic CpG methylation of IL-6

To investigate how DHCA may attenuate IL-6 generation, we tested the effect of DHCA on Toll-like receptor-4 mediated signaling pathways. PBMCs were pretreated with DHCA and stimulated with LPS using the same protocol as during screening. Samples were taken at 0, 30 min, 60 min, 6 h and 16 h following LPS stimulation and cell lysates were subjected to a multiplex ELISA assay. We found that DHCA had no effect on the activation of JNK or p38 as reflected by the lack of changes in phosphorylation of these molecules at any time points (Supplementary Figs. 2a and b). DHCA treatment produced a ~10% decrease in ERK and AKT activation 30 min and 60 min following LPS stimulation but no further changes were observed at 6 h and 16 h (Supplementary Figs. 2c and d). Based on evidence that DNA methylation at a single CpG in the human IL-6 promoter region effectively alters IL-6 expression35, we next tested whether DHCA can modulate IL-6 gene expression through methylation mechanisms. We treated PBMC cells with DHCA and measured the expression of enzymes essential for DNA methylation/demethylation. We found DHCA treatment significantly reduced the expression of the DNA-methyltransferase 1 (DNMT1), an enzyme that plays a key role in methylation maintenance and also in de novo methylation processes (Fig. 2e). To investigate how methylation influences IL-6 gene expression, we employed the CpG-free luciferase reporter system. We cloned a ~280 bp CpG rich DNA segment from the mouse IL-6 promoter into a promoterless CpG-free reporter construct and transfected into N2a cells. We found the CpG-rich IL-6 promoter segment presented no inherent promoter activity as reflected by no differences in luciferase activity compared to the control construct (Fig. 2f). Moreover, treatment with 5-aza-2′-deoxycytidine (AZA-DC, a DNA methylation inhibitor) had no effect on the expression of luciferase (Fig. 2f), confirming methylation does not play any role in IL-6 promoter activity. We then cloned CpG rich DNA segments from IL-6 introns 1, 3 or 4 into the CpG-free luciferase reporter construct with a minimal EF1 promoter and no enhancer activity. Transfection of EF1-luciferase reporter constructs containing IL-6 intron 1 or intron 3 CpG-rich DNA segments significantly increased the luciferase activity, indicating both intronic 1 and 3 CpG-rich sequences present inherent enhancer activity (Fig. 2g). AZA-DC treatment partially reduced luciferase activity of intron 1 and totally abolished the enhancer activity of intron 3, demonstrating the contributions of intron 1 and intron 3 methylation on IL-6 transcription activity (Fig. 2g). Similar to AZA-DC, DHCA treatment also partially attenuated the enhancer activity of intron 1 and totally abolished the enhancer activity of intron 3 (Fig. 2g), suggesting DHCA has inhibitory activity on DNA methylation. In contrast, the CpG-rich region of IL-6 intron 4 presented no observable enhancer activity (Fig. 2g). These data suggest that DNA methylation at the CpG-rich sequences of IL-6 introns 1 and 3 can influence IL-6 expression and DHCA functions similar to a DNA methylation inhibitor that can reduce intronic DNA methylation to lead to attenuated IL-6 expression. To test whether DHCA may influence the expression of other pro-inflammatory factors, we measured the level of secreted cytokines following LPS stimulation (Supplementary Fig. 3). Besides IL-6, DHCA significantly reduced LPS-induced production of cytokines, mostly of pro-inflammatory nature, including granulocyte macrophage-colony stimulating factor (GM-CSF), IL-1β, IL-12 p40 and p70, IL-17 and MCP-1 (Supplementary Fig. 3) while in the absence LPS stimulation, DHCA did not influence cytokine expression profile.

Mal-gluc increases acetylation of Rac1 gene promoter region

Stress-induced reduction of Rac1 expression is associated with a repressed chromatin state surrounding the promoter region of Rac1 in the NAc11. Since Mal-gluc can cross the BBB, we hypothesized that Mal-gluc may promote Rac1 expression, in part, by modulating chromatin acetylation. We first assessed the effect of Mal-gluc on HDACs that play key roles in chromatin deacetylation. Treatment of MSN-enriched primary cultures with Mal-gluc significantly reduced the expression of HDAC2, but had no observable effect on other class I or class II HDACs (Fig. 2h). Using site directed quantitative chromatin immunoprecipitation (qCHIP), we examined the permissive histone H3 acetylation (AcH3) in Mal-gluc treated MSN-enriched primary cultures. Compared to vehicle, Mal-gluc significantly increased permissive acetylation of Rac1 across the region surveyed (Fig. 2i) while having no effect on the repressive trimethylation of the Rac1 promoter (Fig. 2j). Consistent with this observation, Mal-gluc treatment did not affect the expression of enzymes involved in de novo methylation and demethylation process (Supplementary Fig. 4).

In vivo safety and dose finding

We then initiated dose response and safety studies in mice to determine testing dosages and safety of DHCA and Mal-gluc. C57BL/6 mice were treated with either DHCA (doses ranging from 50 µg to 50 mg/kg-BW/day) or Mal-gluc (doses ranging from 50 ng to 5 mg/kg-BW/day) for 2 weeks to simulate long-term administration. Mice treated with DHCA were challenged with intraperitoneal (i.p.) injection of 0.4 mg/kg-BW LPS and plasma levels of IL-6 were measured 6 h post injection. Pretreatment with DHCA led to a dose-dependent suppression of IL-6 and that the groups treated with 5 and 50 mg/kg-BW/day showed the most significant reductions (Fig. 3a). In parallel, we measured Rac1 mRNA in the NAc of animals treated with Mal-gluc by real-time PCR and found a dose-response increase of Rac1 expression with 500 ng/kg-BW/day and 5 µg/kg-BW/day having the strongest promotion of Rac1 (Fig. 3b). Following common discovery-stage practices, we measured the plasma levels of alkaline phosphatase (ALP), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) for liver function and blood urea nitrogen (BUN) for renal function. We found neither phytochemical induced significant changes in any of the indexes (Supplementary Fig. 5).

Fig. 3 In vivo dose-finding and prophylactic effect of DHCA/Mal-gluc in promoting resilience to RSDS. a IL-6 levels in plasma 6 h post LPS challenge in mice treated with various doses of DHCA for 2 weeks (one-way ANOVA, F 5,20 = 12.50, P < 0.0001, n = 3–5 animals per group). b Levels of Rac1 mRNA in the NAc from mice treated with various doses of Mal-gluc for 2 weeks (one-way ANOVA, F 6,22 = 4.22, P = 0.01, n = 3–4 animals per group). c Schematic design of the experiment. d Representative heatmaps and scatter plots of social avoidance behavioral test in mice treated with vehicle or DHCA/Mal-gluc for 2 weeks with or without RSDS (one-way ANOVA, F 3,64 = 2.79, P = 0.048). e Sucrose preference test performed 48 h and 72 h following the SI testing (one-way ANOVA, F 3,38 = 5.53, P = 0.003 for 48 h and F 3,38 = 6.73, P = 0.001 for 72 h). f Plasma levels of IL-6 24 h following the last defeat (one-way ANOVA, F 3,35 = 6.25, P = 0.002, n = 7,8,11,10 mice). g Rac1 expression 48 h following the last defeat (one-way ANOVA, F 3,37 = 4.81, P = 0.007). All graphs represent mean ± s.e.m., *P < 0.05, **P < 0.01 Full size image

Prophylactic treatment of DHCA/Mal-gluc promotes resilience

Based on the dose–response studies, we chose 5 mg/kg-BW/day DHCA and 500 ng/kg-BW/day Mal-gluc for the preclinical studies. We tested the prophylactic effect by treating mice with a mixture of DHCA/Mal-gluc for 2 weeks prior to and throughout RSDS (Fig. 3c). We found that RSDS vehicle group had significantly lower SI ratios compared to unstressed mice while RSDS mice with DHCA/Mal-gluc treatment had significantly higher SI ratios (Fig. 3d). Following SI testing, mice were subjected to a sucrose preference test to evaluate the treatment effect on stress-induced anhedonia. We found that RSDS vehicle group had significantly reduced sucrose consumption compared to unstressed mice while DHCA/Mal-gluc treated RSDS mice had sucrose consumption similar to unstressed animals measured 48 and 72 h following the SI testing (Fig. 3e). These data suggest that DHCA/Mal-gluc treatment can prophylactically promote resilience against RSDS-induced anhedonia and social avoidance, both of which are key symptoms of depression in humans.

We next measured the plasma level of IL-6 following the defeat. RSDS led to a significantly higher induction of peripheral IL-6 and that treatment with DHCA/Mal-gluc significantly reduced plasma levels of IL-6 (Fig. 3f). Consistent with the in vitro studies (Fig. 2e), PBMCs isolated from stressed mice with DHCA/Mal-gluc treatment had significantly lower mRNA expression of both IL-6 and DNMT1 compared to vehicle-treated stressed mice (Supplementary Fig. 6). Examination of Rac1 expression in the NAc revealed that RSDS led to reduced Rac1 expression compared to the unstressed mice and DHCA/Mal-gluc treatment significantly prevented this decrease (Fig. 3g). These studies confirmed that treatment with DHCA/Mal-gluc led to the engagement of the intended targets.

To test whether DHCA/Mal-gluc-mediated modulation of IL-6 and Rac1 can lead to synaptic structural changes in the NAc, we measured the number of PSD-95 puncta in the NAc shell. Consistent with previous findings14, we found that RSDS significantly increased the number of PSD-95 immunoreactive puncta compared to the vehicle-treated unstressed mice (Fig. 4a). DHCA/Mal-gluc treatment significant reduced the number of puncta and the level was comparable to that from unstressed mice (Fig. 4a). To test the effect of DHCA/Mal-gluc on RSDS-induced synaptic functional changes, we measured the miniature excitatory postsynaptic currents (mEPSCs) in dopamine receptor D2 (Drd2) neurons in the NAc following RSDS using Drd2-EGFP transgenic mice. D2 neurons were selected because RSDS increases mEPSCs specifically on D2, but not D1 neurons in susceptible mice32. Drd2-EGFP mice were treated with DHCA/Mal-gluc for 2 weeks prior to and throughout the RSDS and subjected to SI testing. As expected, DHCA/Mal-gluc treatment significantly reduced the social avoidance phenotype (Fig. 4b). Electrophysiology recordings of mEPSCs of D2 neurons in the NAc shell showed that DHCA/Mal-gluc treatment significantly reduced mEPSC frequency compared to the vehicle treatment (Fig. 4c, d) with no difference in the mEPSC amplitude (Fig. 4e). Collectively, these data suggest that DHCA/Mal-gluc treatment may attenuate depression-like behavior in part through modulation of MSN synaptic structure and function in the NAc.

Fig. 4 Prophylactic treatment with DHCA/Mal-gluc reverses RSDS-induced synaptic structural and functional alteration in the NAc. a Immunochemistry quantification of PSD95 puncta in NAc (one-way ANOVA, F 3,22 = 4.50, P = 0.015). Inset; representative images of PSD95 puncta, scale bar = 10 µm. b–e DHCA/Mal-gluc treatment regulates synaptic transmission in D2 neurons in the shell of NAc following RSDS. b Social avoidance test in the Drd2 mice (two-tailed unpaired t-test, t 21 = 2.114, P = 0.047). c Representative traces of mEPSCs. d mEPSCs frequencies (two-tailed unpaired t-test, t 46 = 2.890, P = 0.0059). e mEPSCs amplitudes (two-tailed unpaired t-test, t 46 = 0.5008, P = 0.6189). All graphs represent mean ± s.e.m., *P < 0.05, **P < 0.01 Full size image

In parallel study, we tested the effect of single target compounds by treating mice with either 5 mg/kg-BW/day DHCA or 500 ng/kg-BW/day Mal-gluc prior to RSDS. We found that neither treatment significantly promoted resilience (Supplementary Fig. 7a) or alleviate anhedonia (Supplementary Fig. 7b). These data indicate that combination treatment simultaneously targeting Rac1 and IL-6 is necessary for preventing stress susceptibility to RSDS.

Therapeutic treatment of DHCA/Mal-gluc attenuates depression

To test the therapeutic efficacy of DHCA/Mal-gluc, we induced depression-like behavior using two different methods. In the first set of studies, mice were subjected to RSDS followed by SI test. All susceptible mice with SI ratio <0.8 were randomly grouped into vehicle or DHCA/Mal-gluc group. Following two weeks treatment, all mice were subjected to SI re-testing without further stress exposure (Fig. 5a). We did not find statistical differences in the overall SI ratio (Fig. 5b), however, we found that 25% of the vehicle group displayed a resilient phenotype upon retesting whereas over 50% of the mice from the DHCA/Mal-guc group became resilient (Fig. 5b). In a parallel study to compare the treatment efficacy with the tricylic antidepressant Imipramine, we treated the susceptible mice with Imipramine through i.p. injection for 35 days. We found that about 50% of the susceptible mice treated with Imipramine displayed a resilient phenotype upon retesting (Supplementary Fig. 8), suggesting that the efficacy of DHCA/Mal-gluc is comparable to that of Imipramine in treating RSDS-induced social avoidance phenotype. We then examined the effect of DHCA/Mal-gluc on RSDS-induced anhedonia and an ethologically relevant self-neglect phenotype. We found the average sucrose consumption was significantly higher in DHCA/Mal-gluc group compared to the vehicle group (Fig. 5c). Using the splash test, a measure of stress induced decreased self-care that is only reversible by chronic standard antidepressant treatment36, we found that treatment with DHCA/Mal-gluc significantly increased the time spent grooming following aerosol delivery of a 10% sucrose solution to the fur (Fig. 5d). We next measured the levels of IL-6 in plasma and Rac1 in the NAc after behavioral testing. We found that circulating IL-6 was back to baseline levels two weeks after the defeat regardless of treatment status. This is largely consistent with our previous studies30. We found DHCA/Mal-gluc treatment significantly increased Rac1 expression in the NAc compared to the vehicle-treated group (Fig. 5e) suggesting that the therapeutic treatment indeed engages Rac1 in the NAc.

Fig. 5 Therapeutic effect of DHCA/Mal-gluc in treating stress-induced depression. a Schematic design of the experiment. b–d Behavioral tests in stress susceptible mice following 2 weeks vehicle or DHCA/Mal-gluc treatment. b Social avoidance behavioral test (two-tailed unpaired t-test, t 15 = 1.039, P = 0.3152). c Sucrose preference test (two-tailed unpaired t-test, t 16 = 2.406, P = 0.029) and d splash test (two-tailed unpaired t-test, t 16 = 2.539, P = 0.021). e Rac1 expression in the NAc (two-tailed unpaired t-test, t 14 = 3.637, P = 0.003). f Schematic design of the experiment. g, h Behavioral tests following sub-threshold defeat in BM chimeras with BM reconstructed from naive mice or susceptible mice with or without DHCA/Mal-gluc treatment. g Social avoidance test (one-way ANOVA, F 2,52 = 4.58, P = 0.015) and h sucrose preference test (one-way ANOVA, F 2,56 = 6.17, P = 0.004). i Plasma level of IL-6 24 h after the sub-threshold defeat (one-way ANOVA, F 2,49 = 6.98, P = 0.002). j–l DHCA/Mal-gluc treatment suppressed stress-induced increase of inflammatory Ly6Chi of monocytes and neutrophils in BM chimeras with BM reconstructed from susceptible mice. j Percentage of live leukocytes derived from the donor (one-way ANOVA, F 2,28 = 3.13, P = 0.144). k Frequency of Ly6Chi monocytes of donor origin. Numbers represent percentages of live leukocytes (one-way ANOVA, F 2,29 = 6.18, P = 0.006) and l frequency of neutrophils of donor origin. Numbers represent percentages of live leukocytes (one-way ANOVA, F 2,28 = 5.377, P = 0.011). m, n Plasma levels of G-CSF and GM-CSF 24 h after the sub-threshold defeat (one-way ANOVA, F 2,25 = 5.189, P = 0.0138 for G-CSF, F 2,25 = 6.103, P = 0.0075 for GM-CSF). All graphs represent mean ± s.e.m., *P < 0.05, **P < 0.01 Full size image

We also induced depression-like behavior in naive animals via a BM transplant from susceptible mice as a model of enhanced systemic inflammation30. As indicated in Fig. 5f, 4-week-old CD45.2+ C57BL/6 recipient mice were irradiated and reconstituted with BM hematopoietic progenitors isolated from stress susceptible (average SI ratio of 0.46 from two mice) or unstressed control (average SI ratio of 1.81) donors. Following 3-week recovery, recipient mice were treated with vehicle or DHCA/Mal-gluc for 2 weeks before being subjected to a sub-threshold defeat stress that is not sufficient to induce social avoidance in control BM mice11, 30. As expected, vehicle-treated susceptible BM chimeras demonstrated increased social avoidance behavior compared to CTRL chimeras following sub-threshold defeat (Fig. 5g). Susceptible BM chimeras with DHCA/Mal-gluc treatment had a significantly decreased social avoidance behavior (Fig. 5g). The sucrose preference test showed that susceptible BM chimeras had significantly lower sucrose consumption compared to the CTRL chimeras and that this effect was completely reversed by DHCA/Mal-gluc treatment (Fig. 5h). Measurements of plasma IL-6 following sub-threshold defeat revealed that vehicle-treated susceptible BM chimeras had significantly higher levels of IL-6 compared to the CTRL BM chimeras30, while treatment with DHCA/Mal-gluc significantly lowered IL-6 levels (Fig. 5i).

To determine the level of donor chimerism, blood was collected for flow cytometry analysis after the behavioral testing. We found that over 90% of the viable leukocytes were derived from the donor progenitor cells (Fig. 5j). Notably, DHCA/Mal-gluc treatment of susceptible BM chimeras significantly inhibited stress-induced increases of donor-derived inflammatory Ly6Chi monocytes and neutrophils compared to vehicle-treated susceptible BM mice (Fig. 5k, l). Consistent with previous findings that cells from stressed or non-stressed donor do not affect BM cells reconstitution37, we found no differences in the frequency of monocytes or neutrophils between the susceptible chimeras and the control chimeras prior to the defeat (Supplementary Figs. 9a and b) suggesting the increases of inflammatory monocytes and neutrophils were triggered by the sub-threshold defeat. We then measured the plasma levels of GM-CSF which stimulates the proliferation and release of granulocytes and monocytes from BM, and G-CSF, an important cytokine for the proliferation and differentiation of neutrophils, 24 h after the sub-threshold defeat. We found both G-CSF and GM-CSF were significantly higher in the vehicle-treated susceptible BM chimeras compared to the CTRL BM chimeras while DHCA/Mal-gluc treatment almost normalized the levels of both growth factors to that of the CTRL BM chimeras (Fig. 5m, n). These data suggest that DHCA/Mal-gluc can attenuate stress-induced upregulation of G-CSF and GM-CSF and subsequently the proliferation and release of monocytes and neutrophils from BM.

DHCA/Mal-gluc treatment itself did not have any effect on behavioral changes or circulating IL-6 in CTRL BM chimeras following sub-threshold defeat. Neither were there any changes in the number of inflammatory Ly6Chi monocytes or neutrophils (Supplementary Fig. 10).

DHCA/Mal-gluc in variable stress (VS) model of depression

RSDS is one of the best-established models for depression. However, no single model recapitulates all aspects of human depression. The utility and characterization of RSDS in females are also limited. To investigate whether DHCA/Mal-gluc can produce antidepressant responses to other stress paradigms, we used the VS model which has been shown to induce depression- and anxiety-like phenotypes in both male and female mice36, 38. Male mice were treated with DHCA/Mal-gluc for 14 days and subjected to a 21-day chronic variable stress (CVS) consisting of alternating foot shock, tail suspension and restrain (Fig. 6a). Following CVS, mice were subjected to a battery of behavior tests including the splash test, novelty suppressed feeding (NSF), forced swim test (FST), and sucrose preference test. As expected, CVS male mice groomed significantly less when sprayed with 10% sucrose solution compared to the non-CVS mice while DHCA/Mal-gluc treatment completely reversed CVS-induced self-neglect behavior (Fig. 6b). NSF test was used to examine an anxiety component of stress-induced behavior39. Stressed mice exhibited longer latency to feed compared to the non-stressed mice following overnight food deprivation. Notably, DHCA/Mal-gluc treatment significantly reduced the latency to eat (Fig. 6c). In a parallel control study, when food was provided in their home cage, there was no difference in feeding latency (Fig. 6c, inset). Next, FST was used to measure passive vs. active coping response. We found CVS mice spent significantly more time immobile compared to the non-stressed mice. Treatment with DHCA/Mal-gluc significantly reduced the time of floating (Fig. 6d). We found CVS did not induce anhedonia behavior using the sucrose preference test (Fig. 6e).

Fig. 6 Prophylactic treatment with DHCA/Mal-gluc attenuates variable stress-mediated depression and anxiety phenotypes in both male and female mice. a Schematic design of the experiment. b–e Behavioral responses of male mice following 21 days of CVS. b Splash test (one-way ANOVA, F 3,38 = 4.61, P = 0.008). c NSF test (one-way ANOVA, F 3,39 = 3.23, P = 0.034). d FST (one-way ANOVA, F 3,39 = 10.30, P < 0.0001) and e sucrose preference test (one-way ANOVA, F 3,37 = 1.65, P = 0.197). f–i Behavioral responses of female mice following 6 days of SCVS. f Splash test (one-way ANOVA, F 3,39 = 5.33, P = 0.004). g NSF test (one-way ANOVA, F 3,39 = 5.22, P = 0.004); h FST (one-way ANOVA, F 3,39 = 0.581, P = 0.631) and i sucrose preference test (one-way ANOVA, F 3,37 = 3.312, P = 0.032). All graphs represent mean ± s.e.m., *P < 0.05, **P < 0.01, ***P < 0.001 Full size image

While 21 days of stress is necessary to induce depression-like behavior in male mice38, female mice are more susceptible to variable stress and express a depression-associated phenotype after only 6 days of stress, termed subchronic variable stress (SCVS)36, 40. Similar to male mice, DHCA/Mal-gluc treatment significantly reduced depression-like behavior of self-neglect and anxiety in the splash and NSF tests (Fig. 6f, g). However, SCVS did not induce significant passive coping behavior or anhedonia in female mice (Fig. 6h, i).

In vitro toxicity and drug-like properties

Based on the efficacy of DHCA/Mal-gluc in treating stress-induced depression, we initiated an in vitro toxicity and drug-like properties studies to investigate the potential of developing these two phytochemicals as novel therapies. MTT assay and LDH assay showed that these two phytochemicals have no general cytotoxicity (Table 1). At low concentrations, DHCA and Mal-gluc had no detectable effects on cell number, nuclear size, DNA structure, cell membrane permeability, mitochondrial mass, mitochondrial membrane potential or cytochrome C release in high content screening (HCS)41 using HepG2 cells (Table 1). At high concentrations, Mal-gluc reduced cell number, increased nuclear size, reduced cell membrane permeability, and increased mitochondrial mass, with calculated EC50s beyond physiological concentration (Table 1). DHCA, at high concentrations also decreased cell membrane permeability, increased mitochondrial mass and cytochrome C release (Table 1). IC50 of both phytochemicals for inhibition of the human Ether-à-go-go-related gene (hERG)42 was >25 µM (Table 1). Based on common discovery-stage cutoff criteria that HCS cytotoxicity with EC50 > 50 μM and inhibition of hERG with IC50 > 20 μM are acceptable, both phytochemicals are deemed to possess acceptable cytotoxicity/cardiotoxicity properties.

Table 1 In vitro toxicity characteristics and drug-like properties of DHCA and Mal-gluc Full size table

For drug-like properties, we assessed the two phytochemicals for plasma stability43, brain stability44, plasma protein binding45 and inhibition of cytochrome P450s (CYPs) (Table 1). Common discovery-stage cutoff criteria used for classifying individual drug-like properties as acceptable are: T1/2 > 30 min for plasma stability, T1/2 > 30 min for brain stability, plasma protein binding >2% free (i.e., <98% bound fraction) and IC50 > 10 µM for any CYP isoform. Thus, both phytochemicals are deemed to have acceptable drug-like properties.

Monoaminergic receptor and transporter binding activities

Currently available antidepressants are believed to act by modifying the activity of the brain monoaminergic system46, 47. We evaluated whether Mal-gluc and DHCA directly interact with the monoaminergic system using a radioligand binding assay. Among the 37 receptors and transporters tested (Table 2), we found that DHCA has no significant binding with any of them at concentrations as high as 10 µM (Table 2). Mal-gluc, at 10 µM concentration, binds weakly to 5-HT1D and 5-HT3 with a calculated inhibitory constant (Ki) of 4.3 µM for 5-HT3 and >10 µM for 5-HT1D, suggesting Mal-gluc does not have significant effect on the serotonin receptors at physiological concentrations. Collectively, these data suggest that neither phytochemical directly interacts with receptors/transporters in the brain that are known to play key roles in the pathogenesis of depression.