The hypothalamic steroidogenic pathway mediates susceptibility to inflammation-evoked depression in female mice

Abstract

Background: Depression is two-to-three times more frequent among women. The hypothalamus, a sexually dimorphic area, has been implicated in the pathophysiology of depression. Neuroinflammation-induced hypothalamic dysfunction underlies behaviors associated with depression. The lipopolysaccharide (LPS)-induced mouse model of depression has been well-validated in numerous laboratories, including our own, and is widely used to investigate the relationship between neuroinflammation and depression. However, the sex-specific differences in metabolic alterations underlying depression-associated hypothalamic neuroinflammation remain unknown.

Methods: Here, we employed the LPS-induced mouse model of depression to investigate hypothalamic metabolic changes in both male and female mice using a metabolomics approach. Through bioinformatics analysis, we confirmed the molecular pathways and biological processes associated with the identified metabolites. Furthermore, we employed quantitative real-time PCR, enzyme-linked immunosorbent assay, western blotting, and pharmacological interventions to further elucidate the underlying mechanisms.

Results: A total of 124 and 61 differential metabolites (DMs) were detected in male and female mice with depressive-like behavior, respectively, compared to their respective sex-matched control groups. Moreover, a comparison between female and male model mice identified 37 DMs. We capitalized on biochemical clustering and functional enrichment analyses to define the major metabolic changes in these DMs. More than 55% of the DMs clustered into lipids and lipid-like molecules, and an imbalance in lipids metabolism was presented in the hypothalamus. Furthermore, steroidogenic pathway was confirmed as a potential sex-specific pathway in the hypothalamus of female mice with depression. Pregnenolone, an upstream component of the steroid hormone biosynthesis pathway, was downregulated in female mice with depressive-like phenotypes but not in males and had considerable relevance to depressive-like behaviors in females. Moreover, exogenous pregnenolone infusion reversed depressive-like behaviors in female mice with depression. The 5α-reductase type I (SRD5A1), a steroidogenic hub enzyme involved in pregnenolone metabolism, was increased in the hypothalamus of female mice with depression. Its inhibition increased hypothalamic pregnenolone levels and ameliorated depressive-like behaviors in female mice with depression.

Conclusions: Our study findings demonstrate a marked sexual dimorphism at the metabolic level in depression, particularly in hypothalamic steroidogenic metabolism, identifying a potential sex-specific pathway in female mice with depressive-like behaviors.

Keywords: Depression; Hypothalamus; Neuroinflammation; Neurosteroid biosynthesis; Pregnenolone; Sex.

Fig. 1

Assessment of the LPS-induced depressive-like behaviors and hypothalamic inflammatory cytokines in female and male mice. A Experimental timeline and design. B–E Body weight changes (B) and sucrose preference test (C), tail suspension test (D), and forced swimming test (E) results in female and male mice after LPS exposure, n = 10 mice/group. F, G The mRNA (F) and protein (G) levels of hypothalamic cytokines (TNF-α, IL-1β, and IL-6), n = 5 (F) n = 6 (G) mice/group. All data are presented as means ± SEM and were analyzed by two-way ANOVA (sex × treatment) followed by Bonferroni's post hoc tests. *P < 0.05, **P < 0.01, ***P < 0.001. LPS lipopolysaccharide-treated group, CON saline-treated group, BW body weight, SPT sucrose preference test, TST tail suspension test, FST forced swimming test

Fig. 2

Metabolomics analysis of the hypothalamus from male and female mice with LPS-induced depressive-like behavior compared to their control counterparts. A 3D view of the OPLS-DA model showing four clearly separated groups, including saline-treated male controls (CON-Male, purple), LPS-treated depressive-like male mice (LPS-Male, green), saline-treated female controls (CON-Female, blue), and LPS-treated depressive-like female mice (LPS-Female, turquoise). B The OPLS–DA model shows a more noticeable separation between t each group pair. The colours correspond to those in (A). C The permutation test suggests the validity of the OPLS–DA model between each group pair, as the Q² and R² values yielded by the permutation test were higher than their original values

Fig. 3

Sex-specific hypothalamic metabolic signatures of male and female mice with LPS-evoked depressive-like behavior. A–E Hypothalamic metabolic signatures of saline-treated male and female controls. A Comprehensive biochemical classification of all identified differential metabolites (DMs) was clustered and visualized as a pie chart. B Lipid subgroups and lipid molecule counts according to the annotated lipid classifications of the LIPID MAPS and HMDB databases. C–E Levels of lipid metabolites belonging to glycerophospholipids (C), fatty acyls (D), sterol lipids, and sphingolipids (E) in CON-Male mice relative to CON-Female mice. F–J Hypothalamic metabolic signatures in saline- and LPS-treated male mice. F Pie chart depicting the biochemical classification of all DMs. G Lipid subgroups and lipid molecule counts based on LIPID MAPS and HMDB annotations. H–J Glycerophospholipids (H), fatty acyls (I), sterol lipids, and sphingolipids (J) levels in the LPS-Male group compared to the CON-Male group. K–N Hypothalamic metabolic signatures in saline- and LPS-treated female mice. K Biochemical classification of all DMs. L Lipid subgroups and lipid molecule counts. M, N Glycerophospholipids (M), fatty acyls, sterol lipids and sphingolipids (N) in the LPS-Female vs. CON-Female. Fold changes in the lipids are expressed as z-scores; a positive value indicates a higher level, whereas a negative value indicates a lower concentration in each group compared to the corresponding control group. LPA lysophosphatidic acid, PA phosphatidic acid, LPC lysophosphatidylcholine, PC phosphatidylcholine, LPE lysophosphatidylethanolamine, PE phosphatidylethanolamine, LPG lysophosphatidylglycerol, PG phosphatidylglycerol, LPI lysophosphatidylinositol, PI phosphatidylinositol, LyPS lysophosphatidylserine, PS phosphatidylserine, FA fatty acyls; sterol lipids, ST; and SP sphingolipids

Fig. 4

The hypothalamic steroid hormone biosynthesis is significantly affected by inflammation, particularly in female mice. A–E Hypothalamic metabolic signatures of LPS-treated female and male mice. A Biochemical classifications of all DMs. B Lipid subgroups and lipid molecule counts. C–E Glycerophospholipids (C), fatty acyls (D), sterols, and sphingolipids (E) in LPS-Female vs. LPS-Male. Relative levels of the lipids by z-scores, a positive value indicates a higher level, whereas a negative value indicates a lower concentration in the LPS-Female group. F, G Implications of DMs in biological processes (F) and metabolic pathways (G) in the LPS-Female vs. LPS-Male comparison based on MetaboAnalyst 5.0 analyses. H Venn plot of significantly affected metabolic pathways in LPS-treated female and male mice. Perturbations of steroid hormone biosynthesis are apparent in the hypothalamus of the female depression model. I Venn diagram of DMs from different comparisons. Red boxes represent increased levels, and green boxes represent decreased levels. FA fatty acyls, LPG lysophosphatidylglycerol, LPI lysophosphatidylinositol, MG monoacylglycerol, PC phosphatidylcholine, PG phosphatidylglycerol, PS phosphatidylserine, SP sphingolipid, ST sterol lipid, TG triacylglycerol

Fig. 5

Verification of perturbations in steroidogenic pathways in the hypothalamus of LPS-induced depressive-like female mice. A, B Heatmap (A) and scatter plot (B) of the detected steroid metabolites in the hypothalamus of LPS-induced depressive-like mice and sex-matched controls. A Raw measurement mean was converted into z-scores, with downregulation denoted by blue, and upregulation indicated by red. B Relative levels of steroid metabolites were determined by comparing them to the raw measurement mean of the CON-Male group. C Metabolite–protein interaction network of the steroid biosynthesis pathway, according to the KEGG annotations. Differential steroids between LPS-Female and CON-Female mice are labelled with red (increased) and green (decreased) in the network, and essential proteins and unchanged steroid metabolites are designated. D, E mRNA transcriptional (D) and protein (E) levels of the key proteins involved in steroidogenic pathways, n = 6 (D) and n = 4 (E) mice/group. All data are presented as means ± SEM and were analyzed by two-way ANOVA followed by Bonferroni's post hoc tests. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

Pregnenolone supplements reversed hypothalamic inflammation and depressive-like behaviors in female mice. A Schematic illustration of the central pregnenolone (Preg) delivery and subsequent behavioral tests in female mice. Blue arrows show the injection phases. Schematic representation of the injection site in the third ventricle stained with Evans blue (bottom right, Scale bar 2 mm). B Transcriptional levels of hypothalamic Tnf, Il1b, and Il6, after i.c.v. administration of artificial cerebrospinal fluid (ACSF) or pregnenolone (Preg), n = 6 mice/group. C, D Recorded parameters to assess sucrose preference in the SPT (C) and immobility in the FST (D) after i.c.v. administration of ACSF or pregnenolone, n = 6 mice/group. All data are presented as means ± SEM and were analyzed using one-way ANOVA with Bonferroni's post hoc tests. *P < 0.05, **P < 0.01, ***P < 0.001. AP anterior–posterior, ML medial–lateral, DV dorsal–ventral, SPT sucrose preference test, FST forced swimming test; i.c.v., intracerebroventricular

Fig. 7

Inhibition of 5α-reductase attenuates hypothalamic inflammation and depressive-like behaviors in female mice. A Schematic illustration of dutasteride (a 5α-reductase inhibitor) or vehicle administration via bilateral infusion into the hypothalamus 2 h before LPS injection and subsequent behavioral and biochemical tests in female mice. Schematic representation of the injection site in the hypothalamus stained with Evans blue (bottom right, Scale bar 2 mm). B Hypothalamic levels of the three neurosteroids, pregnenolone, progesterone, and allopregnanolone, in female mice, n = 6–8 mice/group. C Transcriptional levels of hypothalamic Tnf, Il1b, and Il6 expression, n = 6–8 mice/group. D, E Behavioral assessments. Sucrose preference (D) and immobility in the FST (E), n = 6–8 mice/group. All data are presented as means ± SEM and were analyzed using one-way ANOVA with Bonferroni's post hoc tests. *P < 0.05, **P < 0.01, ***P < 0.001