Portulaca oleracea Extract Modulates Diet-Dependent Neuroplasticity in a Murine Model of MCD-Induced NAFLD and Depression

Abstract

Non-alcoholic fatty liver disease (NAFLD) is increasingly recognized as a systemic condition with neuropsychiatric comorbidities, including depression. Growing evidence for the neuroprotective, antidepressant, and anxiolytic potential of Portulaca oleracea (PO) extract, provides a compelling rationale for investigating its effects in the interaction between dietary models of NAFLD and vulnerability to stress-related disorders. Fifty-four 14- to 18-week-old male and female C57BL/6N mice were distributed in two equal groups and fed either a methionine- and choline-deficient diet (MCD) or a methionine- and choline-controlled diet (MC). Subsequently, half of each group was subjected to chronic unpredictable mild stress (CUMS) and PO treatment. MCD caused significant weight loss, whereas MC promoted weight gain. Behaviorally, MCD induced anhedonia- and anxiety-like behaviors, worsened by CUMS. MC diet reduced CUMS-induced anhedonia, though anxiety-like behavior emerged only under stress. Recognition memory was impaired in stressed MCD-fed mice, while MC-fed mice showed enhanced novel object preference. At the cellular level, MCD suppressed hippocampal microglia and caused cortical astrocyte dysfunction, whereas the MC diet promoted cortical neurogenesis potentiated through PO, abolished by chronic stress. These findings underscore the impact of dietary composition on PO's systemic effects under chronic stress and support a mechanistic link between NAFLD-related dysfunction and depression-like phenotypes.

Keywords: CUMS; MC; MCD; NAFLD; Portulaca oleracea; depression; neurogenesis; neuroprotection.

Figure 1

(a) Body weight evolution during the experiment. Anhedonia-like behavior assessed by Sucrose Preference Test in mice which were fed (b) MCD or (c) MC diet. (d) Diet-dependent comparison of percent change in sucrose preference. The plots show mean values ± SD, * p < 0.05, ** p < 0.01. CUMS: Chronic unpredictable mild stress; MC: Methionine- and choline-controlled diet; MCD: Methionine- and choline-deficient diet; PO: Portulaca oleracea; SD: Standard deviation; VEH: Vehicle.

Figure 2

Anxiety-like behavior using the open field test in mice which were fed either (a) an MCD or (b) an MC diet. (c) Representative images depicting trajectories of animals within the testing arena at baseline and after treatment. (d) Diet-dependent comparison of percent changes in anxiety-like behavior. The plots show mean values ± SD, * p < 0.05, ** p < 0.01, and **** p < 0.0001.

Figure 3

Memory performance assessed using the Novel Object Preference test in mice which were fed either (a) MCD or (b) MC diet. (c) Representative images depicting exploration trajectories within the testing arena at baseline and following treatment. (d) Diet-dependent analysis of percent changes in novel object preference. The plots show mean values ± SD, * p < 0.05, ** p < 0.01.

Figure 4

Quantification of Iba1-positive microglia in (a) the cortex, and in (c) the hippocampus from mice included in the study groups. Representative microscopic images for the Iba1 positivity in the cortex (b) and in the hippocampus (d). Microglia were labeled with Iba1 (green) and cell nuclei were labeled with DAPI (blue). The graphs show mean values ± SD, * p < 0.05, ** p < 0.01. Scale bars 50 μm. DAPI: 4′,6-Diamidino-2-phenylindole; Iba1: Ionized calcium binding adaptor molecule 1.

Figure 5

Comparison of GFAP-positive signal in (a) the cortex and in (c) the hippocampus of mice included in the study groups. Representative microscopic images depicting GFAP positivity in the cortex (b) and in the hippocampus (d). Astrocytes were labeled with GFAP (yellow), and cell nuclei with DAPI (blue). The plots show mean values ± SD, * p < 0.05, ** p < 0.01. Scale bars 50 μm. GFAP: Glial fibrillary acidic protein.

Figure 6

Comparison of (a) cortical and (c) hippocampal neurogenesis by quantitative morphometry of immunofluorescent stains for NeuN in mouse brains. Representative microscopic images depicting NeuN positivity in the cortex (b) and in the hippocampus (d). Neurons were labeled with NeuN (red), and cell nuclei were labeled with DAPI (blue). The plots show mean values ± SD, * p < 0.05, ** p < 0.01. Scale bars 50 μm. NeuN: Neuronal nuclei.

Figure 7

Experimental design. The diagram shows the stages of the study and their sequence. D: Day.

Figure 8

Portulaca oleracea extract preparation. (a) Representative UHPLC–PDA chromatogram (detection at 265 nm) showing the separation of FMOC-derivatized methionine standard. The peak corresponding to unreacted FMOC reagent (or its hydrolysis product) is observed at a t_R of 5.512 min, while the peak for FMOC–methionine elutes at t_R 7.127 min. (b) Positive ion electrospray mass spectrum obtained for the peak identified as FMOC–methionine in (a). The spectrum confirms the identity of the derivatized complex, showing the characteristic protonated molecular ion [M + H]⁺ for FMOC–methionine at m/z 372. FMOC: Fluorenylmethyl chloroformate; t_R: Retention time; UHPLC–PDA: Ultra-high-performance liquid chromatography–Photodiode array.