Betaine Inhibits NLRP3 Inflammasome Hyperactivation and Regulates Microglial M1/M2 Phenotypic Differentiation, Thereby Attenuating Lipopolysaccharide-Induced Depression-Like Behavior

Abstract

Depression is one of the most important mental illnesses and is closely related to inflammation. Betaine is a natural product with an anti-inflammatory and antioxidant activities. However, the mechanism by which betaine ameliorates depression-like behaviors induced by lipopolysaccharide (LPS) is poorly understood. The purpose of this study was to investigate the neuroprotective effect of betaine on LPS-induced depression-like behavior in mice and its mechanism of action. ICR mice were randomly divided into four groups: the control group, the LPS model group (0.83 mg/kg), the positive drug group (MIDO, 50 mg/kg), and the betaine group (5% and 1% in drinking water). The betaine group was administered for 21 days, and on the 22nd day, except for the blank group, LPS (0.83 mg/kg) was intraperitoneally injected to establish a lipopolysaccharide-induced mice depression-like model. Twenty-four hours after LPS injection, the tail suspension test (TST), open field test (OFT), and sucrose preference test (SPT) were performed to evaluate the effect of betaine on LPS-induced depressive behavior in mice. After the behavioral study, the mouse brain, hippocampus, and serum were taken for detection. The expressions of cytokines and inflammatory mediators were detected by ELISA, HE staining, immunofluorescence, immunohistochemistry, and western blotting. Western blotting was used to detect the protein expression levels of the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), caspase-1, and ASC, the protein expression levels of the microglial polarization markers COX-2, inducible nitric oxide synthase (iNOS), and CD206. The results showed that betaine significantly ameliorated the depression-like behavior in LPS-induced mice, significantly attenuated the production of proinflammatory cytokines and increased the release of an anti-inflammatory cytokines. Betaine decreased the expression of the NLRP3 inflammasome, decreased the expression of M1 polarization markers, tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), COX-2, and iNOS and promoted the expression of M2 polarization marker CD206. Our study suggests that betaine may promote the transition of microglia from the M1 to the M2 phenotype by inhibiting NLRP3 inflammasome activation, thereby attenuating lipopolysaccharide-induced depression-like behavior.

Figure 1

Chemical structure of betaine.

Figure 2

Effects of betaine on food intake, body weight, and depressive behavior in LPS-induced depression-like mice. (a) Experimental flow chart. (b) Changes in body weight of mice, 24 hours after LPS injection. (c) Changes in food intake in mice, 24 hours after LPS injection. (d) Influence of betaine on sucrose preference test. (e) Influence of betaine on tail suspension test. (f) Influence of betaine on open field test. The results were expressed as median ± interquartile range; ^###P < 0.01 compared to the normal control group; ^∗∗P < 0.01 and ^∗∗∗P < 0.001 compared to the model group, n = 12.

Figure 3

HE staining to observe the morphological changes of LPS-induced mice hippocampal CA1, CA3, and DG regions, n = 6. Red arrows indicate the irregular arrangement of neurons with enlarged pericellular spaces, black arrows indicate the neuronal pyknosis and the deepened coloration.

Figure 4

Effects of betaine on LPS-induced inflammatory cytokine production in mice hippocampus. (a–e) The expression levels of IL-1β, IL-6, IL18, TNF-α, and IL-10 in mouse hippocampus were determined by ELISA, with minocycline as the positive control. The results were expressed as mean ± SEM; ^##P < 0.01 and ^###P < 0.001 compared to the normal control group; ^∗P < 0.05 and ^∗∗P < 0.01 compared to the model group, n = 4.

Figure 5

Determination of serum C-reactive protein (CRP) and corticosterone (CORT) levels in mice by ELISA. (a) Expression of corticosterone content in tissues. (b, c) Levels of C-protein in serum and hippocampus. ^##P < 0.01 compared to the normal control group; ^∗P < 0.05 and ^∗∗P < 0.01 compared to the model group, n = 6.

Figure 6

Immunohistochemical labeling of M1 polarization marker COX-2 and M2 polarization marker CD206 in mice CA1 and CA3 brain regions. Black arrows indicate positive immunohistochemical staining for M1/M2 polarization markers. (a) Typical immunohistochemical results for the effects of betaine on CD206 positive cells in mouse hippocampus. (b) Statistics on the number of CD206 positive cells. (c) Typical immunohistochemical results for the effects of betaine on COX-2 positive cells in mouse hippocampus. (d) Statistics on the number of COX-2 positive cells. The results were expressed as mean ± SEM; ^##P < 0.01 and ^###P < 0.001 compared to the normal control group; ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 compared to the model group, n = 6.

Figure 7

Betaine reduces the expression of NLRP3 inflammasome in mice GD brain region. Immunostaining with anti-NLRP3 (green) and anti-Iba1 (microglia activation marker, (red) antibodies, and nuclei stained with DAPI (blue). (a) Representative images of NLRP3 fluorescence intensity after stimulation with LPS. (b) Analysis of NLRP3 fluorescence intensity in brain regions. (c) Analysis of Iba1 fluorescence intensity in brain regions. (d) Colocalization analysis of NLRP3 and Iba1. The results were expressed as mean ± SEM; ^##P < 0.01 and ^###P < 0.001 compared to the normal control group; ^∗P < 0.05 and ^∗∗∗P < 0.001 compared to the model group, n = 6.

Figure 8

The effects of betaine on LPS-induced NLRP3/TLR4/NF-κB signal transduction in mice. Western blot analysis of (a) CD206/iNOS/COX-2 protein levels, (b) P-NF-κB/MyD88/TLR4 protein levels, and (c) NLRP3, caspase-1, and ASC protein levels; data were presented as mean ± SEM; ^#P < 0.05 and ^##P < 0.01 compared to the normal control group; ^∗P < 0.05 and ^∗∗P < 0.01 compared to the model group, n = 4.