Ginsenoside Rh2 attenuates microglial activation against toxoplasmic encephalitis via TLR4/NF-κB signaling pathway

Abstract

Background: Ginsenoside Rh2 (GRh2) is a characterized component in red ginseng widely used in Korea and China. GRh2 exhibits a wide range of pharmacological activities, such as anti-inflammatory, antioxidant, and anticancer properties. However, its effects on Toxoplasma gondii (T. gondii) infection have not been clarified yet.

Methods: The effect of GRh2 against T. gondii was assessed under in vitro and in vivo experiments. The BV2 cells were infected with tachyzoites of T. gondii RH strain, and the effects of GRh2 were evaluated by MTT assay, morphological observations, immunofluorescence staining, a trypan blue exclusion assay, reverse transcription PCR, and Western blot analyses. The in vivo experiment was conducted with BALB/c mice inoculated with lethal amounts of tachyzoites with or without GRh2 treatment.

Results and conclusion: The GRh2 treatment significantly inhibited the proliferation of T. gondii under in vitro and in vivo studies. Furthermore, GRh2 blocked the activation of microglia and specifically decreased the release of inflammatory mediators in response to T. gondii infection through TLR4/NF-κB signaling pathway. In mice, GRh2 conferred modest protection from a lethal dose of T. gondii. After the treatment, the proliferation of tachyzoites in the peritoneal cavity of infected mice markedly decreased. Moreover, GRh2 also significantly decreased the T. gondii burden in mouse brain tissues. These findings indicate that GRh2 exhibits an anti-T. gondii effect and inhibits the microglial activation through TLR4/NF-κB signaling pathway, providing the basic pharmacological basis for the development of new drugs to treat toxoplasmic encephalitis.

Keywords: Ginsenoside Rh2; Microglia; TLR4; Toxoplasma gondii; Toxoplasmic encephalitis.

Fig. 1

GRh2 cytotoxicity in BV2 cells. BV2 cells treated with GRh2 (5–40 μM) or SD (100 μg/mL) for 24 h and the cell viability was measured using the MTT assay. Three independent experiments were conducted in triplicate, and data were expressed as the mean ± standard deviation. ^##p < 0.01 vs. N group. SD, sulfadiazine.

Fig. 2

Microscopic observation of tachyzoites and BV2 cells. (A) BV2 cells cocultured with different parasites ratio. (i) Normal BV2 cells without T. gondii infection. (ii) The parasite:host ratio was 1:1. (iii) The parasite:host ratio was 5:1. (iv) The parasite:host ratio was 10:1. (B) BV2 cells cocultured with different infection duration. (i) Normal BV2 cells. (ii) For 24-h culture. (iii) For 36-h culture. (iv) For 48-h culture. The red arrow indicated T. gondii tachyzoite, the black arrow indicated the sensitized cell, and the yellow arrow indicated the normal cell.

Fig. 3

The inhibition effects of GRh2 against T. gondii in the in vitro infection study. (A) Direct inhibition rate on tachyzoites. (B) Morphological characteristics during a microscopy analysis. The red arrow indicated T. gondii tachyzoite, the black arrow indicated the sensitized cell, and the yellow arrow indicated the normal cell. (C) Immunofluorescence of T. gondii–infected BV2 cells. The T. gondii was indicated in green, whereas the DAPI stain (blue) indicated the location and size of nuclei. (D) and (E) The living host cells viability and extracellular T. gondii inhibition rate. (F) RT-PCR analysis for determining SAG1 and T.g.HSP70 mRNA expression in the BV2 cells. Three independent experiments were conducted in triplicate, and data were expressed as the mean ± standard deviation. ^##p < 0.01 vs. N group; ^∗p < 0.05 and ^∗∗p < 0.01 vs. CN group. RT-PCR, reverse transcription PCR; SD, sulfadiazine.

Fig. 4

Anti–T. gondii activity of GRh2 in BALB/c mice. (A) Treatment effects on the survival of T. gondii–infected BALB/c mice. (B) QC-PCR analysis for determining T. gondii abundance in the brain. (C) Tachyzoites inhibition rate in the peritoneal cavity. (D) Immunofluorescence of T. gondii–infected PECs. The T. gondii was indicated in green, whereas the DAPI staining (blue) indicated the location and size of nuclei. Three independent experiments were conducted in triplicate, and data were expressed as the mean ± standard deviation. ^∗p < 0.05 and ^∗∗p < 0.01 vs. CN group. QC-PCR, quantitative competitive PCR; SD-Na, sulfadiazine sodium; gDNA, genomic DNA.

Fig. 5

The effects of GRh2 on antimicroglia activation after T. gondii infection. Western blot analysis of Iba-1 protein expression in (A) BV2 cells and (B) mouse brain tissues. (C) The effects of GRh2 on Iba-1expression in T. gondii–infected BV2 cells by immunofluorescence assay. Three independent experiments were conducted in triplicate, and data were expressed as the mean ± standard deviation. ^#p < 0.01 vs. N group; ^∗∗p < 0.01 vs. CN group. SD, sulfadiazine; SD-Na, sulfadiazine sodium.

Fig. 6

The effects of GRh2 on inflammatory mediator production and neuronal morphology after T. gondii infection. Western blot analysis was completed to analyze iNOS, IFN-γ, and TNF-α expression in (A) BV2 cells and (B) mouse brain tissues. (C) The nitrite concentration was determined for the BV2 cells culture supernatants or mice serum based on the ELISA. (D) and (E) Neuronal morphology and density in the cortex and hippocampus by Nissl staining. Three independent experiments were conducted in triplicate, and data were expressed as the mean ± SD. ^##p < 0.01 vs. N group; ^∗∗p < 0.01 vs. CN group. IFN-γ, Interferon-γ; iNOS, inducible nitric oxide synthase; TNF-α, tumor necrosis factor α; ELISA, enzyme-linked immunosorbent assay.

Fig. 7

The effects of GRh2 on TLR4-mediated signal after T. gondii infection. Western blot analysis was completed to analyze (A) the TLR4, TRIF, and MyD88 expression in BV2 cells and (B) the TLR4 expression in mouse brain tissues. (C) Immunohistochemical double labeling for TLR4 (red) and Iba1 (green) in the cortex. The areas in the square were shown below at higher magnification. Three independent experiments were conducted in triplicate, and data were expressed as the mean ± standard deviation. ^##p < 0.01 vs. N group; ^∗∗p < 0.01 vs. CN group. SD-Na, sulfadiazine sodium.

Fig. 8

The effects of GRh2 on IκBα degradation and NF-κB p65 nuclear translocation after T. gondii infection. Western blot analysis was completed to analyze NF-κB p65, p-NF-κB p65, IκBα, and p-IκBα expression in (A) BV2 cells and (B) mouse brain. (C) Representative confocal microscopic images showing labeling for Iba-1 (green) in the BV2 cells. (D) Immunohistochemical double labeling for p-NF-κB p65 (red) and Iba1 (green) in the cortex. The areas in the square were shown below at higher magnification. Three independent experiments were conducted in triplicate, and data were expressed as the mean ± standard deviation. ^##p < 0.01 vs. N group; ^∗∗p < 0.01 vs. CN group. SD, sulfadiazine; SD-Na, sulfadiazine sodium.

Fig. 9

A proposed mechanism of GRh2 against TE by attenuating microglial activation via TLR4/NF-κB signaling pathway. TE, toxoplasmic encephalitis; IFN-γ, Interferon-γ; iNOS, inducible nitric oxide synthase; TNF-α, tumor necrosis factor α.