Rivaroxaban Modulates TLR4/Myd88/NF-Kβ Signaling Pathway in a Dose-Dependent Manner With Suppression of Oxidative Stress and Inflammation in an Experimental Model of Depression

Abstract

Depression is a common mental illness leading to upset or anxiety, with a high incidence rate in the world. Depression can lead to suicidal thoughts and behavior. The present study aimed to evaluate the effect of the direct oral anticoagulant rivaroxaban (RVX), in the model of depression induced by chronic unpredicted mild stress (CUMS) in rats. Fifty-six male Wister rats were randomly divided into seven experimental groups (8 rats/group); Group 1: Control group given vehicle per oral (p.o.), Group 2: RVXL-control group (received rivaroxaban 20 mg/kg/day, p.o..), Group 3: RVXH-control group (received rivaroxaban 30 mg/kg/day, p.o.), Group 4: chronic unpredictable mild stress (CUMS) group, Group 5: FLX-treated CUMS group (received fluoxetine 10 mg/kg/day, p.o..), Group 6: RVXL-treated CUMS group (received rivaroxaban 20 mg/kg/day, p.o.), and Group 7: RVXH-treated CUMS group (received rivaroxaban 30 mg/kg/day, p.o.). The rats received the drugs from the first day of the experiment and continued till 4 weeks-the duration of the study. The following were measured: monoamine neurotransmitters, malondialdehyde (MDA), total nitrite/nitrate (NOx), reduced glutathione (GSH), superoxide dismutase (SOD), Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), nuclear factor-kappa B (NF-κB), tumor necrosis factor-α (TNF-α), brain-derived neurotrophic factor (BDNF), and vascular endothelial growth factor-A (VEGF-A). A forced swimming test (FST) was done. Furthermore, histological changes and glial fibrillary acidic protein (GFAP) immunoexpression were evaluated. CUMS showed a significant decrease in hypothalamic neurotransmitters, hippocampal GSH, SOD, BNDF, and VEGF-A with a significant increase in hippocampal MDA, NOx, NF-kβ, Myd88, TLR4, TNF-α, and GFAP immunoexpression. RVX showed significant improvement in all parameters (p -value < 0.0001). In conclusion, RVX in a dose-dependent manner possesses potent ameliorative effects against depression by reducing the oxidative stress and inflammatory process, through the regulation of the TLR4/Myd88/NF-kβ signaling pathway.

Keywords: MyD88; NF-kβ; TLR4; depression; rivaroxban.

FIGURE 1

Diagrammatic scheme of the experimental design.

FIGURE 2

Effect of RVX at different doses on BNDF (Figure 1A) and VEGF-A (Figure 1B) in the hippocampal tissue. The findings are displayed as mean ± S.E.M. Significant difference at p < 0.05. ^asignificant difference compared to the control, ^bsignificant difference compared to the FLX group, ^csignificant difference compared to the CUMS group, and ^dsignificant difference compared to RVXH (BNDF: p < 0.0001, and p < VEGF-A: 0.0004). BNDF, brain-derived neurotrophic factor; CUMS, chronic unpredictable mild stress; FLX, fluoxetine; RVX L, rivaroxaban low dose; RVX H, rivaroxaban high dose; and VEGF-A, vascular endothelial growth factor-A.

FIGURE 3

The findings are displayed as mean ± S.E.M. Significant difference at p < 0.05. ^asignificant difference compared to the control, ^bsignificant difference compared to the FLX group, ^csignificant difference compared to the CUMS group, and ^dsignificant difference compared to the RVXH (p < 0.0001). CUMS, chronic unpredictable mild stress; FLX, fluoxetine; NFkB, nuclear factor Kappa B; MyD88, myeloid differentiation factor 88; RVX L, rivaroxaban low dose; RVX H, rivaroxaban high dose; TLR4, Toll-like receptor 4; and TNF-α, tumor necrosis factor-α.

FIGURE 4

Effect of RVX at different doses on behavior (FST). The findings are displayed as mean ± S.E.M. Significant difference at p < 0.05. ^aSignificant difference compared to the control, ^bsignificant difference compared to the FLX group, ^csignificant difference compared to the CUMS group, and ^dsignificant difference compared to RVXH. p value <0.0001. CUMS, chronic unpredictable mild stress; FLX, fluoxetine; RVX L, rivaroxaban low dose; and RVX H, rivaroxaban high dose.

FIGURE 5

Representative photomicrographs of the CA3 zone of the hippocampus (A––D) Control, RVXL, RVXH, and FLX groups showing normal densely packed pyramidal neurons with vesicular nuclei and prominent nucleoli (thick arrow), neurophil (n), and glial cells (blue arrow). (E) The CUMS group showing shrunken elongated pyramidal neurons with darkly stained nuclei (thin arrows). The structure in the circle may indicate fused microglia. (F) The RVXL/CUMS group showing a normal morphological appearance of the pyramidal neurons (thick arrow), while the thin arrow reveals a shrunken elongated pyramidal neuron with a darkly stained nucleus. (G) RVXH/CUMS group showing normal neurons (thick arrow), neurophil (n), and glial cells (blue arrow). H&E x400, scale bars = 50 μm ^aSignificant difference compared to the control, ^bsignificant difference compared to the FLX group, ^c significant difference compared to the CUMS group and ^dsignificant difference compared to the RVXH. p value <0.0001. CUMS, chronic unpredictable mild stress; FLX, Fluoxetine; RVX L, rivaroxaban low dose; and RVX H, rivaroxaban high.

FIGURE 6

GFAP immunoreactivity. (A––D) Control, RVXL, RVXH, and FLX groups showing small-sized faintly stained astrocytes with few processes (arrows). (E) The CUMS group, displaying enlarged densely stained astrocytes with ramifying processes (arrows). (F) The RVXL/CUMS group, showing less densely stained astrocytes with fewer processes (arrows). (G) The RVXH/CUMS group, displaying more or less normal astrocytes (arrows). GFAP immunostaining x400, scale bars = 50 μm ^aSignificant difference compared to the control, ^bsignificant difference compared to the FLX group, ^c significant difference compared to the CUMS group, and ^d significant difference compared to the RVXH. p value <0.0001. CUMS, chronic unpredictable mild stress; FLX, fluoxetine; RVX L, rivaroxaban low dose; and RVXH, rivaroxaban high.