Treatment with lysophosphatidic acid prevents microglial activation and depression-like behaviours in a murine model of neuropsychiatric systemic lupus erythematosus

Abstract

Neuropsychiatric systemic lupus erythematosus (NPSLE) is an incurable disease characterised by neuropsychiatric symptoms, particularly depression. Novel therapeutic options for NPSLE are urgently needed. Several previous reports have suggested that both microglial activation and impaired neurogenesis may be involved in the progression of depression. In contrast, the administration of lysophosphatidic acid (LPA) ameliorates depression and anxiety. Therefore, in the present study, we determined whether treatment with LPA affects microglial activation, impaired neurogenesis, and abnormal behaviour in MRL/lpr mice. In both tail suspension test and forced swim test, the MRL/lpr mice exhibited a significant increase in total immobility time compared with MRL/+ mice. Treatment with LPA significantly suppressed the prolonged immobility time in MRL/lpr mice. In contrast, pretreatment with ki16425 (a specific antagonist of LPA receptor 1 and 3) significantly reversed the effects of LPA. Furthermore, MRL/lpr mice exhibited impairments in spatial working memory and visual cognitive memory, which were suppressed by LPA treatment. The expression levels of TMEM119, CD68, GFAP, and caspase-3 in the hippocampus and prefrontal cortex of MRL/lpr mice were significantly higher than those in MRL/+ mice. Treatment with LPA inhibited these increases in MRL/lpr mice. Pretreatment with ki16425 reversed LPA-mediated inhibition of microglial activation. The quantity of sodium fluorescein that leaked into the brain tissues in MRL/lpr mice were significantly higher than that in MRL/+ mice. Treatment with LPA tended to decrease the sodium fluorescein leakage. These findings suggest that treatment with LPA may regulate microglial activation, which is important in the pathogenesis of NPSLE, as well as blood-brain-barrier weakening and abnormal behaviour.

Keywords: LPA; NPSLE; depression; microglia; neuroinflammation.

Graphical abstract

Figure 1:

Effect of LPA on depression-like behaviours in MRL/lpr mice. The tail suspension tests (A and B) and forced swimming tests (C) were performed to evaluate depression-like behaviours in MRL/lpr mice. Bar graphs show the quantitative data of the total time of immobility and latency to the bout of immobility in the vehicle-treated MRL/+ group (control-vehicle; n = 12), vehicle-treated MRL/lpr group (lpr-vehicle; n = 12), or LPA-treated MRL/lpr group (lpr-LPA; n = 11). The data are expressed as mean ± SEM. *P < 0.05, **P < 0.01 versus control-vehicle; #P < 0.05 versus lpr-vehicle; ##P < 0.01 versus lpr-vehicle.

Figure 2:

Effect of an LPA receptor antagonist on depression-like behaviours in LPA-treated MRL/lpr mice. The tail suspension tests (A and B) and forced swimming tests (C) were performed to evaluate depression-like behaviours in the MRL/lpr mice. Bar graphs show the quantitative data of the total time of immobility and latency to the bout of immobility in the vehicle-pretreated + vehicle-treated MRL/lpr group (lpr-vehicle-vehicle; n = 12), vehicle-pretreated + LPA-treated MRL/lpr group (lpr-vehicle-LPA; n = 12), or Ki16425-pretreated + LPA-treated MRL/lpr group (lpr-Ki16425-LPA; n = 12). The data are expressed as mean ± SEM. *P < 0.05, **P < 0.01 versus lpr-vehicle-vehicle; #P < 0.05 versus lpr-vehicle-LPA; ##P < 0.01 versus lpr-vehicle-LPA.

Figure 3

Effects of LPA on spatial working memory and visual cognitive memory in MRL/lpr mice. The Y-maze tests (A) and object recognition tests (B) were respectively performed to evaluate spatial working memory and visual cognitive memory in MRL/lpr mice. Bar graphs show the quantitative data of the correct alternation and recognition index in the vehicle-treated MRL/+ group (control-vehicle; n = 12), vehicle-treated MRL/lpr group (lpr-vehicle; n = 12), and LPA-treated MRL/lpr group (lpr-LPA; n = 11). The data are expressed as mean ± SEM. *P < 0.05 versus control-vehicle; #P < 0.05 versus lpr-vehicle.

Figure 4:

Effect of LPA on the expressions of Iba1 and CD68 in MRL/lpr mice. Representative images of immunofluorescence staining of Iba1 (red) and CD68 (green) in prefrontal cortex (A) and sub-granular zone of hippocampus (C) in vehicle-treated MRL/+ (control-vehicle), vehicle-treated MRL/lpr (lpr- vehicle), or LPA-treated MRL/lpr (lpr-LPA) groups. White bar = 100 μm. Bar graphs show quantitative analysis of CD68-positive rates in the prefrontal cortex (B) and sub-granular zone of the hippocampus (D) in vehicle-treated MRL/+ group (control-vehicle; n = 6), vehicle-treated MRL/lpr group (lpr-vehicle; n = 5), or LPA-treated MRL/lpr group (lpr-LPA; n = 5). The data are expressed as mean ± SEM. *P < 0.05 versus control-vehicle; #P < 0.05 versus lpr-vehicle.

Figure 5:

Effect of an LPA receptor antagonist on the expressions of Iba1 and CD68 in LPA-treated MRL/lpr mice. Representative images of immunofluorescence staining of Iba1 (red) and CD68 (green) in prefrontal cortex (A) and sub-granular zone of hippocampus (C) in vehicle-pretreated + vehicle-treated MRL/lpr group (lpr-vehicle-vehicle), vehicle-pretreated + LPA-treated MRL/lpr group (lpr-vehicle-LPA), or Ki16425-pretreated + LPA-treated MRL/lpr group (lpr-Ki16425-LPA). White bar = 100 μm. Bar graphs show quantitative analysis of CD68-positive rates in the prefrontal cortex (B) and sub-granular zone of the hippocampus (D) in vehicle-pretreated + vehicle-treated MRL/lpr group (lpr-vehicle-vehicle; n = 5), vehicle-pretreated + LPA-treated MRL/lpr group (lpr-vehicle-LPA; n = 6), or Ki16425-pretreated + LPA-treated MRL/lpr group (lpr-Ki16425-LPA; n = 5). The data are expressed as mean ± SEM. *P < 0.05, **P <0.01 versus lpr-vehicle-vehicle; ##P < 0.01 versus lpr-vehicle-LPA.

Figure 6:

Effect of LPA on the expression of TMEM119 in MRL/lpr mice. Representative images of immunofluorescence staining of TMEM119 (red) and CD68 (green) in prefrontal cortex (A) and sub-granular zone of hippocampus (C) in vehicle-treated MRL/+ (control-vehicle), vehicle-treated MRL/lpr (lpr-vehicle), or LPA-treated MRL/lpr (lpr-LPA) groups. White bar = 100 μm. Bar graphs show quantitative analysis of CD68-positive rates in the prefrontal cortex (B) and sub-granular zone of the hippocampus (D) in vehicle-treated MRL/+ group (control-vehicle; n = 5), vehicle-treated MRL/lpr group (lpr-vehicle; n = 5), or LPA-treated MRL/lpr group (lpr-LPA; n = 5). The data are expressed as mean ± SEM. *P < 0.05, **P < 0.01 versus control-vehicle; ##P < 0.01 versus lpr-vehicle.

Figure 7:

Effect of LPA on GFAP expression in the hippocampus of MRL/lpr mice. Representative images of immunofluorescence staining of GFAP (red) in the sub-granular zone of hippocampus (A) in vehicle-treated MRL/+ (control-vehicle), vehicle-treated MRL/lpr (lpr-vehicle), or LPA-treated MRL/lpr (lpr-LPA) groups. White bar = 100 μm. Bar graphs show quantitative analysis of GFAP positive rates in the sub-granular zone of the hippocampus (B) in vehicle-treated MRL/+ groups (control-vehicle; n = 5), vehicle-treated MRL/lpr groups (lpr-vehicle; n = 5), or LPA-treated MRL/lpr groups (lpr-LPA; n = 5). The data are expressed as mean ± SEM. **P < 0.01 versus control-vehicle; #P < 0.05 versus lpr-vehicle.

Figure 8:

Effect of LPA on BrdU expression in the hippocampus of MRL/lpr mice. Representative images of immunofluorescence staining of NeuN (red) and BrdU (green) in sub-granular zone of hippocampus (A) in vehicle-treated MRL/+ (control-vehicle), vehicle-treated MRL/lpr (lpr-vehicle), and LPA-treated MRL/lpr groups (lpr-LPA). White bar = 50 μm. The bar graph shows the quantitative analysis of BrdU-positive rates in the sub-granular zone of the hippocampus (B) in vehicle-treated MRL/+ group (control-vehicle; n = 6), vehicle-treated MRL/lpr group (lpr-vehicle; n = 4), or LPA-treated MRL/lpr group (lpr-LPA; n = 6). The data are expressed as mean ± SEM. *P < 0.05 versus control-vehicle, ##P < 0.01 versus lpr-vehicle.

Figure 9:

Effect of LPA on cleaved caspase3 expression and TUNEL staining in MRL/lpr mice. Representative images of immunofluorescence staining of cleaved caspase3 (red) in the sub-granular zone of hippocampus and prefrontal cortex (A) in vehicle-treated MRL/+ (control-vehicle), vehicle-treated MRL/lpr (lpr-vehicle), or LPA-treated MRL/lpr (lpr-LPA) groups. White bar = 100 μm. Bar graphs show quantitative analysis of cleaved caspase3 positive rates in the sub-granular zone of the hippocampus and prefrontal cortex (B) in vehicle-treated MRL/+ group (control-vehicle; n = 5), vehicle-treated MRL/lpr group (lpr-vehicle; n = 6), or LPA-treated MRL/lpr group (lpr-LPA; n = 5), and the average value of TUNEL positive cells in the two sections containing prefrontal cortex (C) in vehicle-treated MRL/+ group (control-vehicle; n = 5), vehicle-treated MRL/lpr group (lpr-vehicle; n = 5), or LPA-treated MRL/lpr group (lpr-LPA; n = 5). The data are expressed as mean ± SEM. **P < 0.01 versus control- vehicle; #P<0.05, ##P<0.01 versus lpr-vehicle.

Figure 10:

Effect of LPA on sodium fluorescein leakage into the brain of MRL/lpr mice. Bar graphs show the quantitative analysis of sodium fluorescein leakage by spectrophotometric measurement in vehicle-treated MRL/+ (control-vehicle; n = 7), vehicle-treated MRL/lpr group (lpr-vehicle; n = 5), or LPA-treated MRL/lpr group (lpr-LPA; n = 5). The data are expressed as mean ± SEM. **P < 0.01 versus control-vehicle.

Figure 11:

Effect of LPA on inflammatory cytokines in hippocampus of MRL/lpr mice. The blots shown are representative data for the expressions of β-actin, IL-1β, and TNF-α (A) of hippocampus from vehicle-treated MRL/+ (control-vehicle), vehicle-treated MRL/lpr (lpr-vehicle), or LPA-treated MRL/lpr (lpr-LPA) groups. Bar graphs show quantitative analysis of the rates of standardized to β-actin in the hippocampus (B) in vehicle-treated MRL/+ group (control-vehicle; n = 8), vehicle-treated MRL/lpr group (lpr-vehicle; n = 8), or LPA-treated MRL/lpr group (lpr-LPA; n = 8). The data are expressed as mean ± SEM. **P < 0.01 versus control-vehicle; #P < 0.05, ##P < 0.01 versus lpr-vehicle.

Figure 12:

Effect of LPA on plasma anti-dsDNA antibody levels in MRL/lpr mice. Bar graphs show the quantitative analysis of plasma anti-dsDNA antibody levels by ELISA in the vehicle-treated MRL/+ group (control-vehicle; n = 8), vehicle-treated MRL/lpr group (lpr-vehicle; n = 8), or LPA-treated MRL/lpr group (lpr-LPA; n = 8). The data are expressed as mean ± SEM. **P < 0.01 versus control-vehicle.