Fluoxetine attenuates neuroinflammation in early brain injury after subarachnoid hemorrhage: a possible role for the regulation of TLR4/MyD88/NF-κB signaling pathway

Abstract

Background: Neuroinflammation is closely associated with functional outcome in subarachnoid hemorrhage (SAH) patients. Our recent study demonstrated that fluoxetine inhibited NLRP3 inflammasome activation and attenuated necrotic cell death in early brain injury after SAH, while the effects and potential mechanisms of fluoxetine on neuroinflammation after SAH have not been well-studied yet.

Methods: One hundred and fifty-three male SD rats were subjected to the endovascular perforation model of SAH. Fluoxetine (10 mg/kg) was administered intravenously at 6 h after SAH induction. TAK-242 (1.5 mg/kg), an exogenous TLR4 antagonist, was injected intraperitoneally 1 h after SAH. SAH grade, neurological scores, brain water content, Evans blue extravasation, immunofluorescence/TUNEL staining, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot were performed.

Results: Fluoxetine administration attenuated BBB disruption, brain edema, and improved neurological function after SAH. In addition, fluoxetine alleviated the number of Iba-1-positive microglia/macrophages, neutrophil infiltration, and cell death. Moreover, fluoxetine reduced the levels of pro-inflammatory cytokines, downregulated the expression of TLR4 and MyD88, and promoted the nuclear translocation of NF-κB p65, which were also found in rats with TAK-242 administration. Combined administration of fluoxetine and TAK-242 did not enhance the neuroprotective effects of fluoxetine.

Conclusion: Fluoxetine attenuated neuroinflammation and improved neurological function in SAH rats. The potential mechanisms involved, at least in part, TLR4/MyD88/NF-κB signaling pathway.

Keywords: Early brain injury; Fluoxetine; Neuroinflammation; Subarachnoid hemorrhage.

Fig. 1

The effects of fluoxetine on mortality, SAH grade, neurological scores, Evans blue extravasation, and brain edema. a The quantification of mortality. b The quantification of SAH grade. n = 28/group. c The quantification of Evans blue extravasation. *p < 0.05 vs sham. ^#p < 0.05 vs SAH+vehicle. n = 6/group. d The quantification of brain water content. **p < 0.01 vs sham. ^#p < 0.05 vs SAH+vehicle. n = 6/group. e The quantification of neurological score. *p < 0.05 vs sham. ^#p < 0.05 vs SAH+vehicle. n = 28/group

Fig. 2

Fluoxetine downregulated MMP-9 expression and prevented degradation of the tight junction proteins in the ipsilateral cortex at 24 h after SAH. a Representative western blot bands of MMP-9, occludin, claudin-5, and ZO-1. b Densitometric quantification of MMP-9. **p < 0.01 vs sham. ^##p < 0.01 vs SAH+vehicle. n = 6/group. c Densitometric quantification of occludin. **p < 0.01 vs sham. ^#p < 0.05 vs SAH+vehicle. n = 6/group. d Densitometric quantification of claudin-5. **p < 0.01 vs sham. ^##p < 0.01 vs SAH+vehicle. n = 6/group. e Densitometric quantification of ZO-1. **p < 0.01 vs sham. ^#p < 0.05 vs SAH+vehicle. n = 6/group

Fig. 3

Fluoxetine decreased the mRNA levels of pro-inflammatory cytokines. a The quantification of TNF-α mRNA levels. **p < 0.01 vs sham. ^##p < 0.01 vs SAH+vehicle. n = 5/group. b The quantification of IL-1β mRNA levels. **p < 0.01 vs sham. ^#p < 0.05 vs SAH + vehicle. n = 5/group. c The quantification of IL-6 mRNA levels. **p < 0.01 vs sham. ^##p < 0.01 vs SAH+vehicle. n = 5/group. d The quantification of CD86 mRNA levels. **p < 0.01 vs sham. ^##p < 0.01 vs SAH+vehicle. n = 5/group

Fig. 4

Fluoxetine decreased the number of Iba-1-positive microglia/macrophages in the ipsilateral cortex at 24 h after SAH. a Representative Iba-1 staining. b The quantification of Iba-1-positive cells. **p < 0.01 vs sham. ^##p < 0.01 vs SAH+vehicle. Scale bar = 100 μm. n = 5/group

Fig. 5

Fluoxetine inhibited neutrophil infiltration in the ipsilateral cortex at 24 h after SAH. a Representative MPO staining. b The quantification of MPO-positive cells. **p < 0.01 vs sham. ^##p < 0.01 vs SAH+vehicle. Scale bar = 50 μm. n = 5/group

Fig. 6

Fluoxetine attenuated neuronal apoptosis in the ipsilateral cortex at 24 h after SAH. a Representative TUNEL staining. b The percentage of TUNEL-positive neurons. ***p < 0.01 vs sham. ^##p < 0.01 vs SAH+vehicle. Scale bar = 100 μm. n = 5/group

Fig. 7

Fluoxetine downregulated the protein levels of TLR4 and MyD88, but increased the nuclear protein levels of NF-κB p65 in the ipsilateral cortex at 24 h after SAH. a Representative western blot bands of TLR4, MyD88, and nuclear NF-κB p65. b Densitometric quantification of TLR4. **p < 0.01 vs sham. ^##p < 0.01 vs SAH+vehicle. n = 6/group. c Densitometric quantification of MyD88. **p < 0.01 vs sham. ^##p < 0.01 vs SAH+vehicle. n = 6/group. d Densitometric quantification of nuclear NF-κB p65. **p < 0.01 vs sham. ^##p < 0.01 vs SAH+vehicle. n = 6/group

Fig. 8

Combined administration of fluoxetine and TAK-242 did not enhance the effects of fluoxetine in the expression of TLR4, MyD88, nuclear NF-κB, and pro-inflammatory cytokines, BBB disruption, and neurological function. a Representative western blot bands of TLR4, MyD88, and nuclear NF-κB p65. b Densitometric quantification of TLR4. **p < 0.01 vs SAH+vehicle. n = 6/group. c Densitometric quantification of MyD88. **p < 0.01 vs SAH+vehicle. n = 6/group. d Densitometric quantification of nuclear NF-κB p65. **p < 0.01 vs SAH+vehicle. n = 6/group. e The quantification of TNF-α mRNA levels. **p < 0.01 vs SAH+vehicle. n = 5/group f The quantification of IL-1β mRNA levels. *p < 0.05 vs SAH+vehicle. n = 5/group. g The quantification of IL-6 mRNA levels. **p < 0.01 vs SAH+vehicle. n = 5/group. h The quantification of Evans blue extravasation. **p < 0.01vs SAH+vehicle. n = 6/group. i The quantification of neurological score. *p < 0.05 vs sham. ^#p < 0.05 vs SAH + vehicle. n = 17/group

Fig. 9

The potential molecular mechanisms of anti-inflammatory effects of fluoxetine through TLR4/MyD88/NF-κB signaling pathway