Melatonin protects against blood-brain barrier damage by inhibiting the TLR4/ NF-κB signaling pathway after LPS treatment in neonatal rats

Abstract

Hypoxic-ischemic and inflammatory (HII) induces the disruption of blood-brain barrier (BBB) which leads to inflammatory responses and neuronal cell death, resulting in brain secondary damage. Previous studies showed that melatonin produced potent neuroprotective effects in neonatal hypoxic-ischaemic models. However, the relationship between BBB disruption and melatonin in HII was still unclear. The present study therefore investigated the beneficial effects of melatonin on BBB after HII and the underlying mechanisms. HII animal model was conducted by receiving lipopolysaccharide followed by 90 min hypoxia-ischaemia in postnatal day 2 Sprague-Dawley rat pups. Melatonin was injected intraperitoneally 1 h before lipopolysaccharide injection and then once a day for 1 week to evaluate the long-term effects. In this study, we demonstrated that melatonin administration inhibited the disruption of BBB permeability and improved the white matter recovery in HII model rats. Melatonin significantly attenuated the degradation of junction proteins and the neuroprotective role was related to the inhibition of microglial toll-like receptor 4/ nuclear factor-kappa B signaling pathway both in vivo and in vitro. Taken together, our data demonstrated that therapeutic strategies targeting inflammation might be suitable for the therapy of preserving BBB integrity after HII.

Keywords: TLR4/NF-κB; blood-brain barrier; hypoxic-ischemic and inflammatory; melatonin; white matter injury.

Figure 1. Melatonin improved white matter recovery and reduced BBB permeability after neonatal HII

A. MBP staining in the white matter of callosum and striatum regions at 21 d after HII. Scale = 400 μm (callosum) and 200 μm (striatum). B. HE staining in the brain of cortex region at 24 h after HII. Scale bar = 100 μm. C. BBB permeability was evaluated by serum IgG extravasation in rats 24 h after HII. Scale bar = 200 μm. D. Analysis of the immunohistochemistry results from A. **P < 0.01, ***P < 0.001 versus the Sham group. ^#P < 0.05 versus the HII group. Mean values ± SEM, n = 6 rats per group. E. Quantification of BBB permeability data from C by software Image-pro Plus. **P < 0.01 versus the Sham group. ^##P < 0.01 versus HII group. Mean values ± SEM, n = 5 rats per group.

Figure 2. Melatonin prevented the loss of tight junction and adherens junction proteins after neonatal HII

A. Representative micrographs showing double immunofluorescence with Claudin-5 (green) and CD31 (endothelial cell marker, red), nuclei were labeled with DAPI (blue) in each group. Scale bar = 75 μm. B. Representative western blots of adherens junction proteins (β-Catenin and P120) and tight junction proteins (Occludin and Claudin-5) in the sham, HII model and HII model treated melatonin groups 24 h after HII. C. Quantification of western blot data from B. *P < 0.05, **P < 0.01, ***P < 0.001 versus the Sham group. ^#P < 0.05, ^###P < 0.001 versus HII group. Mean values ± SEM, n = 5 rats per group.

Figure 3. Melatonin prevented the loss of pericytes after neonatal HII

A. Representative micrographs showing double immunofluorescence with Desmin (green) and PDGFRβ (red), nuclei were labeled with DAPI (blue) in each group. Scale bar = 75 μm. B. Representative western blots of pericyte markers PDGFRβ and Desmin in the sham, HII model and HII model treated melatonin groups 24 h after HII. C. Quantification of western blot data from B. **P < 0.01, ***P < 0.001 versus the Sham group. ^#P < 0.05, ^###P < 0.001 versus HII group. Mean values ± SEM, n = 5 rats per group.

Figure 4. Melatonin decreased astrogliosis and microgliosis after neonatal HII

A. Representative micrographs showing double immunofluorescence with GFAP (red) and Iba-1 (microglia marker, green), nuclei were labeled with DAPI (blue) in each group. Scale bar = 75 μm. B. Representative western blots of astrocytes marker GFAP in the sham, HII model and HII model treated melatonin groups 24 h after HII. C. Quantification of western blot data from B. ***P < 0.001 versus the Sham group. ^###P < 0.001 versus HII group. Mean values ± SEM, n = 5 rats per group.

Figure 5. Melatonin suppressed TLR4/NF-κB signaling pathway proteins expression after neonatal HII

A. Representative western blots of TLR4, NF-κB, p-IKB-α and IKB-α in the sham, HII model and HII model treated melatonin groups 24 h after HII. B. Quantification of western blot data from A. *P < 0.05, ***P < 0.001 versus the Sham group. ^#P < 0.05 versus HII group. Mean values ± SEM, n = 5 rats per group. C. Representative western blot and quantification data of TNF-α in the sham, HII model and HII model treated melatonin groups 24 h after HII. β-actin was used as loading control. **P < 0.01 versus the Sham group. ^##P < 0.01 versus HII group. Mean values ± SEM, n = 5 rats per group. D. RT-PCR analysis of RNA extracts from lesion side brains. Data were expressed as fold change versus sham-operated control. ^#P < 0.05, ^##P < 0.001 versus HII group. Mean values ± SEM, n = 6 rats per group.

Figure 6. Melatonin attenuated the decreases of tight junction and adherens junction proteins induced by LPS in the transwell co-culture of BV-2 cells and HUVECs

A. Representative western blots of tight junction and adherens junction proteins (β-Catenin, P120 and Occludin) in each group of HUVECs. B., C. and D. Quantification of western blot data from A. *P < 0.05, **P < 0.01, ***P < 0.001 versus the Control group. ^#P < 0.05, ^##P < 0.01 versus LPS group. Mean values ± SEM, n = 5 per group.

Figure 7. Melatonin decreased the expression of TLR4 protein induced by LPS in BV-2 cells

A. Immunofluorescence staining of TLR4 (green) in BV-2 cells treated with LPS for 24 h, nuclei were labeled with DAPI (blue). Scale bar = 75 μm. B. Representative western blot of protein TLR4 in each group of BV-2 cells. C. Quantification of western blot data from B. **P < 0.01 versus the Control group. ^#P < 0.05 versus LPS group. Mean values ± SEM, n = 5 per group.

Figure 8. Melatonin inhibited NF-κB translocation induced by LPS in BV-2 cells

A. Immunofluorescence staining for NF-κB translocation of BV-2 cells treated with LPS for 24 h, nuclei were labeled with DAPI (blue). Scale bar = 75 μm. B. Representative western blots of proteins p-IκB-α and IκB-α in each group of BV-2 cells. C. Quantification of western blot data from B. *P < 0.05, ***P < 0.001 versus the Control group. ^#P < 0.05, ^###P < 0.001 versus LPS group. Mean values ± SEM, n = 5 per group.

Figure 9. A model illustrated the BBB protective effects of melatonin after HII

Glial activation and TLR4/NF-κB inflammation signaling pathway were two mutually potentiating mechanisms leading to BBB disruption in hypoxic-ischemic and inflammatory (HII) injury of the developing brain. TLR4/NF-κB signaling pathway-induced inflammation activation contributed to ECs and pericytes damage after HII, which was inhibited by melatonin.