Melatonin Stimulates the SIRT1/Nrf2 Signaling Pathway Counteracting Lipopolysaccharide (LPS)-Induced Oxidative Stress to Rescue Postnatal Rat Brain

Abstract

Aims: Lipopolysaccharide (LPS) induces oxidative stress and neuroinflammation both in vivo and in vitro. Here, we provided the first detailed description of the mechanism of melatonin neuroprotection against LPS-induced oxidative stress, acute neuroinflammation, and neurodegeneration in the hippocampal dentate gyrus (DG) region of the postnatal day 7 (PND7) rat brain.

Methods: The neuroprotective effects of melatonin against LPS-induced neurotoxicity were analyzed using multiple research techniques, including Western blotting, immunofluorescence, and enzyme-linked immunosorbent assays (ELISAs) in PND7 rat brain homogenates and BV2 cell lysates in vitro. We also used EX527 to inhibit silent information regulator transcript-1 (SIRT1).

Results: A single intraperitoneal (i.p) injection of LPS to PND7 rats significantly induced glial cell activation, acute neuroinflammation, reactive oxygen species (ROS) production and apoptotic neurodegeneration in hippocampal DG region after 4 h. However, the coadministration of melatonin significantly inhibited both LPS-induced acute neuroinflammation and apoptotic neurodegeneration and improved synaptic dysfunction in the hippocampal DG region of PND7 rats. Most importantly, melatonin stimulated the SIRT1/Nrf2 (nuclear factor-erythroid 2-related factor 2) signaling pathway to reduce LPS-induced ROS generation. The beneficial effects of melatonin were further confirmed in LPS-stimulated BV2 microglia cell lines in vitro using EX527 as an inhibitor of SIRT1. LPS-induced oxidative stress, Nrf2 inhibition, and neuroinflammation are SIRT1-dependent in BV2 microglia cell lines.

Conclusion: These results demonstrated that melatonin treatment rescued the hippocampal DG region of PND7 rat brains against LPS-induced oxidative stress damage, acute neuroinflammation, and apoptotic neurodegeneration via SIRT1/Nrf2 signaling pathway activation.

Keywords: Lipopolysaccharide; Melatonin; Neuroinflammation; Nuclear factor-erythroid 2-related factor 2; Reactive oxygen species; Silent information regulator transcript-1.

Figure 1

Melatonin attenuated LPS‐induced activated glial cells in the hippocampus of developing rat brain. Representative photomicrographs of the immunofluorescence analysis and the relative integral optical density (IOD) histogram of (A) astrocytes (GFAP)‐positive cells and (B) microglia (Iba‐1)‐positive cells in the DG region of the rats in the experimental groups. The images are representative of immunostaining obtained from sections prepared from at least animal animals per group. The panels represent the hippocampal DG region of the young rat brain immunostained with GFAP (green) and Iba‐1 (green) and counterstained with DAPI (blue), respectively. Significance; ^a P < 0.01, ^y P < 0.05.

Figure 2

Beneficial effect of melatonin against LPS‐induced neuroinflammation and synaptic deficits in immature rat brain. Immunoblots of pro‐inflammatory markers, including (A) phospho‐NF‐κB, COX2, and iNOS proteins. (B) The ELISA histogram of NF‐κBp65 (total) in the brain homogenates of the experimental groups. The assay was conducted according to the manufacturer's instructions. (C) Immunoblot of synapse‐related proteins, such as synaptophysin (presynapse) and PSD95 (postsynapse), in the hippocampus of the postnatal day 7 rat brain. The bands were quantified using Sigma Gel software and density histograms (expressed in arbitrary units, i.e., A.U) relative to the control using GraphPad Prism software. The values represent the means ± SEM for the indicated proteins (n = 5 animals per group). Significance; ^aP < 0.01, 0.05 and ^x,yP < 0.01, 0.05, respectively.

Figure 3

Melatonin abrogated LPS‐induced oxidative stress and apoptotic neurodegeneration in the hippocampus of developing rat brain. (A) The histogram of the ROS assay conducted with brain homogenates from rats in the experimental groups. The assay was repeated three times with the same results. (B) The immunostaining images and respective relative IOD histograms of 8‐OxoG in experimental groups. (C) The Western blot analysis of caspase‐3 and PARP‐1 proteins in the hippocampus of PND7 rat brains, following LPS and melatonin treatment. The relative integrated density for above‐mentioned proteins is depicted in the histograms. The membranes were redeveloped for β‐actin and used as a loading control. (D) The immunofluorescence images and respective relative IOD histograms of FJB‐positive neuronal cells in the DG region of immature rat brains. The density values are expressed in arbitrary units as the means ± SEM for the indicated proteins (n = 5 animals per group). The details are shown in the Methods section. Significance; ^a,bP < 0.01, 0.05 and ^yP < 0.05, respectively.

Figure 4

Melatonin stimulated the SIRT1/Nrf2 signaling pathway against LPS in the hippocampus of the developing rat brain. (A) The Western blot analysis of SIRT1, Nrf2, and HO‐1 proteins in the hippocampus of PND7 rat brains following LPS and melatonin treatment. The relative integrated density for the above‐mentioned proteins is depicted in the histograms. The membranes were redeveloped for β‐actin and used as a loading control. (B) The histogram of SIRT1 activity in the hippocampal brain homogenates of young rats subjected to LPS and melatonin treatment. The assay was repeated three times. (C) The immunostained images of Nrf2 (green) protein counterstained with DAPI (blue) in the hippocampal DG region of PND7 rat brains. The results are expressed as the means ± SEM. (n = 5). Significance; ^a P < 0.01 and ^x,yP < 0.01, 0.05, respectively.

Figure 5

Melatonin inhibited LPS‐stimulated BV2 cell lines and activated SIRT1 in vitro. The immunostaining images of (A) SIRT1 (green) and Iba‐1 (red) and (B) Nrf2 (green) counterstained with DAPI (blue) in LPS‐stimulated BV2 cell lines in vitro. BV2 cells were cultured on chamber slides and treated for 4 h. The results represent three repeated experiments. All relevant details are shown in the Material and Methods section.

Figure 6

Melatonin reduced LPS‐induced oxidative stress in BV2 cells in vitro. (A) The immunostaining images of 8‐OxoG (green) and DAPI (blue) in BV2 cells treated with LPS and melatonin for 4 h. (B) The histogram of ROS assay conducted in BV2 cell lines cultured in 96‐well plates and treated with LPS, melatonin with or without EX527 (SIRT1 inhibitor) for 4 h. The assay was repeated in triplicate. Significance; ^a,bP < 0.01, 0.05 and ^y P < 0.05, respectively.

Figure 7

Melatonin stimulated the SIRT1/Nrf2 signaling pathway against LPS in BV2 cells in vitro. (A) The Western blot analysis and relative integrated density histograms of SIRT1, Nrf2, and Caspase‐3 proteins after LPS and melatonin treatment, with or without EX527 treatment for 4 h in BV2 cell lines. β‐Actin was used as a loading control. (B) The histogram of SIRT1 activity in cell lysates subjected to LPS and melatonin, with or without EX527 treatment for 4 h in BV2 cell lines. The assay was repeated for three times, and the values represent the means ± SEM. Significance;^a,bP < 0.01, 0.05 and ^y P < 0.05, respectively.

Figure 8

Melatonin reduced LPS‐activated proinflammatory markers in BV2 cell lines in vitro. (A) The Western blot analysis and relative integrated density histograms of p‐NF‐κB and COX2 proteins after LPS, melatonin, and with or without EX527 treatment for 4 h in BV2 cell lines. β‐Actin was used as a loading control. (B) The histogram of NF‐κBp65 activity in cell lysates subjected to LPS, melatonin, and with or without EX527 treatment for 4 h in BV2 and HT22 cell lines, respectively. (C) The histogram of COX2 in BV2 and HT22 cell lines, respectively, cultured in 96‐well plate subjected to LPS and melatonin, with or without EX527 treatment for 4 h. These assays were repeated three times, and the values are expressed as the means ± SEM. Significance; ^a,bP < 0.01, 0.05 and ^x,yP < 0.01, 0.05, respectively.

Figure 9

Schematic diagram suggested a neuroprotective mechanism for melatonin against LPS‐induced ROS, acute neuroinflammation, and neurodegeneration. The diagram depicts a potential mechanism by which melatonin prevents LPS‐induced oxidative stress, acute neuroinflammation, and neurodegeneration via a SIRT1/Nrf2 signaling pathway in the hippocampal dentate gyrus region of PND7 rat brains.