Anti-inflammatory Effect Of N-Acetyl-5-Methoxytryptamine(Melatonin) On Neuro-inflammatory Reactions Via Inhibition Of High Mobility Group Box1(HMGB-1) Release In Microglial BV-2 Cells

Background: Stroke,majority are the ischemic,is the most common cause of death and serious,long-term disability worldwide.In central nervous system,the secondary neuroinflammation contributed by microglial activation is a consequential response observed in the pathogenesis of ischemic stroke.High-mobility group box-1(HMGB-1),a non-histone nuclear protein,interacts with immune cells,such as microglia and leads to a cascade amplification of the secondary neuroinflammatory responses,which are related to brain damage later.N-acetyl-5-methoxytryptamine(melatonin)is a neuro-hormone,well-known as its antioxidative and anti-inflammatory effects.However,until now,more findings are required for better understanding about anti-inflammatory effect of melatonin on HMGB1 and HMGB1-triggered pathway in microglial activation.Therefore,in this study we aimed to investigate whether melatonin can inhibit LPS-induced microglial inflammation by regulating HMGB1 nucleocytoplasmic translocation and release.Methods: CCK-8 assay was used to determine the effect of melatonin on the viability of BV-2 microglial cells under the action of LPS and to select the optimal concentration of drug intervention.Quantitative real-time polymerase chain reaction assay(qRT-PCR)and enzymelinked immunosorbent assays(ELISAs)were used to measure the effect of melatonin on the levels of m RNA and the protein expression of HMGB1 and other inflammatory cytokines TNF-α,IL-6 and IL-1β in LPS-induced BV-2 microglia.HMGB1 nucleocytoplasmic translocation and release in BV2 microglial cells were examined by immunofluorescence assay and western blot assay.Moreover,western blot assay was used to determine HMGB1 downstream TLR4/MyD88/NF-kB pathway related proteins and SIRT1 expression levels.Results: According to the CCK-8 assay results,the optimal drug concentration of melatonin for the experiment was 1μmol/ml and no obvious cytotoxic effect was observed on BV-2microglia cells.Compared with the control group,in the LPS-induced inflammation group proinflammatory cytokines of TNF-α,IL-6 and IL-1β were significantly increased(p<0.05),but after melatonin administration the expression levels of TNF-α,IL-6 and IL-1β were decreased significantly(p<0.05).After BV-2 microglial cells were stimulated by LPS,the content of HMGB1 in the cytoplasm was significantly increased(p<0.05),and melatonin pre-treatment can significantly reduced LPS-induced HMGB1 nucleocytoplasmic translocation and release.Moreover,melatonin intervention up-regulated LPS-induced decrease of SIRT1 expression and down-regulated LPS-induced enhancement of TLR4,MyD88 and NF-kB protein expression.The results between control group and LPS-induced inflammation group;LPS-induced inflammation group and melatonin treatment group with p<0.05 were considered as a statistically significant.Conclusions: In summary,melatonin can suppress LPS-triggered BV-2 microglial activation-mediated neuro-inflammation through interfering the TLR4/MyD88/NF-kB signaling pathway by inhibiting the high expression and release of HMGB1 and up-regulate SIRT1 expression.Furthermore,melatonin pre-administration notably reversed LPS-induced expression of pro-inflammatory cytokines(i.e.,TNF-α,IL-6 and IL-1β).Therefore,our findings suggest that melatonin may act as a promising therapeutic agent for ischemic stroke by reducing neuroinflammation targeting microglial activation and HMGB1 translocation and release.