HMGB-1 Induces Neuroinflammation By Activating Mast Cells And This Effect Is Associated With RAGE/NF-?B Signaling Pathway

Background: Postoperative cognitive dysfunction(POCD)is a central nervous system(CNS)complication in aged patients who have received anesthesia and surgery.Patients with POCD often present with mental disorders,anxiety,personality changes,mental decline and hypomnesis for weeks or months which have a pernicious impact on postoperative rehabilitation.The pathological mechanism behind POCD has not yet been fully clarified,and studies shown that the neuroinflammation induced by surgery play an important role in the occurrence and development of POCD.Surgical trauma can activate body's innate immune system which resulting in the activation of peripheral immune cells and the release of large numbers of cytokines.After immunization signal was transferred to the brain from blood circulation,the cerebral immune cells could be activated which resulting the release of inflammatory mediators,and damage of hippocampal neurons,and cognitive function dysfunction.The occurrence and development of neuroinflammation is a very complicated process and many factors were involved in.In recent years,the role of brain mast cells(MCs)in neuroinflammation has gained extensive attention from researchers.Brain MCs are located around the blood-brain barrier(BBB)and in the brain parenchyma side of thalamus,hypothalamus,hippocampus,choroidal plexus pia mater,dura mater and.They play an early role in the development of neuroinflammation by acting on astrocytes,microglia,vascular endothelial cells via their pre-stored and re-synthetic inflammatory mediators,such as histamine,trypsin,serotonin,TNF(Tumor necrosis factor),IL-1?(Interleukin-1?),etc.Our previous study found that the number of MCs in the brain of SD rats after tibial fracture surgery significantly increased,and mast cell stabilizer cromoglycate(cro)can inhibit peripheral surgery induced neuroinflammation,BBB broken,and cognitive decline.The results suggested that MCs may play an important role in the surgery-induced neuro-inflammation.Brain MCs locate on the brain side of the BBB,acting as first responders to peripheral inflammatory signals and cause neuroinflammation by interacting with neurons,glial,and microvascular endothelial cells via preformed and newly synthesized reactive chemicals.But,how peripheral surgery induces brain MCs activation remains unclear.Surgery stimulate the release of large numbers of inflammatory mediators into the blood circulation,and among which,high mobility group box-1(HMGB-1)is a potent pro-inflammatory factor.HMGB-1 is reported to be involved in the development of various neurodegenerative diseases and plays an important role in the occurrence of central inflammation and cognitive decline.HMGB-1 is highly expressed in the nucleus,cytosol,and extracellular fluid and released by injured and infected cells.HMGB-1 has the cytokine-like ability to activate immune cells by interacting with the receptors expressed on the cell surface,such as the Receptor of Advanced Glycation Endproducts(RAGE).RAGE,a membrane-bound receptor can be activated by a large of ligands,such as Advanced Glycation End Products(AGEs),HMGB-1,? amyloid,and S100B/S100A12.After RAGE binding,all of them are able to activate NF-?B signaling pathway,which has been considered to play an important role in many Central Nervous System(CNS)pathological processes such as neuro-inflammation,neuro-degeneration and memory impairment.Although NF-?B activity has been demonstrated in any CNS cells,current studies have been focused on the function of RAGE/NF-?B signaling pathway and its ligands in neurons and glial cells.So,it remains unclear whether HMGB-1 causes the neuroinflammation by activating brain MCs through RAGE/NF-?B signaling pathway in the course of neuroinflammation induced by peripheral surgery.Objective: To investigate whether HMGB-1 induces neuroinflammation by activating brain MCs and whether this effect is related to RAGE/NF-?B signaling pathways.Methods: In vitro study,we used P815 MCs to observe the effect of HMGB-1 on mast cell activation and evaluate its underlying mechanism.1.After incubating P815 MCs with 10,50,100,and 200 ng/ml of r HMGB-1 for 24 or 48 hours,respectively,the effect of HMGB-1 on P815 MCs viability was examined using the WST-8 assay.2.After incubating P815 MCs with 100 ng/ml HMGB-1,1 ?g/ml lipopolysaccharide(LPS,stimulator of MCs inflammatory mediator release)and 1 ?g/ml C48/80(MCs degranulation stimulator).P815 MCs were divided into three groups: CONTROL group,LPS group,C48/80 group.ELISA,immunofluorescence and real-time PCR were used to detect the release of tryptase and the synthesis and release of inflammatory cytokines TNF and IL-1? from P815 MCs.3.After incubating P815 MCs with 100 ng/ml HMGB-1,the expression of TLR2(Toll-like receptor 2),TLR4(Toll-like receptor 4)on the surface of P815 MCs were detected by western-blot and immunofluorescence.4.RAGE-Si RNA was used to silence the expression of RAGE on the surface of P815 MCs.P815 MCs were divided into four groups: negative control(NC)group,RAGE-si RNA group,HMGB-1 group,and HMGB-1+RAGE-si RNA.Then we observed whether activation of P815 MCs is inhibited after RAGE receptor silencing by RAGE-Si RNA.Subsequently,Western-blot was used to detect the level of phosphorylation of the NF-?B signaling pathway.In vivo study,male Sprague-Dawley(SD)rats received intracerebroventricular injection of cromoglycate(MCs stabilizer)or sterile saline 30 min ahead of intraperitoneal injection of HMGB-1.Male SD rats were divided into 4 groups: simple intracerebroventricular injection of sterile saline(CONTROL group),simple lateral ventricle injection of cro(CRO group),intraperitoneal injection HMGB-1 30 minutes after intracerebroventricular injection of sterile saline(HMGB-1 group),intraperitoneal injection HMGB-1 30 minutes after intracerebroventricular injection of cro(CRO+HMGB-1 group).The number of mast cells in hippocampal CA1 area,secretion of inflammatormediators,changes in blood brain barrier(BBB)permeability,and activation of RAGE/NF-?B signaling pathway were examined.Results: 1.Incubation of P815 MCs with 10,50,100,or 200 ng/ml of HMGB-1 for 24 or 48 h did not affect MCs viability.2.Compared with the CONTROL group,C48/80(P <0.01)and HMGB-1(P <0.01)both stimulated the degranulation with pre-stored tryptase of P815 MCs.Compared with the CONTROL group,the expression of TNF m RNA in P815 MCs reached the peak at 6 h(P < 0.01)after HMGB-1 stimulation,and the expression of IL-1 ? m RNA peaked at 12 h(P <0.05)after HMGB-1 stimulation.HMGB-1 significantly increased the protein level of TNF(P < 0.05)and IL-1?(P < 0.01)from P815 MCs.3.Compared with the NC group,HMGB-1 induced the upregulation of RAGE on the surface of P815 MCs(P < 0.05).RAGE-si RNA could significantly downregulate the expression of RAGE on the surface of P815 MCs(P < 0.01).4.Compared with the NC group,the RAGE-si RNA group had no significant change,but the levels of tryptase,TNF and IL-1? were significantly increased in the HMGB-1 group(P < 0.05).Compared with the HMGB-1 group,the expression of tryptase,TNF and IL-1? in RAGE-si RNA+HMGB-1 group was significantly decreased(P < 0.05).5.Compared with NC group,HMGB-1 stimulation significantly increased phosphorylation of P65 and ik B? which are invovoled in RAGE/NF-?B signaling(P < 0.05).Compared with HMGB-1 group,Phosphorylation of P65 and ik B? was significantly decreased in RAGE-si RNA+HMGB-1 group(P < 0.05).6.In vivo study,compared with the CONTROL group,there is no significant change in CRO group.The number of MCs in the CA1 area of the rat hippocampus in the HMGB-1 group was significantly increased(P < 0.05),and the expression of TNF and IL-1? in the hippocampus was increased(P < 0.01).The level of tight junction protein occludin decreased(P < 0.05)and level of albumin increased(P <0.01).The level of P-P65(P <0.05)and P-ik B?(P <0.01)increased.Pre-i.p.injection of mast cell stabilizer cromoglycate could inhibit these effects induced by i.p injection of HMGB-1.Conclusions: The results above suggest that HMGB-1 can activate P815 MCs to release inflammatory mediators and activate brain MCs to induce the occurrence of neuroinflammation.This effect is related to RAGE/NF-?B signaling pathways.