| BackgroudStroke is a common clinical disease among the middle-aged and old people in China,ischemic stroke accounts for 70%-80% of the incidence of stroke.The pathophysiological mechanism of cerebral ischemia/reperfusion(I/R)injury is very complex,including oxidative stress response,intracellular calcium overload,toxic effect of excitatory amino acids,apoptosis,mitochondrial dysfunction,inflammatory response and so on.The inflammatory response plays an important role in the development of the disease.Pathophysiological cascades involving inflammation are triggered by cerebral ischemia/reperfusion-induced neuronal death,which is one of the main factors causing secondary brain injury.Astrocytes,as the most widely distributed and numerous cells in the brain,astrocytes are not only the main cells involved in the early inflammatory response after cerebral ischemia,but also play an important role in protecting neurons and repairing tissue damage.Therefore,it is of great significance to understand the mechanism of the astrocytic inflammatory response,it may be an efficacious therapeutic strategy to reduce the inflammatory response after cerebral I/R injury.Recently,DJ-1(also known as PARK7),as a multifunctional protein,is abundantly expressed in reactive astrocytes during neurodegenerative diseases such as Parkinson's disease and stroke.Our previous studies also showed that DJ-1 was intensely expressed in reactive astrocytes after cerebral I/R injury,and these DJ-1 play important roles in neuroprotection.Recently,DJ-1 has been found to play an important role in anti-inflammatory.However,it is unclear that the role of DJ-1 in anti-inflammatory in cerebral I/R injury and its potentional mechenism.ObjectiveTo explore the anti-inflammatory protective effect of DJ-1 in cerebral I/R injury and its potentional mechenism.Methods(1)In this study,we used transient middle cerebral artery occlusion(MCAO)/ reperfusion in male adult SD rats in vivo and oxygen-glucose deprivation/reoxygenation(OGD/R)in primary astrocyte cultures in vitro to mimic cerebral ischemia-reperfusion insult.(2)The MCAO/R model of SD rats was established.After 4 h,8 h,12 h,and 48 h of reperfusion,western blot were used to detect the highest expression of DJ-1.(3)The OGD/R was established.The astrocytes were treated with glucose-free DMEM in an incubator with 94% N2,1% O2,and 5% CO2 for 1,2,3,4,5,and 6 h at 37 °C.After 24 h of reoxygenation,Western blot was used to detect the highest expression of DJ-1(4)Negative siRNA and DJ-1 siRNA were injected into the left lateral ventricle 24 hours before the MCAO.After DJ-1 interference,neurological deficit scores,infarct volume,and ischemic lesion morphological alterations were measured to evaluate the cerebral injury.(5)Three days before OGD/R,lentivirus was added to the culture medium.The CCK-8 assay was conducted to evaluate the astrocytes viability after OGD/R.(6)Western blot was used to detect the expression of SHP-1,TRAF6,NLRX1 and inflammatory cytokines TNF-?,IL-1 ?,IL-6.The levels of TNF-?,IL-1 ? and IL-6 were detected by ELISA.(7)The subcellular localization of NLRX1 was observed by immunofluorescence staining.CO-IP was used to detect the interactions between NLRX1 and TRAF6 and between TRAF6 and SHP-1.(8)One month before MCAO,AAV was injected into the left lateral cerebral cortices of the rats,and intraperitoneally injected 1 mg/kg TPI-1 into MCAO rats after they awoke.Three days before OGD/R,lentivirus was added to the culture medium,and TPI-1(10 ng/ml)was added to the culture medium after OGD.(9)Virus and TPI-1 were used to overexpress DJ-1 and inhibit SHP-1.Then,western blot was used to detect the expression of SHP-1,TRAF6,NLRX1 and inflammatory cytokines TNF-?,IL-1 ?,IL-6.The levels of TNF-?,IL-1 ? and IL-6 were detected by ELISA.(10)Virus and TPI-1 were used to overexpress DJ-1 and inhibit SHP-1.CO-IP was used to detect the interactions between NLRX1 and TRAF6 and between TRAF6 and SHP-1.Results(1)After MCAO/R,western blot showed that higher levels of DJ-1 at reperfusion 24 h when compared with the sham group.After OGD/R,western blot shows that DJ-1 was upregulated in the 5-h OGD group compared with the controls.(2)Knockdown DJ-1 aggravated behavioral deficits,increased the infarct volume and increased the damage of neurons.At the same time,the astrocyte viability was significantly reduced after DJ-1 interference after OGD/R.(3)The expression of DJ-1,SHP-1,TRAF6 and NLRX1 and inflammatory cytokines TNF-?,IL-1 ?,IL-6 was significantly increased after cerebral I/R injury.After treatment with DJ-1 siRNA,the expression of DJ-1,SHP-1 was decreased,the expression of TRAF6 and inflammatory cytokines was further upregulated,however,changes in DJ-1 had no effect on the levels of NLRX1.At the same time,NLRX1 disassociated from TRAF6 after cerebral I/R injury.Interestingly,DJ-1 interference promoted the interaction between NLRX1 and TRAF6.On the contrary,SHP-1 was associated with TRAF6 after cerebral I/R injury.but these interactions were significantly inhibited after treatment with DJ-1 siRNA.(3)The overexpression of DJ-1 reduced the expression of TRAF6 and cytokines TNF-?,IL-1?,and IL-6,while increased SHP-1 expression.After treatment with an SHP-1 inhibitor,the expression of TRAF6 and cytokines TNF-?,IL-1?,and IL-6 were increased,SHP-1 expression was decreased.Interestingly,changes in DJ-1 or SHP-1 levels had no effect on the levels of NLRX1.In the subsequent experiments,the overexpression of DJ-1 facilitated the interaction between SHP-1 and TRAF6,which was inhibited after treatment with an SHP-1 inhibitor.However,NLRX1 disassociated from TRAF6 upon DJ-1 overexpression,and SHP-1 inhibition promoted the interaction between NLRX1 and TRAF6.ConclusionDJ-1 play an important role in anti-inflammatory by inducing the dissociation of NLRX1 and TRAF6 in cerebral I / R injury.The mechanism of DJ-1 induces the dissociation of NLRX1 from TRAF6 by activating SHP-1 to facilitate the interaction between SHP-1 and TRAF6. |
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