NDRG2 Is Involved In NF-?B-mediated Inflammation Induced By Ischemia Stroke

Stroke is a devastating illness that is second only to cardiac ischaemia as a cause of death worldwide and is responsible for nearly 6 million deaths per year.Emerging evidence suggests that neuroinflammation plays a critical role in the pathophysiology of post-ischemia brain injury and worsens neuronal injury.Inhibiting inflammation,which induces remarkable protective effects in the ischemia brain,has recently emerged as a promising therapeutic strategy.Studies of post-ischemia neuroinflammation mainly focused on microglia in previous papers.However,astrocyte,the most abundant cell type in CNS,plays a critical role in ischemia-induced neuroinflammation,and identified as a potential cell target against inflammation after stroke.Our previous researches demonstrated that NDRG2 mainly expressed in astrocytes and acts a pivotal role in the pathology of cerebral ischemia injury.Inhibiting astrocytic NDRG2 expression decreased the activation of astrocyte and apoptosis.Whether NDRG2 involved in neuroinflammation induced by ischemia is unclear.In order to explore the role of astrcytic NDRG2 in NF-?B mediated inflammation after global cerebral ischemia,we designed a series of experiements as follows.Experiment ?: The cellular localization of NDRG2 and NF-?B[Objective] To detect the cellular localization of NDRG2 and NF-?B in CA1 region[Methods] 1.6 adult male C57 mice were randomly divided into two groups,namely the Con and GCI/R group.Detect the localization of NDRG2 and NF-?B in hippocampal CA1 region 24 hours after ischemia-reperfusion with immunofluorescent double staining.2.Culture primary astrocytes in vitro,and detect the cellular localization of NDRG2 and NF-?B in astrocytes through immunofluarescense staining.[Results] 1.Immunofluarescense staining showed that NDRG2 and NF-?B co-localized with GFAP(glial Fibrillary acidic protein),an astrocyte marker,within CA1 region of hippocampus,respectively.Additionally,NDRG2 overlapped with NF-?B in hippcampal CA1 region and increased after GCI/R compared to Con group.2.NDRG2 and NF-?B co-localized in primary astrocytes,mainly localized in cytoplasm while rarely colocalized with DAPI staining in normal conditioning.[Conclusion] NDRG2 and NF-?B highly expressed and colocalized in hippcampal CA1 region astrocytes,mainly in cytoplasm.Furthermore,the colocalization increased in ischemia injured brain.Experiment ?: Ischemia injury induced significant increases in NF-?B and NDRG2 expression levels and a severe inflammatory response[Objective] To explore the expression of NDRG2,NF-?B and inflammatory cytokines of hippocampal CA1 region after globle cerebral ischemia.[Methods] 1.32 adult male C57 mice were randomly divided into 8 groups: Con,GCI/R 0 h,GCI/R 2 h,GCI/R 6 h,GCI/R 12 h,GCI/R 24 h,GCI/R 48 h and GCI/R 72 h groups.Detect the expression of NDRG2 and NF-?B with Western blot and TNF-?,IL-6 and IL-1? with ELISA in hippocampal CA1 region at 0 h,2 h,6 h,12 h,24 h,48 h and 72 h after reperfusion,respectively.2.Primary astrocytes were randomly divided into 6 groups,namly the Con,OGD/R 0 h,OGD/R 2 h,OGD/R 6 h,OGD/R 12 h and OGD/R 24 h.All groups except Con group suffered to 4 hours OGD(Oxgen-Glucose Deprivation)injury,and then detect the NDRG2 and NF-?B protein levels in astrocytes by Western blot and TNF-?,IL-6 and IL-1? in medium by ELISA at different time point(0 h,2 h,6 h,12 h and 24 h)after 4 hours OGD,respectively.[Results] 1.Western blot showed that NDRG2 and NF-?B protein in hippocampal CA1 region increased immediately at 0 h after reperfusion,and maintained at a high level up to 72 h after reperfusion(*p<0.05,**p<0.01).Compared to Con group,inflammatory cytokines TNF-? and IL-6 of hippocampal CA1 region dramatically increased after reperfusion(*p<0.05,**p<0.01,***p<0.001),while IL-1? expression has no significant difference between two groups.2.Compared to Con group,NDRG2 and NF-?B in astrocytes significantly upregulated after reoxgenation(*p<0.05,**p<0.01),especially in OGD/R 0 h and OGD/R 24 h group.ELISA showed that the increasing of TNF-? and IL-6 in medium began at 0 h and 2 h after reoxgenation respectively,and reached peak at OGD/R 24 h(*p<0.05,**p<0.01,***p<0.001),whereas the levels of IL-1? did not change except OGD/R 24 h(*p<0.05).[Conclusion] Global cerebral ischemia injury triggered the increasing of NDRG2 and NF-?B in hippocampal CA1 region,upregulated inflammatory cytokines levels.Experiment ?:Inhibition of NF-?B downregulate the NDRG2 expression induced by ischemia,alleviate neuroinflammation and protect neuron from ischemia injury[Objective] To study the effects of PDTC on expression of NDRG2,TNF-? and IL-6,and neuronal damage induced by ischemia[Methods] 1.24 adult male C57 mice were randomly divided into 8 groups: Con,GCI/R 1 d,GCI/R 2 d,GCI/R 3 d,PDTC + Con,PDTC + GCI/R 1 d,PDTC + GCI/R 2 d,PDTC +GCI/R 3 d.Mice in PDTC groups administrated PDTC ip 20 min before occlusion of bilateral common carotid artery,while mice in Vehicle groups administrated equal volume salt water.Detect the expression of NDRG2,TNF-? and IL-6 in hippocampal CA1 region at different time after reperfusion with Western blot and ELISA,respectively.2.Primary astrocytes were randomly divided into 2 groups,namely the Vehicle group and PDTC group.Astrocytes in PDTC groups treated with PDTC 1 h prior to OGD injury,while astrocytes in Vehicle groups accepted equal volume solution without PDTC.Detect the protein of NDRG2 in astrocytes,TNF-? and IL-6 in medium with Western blot and ELISA,respectively.3.In vivo,27 adult male C57 mice were randomly divided into Vehicle group and PDTC group.Detect ischemia induced neuronal damage with HE staining,TUNEL staining and Neu N staining at 3 d or 7 d after reperfusion.In vitro,primary astrocytes administrated with PDTC 1 h,and then Co-culture with primary neurons.Detect neuron apoptosis and Cleaved-caspase3 expression at OGD/R 24 h with Flow cytometry and Western blot,respectively.[Results] 1.Compared to GCI/R group,PDTC significantly down-regulate the increasing of NDRG2 induced by ischemia(#p<0.05).ELISA showed that PDTC decreased the levels of TNF-? and IL-6 induced by ischemia injury(#p<0.05).2.Western blot showed that PDTC dramatically reduced NDRG2 expression at OGD/R 12 h and OGD/R 24 h compared to Vehicle group(#p<0.05).Consistent with expression of NDRG2 in astrocytes,the levels of TNF-? and IL-6 in medium increased after reoxgenation,while PDTC administration decreased the levels of TNF-? and IL-6 in medium(#p<0.05).3.In animal experiments,HE staining showed that the neurons remained intact in the Con group,and abundant nuclei centered in the cytoplasm were clearly stained.In contrast,neurons were severely damaged and lacked a normal morphology after GCI/R,while PDTC treatment reduced the neuronal damage.TUNEL staining revealed that the PDTC substantially decreased the proportion of TUNEL-positive cells compared to the GCI/R group(*p<0.05).Neu N staining demonstrated that the proportion of Neu N-positive cells in the hippocampal CA1 region significantly decreased at 7 d after GCI/R(**p<0.01),while PDTC treatment dramatically increased the proportion of Neu N-positive cells(##p<0.01).In vitro experiments,the apoptotic index of the neurons in the OGD group decreased at 24 h after reoxygenation(***p<0.001),whereas pretreatment of astrocytes with PDTC dramatically attenuated OGD/R-induced neuronal apoptosis(###p<0.001).Cleaved caspase-3 expression in neurons was significantly increased at 24 h after reoxygenation(**p<0.01),while pretreatment of astrocytes with PDTC markedly decreased cleaved caspase-3 expression in neurons(##p<0.01).[Conclusion] PDTC down-regulate NDRG2 expression and relief inflammation response induced by ischemia,showing a neuroprotection against ischemia.Experiment ?: Down-regulation of NDRG2 expression alleviated the inflammation induced by OGD/R[Objective] To explore the role of NDRG2 in inflammation induced by OGD[Methods] 1.Detect the expression of NDRG2 protein in lentivirus transfected astrocytes cell lines MA1800 cell to ensure we have down-and up-regulated NDRG2 in MA1800 cell successfully through Western blot.2.Detect the levels of TNF-?,IL-6 and IL-1? in NDRG2 up-and down-regulation cell medium at 24 h after OGD by ELISA.[Results] 1.NDRG2 expression increased in LV-NDRG2 group(**P<0.01),and decreased in sh RNA-NDRG2 group(#P<0.01).2.sh RNA-NDRG2 significantly decreased the levels of TNF?,IL-6 and IL-1? compared to the sh RNA-Con group(*p<0.05).In contrast,overexpression of NDRG2 expression by LV-NDRG2 markedly increased the levels of TNF? and IL-6 but not IL-1? compared to the LV-Con group(#P<0.01).[Conclusion] NDRG2 regulate the expression of TNF? and IL-6 in astrocytes after ischemia injury.