Regulation Mechanism Of Iron Metabolism In A1 And A2 Reactive Astrocytes

Parkinson’s disease(PD)is a common neurodegenerative disease,of which pathological feature is the loss of dopaminergic neurons in the substantia nigra compact zone of the midbrain.Iron deposition in substantia nigra may be involved in the injury of dopaminergic neurons of PD.In the previous studies,it was found that iron metabolism in astrocytes played an important role in iron accumulation in substantia nigra of PD animal model.In astrocytes,iron is transferred into the cells mainly by divalent metal ion transfer protein 1(DMT1),and the iron entering the cell is either stored in ferritin or transferred out of the cell through ferroportinl(FPN1).Ferritin has two subunits,light chain ferritin(L-ferritin)and heavy chain Ferritin(H-Ferritin),and their expression levels can be used to assess intracellular iron content.Therefore,the level of ferritin can reflect the content of iron in cells.Iron regulatory protein(IRPs)and hypoxia inducible factor(HIF s)are involved in the regulation of iron transporter.There are activation and proliferation of astrocytes in the pathogenesis of PD.The activation of astrocytes,also known as reactive glial hyperplasia,produces reactive astrocytes,which are divided into A1 and A2 types.Studies have shown that in central nervous system activated microglia can induce astrocytes to Alreactive astrocytes by secreting proinflammatory factors TNF α,IL-1β and C1q alone or in combination.A1 astrocytes are harmful to neurons and oligodendrocytes.Complement component 3(C3)and guanine nucleotide binding protein 2(GBP2)can be used as characteristic markers of A1 reactive astrocytes.In the ischemia-reperfusion model made by middle cerebral artery occlusion(MCAO),astrocytes are induced as type A2 reactive astrocytes,and type A2 astrocytes have neuroprotective effect.Pentraxin-3(PTX3)and S100 calcium binding protein A10(S100a10)can be used as characteristic markers of A2 reactive astrocytes.However,the changes of iron metabolism in different types of reactive astrocytes have not been reported.Objective:To observe the changes of DMT1 and FPN1 expression in different types of reactive astrocytes,so as to reveal the differences and possible mechanisms of iron metabolism in different types of reactive astrocytes.Methods:In this study,exogenous TNFα was used on primary cultured astrocytes to prepare A1 reactive astrocytes,oxygen glucose deprivation/reoxygenation(OGD/R)model was used to prepare A2 reactive astrocytes.CCK8 was used to observe the effect of TNFα on the cell viability of primary cultured astrocytes.Real-time fluorescence quantitative PCR was used to detect the mRNA expression of the phenotypic markers of two types of reactive astrocytes.And western blot was used to detect the expressions of iron metabolism related proteins in primary cultured astrocytes.Results:1.The regulation mechanism of TNFα on iron metabolism in astrocytes.(1).Primary cultured astrocytes were treated with different concentration of TNFα and the cell viability was determined by CCK8 assay.When the concentration of TNFαwas less than 60ng/mL,the cell viability was unchanged compared with control.Therefore,40 ng/mL was selected as the concentration to be used in subsequent experiments.(2).Real time PCR was applied to detect C3,GBP2,S100a10 and PTX3 mRNA levels in primary cultured astrocytes treated with TNFα for 24h.The expression of C3 mRNA significantly increased in TNFα group compared with control group(P<0.001,n=6).The expression of GBP2 mRNA significantly increased in TNFαgroup compared with control group(P<0.001,n=6).The expression of S100a10 mRNA significantly decreased in TNFα group compared with control group(P<0.01,n=6).The expression of PTX3 mRNA significantly decreased in TNFαgroup compared with control group(P<0.01,n=6).The results showed that after TNFα treatment,astrocytes were activated to A1 type.(3).Western blot was applied to detect HIF-1α,HIF-2α,IRP1,DMT1,FPN1,H-Ferritin and L-Ferritin protein levels in primary cultured astrocytes treated with TNFα for 24h.The expression of HIF-1α protein did not change in TNFα group compared with control group.The expression of HIF-2a protein significantly increased in TNFα group compared with control group(P<0.01,n=6).The expression of IRP1 protein significantly increased in TNFα group compared with control group(P<0.001,n=6).The expression of DMT1 protein significantly increased in TNFαgroup compared with control group(P<0.01,n=6).The expression of FPN1 protein significantly increased in TNFα group compared with control group(P<0.001,n=6).The expressions of H-Ferritin and L-Ferritin did not change in TNFα group compared with control group.The results showed that the expressions of iron import protein and iron export protein increased,iron transport rate increased,but the iron content did not change in astrocytes treated by TNFα.(4).Western blot was applied to detect HIF-2α,IRP1,DMT1,FPN1,H-Ferritin and LFerritin protein levels in primary cultured astrocytes pretreated with HIF-2a inhibitor for 24h and then co-treated with TNFα for 24h.The expression of HIF2α significantly decreased in HIF-2α inhibitor/TNFα group compared with TNFαgroup(P<0.01,n=6).The expression of IRP1 did not change in HIF-2α inhibitor/TNFα group compared with TNFα group.The expressions of DMT1 and FPN1 significantly decreased in HIF-2a inhibitor/TNFα group compared with TNFαgroup(P<0.05,P<0.001,n=6).The expressions of H-Ferritin and L-Ferritin significantly increased in the HIF-2α inhibitor/TNFα group compared with control group(P<0.001,n=6).The results showed that inhibition of HIF-2α could block the upregulation of iron import protein DMT 1 and iron export protein FPN1,as well as the increased iron traffic in astrocytes induced by TNFα.(5).Western blot was applied to detect HIF-2α,IRP1,DMT1,FPN1,H-Ferritin and LFerritin protein levels in primary cultured astrocytes pretreated with rottlerin and then co-treated with TNFα.The expression of HIF-2a significantly decreased in the PKCδ inhibitor/TNFα group compared with TNFα group(P<0.001,n=6).The expression of IRP1 significantly decreased in the PKCδ inhibitor/TNFα group compared with the TNFα group(P<0.05,n=6).The expressions of DMT1 and FPN1 significantly decreased in PKCδ inhibitor/TNFα group compared with TNFα group(P<0.01,n=6).The expression of H-Ferritin significantly increased in the PKCδ inhibitor/TNFα group compared with TNFα group(P<0.001,n=6).The expression of L-Ferritin did not change in the PKCδ inhibitor/TNFα group compared with the TNFα group.The results showed that inhibition of PKCδ could block the activation of HIF-2α and IRP1,the upregulation of iron import protein DMT1 and iron export protein FPN1,as well as the increased iron traffic in astrocytes induced by TNFα.(6).Real time PCR was applied to detect C3 and S100a10 mRNA levels in primary cultured astrocytes pretreated with HIF-2α inhibitor or rottlerin then co-treated with TNFα.The expression of C3 mRNA significantly increased in HIF-2αinhibitor/TNFα group compared with control group(P<0.001,n=6).The expression of S100a10 mRNA significantly decreased in HIF-2a inhibitor/TNFαgroup compared with control(P<0.01,n=6).The expression of C3 mRNA significantly increased in PKCδ inhibitor/TNFα group compared with control group(P<0.001,n=6).The expression of S100a10 mRNA significantly decreased in the PKCδ inhibitor/TNFα group compared with the control group(P<0.01,n=6).The results showed that HIF-2a inhibitor or rottlerin could not block the formation of A1 type astrocytes induced by TNFα.2.The regulation mechanism of OGD/R treatment on iron metabolism in astrocytes.(1).Real time PCR was applied to detect C3 and PTX3 mRNA levels in primary cultured astrocytes treated with reoxygenation for 3h,6h,12h after OGD for 3h.The expression of C3 mRNA significantly decreased in primary cultured astrocytes when treated with reoxygenation for 3h(P<0.05,n=6)and 6h(P<0.001,n=6)after OGD for 3h compared with the control group.The expression of PTX3 mRNA significantly increased in primary cultured astrocytes when treated with reoxygenation for 6h after OGD for 3h compared with the control group(P<0.001,n=6).Therefore,reoxygenation for 6 h after OGD for 3 h was selected as the OGD/R treatment scheme in subsequent experiments.(2).Real time PCR was applied to detect C3,GBP2,S100a10 and PTX3 mRNA levels in primary cultured astrocytes treated OGD/R.Expression of C3 mRNA significantly decreased after OGD/R treatment compared with the control group(P<0.001,n=6).Expression of GBP2 mRNA significantly decreased after OGD/R treatment compared with the control group(P<0.0001,n=6).Expression of S100a10 mRNA significantly decreased after OGD/R treatment compared with the control group(P<0.05,n=6).Expression of PTX3 mRNA significantly increased after OGD/R treatment compared with the control group(P<0.001,n=6).The results showed that OGD/R treatment could activate astrocytes into A2 type.(3).Western blot was applied to detect HIF-1α,HIF-2α,IRP1,DMT,H-Ferritin and LFerritin protein levels in primary cultured astrocytes treated with OGD/R.Expression of HIF-1α protein significantly decreased after OGD/R treatment compared with the control group(P<0.001,n=6).Expression of HIF-2α,IRP1,DMT1,FPN1,H-Ferritin and L-Ferritin protein did not change after OGD/R treatment compared with the control group.Expression of IRP1 protein did not change after OGD/R treatment compared with the control group.The results showed that the iron metabolism of astrocytes did not change after OGD/R treatment.Conclusion:The above results showed that TNF a could activate primary cultured astrocytes into A1 reactive astrocytes.The expression of DMT1 and FPN1 in A1 reactive astrocytes increased,and iron traffic increased as well.Therefore,there was no significant change in intracellular iron content.The changes of DMT 1 and FPN1 in primary cultured astrocytes induced by TNF a may be related to the upregulation of HIF-2α,which might be induced by the activation of PKCδ.Increased iron traffic rate in A1 astrocytes might lead to iron deposition in adjacent neurons,thus neurons would be damaged.OGD/R could activate primary cultured astrocytes into A2 reactive astrocytes,but the expression of DMT1 and FPN1 in A2 astrocytes remained unchanged.There was no significant change in iron transport ability in A2 types,which might not be related to neuronal iron deposition.Therefore,the changes of iron metabolism in astrocytes are related to the different activation phenotypes of astrocytes,and the changes of iron metabolism in A1 and A2 reactive astrocytes are different.This study provides a theoretical and experimental basis for the mechanism of iron accumulation in PD.