Effects Of 17 ?-estrogen On Iron Metabolism Of BV2 Microglia

Parkinson's disease?PD?is a common neurodegenerative disorder characterized in its late phase by the sustained loss of dopaminergic neurons in the substantia nigra pars compacta?SNpc?.The mechanisms underlying PD pathogenesis have not been revealed yet.Multiple factors might be involved,such as genetic mutation,environmental factors,aging,et al.A growing body of research has confirmed that nigral iron accumulation was involved in the death of DA neurons in PD.Brain iron could be taken up by divalent metal transporter1?DMT1?.The iron transported in the cells may be retainted or exported by ferroportin 1?FPN1?,which is the only iron exporter described so far.DMT1 and FPN1are regulated by iron regulatory proteins?IRPs?,hepcidin and hypoxia inducible factors?HIFs?.There are gender differences in PD,and the incidence of PD in men is higher than women.Epidemiological studies demonstrated that estrogen affects iron metabolism in peripheral tissues,postmenopausal women harbor a higher level of body iron than premenopausal women.In addition to anemia,iron deposition was observed in bone tissue of ovariectomized rats,and iron chelating agents could mitigate bone loss,which were caused by iron deposition.These studies indicated that estrogen played a regulatory role on iron metabolism.Estrogen's actions are mainly mediated through estrogen receptor??ER??,estrogen receptor??ER??and G protein-coupled estrogen receptor?GPER?.It was ER?but not ER?expressed in BV2 microglia.Besides iron accumulation,activated microglia occurs in the substantia nigra of PD.In particular,the reactive microglia contain high levels of iron in PD patients.Estrogen have anti-inflammatory and anti-apoptotic effects on microglia.Our previous studies reported that in astrocytes,17?-estrogen?E₂?increased the levels of DMT1 and FPN1through upregulating HIF-1?.However,in VM neurons,E₂ decreased the level of DMT1,increased the level of FPN1 by down-regulating IRP1.Then,how does E₂ affect iron metabolism in microglia?What kind of estrogen receptor is involved?In this study,the expressions of DMT1,FPN1,IRP1,hepcidin and HIF-1?were investigated when BV2microglia were treated with E₂,ER?inhibitor PHTPP and GPER inhibitor G15.The results were as follow:1.When BV2 cells were treated with 10 nM E₂ for 24h,the protein levels of DMT1 and FPN1 in the experimental group were significantly increased compared to the control group?DMT1:P<0.001,n=3;FPN1:P<0.01,n=4?.2.When BV2 cells were treated with 10 nM E₂ for 24h,the protein levels of FT-L and FT-H in the experimental group were significantly increased compared to the control group?FT-L:P<0.05,n=4;FT-H:P<0.05,n=4?.3.When BV2 cells were treated with 10 nM E₂ for 24h,the protein level of IRP1 in the experimental group was not changed compared to the control group?P>0.05,n=5?.4.When BV2 cells were treated with 10 nM E₂ for 24h,the protein level of Hepcidin in the experimental group did not change compared to the control group?P>0.05,n=12?.5.When BV2 cells were treated with 10 nM E₂ for 24h,the protein level of HIF-1?in the experimental group was significantly increased compared to the control group?P<0.001,n=6?.6.The protein levels of DMT1 and FPN1 were both significantly increased in 10 nM E₂treated BV2 cells for 24h compared to the control group?DMT1:P<0.001,n=3;FPN1:P<0.01,n=5?.When BV2 cells were treated with 1?M G15,the protein levels of DMT1and FPN1 were significantly decreased compared to the E₂ group?DMT1:P<0.001,n=3;FPN1:P<0.05,n=5?.When BV2 cells were pretreated with 1?M G15 for 1h and then 10nM E₂ for 24h,the protein levels of DMT1 and FPN1 were significantly decreased compared to E₂ group?DMT1:P<0.01,n=3;FPN1:P<0.01,n=5?.7.The protein levels of FT-L and FT-H were both significantly increased in 10 nM E₂treated BV2 cells for 24h compared to the control group?FT-L:P<0.05,n=6;FT-H:P<0.01,n=5?.When BV2 cells were treated with 1?M G15,the protein levels of FT-L and FT-H were significantly decreased compared to the E₂ group?FT-L:P<0.05,n=6;FT-H:P<0.05,n=5?.When BV2 cells were pretreated with 1?M G15 for 1h and then 10 nM E₂for 24h,the protein levels of FT-L and FT-H were significantly decreased compared to E₂group?FT-L:P<0.05,n=6;FT-H:P<0.05,n=5?.8.The protein levels of DMT1 and FPN1 were both significantly increased in 10 nM E₂treated for BV2 cells for 24h compared to the control group?DMT1:P<0.01,n=5;FPN1:P<0.05,n=4?.When BV2 cells were treated with 1?M PHTPP,the protein levels of DMT1and FPN1 were significantly decreased compared to the E₂ group?DMT1:P<0.01,n=5;FPN1:P<0.01,n=4?.When BV2 cells were pretreated with 1?M PHTPP for 1h and then10 nM E₂ for 24h,the protein levels of DMT1 and FPN1 were significantly decreased compared to E₂ group?DMT1:P<0.01,n=5;FPN1:P<0.01,n=4?.9.The protein level of HIF-1?was increased in 10 nM E₂ treated BV2 cells compared to the control group?P<0.01,n=3?.When BV2 cells were treated with 1?M PHTPP,the protein level of HIF-1?was decreased compared to the E₂ group?P<0.05,n=3?.When BV2 cells were pretreated with 1?M PHTPP for 1h and then 10 nM E₂ for 24h,the protein level of HIF-1?was significantly decreased compared to E₂ group?P<0.05,n=3?.In conclusion,our data indicate that 10 nM E₂ increase the protein expressions of DMT1,FPN1,FT-L and FT-H in BV2 microglia,as well as cellular iron content,which are not related to the regulation of IRP1 and hepcidin,but related to the upregulation of HIF-1?.In addition,E₂ regulates the expression of iron-related proteins in BV2 microglia through both ER?and GPER.This study clarify the cellular and molecule mechanism of estrogen on microglia iron metabolism,thus,providing an experimental basis for understanding the brain iron metabolism.