Effect Of Iron Level On Lcn2 Protein Expression Induced By Lipopolysaccharide And α-synuclein In Astrocytes

Parkinson’s disease(PD)is the second most common neurodegenerative disease.The main pathological feature is the progressive loss of dopaminergic neurons in the substantia nigra(SN)and the reduced dopamine(DA)levels in the striatum(Str),causing movement symptoms.The etiology of PD pathology is still unclear.Oxidative stress,neuroinflammation,mitochondrial dysfunction,abnormal protein aggregation caused by genetic factors,environmental factors,and aging,might participate in the degeneration of dopaminergic neurons.In recent years,increasing evidence indicates that activated microglia and astrocytes trigger persistent neuroinflammatory reactions,which are one of the causes of PD dopaminergic neuron degeneration.In the disease state of PD,the activation of astrocytes can induce the release of pro-inflammatory cytokines and take up α-synuclein(α-syn),which play crucial roles in neurodegeneration.Iron is the most abundant redox active metal in the brain and is an essential trace element for various physiological processes,including oxygen transport(through hemoglobin),redox reactions,neurotransmitter synthesis,myelin production,and mitochondrial functions.However,iron easily releases electrons and generates reactive oxygen species(ROS),excessive iron can cause mitochondrial dysfunction,oxidative stress and ferroptosis.Iron and dopamine act as a toxic couple,contributing to the vulnerability of dopaminergic neurons in PD.Astrocytes are indispensable elements of neurovascular units that establish the relative independence of iron balance in the brain by constituting the blood brain barrier(BBB).Astrocytes also can regulate synaptic activity by buffering iron in the synaptic environment.Hepcidin is a regulator of brain iron homeostasis.Intravenous injection of lipopolysaccharide(LPS)induced the expression of hepcidin in peripheral organs as well as in the brain.In the brain,hepcidin is mainly expressed in astrocytes,and could be up-regulated by inflammatory insults.When treated with hepcidin peptides or infected with hepcidin-expressing adenovirus,astrocytes showed a marked reduction in iron uptake and release.Therefore,the regulation of hepcidin in astrocytes plays a key role in controlling the transport of iron through BBB and brain iron metabolism.Lipocalin 2(Lcn2)is a member of the secreted lipocalin family.As a bacteriostatic factor,under normal physiological conditions,Lcn2 can transport iron through mammalian siderophore and inhibit microorganisms from mammalian hosts to obtain iron and therefore inhibit the growth of bacteria.As an important regulator of neuroinflammation and iron homeostasis in the central nervous system,Lcn2 is mainly released by glial cells and acts on the surrounding microenvironment.Under physiological conditions,the normal brain expresses low Lcn2,but it can be significantly upregulated by injury or inflammation.In the SN and Str of MPTP-treated mice,Lcn2 is mainly expressed in reactive astrocytes and some activated microglia.Increased levels of Lcn2 lead to neurotoxicity and neuroinflammation,then impaired SN-Str dopaminergic projection and abnormal motor behavior in mice.These symptoms are improved in Lcn2 knockout mice.When treated with exogenous iron,Lcn2-induced neurotoxicity was aggravated in vivo,however,attenuated with iron chelator deferoxamine(DFO)treatment.The regulation of Lcn2 gene expression is mainly at the transcription level.Nuclear factor-kappa B(NF-κB)is the main regulator of inflammatory gene expression and the most important regulator of Lcn2 gene transcription.In this study,western blotting,enzyme-linked immunosorbent assay,and quantitative real-time PCR were used to observe the levels of proinflammatory cytokines with treatment with two proinflammatory insults,LPS and α-syn,in primary cultured astrocytes.Tumor necrosis factor-alpha(TNF-α),Interleukin 1-beta(IL-1β)expression and release,and hepcidin expression were observed in these cells.The iron overload reagent ferric ammonium citrate(FAC)and the iron chelator reagent(DFO)were used to alter iron levels in primary cultured astrocytes.We are interested in the effect of changes in intracellular iron levels on LPSinduced Lcn2 expression,and investigate whether autophagy,proteasome and NF-κB pathway are related to DFO inhibiting LPS-induced Lcn2 protein expression.The results were as follows: 1.In primary astrocytes,compared with the control group,0.2 μg/mL and 1μg/mL LPS significantly induced up-regulation of TNF-α and IL-1β mRNA expression and increased protein release(P <0.01,P <0.001),5 μg/mL α-syn significantly induced up-regulation of TNF-α mRNA expression(P <0.001)and increased protein release(P <0.01),and upregulation of IL-1β mRNA expression(P <0.001),but does not affect the release.Compared with the control group,FAC or DFO treatment or pretreatment did not affect TNF-α,IL-1β mRNA expression and protein release.Compared with 1μg / mL LPS group,FAC or DFO pretreatment inhibited 1μg/mL LPS-induced up-regulation of TNF-α and IL-1β mRNA expression(P <0.001,P <0.01)but had no effects on release.Compared with 5μg/mL α-syn group,DFO pretreatment inhibited 5μg/mL α-syn-induced up-regulation of TNF-α mRNA expression(P <0.01)but had no effects on release,and FAC or DFO pretreatment had no effects on 5μg/mL α-syn induced IL-1β mRNA expression and release.The results indicate that inflammatory factors LPS or α-syn can cause obvious inflammation.Although FAC or DFO pretreatment can inhibit LPS-induced up-regulation of TNF-α and IL-1β mRNA expression,it can cause TNF-α caused by LPS or α-syn,IL-1β protein release has no effect.These results suggest that intracellular iron levels are not significant to the inflammatory response caused by the inflammatory factors LPS or α-syn.2.In primary astrocytes,compared with the control group,0.2 μg/mL,1 μg/mL LPS increased the level of hepcidin mRNA(P <0.01,P <0.05);FAC or DFO treatment alone or pretreatment did not affect hepcidin mRNA levels.Compared with the 1μg/mL LPS group,FAC or DFO pretreatment inhibited 1μg/mL LPS-induced up-regulation of hepcidin mRNA level(P <0.05).The 1μg/mL,5μg/mL α-syn treatment alone had no effects on the level of hepcidin mRNA.However,compared with the 5μg/mL α-syn group,FAC pretreatment inhibited 5 μg/mL α-syn-induced hepcidin mRNA levels(P <0.05);DFO pretreatment increased 5 μg/mL α-syn hepcidin mRNA levels(P <0.001).The results indicate that the expression of hepcidin mRNA is regulated by the inflammation factor LPS,and the intracellular iron level can change the regulation of hepcidin mRNA expression by the inflammation factor LPS or α-syn.3.In primary astrocytes,compared with control group,0.2 μg/mL,1 μg/mL LPS treatment upregulated ferritin expression(P <0.05)and 1μg/mL,5μg/mL α-syn treatment up-regulated ferritin expression(P <0.05,P <0.01),FAC or DFO treatment alone could up-regulate/downregulate ferritin expression level(P <0.05,P <0.001).Compared with the 1 μg/mL LPS group,FAC or DFO pretreatment up-regulated or down-regulated 1 μg/mL LPS-induced ferritin expression(P <0.05).Compared with 5 μg/mL α-syn group,FAC or DFO pretreatment upregulated or down-regulated ferritin protein expression(P <0.01,P <0.05).The results indicate that the expression of ferritin is regulated by inflammatory factors and intracellular iron levels.4.In primary astrocytes,compared with the control group,0.2 μg/mL,1 μg/mL LPS treatment alone,FAC or DFO pretreatment and 1 μg/mL LPS co-treatment reduced the ratio of hepcidin mRNA to ferritin protein(P < 0.05).Compared with the control group,the 1 μg/mL,5 μg/mL α-syn treatment alone reduced the ratio of hepcidin mRNA to ferritin protein(P <0.05,P <0.01).Compared with the 5 μg/mL α-syn group,FAC pretreatment and co-treatment with 5 μg / mL α-syn decreased the ratio of the hepcidin mRNA to ferritin protein(P <0.01),and after DFO pretreatment and co-treatment with 5 μg/mL α-syn increased the ratio of hepcidin mRNA to ferritin protein(P <0.001).The results indicate that intracellular iron levels do not affect hepcidin mRNA levels,inflammatory factors and intracellular iron levels affect the expression of ferritin,the ratio of hepcidin mRNA to ferritin can better reflect the inflammation of primary astrocytes factors and dual regulation of intracellular iron levels.5.In primary astrocytes,compared with the control group,0.2 μg/mL,1 μg/mL LPS significantly induced the expression of Lcn2 protein(P <0.01),5 μg/mL α-syn significantly induced the expression of Lcn2 protein(P <0.001),FAC or DFO treatment or pretreatment had no effects on Lcn2 protein expression.Compared with the 1 μg/mL LPS group,FAC pretreatment did not change 1 μg/mL LPS-induced up-regulation of Lcn2 protein expression,and DFO pretreatment reduced 1 μg/mL LPS-induced up-regulation of Lcn2 protein expression(P <0.01).Compared with 5 μg/mL α-syn group,FAC or DFO pretreatment did not change 5 μg/mL α-syn-induced up-regulation of Lcn2 protein expression.In BV2 microglia,compared with the control group,1μg/mL LPS obviously induced the expression of Lcn2 protein(P <0.001).FAC or DFO treatment or pretreatment had no effects on Lcn2 protein expression.Compared with 1μg/mL LPS group,FAC,DFO pretreatment did not change 1 μg/mL LPS-induced up-regulation of Lcn2 protein expression.The results indicate that inflammatory factors LPS or α-syncan induce the up-regulation of Lcn2 expression,and DFO could exert the inhibitory effects of DFO on LPS-induced up-regulation of Lcn2 in primary astrocytes.6.In primary astrocytes,compared with LPS group,pretreatment with the autophagy inducer Rapamycin(RAPA)inhibited LPS-induced up-regulation of Lcn2 protein expression(P <0.05).FAC or DFO co-pretreatment can also inhibit LPS-induced up-regulation of Lcn2 protein expression(P <0.001).Compared with the DFO+LPS group,RAPA had no effect on the effect of DFO on inhibiting LPS-induced upregulation of Lcn2 protein.Compared with the LPS group,autophagy inhibitor Chloroquine(CQ)pretreatment did not change LPSinduced up-regulation of Lcn2 protein,and co-pretreatment with FAC did not change LPSinduced up-regulation of Lcn2 protein expression.Compared with the DFO+LPS group,CQ also had no effect on the effect of DFO on inhibiting LPS-induced upregulation of Lcn2 protein expression.The results indicate that autophagy might not participate in the inhibitory effects of DFO on LPS-induced up-regulation of Lcn2.7.In primary astrocytes,compared with the LPS group,proteasome inhibitor MG132 blocked the LPS-induced up-regulation of Lcn2 protein expression by either pretreatment alone or copretreatment with FAC or DFO(P <0.001).At the same time,compared with the control group,LPS can increased the p-NF-κB/NF-κB ratio(P <0.01),and compared with the LPS group,MG132 pretreatment can block the increased of the LPS-induced p-NF-κB/NF-κB ratio(P <0.05).However,DFO pretreatment did not decrease the LPS-induced p-NF-κB/NF-κB ratio.The ubiquitin proteasome and NF-κB pathway may not participate in the inhibitory effect of DFO on LPS-induced Lcn2 upregulation.The above results indicate that both LPS and α-syn can induce inflammatory response in primary astrocytes,which is manifested by increased proinflammatory cytokine TNF-α mRNA expression and increased protein release.LPS or α-syn can cause obvious inflammation.Although FAC or DFO pretreatment can inhibit LPS-induced up-regulation of TNF-α and IL-1β mRNA expression,it has no effect on the release of TNF-α and IL-1β proteins caused by LPS or α-syn.The expression of hepcidin mRNA is regulated by the inflammatory factor LPS,and the intracellular iron level can change the expression regulation of the inflammatory factor LPS or α-syn on hepcidin mRNA and its ratio with ferritin.LPS can obviously induce the protein expression of Lcn2,and the iron deficiency state in the cell can inhibit the upregulation of Lcn2 protein expression in primary astrocytes induced by LPS.Autophagy,ubiquitin proteasome and NF-κB pathway may not be involved in the inhibitory effect of DFO on LPS-induced upregulation of Lcn2 protein.This experiment will clarify the expression regulation mechanism of astrocyte Lcn2 under PD disease state affected by inflammatory factors and intracellular iron levels,and provide experimental support for its participation in PD pathogenesis and as a possible drug treatment target.