The Study Of Lanthanum Chloride Induced Synthesis/transport Disorders Of Lactate And Overly Excitatory In Astrocytes,and Its Impairments On Neurons

Objective:Rare ea rth elements?REEs?are wid ely discovered and used.Epidemi ological surveys in several major REE mines in China showed that children's mean intelligence-quot ient?IQs?and learning-me mory abilities were much lower than those in the co ntrol areas.Therefore,REEs have obvious neurotoxicity.As a ki nd of light REEs,lanthan um?La?could cross the blood-brain bar rier?BBB?and ultim ately accumu late in the brain,and the central ne rvous system?CNS?is more sens itive to La than other org ans and tissues.Moreover,La owns lively chemical properties and strong biological activity.There fore,La is often used as the repress entative of REEs to investi gate its neurotox icity.Previous studies have shown that La had a strong toxicity on the CNS,as well as,inhibited multiple signaling pathways,caused nerve cell damage and apoptosis,and resulting in the decrease of learning-memory abilities in experimental animals.However,the mecha nism underlyin g the neuroto xicity of La is still unclear.Astrocytes are the most abundant and widely distributed glial cell types in the brain,and play a very important role in mainta ining the functions of CNS,such as energy metabolism,antioxidant defense,inflammatory responses,neuronal excitability and synaptic plasticity.Noticeably,astrocytes is considered as a neural“network inte grator”which regulates the protrusion plasticity and neuronal excitability by releasing multiple astrocytic gliatransmitters,and the protrusions in a single astrocyte can be connected to dendrites and axons of up to 10⁵ neurons.BBB mainly composed by vascular endothelial cells,pericytes and astrocytic endfoots.La,which enters into the brain with blood flow,will contact with astrocytes prior to neurons.Neuron is a kind of differentiated cell and requires a significant amount of energy to perform their various functions.It is reported that the lactate derived from astrocytes is a vital energy substrate for neurons during LTM formation.Astrocytes are the only cell type that can synthesize glycogen as a reserve of brain energy.In cases of LTM formation,the astrocytes can rapidly decompose glycogen into lactate and transport this energy substrate to neurons by way of monocarboxylic acid transporters?MCTs?,which is called"astrocyte-neuron lactate transport?ANLT?".Studies indicated that the inhibition of lactate production in astrocytes and/or ANLT could obviously impair the LTM.Under normal physiological conditions,the moderate activa tion of astrocyte plays an important role in the regulation of neuronal signaling and synaptic plasticity.However,overexpr ession of the N-methyl-D-aspa rtate receptor?NMDAR?on neuronal posts ynaptic membrane caused neuronal excitotoxicity,such as the calcium overload,excessive production of free radical,cellular edema,neuronal apoptosis or even death,and ultimately damaged learning-memo ry abilities or caused neurode generative diseases.Therefore,lactate production/transport and chemical excitability production/transmitting of astrocytes are closely related to neuronal excitotoxicity.In this study,the in vitro cell models were established to study the effects of La on the production/transport of lactate and chemical excitability production/transmitting were investigated,as well as its roles on neuronal excitotoxicity.On this basis,in order to further explore the detrimental effects of astrocytic dysfunction on neurons.The interventions of Rolipram,Forskolin,MK-801,DAAO and DCKA on the corresponding role to verify the relevant experimental results.This study provides some novel clues for clarifying the mechanism underlying the neurotoxicity of La.Methods:part?,Lanthanum chloride reduces lactate production in primary culture rat cortical astrocytes and suppresses primary co-culture rat cortical astrocyte-neuron lactate transport:first,the primary culture astrocytes were treated with 0,0.125 mmo l/L,0.25mmol/L,0.5 mmol/L and 1.0 mmol/L LaCl₃ in serum-free DME M for 6 h,12 h,24 h,48h.Then,the survival rates were measured by methylthiazolyl tetrazolium?MTT?.Second,the primary culture astr ocytes were treated with 0.125 mmol/L,0.25 mmol/L and 0.5mmol/L LaCl₃ for 24 h,and the following experime nts were carried out:?1?Morphological examination was performed,?2?The mRNA and protein expression levels of glucose transporter GLUT1,GS,GP,MCT1,MCT4,LDHA and LDHB were measured by real-time quantitate ve PCR?RT-PCR?and We stern Blot?WB?,?3?The activities of GP and LDH,and the concentrations of lactate were detected by assay kit,?4?The GS/p-GS protein expression ratio was detected by WB.Second,the primary neurons were co-cultured with the primary astrocytes in the transwell dishes?Corning,USA?,neurons were seeded in the lower chamber,and the astrocytes were seeded in the upper layer.The co-culture cells were treated with 0.125 mmol/L,0.25 mmol/L and 0.5 mmol/L LaCl₃ for 24 h.Then,the mRNA and protein expression levels of MCT2 in neurons were measured by RT-PCR and WB.Finally,the primary astrocytes were pretreated with FSK and ROL.The astrocytes were separately pretreated with FSK?20?mol/L,40?mol/L?or ROL?20?mol/L,40?mol/L?for 6 h and then treated with 0.5 mM LaCl₃ for 24 h.The activity of GP and the concentration of lactate were detected by assay kit,and the GS/p-GS protein expression ratio was detected by WB.Part?,Lanthanum chloride induced astrocyte to be overly excitatory:first,the astrocytes were treated with 0.125 mmol/L,0.25 mmol/L and 0.5mmol/L LaCl₃ for 24h,and the followed experiments were carried out:?1?Morphological examination was performed,?2?The membrane damages were measured by lactate dehydrog enase?LDH?release,?3?The apoptosis rates were detected by flow cytometry with Anne xin V/FITC kit,?4?The mRNA and protein expression levels of Cx43,Cx30,mGluR₅ and PLC were measured by real-time quantitative PCR?RT-PCR?and Western blot?WB?,?5?The[Ca²⁺]_i levels were detected by flow cytometry with fluo-3/AM kit,?6?The IP₃concentrations were detected by ELISA kit,?7?The D-serine and Glu levels were detected by high performance liquid chrom atography?HPLC?.Part?,the effects of Lanthanum induced over-excitation of astrocytes on neurons:first,the primary culture astrocytes were treated with 0,0.125 mmol/L,0.25 mmol/L and0.5 mmol/L La Cl₃ for 24 h,and then collected the culture supernatants by centrifugation and hereafter denoted as CM?control?,CM?0.125 mM LaCl₃?,CM?0.25 mM La Cl₃?and CM?0.5 mM La Cl₃?,as well as collectively called as the CM(La³⁺).The blank DMEM was denoted as control?blank?.The primary culture neurons were treated with the CM?control,0.125 mM,0.25 mM and 0.5 mM LaCl₃?for 48 h,and the followed experiments were carried out:?1?The mRNA and protein expression levels of NR1,NR2A and NR2B were measured by the RT-PCR and WB,?2?The[Ca²⁺]_i levels were detected by the flow cytometry with fluo-3/AM kit,?3?The apoptosis rates were detected by the flow cytometry with Annexin V/FITC kit.the primary astrocytes were treated with0 and 0.5mM LaCl₃ for 24 h and the supernatants discarded,then the astrocytes were gently washed twice with D-hanks and treated with the serum-free DMEM for 24 h,ultimate collected the supernatants and denoted as CM?blank?and CM?La-free?.MK-801?Sigma,USA?,DAAO?Sigma,USA?and DAAO?Sigma,USA?were prepared with CM?La-free?according to the protocol given by the suppliers.The primary neurons were treated with?10?mol/L,20?mol/L?DCKA,?40?mol/L,100?mol/L?MK-801and?20 UN/L,40 UN/L?DAAO for 48h,the followed experiments were carried out:?1?The[Ca²⁺]i levels were detected by the flow cytometry with fluo-3/AM kit,?2?The apoptosis rates were detected by the flow cytometry with Annexin V/FITC kit.Results:1.The results showed that LaCl₃ treatment significantly downregulated the mRNA and protein expression of glucose transporter 1?GLUT1?,glycogen synthase?GS?,glycogen phosphorylase?GP?,lactate dehydrogenase A?LDHA?,and monocarboxylate transporter 1,2 and 4?MCT 1 2 and 4?;upregulated the mRNA and protein expression of lactate dehydrogenase B?LDHB?;and decreased the glycogen level,total LDH and GP activity,GS/p-GS ratio and lactate contents.2.The results showed that LaCl₃ treatment significantly downregulated the rates of survival and upregulated the mRNA and protein expression of metabotropic glutamate receptor 5?mGLUR₅?,phospholipase C?PLC?,Connexin 43?Cx43?and Cx30;and increased the rates of apoptosis,the concentrations of inositol trisphosphate?IP₃?and[Ca²⁺]_i,the production and release levels of gliatransmitters?Glu and D-serine?.Moreover,the cultured medium?CM?which got from LaCl₃ treated astrocytes could increase the mRNA and protein expression of NMDAR subunits?NR1,NR2A,NR2B?,[Ca²⁺]_i concentrations and apoptosis rates in primary culture rat cortical neurons.3.MK-801,DAAO and DCKA could decrease the expression levels of NMDAR subunits?NR1,NR2A and NR2B?,the concentrations of[Ca²⁺]_i and the apoptosis rates of CM(La³⁺)treated neurons.Conclusion:1.These results suggested that La-induced learning-memory damage was probably related to its suppression of lactate production in astrocytes and ANLT.2.La could inhibit the transport of lactate between astrocytes and neurons by downregulating the expression of MCTs.3.La could induce the over-excitation of astrocytes and the massive synthesis and release of Glu and D-serine.4.The treatment of CM(La³⁺)and CM?La-free?could upregulate the expression levels of NMDAR subunits,the concentrations of[Ca²⁺]_i and apoptosis on neurons.5.CM?La-free?-induced over-activation of NMDAR,upregulation of[Ca²⁺]_i concentrations and apoptosis rates could be antagonized by MK-801,DAAO and DCKA.Suggesting that the excessive synthesis and release of Glu and D-serine induced by the over-excitation of astrocytes play an important role in leading to the excitotoxicity of neurons.