Role And Mechanism Of Microglial Activation On Neurons Damage Induced By Lanthanum

Objective:Rare Earth Elements（REEs）include 15 lanthanides and its kin of scandium and yttrium in the periodic table of elements.They are widely used in various fields such as industry,agriculture,biology and pharmaceutical industry,so the influence on the environment,ecology and human health,as well as the immediate and long-term damage function attract more and more attentions.REEs easily enrich in the natural environment,enter the body through the way such as gastrointestinal,respiratory and accumulate in the tissues and organs,can cause liver,kidney,heart,immune,nerve,endocrine and genetic damage.In recent years the nervous system damage effects of REEs have been paid more attention.Epidemiological investigation show that REEs are likely to be the cause of decrease of intelligence in children.Animal experimental results confirm that REEs can cross the blood-brain barrier and accumulate in the brain,cause damage and dysfunction of the central nervous system,but its mechanism is not exact.Lanthanum has strong biological activity and accumulation effect in the brain,and is stable in environmental medium,so it is often used as a typical rare earth elements to explore the effects on the nervous system.Studies show that lanthanum can induce neurotoxicity and impair cognitive function by influencing the distribution of trace element,promoting oxidative stress,interfering with the Ca²⁺,enzymes and neurotransmitters dynamic balance.More research evidence show that lanthanum can result in developmental defect of the central nervous system,nervous behavior changes,impairment of learning and memory,neurons form and structural abnormalities.Therefore,research on the damage mechanism of learning and memory ability induced by lanthanum has the vital significance to protect people health.Microglia are ubiquitous throughout the CNS,and are the unique permanent resident immune cells in the CNS.Microglia play an important role in defense and tissue repair,and have an important functions such as support,nutrition,protection and repair in the physiological activities of the neurons.Microglia have two states as“resting state”and“activated state”.Microglia in the resting state can quickly transform into the activated state,when the central nervous system suffer from injury,inflammation,or external stimulation.Microglia activation is the main performance of inflammatory response in the brain.The activated microglia have protective effect by secretion of growth factors,cleaning up cell debris and against foreign antigen attack.Microglia provide neurons with a relatively suitable microenvironment,and promote tissue repair.But activation of microglia also cause an increase in the production of inflammatory cytokines such as TNFαand IL1βthat lead to acute inflammatory reaction.Excessive production of inflammatory cytokines generated during neuroinflammation can damage neighbouring neurons.The activated microglia have been thought to be associated with the pathogenesis of a number of the central nervous system diseases,including the central nervous system infection,ischemic stroke,multiple sclerosis,neurodegenerative diseases,and mental illness.In recent years,considerable attention has been drawn to the damage effect of activated microglia in the central nervous system.NF-κB is a critical transcription factor involved in regulating the expression of various pro-inflammatory mediators and has been found to be a promising target for inflammatory diseases.NF-κB activation result in nuclear translocation of p65,whereupon it can influence the expression of numerous immune response genes.These pro-inflammatory mediators have neurotoxic effect,such as TNFαand IL1β,they can cause severe inflammation,and play an important role in the process of neurodegenerative diseases such as AD and PD.So an inflammatory process induced by activated microglia is believed to play an important role in the pathway leading to central neural system injuries.The aim of the present study was to investigate whether lanthanum can active microglia and NF-κB signaling pathway in vivo and in vitro experiments,and whether there is a relationship between hippocampal neurons or learning and memory damage and the excessive production of inflammatory cytokines generated by activated microglia induced by lanthanum.The data of the study will provide theoretical and experimental basis for the prevention of neurotoxic effect induced by lanthanum.Methods:Eighty male and female KM mice（22-25g body weight）were housed under standard laboratory conditions at an environmental temperature of 17-23℃and humidity of 50±5%.The mice were raised on a 12h dark-light cycle circumstance with free access to standard chow diet and drinking water.The mice were observed for 7 days before the formal experiment.The female mice were randomly divided into 4 groups（20 mice per group）,then females and males were mated at 1:1.The discovery of copulatory plug was a sign of mating successfully and the day was pregnancy 0 day.During the 3 weeks period of lactation,the maternal mice were exposed to LaCl₃ in distilled drinking water under one of four doses:0%,0.25%,0.5%or 1%respectively.After weaning,they were permitted free access to food and drinking water（containing 0,0.25%,0.5%,and 1%LaCl₃,respectively）for two months.Morris water maze was used to test learning and memory ability of mice,HE staining was used to observe the pathology changes in hippocampus,real time PCR and immunohistochemistry was used to measure the level of IBA1,TNFα,IL1β,IL6,MCP1,iNOS genes mRNA transcription and protein expression.BV2 microglial cells were cultured at 37℃and 5%CO₂ in DMEM supplemented with 10%FBS and antibiotics.The cells were treated with the various concentrations of LaCl₃ for different time,MTT assays were used to determine cell viability.The cells were treated with 0,0.05,0.1 and 0.2mmol/L for 24h,cell immunofluorescence was used to detected the expression of IBA1 protein,real time PCR and western blot were used to measure TNFα,IL1β,IL6,MCP1 and iNOS genes mRNA transcription and protein expression,Griess method was used to detected the level of NO,cell immunofluorescence and western blot were used to observe NF-κB-regulated protein expression and nuclear translocation.After pretreatment cells with PDTC then the changes of the above indicators were observed again.The newborn KM mice within 24 hours were used to perform neuron culture,NeuN was used to identify the neurons.After establishing conditioned medium and Transwell model with BV2 and neurons co-cultured,Annexin V/PI were used to measure cell apoptotic/necrosis,Heochst immunofluorescence was used to detected neuronal apoptosis rate.After pretreated cells with PDTC then the changes of the above indicators were observed again.Results:1.Effects of LaCl₃ on the spatial learning and memory of mice.In the place navigation test of Morris water maze,the latency and distance travelled of the LaCl₃-treated groups were significantly higher than those of the control group,the paths taken by rats treated with LaCl₃ were messy and purposeless.In the spatial exploration test of Morris water maze,the time spent in the target quadrant and the number of target quadrant crossings of the LaCl₃-treated groups was significantly lower than those of the control group,the mice in the LaCl₃-treated groups performed purposelessly and seldom went into the target quadrant.2.Effects of LaCl₃ on body weight,weight of hippocampus and hippocampal organization coefficient.The hippocampal organization coefficient of the LaCl₃-treated mice increased markedly.But LaCl₃ had no significant effect on weight of hippocampus,the increase of hippocampal organization coefficient was caused by body weight loss of mice.3.Effects of LaCl₃ on neuron damage.Compared with the control group,the hippocampal neurons in the LaCl₃-treated groups were arranged in disorder and cell gap was widened.4.Effects of LaCl₃ on microglia activation.The level of IBA1protein expression was increased and microglia were activated by LaCl₃ exposure in vivo and vitro.5.Effects of LaCl₃ on NF-κB signaling pathway.The level of p-IKK and p-IκB protein expression were increased in cytoplasm of LaCl₃-treated BV2,the p65 protein expression was decreased in cytoplasm,but increased in cell nucleus.6.Effects of LaCl₃on the expression of inflammatory cytokines.In vivo and in vitro,LaCl₃ can increase the mRNA and protein expression of TNFα,IL1β,IL6,MCP1 and NO than those of control group.7.Effect of activated microglia induced by LaCl₃ on neurons.After LaCl₃ treated BV2 for 24h,neurons injury were observed in microglia and hippocampal neurons co-cultured in vitro.The injuries were more serious after LaCl₃ exposure for 48h than for 24h.8.Effect of PDTC on neurons injury caused by LaCl₃-induced microglia activation.PDTC treated can inhibit NF-κB signaling pathway,decrease the release of TNFα,IL1β,IL6,MCP1 and NO,and antagonize neurons damage induced by activated microglia.Conclusion:1.LaCl₃ can impair learning and memory ability of mice.2.In vivo and in vitro,LaCl₃ can active microglia and its NF-κB signaling pathway.3.LaCl₃ can induce microglia to overexpress the inflammatory cytokines such as TNFα,IL1β,IL6,MCP1 and NO.4.The activated microglia can lead to the neurons damage.5.PDTC,a NF-κB signaling pathway inhibitor,can decrease the release of TNFα,IL1β,IL6,MCP1,NO,and antagonize neurons damage induced by activated microglia.