Role Of Hippocampal Astrocytes Autophagy Flux In Lead-induced Neurotoxicity And Its Underlying Mechanism

Background:Lead is a naturally occurring heavy metal environmental and industrial pollutant,it can be absorbed into body through various transmission routes,including air,drinking water,dust,and food.Then lead could distribute in various organs and tissues,and subsequently causes negative effects on health and wellbeing.Existing evidence reveal that serious lead exposure produces multiple organ injury in nervous system,blood system,cardiovascular system,skeletal system,perhaps long-term and irreversible injury in central and peripheral nervous system.Data suggest lead exposure exhibits greater adverse impact on infants and children,lead exposure during infancy are closely associated with children’s mental retardation and cognitive behavioral dysfunction that contributes to academic failure and school dismissal.Therefore,how to effectively control the neurotoxicity of lead exposure and elucidate its pathological and molecular mechanism has always been the hot research direction in publish health field.At present,it’s now recognized that the neurotoxicity of lead exposure was associated to nerve cell injury,oxidative stress,ion channel damage,neurotransmitter and blood-brain barrier disorder.Neuroglia cells account for approximately 90%brain tissue cells and function as supporting cell in nervous system.It plays irreplaceable role in myelin formation,neural cell development,and injured nerve repair.Astrocytes,the most numerous,widely distributed and complex glial cell type in the central nervous system,could affect neuron development and migration,synaptogenesis,transmitter transmission,and homeostasis through producing and releasing various nerve growth factors.It’s widely accepted that astrocytes are more sensitive to lead exposure and accumulate more lead than neuronal cells.Studies demonstrated that astrocytes could effectively protect other neurons from lead might through binding lead with specific protein and accumulating it.Whereas astrocytes over activation further aggravate neuroinflammation and neuron dysfunction via release multiple inflammatory factors.In addition,it has been reported that lead could directly damage astrocytes and subsequently cause endothelial cells injury and destroy blood brain barrier.Autophagy is considered as a compensatory mechanism and plays crucial role in maintaining cell homeostasis and renovation of organelles.Under physiological conditions,damaged cells and intracellular components in neuron cells could be degraded and replaced via autophagy pathway.when autophagy beyond exceeding the threshold value,it could lead to neuroinflammatory response and oxidative stress.Several studies demonstrated that autophagy level in neuron cells was notably enhanced upon lead exposure,the exact role of autophagy in lead neurotoxicity is still unclear.To the best of our knowledge,there is no report about astrocytes autophagy and lead neurotoxicity through literature search at home and abroad.Hence,this study aims to verify whether lead induced autophagy in astrocytes at both in vivo and in vitro levels,and explore the mechanism of lead exposure on autophagy in astrocyte,as well as the interaction and mechanism between lead exposure,neuroinflammatory response and oxidative stress.Methods:1.Construction of lead exposure model:A total 24 healthy male Sprague-Dawley rats were randomly divided into four groups:normal control group,lead exposure group,3-MA（autophagy inhibitor）group,and lead exposure+3-MA group.Rats in normal control group and 3-MA group received normal feed and drinking water,while rats in lead exposure and lead exposure+3-MA group were given drinking water containing 300 ppm of lead acetate for 30 days.Moreover,rats in lead exposure group+3-MA and 3-MA group also received 3-MA（10 mg/kg）via intraperitoneal injection once for two days,the other groups were given an equal volume of normal saline.2.Lead exposure neurotoxicity assessment:The effect of lead exposure on rats spatial learning and memory capacity was evaluated using Morris water maze assay.H&E staining examined the histopathological changes of hippocampal and whole brain tissues.The hippocampal neuron apoptosis was evaluated using TUNEL assay.The activities of SOD and CAT,as well as levels of GSH and MDA in hippocampal tissues were determined using corresponding commercial kit.3.Effect of lead exposure on hippocampal tissues autophagy:Western blotting was used to examine the expression level of autophagy-related proteins,including LC3-I,LC3-Ⅱ,Beclin-1,p62,Akt,p-Akt Ser473,m TOR,and p-m TORSer2448.The expression of LC3and astrocyte specific markers GFAP,as well as the colocalization were examined using IF assay.4.Effect of lead exposure on astrocytes autophagy:Astrocytes in hippocampal tissues were isolated from P1 SD rat and identified by the GFAP-positive rate using IF.The cytotoxicity of various concentrations lead acetate on astrocytes was evaluated using CCK-8 assay.The formation of autophagosome in astrocytes was observed under transmission electron microscopy（TEM）.Western blotting was used to examine the expression level of autophagy-related proteins,including LC3-I,LC3-Ⅱ,Beclin-1,p62,Akt,p-Akt Ser473,m TOR,p-m TORSer2448,p70S6K and p-p70S6K.5.Effect of lead exposure on cytokines and oxidative stress markers:Cell supernatant IL-1β,TNF-αand MPO levels were measured by ELISA kit.Commercial kit evaluated the activity of MDA in astrocytes.6.Data analysis:Data analysis was performed with Graphpad Prism 8.0.The unpaired t-test was used to analyze differences between two groups.Statistical comparisons between multiple groups were analyzed by one-way ANOVA.All values are expressed as means±standard deviation（SD）,P<0.05 was considered significant.Results:1.Neurotoxicity of lead exposure on SD rats:Morris water maze assay revealed that lead exposure obviously damaged spatial learning and memory capacity of rats,as evidenced by the increased escape latency,as well as the decreased of platform across times and the percentage of time in SW quadrant（P<0.01）.H&E staining results showed that obvious nuclear condensation and apoptotic phenomenon were observed in lead exposure group when compared with control.While autophagy inhibitor 3-MA cotreatment abolished such effects of lead exposure.TUNEL assay revealed that lead exposure significantly increased the number of TUNEL-positive cells,which was counteracted by 3-MA（P<0.05）.2.Effect of lead exposure on hippocampal tissues autophagy:Western blotting analysis results demonstrated that lead exposure significantly up-regulated the level of LC3-Ⅱand Beclin-1,and down-regulated p62 when compared with control group.Co-treatment with autophagy inhibitor 3-MA could partially restore the level of LC3-Ⅱ,Beclin-1,and p62.Moreover,lead exposure also obviously reduced the level of p-Akt^Ser473 and p-m TOR^Ser2448,indicating lead exposure enhances autophagy via inactivation Akt/m TOR signaling pathway.IF results revealed that lead exposure resulted in the colocalization of LC3 and GFAP（P<0.05）.3.Effect of lead exposure on cytokines and oxidative stress markers in hippocampal tissues:Compared with control group,lead exposure significantly reduced SOD and CAT activity,and GSH level,while increased MDA level.Autophagy inhibitor 3-MA cotreatment restored these effects of lead（P<0.01）.4.Effect of lead exposure on astrocytes autophagy flux:IF results implied that 98%isolated astrocytes from hippocampal tissue of SD rats exhibited GFAP positive staining.TEM results showed that lead exposure significantly increased the amount of autophagosome in a dose-dependent manner.Western blotting revealed significantly increased LC3-Ⅱand Beclin-1,while decreased p62,p-Akt^Ser473,p-m TOR^Ser2448,and p-p70S6K expression levels in lead exposure group（P<0.01）.5.Effect of lead exposure on cytokines and oxidative stress markers:Commercial kit results showed that lead exposure significantly increased the activity and levels of TNF-α,IL-1β,MPO,and MDA compared to control groups,while pro-treatment cells with 3-MA notably reduced the increase of TNF-α,IL-1β,MPO,and MDA-induced by lead exposure.Moreover,when cells cotreated with Akt/m TOR inhibitor Rapa,the effect of lead exposure on TNF-α,IL-1β,MPO,and MDA was further enhanced（P<0.01）.Conclusion:Lead exposure induced autophagy in the hippocampus of SD rats,which resulted in oxidative stress and activation of astrocytes,and promoted hippocampal neuron injury and apoptosis,resulting in decreased spatial learning and memory ability of rats.Lead exposure could trigger autophagy in astrocytes through enhancing autophagic flux,and inhibition of autophagy by 3-MA effectively alleviated lead neurotoxicity.Autophagy induced by lead exposure further aggravate inflammatory response and oxidative stress in astrocytes.Lead exposure enhanced autophagy in astrocytes through modulating Akt/m TOR signaling pathway.