Study On The Mechanism Of MiR-124 In Lead Neurotoxicity

Purpose and significance of the study Lead is an undegradable environmental poison that can persist in the environment.According to WHO guideline for the clinical management of exposure to lead issued in 2021,lead exposure is a major public health issue,with 900000 people dying from long-term exposure to lead in 2019.Currently,lead exposure and risk assessment mainly rely on blood lead concentration which is an exposure biomarker of lead,but it accounts for only 1% of human lead load.Children with the same blood lead concentration may not face the same risk of neurodevelopmental damage.Therefore,the combination of lead exposure biomarkers and lead effect biomarkers is more conducive to the evaluation of lead toxicity.Brain is the main target organ of lead toxicity,and small amounts of lead exposure can cause a series of physiological and behavioral dysfunction.However,there is limited research on effect biomarkers of lead neurotoxicity.Therefore,there is a crucial need to explore more sensitive biomarkers,conduct in-depth mechanism research,and timely evaluate the neurotoxicity caused by lead exposure.Circulating miRNAs can make preferred organ damage biomarkers due to their accessibility,specificity and sensitivity.For example,miR-122 can serve as a highly specific biomarker for drug-induced liver injury;miR-133 a can serve as a highly sensitive and specific biomarker for acute muscle injury.miR-124 has the potential to become an effect biomarker of central nervous system,since it is the most abundant miRNA in brains,highly specific in central nervous systems,and has the same sequence in humans,mice and rats.In the study of lead exposed workers,it was found that lead exposure could cause a significant increase in circulating miR-124 indicating that circulating miR-124 has the potential to become a biomarker of lead neurotoxicity.The use of circulating miRNAs as biomarkers in clinical needs to complete the following key steps,including discovery,confirmation,validation,and clinical application.Although many circulating miRNAs have been confirmed in different populations and disease conditions,they have not yet been used in clinic,which can be attributed to the differences in detection platforms,standardization strategies,sample types,and sample processing methods,leading to considerable inconsistency and non-repeatability between studies.According to “Biomarker Qualification: Evidentiary Framework” issued in 2018 by FDA,analytical validation and clinical validation are needed to support biomarker qualification.miR-124 plays an important role in the central nervous system,and its feasibility as a brain damage biomarker has been studied in multiple non clinical and clinical trials,but all of them were in the discovery and confirmation stage.This project is aimed to establish and validate the detection method of circulating miR-124 in our laboratory with MCAO models,to study the stability,tissue specificity,and sensitivity of miR-124,and to explore the role of miR-124 in lead acetate neurotoxicity mechanism.Methods and results This study is divided into three parts: Part I: Due to the low expression level of circulating miR-124 in normal rats,which might interfere with evaluation of miR-124 detection methods,MCAO models of SD rats(recognized as models with high expression of miR-124)were established using thread occlusion method.The detection method was validated by comparing with literature results.To establish reliable standardization strategies,two commonly used reference genes(U6 and 5S)and three miRNAs(miR-24,miR-122,and miR-9a)were selected as candidate reference genes and the expression of the candidate reference genes and target gene miR-124 was detected with RT-q PCR in peripheral blood samples.The acquired data was analyzed using ge Norm,Normfinder,Best Keeper,Ref Finder,and comparative delta-Ct statistical models to screen for stable reference genes.To investigate the stability of circulating miR-124,the collected blood samples from MCAO models were centrifuged,aliquoted,and analyzed immediately or stored at room temperature,2-8°C,and-70°C to-90°C before detection.To investigate the tissue specificity of circulating miR-124,hepatotoxicity,cardiotoxicity,nephrotoxicity,and neurotoxicity models were established with acetaminophen(1250 mg/kg,IG),isoprenaline(2.5 mg/kg,IV),gentamycin(80 mg/kg,IM),and lead acetate(600 mg/kg,IG),respectively.The tissue specificity of miR-124 was evaluated by detecting the expression of miR-124 in these models.The analyzed results showed consistently that miR-24 was the most stably expressed reference gene under the condition of this study.The “optimal combination” calculated by ge Norm was consist of miR-24,U6,and 5S.The expression level of the target gene miR-124 was similar when normalized with the most stable reference gene miR-24 or the “optimal combination”.Compared with fresh blood samples,miR-124 was found to be stable at room temperature for 6 hrs,at 2-8°C for 24 hrs,at-70°C to-90°C for 36 days,and for three freeze-thaw cycles.The stability could support the detection needs of laboratory.miR-124 has good tissue specificity since increased miR-124 was noted in neurotoxicity models,while no significant changes were noted in hepatotoxicity,cardiotoxicity and nephrotoxicity models.Part II: In order to investigate the neurotoxicity mechanism of lead acetate and validate the sensitivity of circulating miR-124,SD rats were administered with lead acetate via oral gavage or intraperitoneal injection to establish neurotoxicity models.Functional observation battery test was used to evaluate motor function,coordination function and behavior changes,passive avoidance experiment was used to evaluate learning and memory function,spontaneous activity experiment was used to evaluate exploration function,HE staining was used to detect histopathological changes of brain tissues,and ICP-MS method was used to detect lead content in brain tissues.Neurotoxicity models were evaluated based on the above test results.ELISA method was used to detect oxidative stress,inflammation and apoptosis biomarkers.RT-q PCR method was used to detect the expression of miR-124.The results showed that,in lead acetate groups,decreased activities were noted in clinical observation;decreased body tone,decreased alert level,slight impairment of ambulation activity,abnormal gait,hypoactivity of startle response,and abnormal extensor thrust and air righting reflex were noted in functional observation battery test;decreased inquiry behaviors were noted in spontaneous activity test;increased glial cells and degeneration/necrosis of neurons in brain tissues were noted in histopathological examinations;obvious lead exposure was detected in brain tissues after repeated administration of lead acetate.Rat lead exposure neurotoxicity models were considered to be established successfully.In brain tissues of these models,persistently increased pro-apoptotic protein BAX and caspase-3 were noted,persistently increased proinflammatory cytokine IL-1β and TNF-α and persistently decreased anti-inflammatory cytokine IL-10 were noted,persistently increased SOD in antioxidant system and LPO in oxidation system were noted.Therefore,apoptosis,inflammatory reaction and oxidative stress in brain tissues contributed to lead acetate induced neurotoxicity.Corresponding index changes were also detected in peripheral blood.However,compared with these indexes,miR-124 could detect neurotoxicity with an earlier and more sensitive manner.Part III: Based on the results in the second part,cell apoptosis,inflammatory response,and oxidative stress were involved in the neurotoxicity caused by lead exposure,and lead exposure could cause an increase in miR-124 expression.In this section,we aimed to investigate the relationship between miR-124 and cell apoptosis,inflammatory response,and oxidative stress,as well as the signaling pathways regulated by miR-124.BV-2 microglia were transfected with miR-124 mimic and miR-124 inhibitor,and then treated with lead acetate.The biomarkers of oxidative stress,inflammation and apoptosis were detected by ELISA,the expression of miR-124 was detected by RT q PCR,and the expression of signal pathway proteins was detected by Western blot.The results showed that lead acetate increased the levels of miR-124,caspase-3(apoptosis marker),TNF-α(inflammation marker),SOD and LPO(oxidative stress markers)in BV-2 microglia(P<0.05).In BV-2 microglia,the levels of TNF-α(inflammation marker)and SOD(oxidative stress markers)changed with the increase or decrease of miR-124 expression after transfected with miR-124 mimic or miR-124 inhibitor.Further research found that regulating the expression of miR-124 in lead acetate models could change the expression of signal pathway proteins,including PIK3 CA,PI3K,p-AKT,p-NF-κB p65,NF-κB p65 and Nrf2(P<0.05).Conclusion 1.The results confirmed the importance of selecting suitable reference genes for normalization of target genes to obtain reliable results.The identified reference gene miR-24 and the “optimal combination” were demonstrated to be able to make internal controls for circulating miR-124.Meanwhile,studies have found that circulating miR-124 has good stability and tissue specificity.2.Lead acetate induced rat neurotoxicity via interfering with apoptosis,inflammatory reaction and oxidative stress in brain tissues.Corresponding indexes changes in peripheral blood could evaluate neurotoxicity of lead acetate.However,compared with these indexes,circulating miR-124 could evaluate neurotoxicity with an earlier and more sensitive manner.3.In BV-2 microglia,lead exposure could interfere with cell apoptosis,inflammatory reaction and oxidative stress,which was consistent with the study results in rats.miR-124 participated in lead exposure induced microglial toxicity through regulating of inflammatory response and oxidative stress via NF-κB and Nrf2 signaling pathways.