MiR-199a-5p Regulates Endoplasmic Reticulum Stress In Cadmium-induced Learning And Memory Disfunction

Background:Cadmium（Cd）,a stable and toxic heavy metal,is a major environmental pollutant harmful to human health due to its long half-life（20-30years）,strong bioaccumulation and widespread existence in the natural environment.Cd gets into the body mainly through ingestion,inhalation and skin contact,causing multiple organ toxicity.Studies have found that Cd exposure can change the permeability of the blood-brain barrier and enter the central nervous system through the blood-brain barrier to cause neurotoxicity,but the specific toxic mechanism is unclear.Endoplasmic reticulum stress（ERS）is an important adaptive defense mechanism in cells,which plays an important role in restoring protein folding disorder induced by exogenous harmful factors.However,prolonged or intense ERS can activate apoptosis-related gene-related genes and mediate cell death.micro RNA is a class of endogenous non-coding short RNA,which has been found to be closely related to ERS.In addition,dysregulation of micro RNA expression is often associated with liver and kidney injury induced by cadmium exposure.However,there is no systematic study on whether heavy metal Cd exposure can induce ERS by regulating micro RNA expression,thus causing nerve damage and affecting cognitive function.Objective:In this study,the effects of long-term oral exposure of heavy metal Cd on cognitive impairment in mice were revealed through animal experiments,and the role of miR-199a-5p in regulating endoplasmic reticulum stress in cadmium exposure-induced neurotoxicity was verified through in vitro experiments.Methods:This study was performed at animal and cellular levels.In vivo,16 SPF C57BL/6 mice were randomly selected and divided into 2 groups:control group（free drinking water）;Cd group（3 mg/kg/day,CdCl₂）.8 mice in each group were exposed for 20 weeks,and their body weight was recorded weekly.After 20 weeks Cd treatment,Open Field（OF）and Morris Water Maze（MWM）test were used to detect motor ability and learning and memory ability of mice.The morphology and distribution of neurons in the hippocampal brain tissues of mice were observed by H&E staining and Nissl staining,and the expression level of miR-199a-5p in the hippocampal tissues was detected by QPCR.The mRNA and protein expressions of ERS-related proteins GRP78,IRE1α,p-IRE1α（S724）and Caspase-12 were detected by QPCR and Western Blot.In vitro,human neuroblastoma cells（SH-SY5Y）were treated with different concentrations of CdCl₂（0μM,2.5μM,5μM,10μM,20μM）for24h.Cell viability was detected by CCK8 assay.Annexin V-APC/7AAD assay was used to detect the number of apoptosis cells,and QPCR was used to detect the expression level of miR-199a-5p in hippocampus tissues.The mRNA and protein expressions of ERS-related proteins GRP78,IRE1α,p-IRE1α（S724）and Caspase-12were detected by QPCR and Western Blot,so as to evaluate the damage of SH-SY5Y cells,miR-199a-5p and endoplasmic reticulum stress levels after Cd exposure.Then,lentivirus-infected SH-SY5Y cells were screened to construct stably overexpressed miR-199a-5p cell lines.After 24 hours of stably overexpressed miR-199a-5p cell lines were treated with 10μM CdCl₂,cell viability,apoptosis level,and expression of ERS-related proteins were detected to clarify the role of miR-199a-5p in regulating endoplasmic reticulum stress in cell injury of SH-SY5Y cells induced by Cd exposure.Results:Compared with control mice,mice exposed to Cd showed decreased spatial learning and memory ability,abnormal hippocampal structure,and significantly reduced number of neurons.In addition,the expression level of miR-199a-5p decreased,the expression of ERS-related proteins such as GRP78,IRE1αand p-IRE1α（S724）increased abnormally,and the expression level of Caspase-12 increased.At the cell level,with the increase of Cd treatment time and concentration,the decrease of cell viability appeared dose-dependent effect.The optimal concentration and time of 10μM 24h CdCl₂ treatment were selected to conduct toxicological mechanism study,and it was found that:Compared with the negative control group,the decreased cell viability and increased apoptosis level of miR-199a-5p overexpressed cells were significantly alleviated after CdCl₂ treatment,and the ERS level was also significantly decreased.Conclusion:Long-term Cd exposure down-regulates the level of miR-199a-5p in hippocampal tissue,leading to increased endoplasmic reticulum stress intensity of neurons and inducing apoptosis,thus causing nerve damage and affecting the learning and memory ability of mice.