The Effects Of Maternal Cadmium Exposure For The Influence Of The Cognitive Ability Of Offspring

Objective:Cadmium（Cd）is a heavy metal with strong toxicity and long-term exposure can cause neurological dysfunction,and Cd exposure can impair the development of the central nervous system and affect the cognitive ability of children.However,the molecular mechanisms of cadmium effects on maternal cognition and the effects of maternal cadmium exposure on the cognitive ability of the offspring are still controversial.In this study,we investigated the effects of cadmium exposure on hippocampal tissues of two generations of rats by constructing a maternal rat cadmium exposure model and breeding the littermates.The study used Morris water maze,gene chip technology combined with bioinformatic analysis and various algorithms to investigate the molecular mechanisms of cadmium effects on hippocampal tissues of maternal rats and the effects of maternal cadmium exposure on the cognitive ability of littermates and possibly their possible molecular mechanisms.Methods.Chapter 2:Experimental SD（Sprague-Dawley）rats were randomly divided into three groups and given 6.6 mg/kg Cd Cl₂（high dose）,3.3 mg/kg Cd Cl₂（low dose）and saline for 2,4 and 8 weeks,respectively,followed by execution of the rats,rapid clamping of the skull,stripping of the meninges and removal of hippocampal tissues for cadmium content identification.Subsequently,hippocampal tissues from high-dose and control rats gavaged for 8 weeks were selected to obtain m RNA expression profile data by memory gene microarray technology,and m RNAs most associated with cadmium exposure were screened using weighted gene co-expression network analysis（WGCNA）method.DAVID online tool was used to perform GO（geneontology）and KEGG pathway enrichment analysis to find the possible pathways associated with cadmium neurotoxicity,and multiple algorithms were used to find overlapping genes and target key genes.Chapter 3:Twelve experimental SD rats were prepared,six in each group,half male and half female.Rats in the cadmium gavage group were given 6.6 mg/kg of Cd Cl₂,and rats in the control group were given saline by gavage for 8 weeks.Then the rats in the same group were caged together as males and females and conceived freely,and the experimental rats that were determined to be conceived were separated and given normal water and free food.After natural delivery of the female rats,24offspring（half male and half female）were randomly selected from each group and fed until the litter was born 6 weeks later,and Morris water maze behavioral experiments were performed.Chapter 4:Detection of lnc RNA（long non-coding RNA）and m RNA（Messenger RNA）gene expression in hippocampal tissues of rat offspring from Cd Cl₂ dams exposed to 6.6 mg/kg of cadmium and control（saline）rat litters using gene microarray technology.The differentially expressed genes were screened by differential expression analysis methods and combined with bioinformatics techniques,GO analysis and Pathway analysis were performed on the differentially expressed genes.Subsequently,various algorithms of the cyto Hubba APP plug-in were used to calculate possible overlapping genes,and the expression levels of the screened genes associated with cadmium neurodevelopmental toxicity were verified by RT-q PCR（Quantitative Real-time PCR）and WB（Western Blotting）.Results.1.by long-term cadmium gavage was able to cause cadmium to pass through the rat blood-brain barrier and enter hippocampal tissue.cadmium levels in hippocampal tissue were significantly elevated in cadmium-gavaged rats versus normal rats（P≤0.05）.m RNA expression in hippocampal tissue of cadmium-exposed rats was analyzed by gene microarray combined with weighted gene co-expression network analysis.A total of 34 modules were generated as a result.The most relevant module for the maternal cadmium exposure phenotype was"lightgreen",and the results listed the top 30 items of the GO analysis and KEGG pathway in"lightgreen",which involved The results listed the top 30 genes of GO analysis and KEGG pathway in"lightgreen",among which"endocytosis"and"transcriptional dysregulation in cancer"were more relevant,and the Pmpcb gene was suggested to be the key gene of cadmium effect on maternal rats by various algorithms.2.After 8 weeks of cadmium gavage,the rats were caged together and mated normally,and the littermates were reared for 6 weeks,and the behavioral performance of the littermates in the locomotor navigation experiment,the spatial search experiment and the working memory experiment were compared and analyzed with and without cadmium exposure in the maternal rats.The results showed that in the locomotor navigation experiment,the littermates of cadmium-exposed rats had to swim longer distances to find the platform compared with the control group（P≤0.05）;the latency period of the littermates of cadmium-exposed rats was longer than that of the control group as the number of training days increased,implying that the littermates of cadmium-exposed rats had to take longer time to find the platform compared with the control group as the training increased（P≤0.05）.In the spatial search experiment,the percentage of the distance of stay in the correct quadrant and the time of stay in the correct quadrant were shorter in the pups of Cd-exposed rats than in the control rats（P≤0.05）.The mean swimming speed of Cd-exposed rats was faster（P≤0.05）and the total swimming distance was longer（P≤0.05）than that of the control rat littermates.In the final working memory experiment,the cadmium-exposed rat littermates showed more panic in the water,swam faster（P≤0.05）,and often spun in place.3.The hippocampal tissues of the rat littermates were taken,and the gene microarray technique was used to confirm the existence of significant changes in the expression profiles of lnc RNAs and m RNAs in the hippocampal tissues of cadmium-exposed rats.147 lnc RNAs and 191 m RNAs were identified to be differentially expressed after cadmium neurotoxicity.Enrichment analysis of differentially expressed m RNAs revealed that differentially expressed genes were significantly enriched in metabolic pathways,pancreatic secretion,protein digestion and absorption,and S.aureus infection pathways.Interestingly,the most important pathway in the KEGG pathway enrichment analysis was"pancreatic secretion".Cpa1and Prss1 were identified as Hub genes using different algorithms for screening.The results of RT-q PCR and WB experiments were consistent with the gene chip results.Conclusion.It was shown that long-term cadmium gavage can make cadmium enter the hippocampal tissue through the blood-brain barrier and affect the altered m RNA expression in both maternal and littermate rats,and it has some effects on the cognitive development of the littermate,leading to the decrease of learning and memory ability in the offspring rats.Cd exposure in maternal rats resulted in alteration of"endocytosis"and"transcriptional dysregulation in cancer"pathways in the hippocampus of maternal generation,and the key gene might be Pmpcb.Cd exposure in the mother rats can lead to alterations in the"pancreatic secretion"pathway in the offspring,which can reduce the expression of key genes Cpa1 and Prss1 genes and proteins in the hippocampus of the offspring rats.The target genes and signaling pathways studied in this work will help to explore the mechanism of cadmium neurotoxicity in the litter and provide intervention pathways and targets to improve neurotoxicity.