| ObjectiveLong-term intake of excessive fluoride can be deposited in brain tissues through the blood-brain barrier,resulting in chronic fluorosis and causing central neuropathological changes and cognitive dysfunction.The brain injury caused by chronic fluorosis,especially the changes in the function and molecular level of the hippocampus of brain,is the focus of the research on the mechanism of brain injury caused by chronic fluorosis.At present,oxidative stress theory can provide a more comprehensive and relatively reasonable explanation for the multi-system pathological changes of the body caused by chronic fluorosis.Although the theory is an important supporting theory in the mechanism of brain injury caused by the disease,it still needs to be enriched and further confirmed.In recent years,it has been found that the damage of central nervous system caused by chronic fluorosis is related to the change of autophagy level,which plays a key role in the changed cell signal transduction and cell damage process due to oxidative stress.NRH: quinone oxidoreductase 2(NQO2)is a highly active molecule,which can lead to the production of reactive oxygen species(ROS),aggravate oxidative stress injury and also cause abnormal changes in autophagy.However,in the mechanism of brain injury caused by chronic fluorosis,it is still unclear whether there is a correlation between the high level of oxidative stress and the change of autophagy,and whether there is a key link between these two factors.This topic is mainly concerned the changes of oxidative stress,autophagy and NQO2 in the hippocampus of rat brains caused by chronic fluorosis and their correlations in which whether oxidative stress and autophagy changes are connected through NQO2,so as to explore the occurrence mechanism of brain injury caused by chronic fluorosis.Methods1.Study on rats with chronic fluorosis.(1)Animal model replication:Sprague-Dawley(SD)rats were fed water with different concentrations of fluoride(5,50 or 100 ppm)for 3 months or 6 months,respectively,to replicate the animal model of chronic fluorosis.At the end of the experiment,dental fluorosis was observed,and fluoride contents in blood,urine,bone and brain tissues were determined to evaluate model replication.(2)Animal behavior study: Morris water maze test was used to conduct directional cruise and space exploration experiments to evaluate the effects of long-term fluoride intake on the ability of learning and memory of the experimental animals.(3)Brain histopathology: the general morphological changes of brains were examined by hematoxylin-eosin(HE)staining.Neuronal Nissl staining was used to observe the changes of neuronal Nissl bodies in the hippocampus.(4)Levels of oxidative stress in hippocampus: the content of ROS and malonydialdehyde(MDA)and the activity of superoxide dismutase(SOD)in brain tissues of rats were determined by flow cytometry and biochemical methods.(5)The level of autophagy in the hippocampus: the submicroscopic structure of neurons and the number of autophagosomes in the hippocampus were observed by transmission electron microscopy.Western blotting for the detection of autophagy-related proteins,including mechanistic target of rapamycin(p-m TOR),autophagy related protein 5(ATG5),microtubule-associated proteins 1A/1B light chain 3B(C3-?)and autophagy connectors p62(p62/sequestosome-1,p62)were used in the study.P62 /SQSTM1).The protein expressions of p-m TOR,Atg5 and p62 in the hippocampus of rats were detected by fluorescence immunohistochemistry.(6)Transcriptome and proteome sequencing and bioinformatics analysis of the hippocampal tissues of experimental animals: high-throughput RNA sequencing(RNA-seq)and Tandem Mass spectrometry proteomics(TMT)were used to sequence the hippocampal tissues of rats.Difference analysis and cluster analysis were used to evaluate the effects of high amount of fluoride treatment on the gene and protein levels of the hippocampus.Correlation and intersection analyses were used to obtain the correlated molecules at the gene and protein levels.NQO2,a molecule related to oxidative stress and autophagy,was selected for further study.Transcriptome and proteome sequencing data were confirmed using Quantitative real-time PCR and Parallel reaction monitoring(PRM)proteomics.(7)Correlation between NQO2 expression and autophagy and oxidative stress: Western blot was used to detect the protein expression of NQO2 in animal brain tissues,and Pearson correlation analysis was conducted between NQO2 expression and autophagy related proteins and oxidative stress indexes.2.Correlative mechanism in changed NQO2 level in vitro in cultured SH-SY5 Y cells(humanized neuroblastoma cells).(1)NQO2 expression interfered with RNA interference and small molecule drugs: building a targeted NQO2 in SH-SY5 Y cells interfered by lentivirus transfection or influenced by S29434(NQO2inhibitor)and activator(Menadione)to regulate NQO2 expression in SH-SY5 Y cells exposed to fluoride.(2)Changes of autophagy and oxidative stress after the regulation of NQO2 in SH-SY5 Y cells treated with fluoride: to detect the effects of fluoride treatment on the level of autophagy and oxidative stress under the inhibited or activated state of NQO2.Results1.The rat model with chronic fluorosis successfully established.Different degrees of dental fluorosis appeared in the fluoride exposed group.The fluoride contents in blood,urine,bone and brain tissues were significantly higher than those in the control group,showing a positive correlation with the fluoride doses.2.The decreased ability of learning and memory of rats with chronic fluorosis.The escape latency of rats exposed to fluoride was significantly higher than that of the control group.In generally,the frequency of crossing the platform and the residence time in the target quadrant were decreased.3.Pathological changes of neurons in brain tissues of rats with chronic fluorosis.HE staining showed no significant morphological changes in the hippocampal CA3 region of brain tissues of rats treated with fluoride.Nissl staining showed that the numbers of neuronal Nissl bodies in the hippocampal CA3 region of rats in fluoride treated group were significantly reduced and the staining was shallow.4.The increased level of oxidative stress in brain tissue of rats exposed to fluoride.The contents of MDA and ROS in the hippocampus of rats exposed to fluoride significantly increased,and the activity of SOD obviously decreased.5.The transmission electron microscopy.The cells in the hippocampal CA3 region of rats exposed with fluoride showed the irregular karyotype,the wrinkled and indented nuclear membrane,and the aggregated chromatin.The numbers of autophagosomes in the hippocampus of rats in the fluoride-treated group were significantly increased,but the increase was not observed with the extension of fluoride-treated time.6.Changes in the expression of LC3 II and p62 protein levels in the hippocampus of rats with chronic fluorosis.P-m TOR expression was decreased,while Atg5,LC3 II and p62 expressions were increased,suggesting autophagy flow block and abnormal autophagy level.7.Transcriptomics and proteomics sequencing results.Compared with the control group,the differentially expressed genes were mainly concentrated in the cognitive function,learning and memory ability,long-term synergism and autophagy regulatory signaling pathways.Combined transcriptome and proteomics analysis 13 differential genes with the same trend of change were revealed,among which NQO2 expression showed the highest difference.8.Changes in NQO2 protein expression in brains of rats with chronic fluorosis.Western blot and immunofluorescence histochemical results showed that NQO2 was highly expressed in the hippocampus of rats with chronic fluorosis,which was positively correlated with the fluoride dose.The expression level of NQO2 was significantly correlated with the changes of autophagy-related proteins and oxidative stress.9.The NQO2 regulatory mechanism in cultured cells in vitro.After fluoride exposure,the expressions of LC3 II and p62 were increased,suggesting autophagy flow block and abnormal autophagy level.MDA and ROS contents were significantly increased and SOD activity was decreased,indicating the elevated level of oxidative stress.The expression of NQO2 was regulated by lentivirus interference and specific treatment of small molecule compounds.The results suggested that inhibition of NQO2 expression could restore the level of autophagy flow and reduce the effect of oxidative stress.Conclusions1.Decrease of learning and memory ability of rats with chronic fluorosis may be related to increased oxidative stress level and abnormal autophagy function in the hippocampus.The chronic fluorosis rats showed high content of ROS and MDA in brain tissue.The autophagy initiating protein,p-m TOR,was decreased;the autophagosome-forming related proteins,Atg5 and LC3 II,were increased;and the autophagy lysosomal functional related protein,p62,was increased.2.The cognition impairment of rats with chronic fluorosis was closely related to gene and protein changes in the hippocampus.Differentially expressed genes were mainly clustered in the signaling pathways related to learning and memory ability and brain rewarding.Some of the cognitive-related genetic changes persist and cannot be reversed after long-time fluoride exposure.3.The increase of NQO2 in the hippocampus of rats with chronic fluorosis may promote the increase of oxidative stress level and abnormal autophagy function: the increase of NQO2 was related to the dose of fluoride,and it was closely related to the abnormal autophagy function and the up-regulated level of oxidative stress.4.Fluoride could promote autophagy initiation,which was a compensatory protective mechanism.However,with the accumulation of damage,autophagic flux was blocked,and the function of autophagy clearance was disturbed.However,knockdown of NQO2 could reduce the level of oxidative stress and restore autophagic flux,suggesting that NQO2 may be the critical factor regulating oxidative stress and autophagy function in the brain. |
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