The Role Of SIK2-regulated CRTC1/BDNF/VGF Pathway In Fluoride-induced Neurobehavioral Abnormalities In Offspring Rats

ObjectivesWith the in-depth study of the molecular mechanisms of fluoride neurotoxicity,the damage to the structure and function of nervous system caused by excessive fluoride exposure has become a research hotspot.Childhood is in the stage of rapid brain development and is more sensitive to fluoride exposure which may cause damage to the brain even at lower concentrations.Studies have found that excessive fluoride exposure may cause abnormal neurobehavior in children,leading to behavioral problems such as attention deficit hyperactivity disorder,psychosomatic problems,learning and memory disorders.However,the specific mechanism of these changes caused by fluoride exposure has not been fully elucidated.Studies have found that the CRTC1/BDNF/VGF signaling pathway is closely correlated to multiple neurobehavioral abnormalities.However,is this pathway a key pathway for neurobehavioral abnormalities caused by fluoride exposure? Meanwhile,it is unclear whether SIK2,which is the upstream of CRTC1 molecule,plays a role in fluoride-induced neurobehavioral changes by regulating the CRTC1/BDNF/VGF pathway.This study aimed to explore the role of SIK2-regulated CRTC1/BDNF/VGF pathway changes in fluoride-induced neurobehavioral abnormalities in rats through in vivo and in vitro experiments,and provide new clues for studying of the neurotoxicity mechanism of fluoride.Materials and Methods1.Construction and measurement of in vivo model of rat neurobehaviorHealthy adult SPF Sprague-Dawley(SD)rats were selected to construct an in vivo fluorosis model.After one week of adaptive feeding,they were randomly divided into 4 groups,including 1 control group and 3 exposure groups(Na F concentration of 10 mg/L,50 mg/L and 100 mg/L,respectively),and the exposure began on the day of grouping.The rats were exposed to Na F for 1 week and then mated.Female rats with vaginal embolisms were considered pregnant,and the female rats was close to delivery were kept in separated cages.When being weaned on PND21,fifteen offspring male rats with similar birth dates and weights in each group were randomly selected as the experimental rats for the study,and were continued to be exposed according to the parental exposure concentration,and the exposure was ended on PND 90.Through open field test,elevated plus maze,forced swimming test and tail suspension experiment,the influence of fluoride exposure on rat behavior was detected.2.Fluoride-induced pathological changes in rat brain tissueHE staining was used to observe the pathological changes of rat hippocampus and prefrontal cortex tissue structure and cell morphology caused by exposure to different concentrations of fluoride.3.Construction of in vitro model and screening of fluoride exposure doseHighly differentiated rat adrenal medullary pheochromocytoma cells(PC12 cells)were used to establish an in vitro model of fluoride exposure,the cell viability exposed by different concentrations of Na F(0,10,20,40,80,100,120 mg/L)was detected by CCK8 to determine the subsequent exposure concentration.After 12 h of transfection with 50μM SIK2 small interfering RNA(SIK2-si RNA),PC12 cells were treated with Na F(80 mg/L)for 24h;after 6h of pretreatment with 5μM7,8-dihydroxyacetone(7,8-DHF),PC12 cells were treated with Na F(80 mg/L)for24h.4.Effect of fluoride exposure on the expression of SIK2-VGF pathway related genesThe real-time fluorescent quantitative PCR(RT-PCR)and Western Blot were used to respectively detect the m RNA transcription levels and protein expression levels of SIK2,CRTC1,BDNF and VGF genes in rat hippocampus,medial prefrontal cortex and PC12 cells.The distribution of the CRTC1 and BDNF proteins in hippocampus and prefrontal cortex were observed by immunofluorescence.5.The effect of fluoride exposure on cell apoptosisWestern Blot was used to detect the protein expression levels of apoptotic protein cleaved Caspase 3 in rat hippocampus,medial prefrontal cortex and PC12 cells.FITC/PI double staining combined with flow cytometry was used to detect the effect of fluoride exposure on PC12 cells apoptosis.The distribution of the cleaved Caspase 3 protein in hippocampus and prefrontal cortex was observed by immunofluorescence.6.Statistical analysisThe Excel 2016 software was used for data recording,the Image J software was used for quantitative analysis of protein expression,and SPSS 21.0 and Graph Pad Prism 6.0 were used for statistical analysis and graphing statistics,respectively.One-way ANOVA was used for continuous data comparison between multiple groups,and Dunnett t-test or Least-Significant Difference(LSD)was used for comparison between the two groups.The test level was α=0.05.Results1.Neurobehavioral changes in rats exposed to fluorideIn the open field test,the total activity distance of the male offspring rats in the50 mg/L Na F and 100 mg/L Na F groups was respectively lower than that of the control male rats,and the central distance and central exploration time of the rats in fluoride exposure group were significantly lower than the control group(P<0.05,respectively);In the elevated plus maze,compared with the control group,the rats in each fluoride group had significantly reduced distance of movement in the open arm,the residence time in the open arm,and the number of times to enter the open arm(P<0.05,respectively);In the forced swimming test,compared with the control group,the resting time of the rats in each fluoride exposure group was significantly increased,and the swimming time was significantly decreased(P<0.05,respectively);In the tail suspension experiment,compared with the control group,the resting time of rats in each fluoride exposure group was significantly increased,and the struggling time was significantly reduced(P<0.05,respectively).2.Pathological changes of brain tissue in rats exposed to fluorideThe results of HE staining showed that the nerve cells in the hippocampus and prefrontal cortex of the control rats had complete structures and clear outlines.With the increase of fluoride exposure dose,the number of nerve cells was decreased and sparse,the arrangement was irregular and disordered,and the degree of interstitial edema and looseness were increased.3.Effect of fluoride exposure on the expression of SIK2-VGF pathway related genesIn the hippocampus and medial prefrontal cortex of rats,the m RNA and protein expression levels of CRTC1,BDNF and VGF in the 50 mg/L and 100 mg/L fluoride exposure groups were significantly lower than those in the control group,and the m RNA and protein level of SIK2 was significantly higher than that in the control group(P< 0.05);In PC12 cells,compared with the control group,the m RNA and protein level of SIK2 in the 40 mg/L and 80 mg/L fluoride exposure groups were significantly increased;the m RNA and protein expression levels of CRTC1,BDNF and VGF were significantly decreased(P<0.05).After pre-intervention of SIK2-si RNA,compared with the Na F+vector group,the SIK2 protein level was significantly reduced,and the CRTC1,BDNF and VGF protein levels were significantly increased(P<0.05).After pre-intervention with 7,8-DHF,the expression level of VGF was significantly higher than that of the Na F alone group(80mg/L Na F)(P<0.05).Immunofluorescence experiments showed that the protein expression levels of BDNF in the cytoplasm and CRTC1 in the nucleus of the rat hippocampus and the medial prefrontal cortex were gradually decreased with the increase of fluoride exposure.The protein expression level of CRTC1 in the nucleus of PC12 cells were also gradually decreased with the increase of fluoride exposure.4.The effect of fluoride exposure on cell apoptosisThe results of Western Blot showed that compared with the control group,with the increase of Na F exposure dose,the protein levels of cleaved Caspase 3 were significantly increased in hippocampus,prefrontal cortex and PC12 cells(P<0.05,respectively).Immunofluorescence experiments showed that the protein expression levels of cleaved Caspase 3 of the rat hippocampus and the medial prefrontal cortex were gradually increased with the increase of fluoride exposure.After pre-intervention of SIK2-si RNA,compared with the Na F+vector group,the protein levels of cleaved Caspase 3 were significantly reduced(P<0.05).After pre-intervention with 7,8-DHF,the expression level of cleaved Caspase 3 was significantly lower than that of the Na F alone group(80mg/L Na F)(P<0.05).The apoptosis results showed that with the increase of the exposure dose,the apoptosis rate of PC12 cells was significantly increased(P<0.05).Compared with the alone fluoride-exposed group alone,the apoptosis level was significantly reduced in fluoride-treated group after SIK2-si RNA or 7,8-DHF intervention(P<0.05).Conclusions1.Excessive fluoride exposure may cause pathological changes in hippocampus and medial prefrontal cortex,leading to abnormal neurobehavior in rats.2.Fluoride exposure regulates CRTC1/BDNF/VGF pathway to induce cell apoptosis by changing the expression of SIK2,playing an important role in fluoride-induced neurobehavioral abnormalities in rats.