L-iminoethyl Ornithine Antagonized The Increase Of NO/eNOS Expression Level And Toxicity In Primary Hippocampal Neurons Induced By Excessive Fluorine Exposure In Rats

Objective: For the research of pathogenesis of fluorosis of nerve injury,this experiment on the basis of previous experiments using primary cultured hippocampal neurons,and replication cell model of fluorosis,endothelial nitric oxide synthase(eNOS)inhibitor L-imino ethyl ornithine(L-NIO)on primary fluoride induced cell apoptosis of hippocampal neurons,eNOS and m RNA protein expression and nitric oxide(NO),nitric oxide synthase content(NOS)activity changes affected.Methods:(1)The primary cultured rat hippocampal neurons were identified by immunofluorescence and NeuN and GFAP respectively.(2)Cell toxicity test and drug screening concentration of rat hippocampal neurons cultured rat hippocampal neurons were divided into sodium fluoride(NaF)(0.05,0.1,0.2,0.4 0.5,1,2,4,5 mmol/L)and L-NIO exposure group(1,2,3,4,5,10 ?mol/L)exposure group,after 48 h exposure,the cell Counting Kit-8(CCK-8)determination of cell activity,selected for its concentration NaF and L-NIO.(3)Experimental grouping and method of poisoning: control group(cells without any treatment,the cells with a blank medium);NaF fluoride group: divided into low fluorine group(the concentration of NaF was0.2mmol/L),the high fluoride group(NaF concentration 1mmol/L);L-NIO exposure group(the concentration of L-NIO was 3 ?mol/L);low fluoride group with L-NIO(NaF concentration 0.2 mmol/L,L-NIO concentration is 3 ?mol/L);high fluoride group with L-NIO(NaF concentration 1 mmol/L,L-NIO concentration is 3?mol/L),the culture time is 48 hours.(4)Western blot was used to detect the expression level of eNOS protein in hippocampal neurons of each experimental group;real-time fluorescence quantitative PCR were examined by eNOS in rat hippocampal neurons mRNA expression level;Nitric oxide(NO)content and NOSactivity in the culture medium of hippocampal neurons in rats of each experimental group were detected by nitrate reductase and colorimetric method;detection of cell apoptosis in the hippocampus of rats in each experimental group the rate of flow cytometry;The morphology of hippocampal neurons in each experimental group was observed under the inverted microscope.The ultrastructural changes of hippocampal neurons in each experimental group were observed by transmission electron microscope.Results:(1)The results of immunofluorescence identification of hippocampal neurons in rats: about 90% of the primary cultured hippocampal neurons were identified as neurons.(2)The cytotoxicity test of rat hippocampal neurons showed that the survival rate of rat hippocampal neurons decreased with the increase of NaF and L-NIO concentration.Therefore,the concentration of NaF in subsequent experiments was 0.2 mmol/L and 1 mmol/L,and the concentration of L-NIO was 3?mol/L.(3)Compared with the control group,the cell eNOS protein expression level of high fluorine group was increased,the difference was statistically significant(P<0.05),the cell m RNA expression levels of low fluoride group and high fluoride group were elevated,the difference was statistically significant(P<0.05).Compared with the same concentration of fluoride alone group,the cells eNOS mRNA and protein expression levels of low fluorine group with L-NIO and high fluorine group with L-NIO were lower than that of the low fluoride group and high fluoride group,the difference was statistically significant(P<0.05).Compared with the control group,the NO content and NOS activity in cell culture of low fluoride group and high fluoride group was significantly higher than that of control group,and the NOS activity of L-NIO groupwas lower than the control group,the difference was statistically significant(P<0.05).Compared with the same concentration of fluoride alone group,the content of NO and activity of NOS in cell culture solution of low fluorine group withL-NIO and high fluorine group with L-NIO were lower than those of low fluoride group and high fluoride group,the difference was statistically significant(P<0.05).Compared with the control group,the apoptosis of high fluoride cell rate increased,the difference was statistically significant(P<0.05).Compared with the same concentration of fluoride alone group,the apoptosis rate of low fluorinegroup with L-NIO and high fluorine group with L-NIO were lower than those of low fluoride group and high fluoride group,the difference was statistically significant(P<0.05).(4)Under the transmission electron microscope,the cells crinkled after fluorine staining,the nucleus chromatin was condensed,the coloring was deepened,and the chromatin edge set was seen in some areas.The number of organelles in the cytoplasm decreased with the increase of fluorine concentration,and some mitochondria could be broken or even vacuolated,and the endoplasmic reticulum dilated obviously.Compared with the single fluorine dyed group,the damage of mitochondria and endoplasmic reticulum in the cell was reduced after L-NIO co-culture was added to the cell culture medium.Conclusion: Excessive fluoride can cause ultrastructural damage and increase of apoptosis rate in hippocampus neurons of rats,which may be related to the increase of eNOS protein and mRNA expression level,the increase of NO content and the increase of NOS activity in hippocampus neurons.L-NIO can antagonize ultrastructural damage of rat hippocampal neurons induced by excessive fluoride,increase the expression of eNOS protein and mRNA and increase the rate of apoptosis,and it plays a protective role in fluorosis induced nerve injury.