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1.
Chinese Journal of Endemiology ; (12): 781-789, 2021.
Article in Chinese | WPRIM | ID: wpr-909097

ABSTRACT

Objective:To observe the expression levels of glial fibrillary acidic protein (GFAP), β-tubulin Ⅲ and synaptophsin, and explore the role of tripartite synapse in the mechanism of central nervous system (CNS) injury and the neuroprotective effect of chondroitin sulfate (CS).Methods:One month old clean grade, 48 female Sparague-Dawley rats and 48 male Sparague-Dawley rats, were randomly divided into 8 groups according to body weight (90 - 120 g) by random number table method, with 12 rats in each group, half male and half female. These rats were fed with water containing different concentrations of sodium fluoride (NaF) [ < 0.5 mg/L (control, CN), 10.0 mg/L (low dose fluoride, LF) and 50.0 mg/L (high dose fluoride, HF)]. Some rats were fed directly for 185 days (CN, LF and HF groups). In addition, rats of CN + normal saline (NS), LF + NS, HF + NS groups and LF + CS, HF + CS groups, were intraperitoneally injected with NS or 0.66 mg/kg CS for 5 consecutive days after 180 days of feeding. After the experiment, the pathological changes of hippocampal CA4 of brain tissue in each group were observed by hematoxylin eosin staining under light microscope, and the expression and distribution of GFAP, β-tubulin Ⅲ and synaptophsin in hippocampal CA4 of rats were detected by immunohistochemistry, the expression of GFAP, β-tubulin Ⅲ and synaptophsin at protein level in hippocampus of rats were detected by Western blotting.Results:Under light microscope, eosinophilic change, loss and irregular arrangement of neuron in the hippocampal CA4 were observed in LF, HF, LF + NS and HF + NS groups. The morphology of LF + CS and HF + CS groups was not significantly changed compared with CN group, but was significant changed compared with LF, HF, LF + NS and HF + NS groups. Immunohistochemical results showed that the rates of positive area of GFAP, β-tubulin Ⅲ and synaptophsin in female and male rats in LF and HF groups were significantly decreased than those in CN group ( P < 0.05); the positive area rates of female and male rats in LF + CS and HF + CS groups were higher than those in LF and HF groups, respectively ( P < 0.05). Western blotting results showed that the proten expression levels of GFAP, β-tubulin Ⅲ and synaptophsin of female and male rats in LF and HF groups (LF group: 0.90 ± 0.09, 0.82 ± 0.08, 1.43 ± 0.14, 0.92 ± 0.02, 1.21 ± 0.15, 0.87 ± 0.02, HF group: 0.58 ± 0.14, 0.73 ± 0.03, 0.63 ± 0.06, 0.67 ± 0.03, 0.87 ± 0.04, 0.70 ± 0.05) were lower than those in CN group (1.24 ± 0.08, 1.09 ± 0.10, 2.64 ± 0.30, 1.54 ± 0.09, 1.72 ± 0.10, 1.13 ± 0.06, P < 0.05). Conclusions:The tripartite synapse and extracellular matrix may take part in pathogenesis of the damages of CNS results from chronic fluorosis; CS may reduce the injury to a certain extent.

2.
Article in Chinese | WPRIM | ID: wpr-883671

ABSTRACT

Objective:To explore the DNA methylation patterns and methylation differential genes of patients with coal-burning-borne endemic fluorosis, and to provide a basis for study of the pathogenesis of fluoride-induced body injury.Methods:A case-control study was conducted in Shuicheng County, Liupanshui, Guizhou Province, ten patients with severe fluorosis were selected as the fluorosis group in Douqing Township, where people burning high fluorine coal in open range all year round; and ten people without fluorosis phenotype were selected as the control group in Huaga Township, where firewood was the main fuel. Peripheral blood samples were collected from the two groups of people. Reduced representation bisulfite sequencing (RRBS) technique was used to detect the whole genome DNA methylation pattern ( n = 4) and DNA differentially methylated region (DMR), the DMR differential degree (log 2Ratio) and KEGG pathway enrichment analysis were used to screen the methylation differential genes, and real-time PCR was used to verify the mRNA expression levels of the candidate methylation differential genes( n = 10). Results:The methylation pattern analysis results showed that the methylation levels of all C bases in the genome DNA of the fluorosis group and the control group were (61.53 ± 0.59)% and (62.48 ± 1.53)%, respectively; among them, the methylated levels at CG sites were (63.75 ± 0.65)%, (64.36 ± 1.01)%, at CHG sites were (13.79 ± 0.72)%, (16.69 ± 4.06)%, and at CHH sites were (25.12 ± 1.72)%, (29.77 ± 3.97)%. Compared with the control group, patients in the fluorosis group had 1 000 DMR distributed on different autosomes; and the chromosome 19 was the most with 104 segments. There were 978 DMR-related genes, including 265 hypermethylation genes and 713 hypomethylation genes; KEGG pathway enrichment analysis showed that methylation differential genes were mainly involved in cell metabolism, cancers, and phosphatidylinositol 3-kinase-protein kinase B (PI3K-AKT) and other signaling pathways; combined with the differential degree of DMR, the hypermethylated succinate dehydrogenase complex flavoprotein subunit A pseudogene 3 (SDHAP3, log 2Ratio = 3.487) and hypomethylated nuclear factor κB inhibitor kinase regulatory subunit γ (IKBKG, log 2Ratio =-4.436) were selected as the candidate genes. There were statistically significant differences in the mRNA expression levels of SDHAP3 (0.54 ± 0.08, 1.00 ± 0.00) and IKBKG (1.32 ± 0.39, 1.00 ± 0.00) between fluorosis group and control group ( F = 22.94, 15.09, P < 0.01 or < 0.05). Conclusion:There are a large number of methylation differential genes in the genomes of patients with coal-burning-borne endemic fluorosis and controls, the hypermethylated SDHAP3 and hypomethylated IKBKG may be involved in fluoride induced body injury.

3.
Chinese Journal of Endemiology ; (12): 446-452, 2019.
Article in Chinese | WPRIM | ID: wpr-753522

ABSTRACT

Objective To study the mechanism of central nervous system (CNS) injury in chronic fluorosis and the neuroprotective effect of chondroitin sulfate (CS).Methods Forty-eight female Sprague-Dawley rats weighting 90-120 g were divided into 8 groups according to body weight by random number table,6 rats in each group:control group,drinking tap water freely;low dose and high dose fluoride groups,freely drinking tap water with fluoride content of 10 and 50 mg/L,respectively;control + normal saline (NS),low dose fluoride + NS,and high dose fluoride + NS groups,each group was fed for 180 d,and treated with intraperitoneal injection of 0.66 mg/kg NS for 5 d (once a day);low dose fluoride + CS and high dose fluoride + CS groups,each group was fed for 180 d,0.66 mg/kg CS was injected intraperitoneally for 5 d (once a day).All groups were fed standard nutritive animal feed for 185 d and dissected for brain tissue.The pathologic change was observed after hematoxylin-eosin (HE)staining;the expression levels of phosphorylated extracellular signal-regulated protein kinase 1/2 (phospho-Erk1/2)and glutamate receptors 1,2 (GluR1,GluR2) in the brain cortex were detected by immunohistochemistry;the protein levels of Erk1/2,phospho-Erk1/2,GluR1,and GluR2 in the brain cortex were detected by Western blotting.Results Brain cortex of all rats in the fluoride groups showed eosinophilic degeneration,loss and disordered arrangement of neurons,and the brain morphological changes in each fluoride + CS groups were significantly improved compared with those in the fluoride groups.Immunohistochemistry results showed that compared with the control group [(0.44 ± 0.09)%,(1.49 ± 0.05)%,(2.51 ± 0.54)%],the expression levels of phospho-Erk1/2 [(1.47 ±0.09)%,(1.03 ± 0.05)%],and GluR2 [(2.37 ± 0.06)%,(3.38 ± 0.12)%] in the low dose and high dose fluoride groups were increased,and the expression levels of GluR1 [(1.49 ± 0.02)%,(0.99 ± 0.19)%] were decreased (P < 0.05).Western blotting results showed that compared with the control group (1.00 ± 0.12,1.76 ± 0.33),the protein levels of Erk1/2 (3.10 ± 0.76,1.99 ± 0.01) and phospho-Erk1/2 (3.27 ± 0.25,2.67 ± 0.05) in low dose and high dose fluoride groups were significantly increased (P < 0.05);compared with low dose fluoride group,the protein levels of Erk1/2,and phospho-Erk1/2 (1.30 ± 0.31,2.20 ± 0.34) in low dose fluoride + CS group decreased significantly (P <0.05).Compared with control group (1.86 ± 0.47,1.17 ± 0.27),the protein levels of GluR1 (1.09 ± 0.26,0.61 ± 0.14) in low dose and high dose fluoride groups decreased significantly,while the protein level of GluR2 (1.99 ± 0.42,3.38 ±0.27) increased significantly (P < 0.05);compared with low dose and high dose fluoride groups,the protein levels of GluR2 in low dose fluoride + CS and high dose fluoride + CS groups (1.53 ± 0.41,2.65 ± 0.32) decreased significantly (P < 0.05).The protein level of phospho-Erk1/2 was negatively correlated with GluR1 protein level (r =-0.975,-0.991,P < 0.05) in low dose and high dose fluoride groups,and it was positively correlated with the protein level of GluR2 (r =0.986,0.993,P < 0.05).Conclusion The CNS injury caused by chronic fluorosis may be related to GluR1 and GluR2 activated Erk1/2 signaling pathway,and CS has certain protection to the injury.

4.
Chinese Journal of Endemiology ; (12): 455-460, 2018.
Article in Chinese | WPRIM | ID: wpr-701353

ABSTRACT

Objective To investigate the effects of endothelial nitric oxide synthase (eNOS) specific inhibitor L-iminoethyl ornithine hydrochloride (L-NIO) for all the fluorine in vitro cultivation of SH-SY5Y cells apoptosis,eNOS mRNA and protein expression,and effect of nitric oxide (NO) content and nitric oxide synthase (NOS) activity change.Methods The SH-SY5Y cells cultured in vitro were divided into control group,low fluoride group,high fluoride group,L-NIO group,low fluoride with L-NIO group,high fluoride with L-NIO group,n =3.The control group added equal volume culture liquid with the experimental group,the concentration of sodium fluoride (NaF) in the low fluoride group and the high fluoride group were 0.2 and 2.0 mmol/L,respectively.The L-NIO group added 3 μmol/L L-NIO,the low fluoride with L-NIO group and the high fluoride with L-NIO group were added to 0.2 mmol/L NaF and 3 μmol/L L-NIO,2.0 mmol/L NaF and 3 μmol/L L-NIO,respectively.The incubation time was 48 h.The expression level of eNOS protein in cells was detected by Western blotting.The expression level of eNOS mRNA in cells was detected by Real-time fluorescence quantitative PCR method.The apoptosis of cells was detected by flow cytometry,NO content and NOS activity in cell culture liquid were detected by nitrate reductase and colorimetric assay.Results Compared with the control group (1.000 ± 0.026),the expression of eNOS protein in the low and high fluoride groups (1.108 ± 0.071,1.349 ± 0.057) increased (P < 0.05),and the L-NIO group (0.755 ± 0.148) decreased (P < 0.05);compared with the low fluoride group,the high fluoride group increased and the low fluoride with L-NIO group (0.802 ± 0.115) decreased (P < 0.05);compared with the high fluoride group,high fluoride with L-NIO group (0.988 ± 0.135) decreased (P < 0.05).Compared with the control group (1.000 ±0.018),the expression of eNOS mRNA in the low and high fluoride groups (1.809 ± 0.099,2.416 ± 0.295) increased (P < 0.05),the L-NIO group (0.609-± 0.077) decreased (P < 0.05);compared with the low fluoride group,the high fluoride group was elevated(P < 0.05),the low fluoride with L-NIO group (1.040 ± 0.034) decreased (P < 0.05);compared with the high fluoride group,high fluoride with L-NIO group (1.233 ± 0.152) decreased (P < 0.05).Compared with the control group [(1.66 ± 0.07)%],the cell apoptosis rate in the low and high fluoride groups [(8.81 ± 0.27)%,(17.60 ± 0.20)%] increased,L-NIO group [(1.03 ± 0.04)%] decreased (P < 0.05);compared with the low fluoride group,the high fluoride group increased and the low fluoride with L-NIO group [(6.03 ± 0.10)%] decreased (P < 0.05);compared with the high fluoride group,the high fluoride with L-NIO [(12.12 ± 0.08)%] decreased (P < 0.05).Compared with the control group [(2.773 ± 0.145)μmol/L],the content of NO in the cell culture medium in the low and high fluoride groups [(8.251 ± 1.047),(14.287 ± 1.062) μmol/L] increased (P< 0.05),and the L-NIO group [(1.648 ± 0.155) μmol/L] decreased (P < 0.05);compared with the low fluoride group,the high fluoride group was elevated (P < 0.05),the low fluoride with L-NIO group [(4.622 ± 0.252) μmol/L] decreased (P < 0.05);compared with the the high fluoride group,high fluoride with L-NIO group [(7.899 ± 0.385) μmol/L] decreased (P < 0.05).Compared with the control group [(0.507 ± 0.041) U/ml],the activity of NOS in the cell culture medium in the low and high fluoride groups [(0.772 ± 0.032),(2.258 ± 0.062) U/ml] increased (P < 0.05),and the L-NIO group [(0.346 ±0.015) U/ml] decreased (P < 0.05);compared with the low fluoride group,the high fluoride group was elevated (P <0.05),the low fluoride with L-NIO group [(0.637 ± 0.026) U/ml] decreased (P < 0.05);compared with the the high fluoride group,high fluoride with L-NIO group [(1.161 ± 0.071) U/ml] decreased (P < 0.05).Conclusions Excessive fluoride can lead to overexpression of eNOS protein and mRNA in SH-SY5Y cells,increase of apoptosis rate,increase the content of NO in cell culture and enhance the activity of NOS.After co-culture of L-NIO and fluorine,it can antagonize the damage of fluorine to SH-SY5Y cells and play a certain neuroprotective effect.

5.
Chinese Journal of Endemiology ; (12): 450-454, 2018.
Article in Chinese | WPRIM | ID: wpr-701352

ABSTRACT

Objective To detect the expression of peroxisome proliferator-activated receptor γ (PPARγ) in the brains of rats with chronic fluorosis and elucidate the relationship between PPARγand oxidative stress in chronic fluorosis.Methods According to body weight (100-120 g),sixty healthy SD rats were divided into control group (less than 0.5 mg/L fluoride in drinking water),low fluoride group (5.0 mg/L fluoride in drinking water,prepared by NaF),and high fluoride group (50.0 mg/L fluoride in drinking water) via the random number table method,20 rats in each group (half male and half female).The experiment periods were 3 and 6 months,respectively.Then 24-hour urine samples of rats were collected from each group,all rats were put to death and brain tissues were taken.The fluoride contents in urine and brain tissue were measured with fluoride-ion selective electrode;the levels of PPARγ protein and mRNA in the cortex and hippocampus were determined by Western blotting and Real-time fluorescence quantitative PCR,respectively;and the activities of superoxide dismutase (SOD) and level of malondialdehyde (MDA) in serum were detected by xanthine oxidase method and thiobarbituric acid method;the correlation between PPARγ protein expression and oxidative stress was analyzed.Results After 3 and 6 months of treatment,the contents of fluoride in urine and brain in low fluoride group [(1.57 ± 0.18) mg/L,(3.43 ± 0.70) μg/g;(1.79 ± 0.17) mg/L,(7.40 ± 1.21) μg/g] were higher than those of control group [(1.11 ± 0.17) mg/L,(2.39 ± 0.50) μg/g;(1.02 ± 0.15) mg/L,(2.87 ± 0.82) μg/g,P < 0.05],and the values in high fluoride group [(1.91 ± 0.23) mg/L,(6.70 ± 0.87) μg/g;(2.44 ± 0.51) mg/L,(12.10 ± 1.30) μg/g] were significantly higher than those in low fluoride group (P < 0.05).In high fluoride group after 3 months of treatment,the expression of PPARγprotein [(79.00 ± 3.46)%,(80.35 ± 2.50)%] and mRNA [(79.11 ± 11.18)%,(82.10 ± 9.94)%] in hippocampus and cortex of rat brains were significantly lower than those of low fluoride group [(104.01 ± 5.77)%,(101.17 ± 6.35)%;(112.88 ± 22.15)%,(101.14 ± 8.60)%,P< 0.05];the expression of PPARγprotein [(64.32 ± 10.43)%,(60.20 ± 10.92)%] and mRNA [(41.03 ± 9.93)%,(52.25 ± 11.48)%] in the same brain regions of the rats after 6 months of treatment in high fluoride group were significantly lower than those of control group [(99.99 ± 11.19)%,(100.00 ± 11.30)%;(100.00 ± 10.00)%,(100.00 ± 9.00)%] and low fluoride group [(73.88 ± 3.36)%,(81.50 ± 14.90)%;(76.02 ± 8.65)%,(73.36 ± 7.43)%,P < 0.05].The activities of SOD in serum in low and high fluoride groups after 6 month treatment [(37.94 ± 1.92),(35.54 ± 2.53) U/ml] were significantly lower than that of control group [(41.24 ± 0.66) U/ml,P < 0.05],and the value in high fluoride group was lower than that in low fluoride group (P < 0.05);serum MDA contents in high fluoride group after 3 and 6 month treatment [(8.29 ± 1.49),(11.63 ± 1.04) nmol/mg pr] were higher than those in low fluoride group [(6.39 ± 0.69),(7.50 ± 1.64) nmol/mg pr] and control group [(5.02 ± 0.71),(5.87 ± 1.03) nmol/mg pr,P < 0.05].The correlation analysis results showed the levels of PPARγprotein in hippocampus and cortex of rats were negatively correlated with fluoride contents in brain tissues (3 month:r=-0.769,-0.793;6 month:r =-0.832,-0.870;P < 0.05),positively correlated with SOD activities (3 month:r =0.550,0.826;6 month:r =0.822,0.896;P < 0.05) and negatively correlated with MDA contents (3 month:r =-0.703,-0.609,6 month:r =-0.792,-0.657;P < 0.05) in serum.Conclusions Declined expression of PPARγat protein and mRNA levels has been detected in brains of rats with chronic fluorosis,which might be related to the increase of oxidative stress.PPARγ may be involved in the occurrence of chronic fluorosis.

6.
Chinese Journal of Endemiology ; (12): 278-282, 2018.
Article in Chinese | WPRIM | ID: wpr-701314

ABSTRACT

Objective To investigate the influence of inhibited gene expression of fisson 1 (Fis1) gene on the level of Fis1,mitofusin 1 (Mfn1) and mitochondrial membrane potential in SH-SY5Y cells with fluorine,to study the role of mitochondrial dynamic balance in the pathogenesis of chronic fluorosis.Methods SH-SY5Y cells were cultured in vitro,when adherent cells entered the logarithmic phase,using a group design,they were divided into four groups:blank control group (control),fluoride group [2 mmol/L sodium fluoride (NaF)],fluoride negative control group (2 mmol/L NaF + non-specific siRNA) and the gene-silencing group (2 mmol/L NaF + specific siRNA-Fis1).The protein expression levels of Fis1 and Mfn1 were measured by Western blotting;the mRNA expression levels of Fis1 and Mfn1 were measured by Real-time PCR;and the levels of the mitochondrial membrane potential was detected by mitochondrial membrane potential detection kit.Results Compared with control (1.37 ± 0.18,1.00 ± 0.04;1.57 ± 0.19,1.00 ± 0.04;1.00 ± 0.10),the expression levels of Fisl protein (1.72 ± 0.04) and mRNA (1.48 ± 0.13) in fluoride group were increased,the expression levels of Mfn1 protein (0.87 ± 0.02) and mRNA (0.69 ± 0.07) in fluoride group were decreased,the level of mitochondrial membrane potential (0.76 ± 0.13) was decreased (P < 0.05).Compared with control,the expression levels of Fis1 protein (0.79 ± 0.07) and mRNA (0.06 ± 0.03) in gene-silencing group were decreased,the expression levels of Mfn1 protein (1.71 ± 0.04) and mRNA (1.52 ± 0.05) in gene-silencing group were increased (P < 0.05),the level of mitochondrial membrane potential (0.94 ± 0.01) was decreased.Compared with fluoride group,the expression levels of Fis1 protein and mRNA in gene-silencing group were decreased,the expression levels of Mfn1 protein and mRNA in gene-silencing group were increased,the level of mitochondrial membrane potential in gene-silencing group was increased (P < 0.05).Conclusion Gene expression inhibition of Fis1 gene can reduce the mitochondrial division and damage of mitochondrial membrane potential in SH-SY5Y cells induced by fluoride.

7.
Chinese Journal of Endemiology ; (12): 271-277, 2018.
Article in Chinese | WPRIM | ID: wpr-701313

ABSTRACT

Objective To investigate the changes of extracellular regulated protein kinases (Erk)1/2,phospho-Erk1/2,matrix metalloproteinases (MMP)-2 and MMP-9 in rats with experimental chronic fluorosis and the role of chondroitin sulfate in treatment of rat with experimental chronic fluorosis.Methods Using a group design and cell culture methods,the SH-SY5Y cells were divided into control group (culture medium with 0.0 mmol/L fluoride ion),fluoride group (fluoride ion:4.0 mmol/L) and chondroitin sulfate group (fluoride ion:4.0 mmol/L,chondroitin sulfate:0.4 g/L).The ultrastructural changes of the SH-SY5Y cells were observed through electron microscope after 24 h treatment.The SH-SY5Y cells were cultured for 72 h,the number of cells survived in three groups.were detected after stained by trypan blue.Fifteen clean grade SD rats with body weight of 100-120 g were divided into control group (tap water:fluorine content less than 0.5 mg/L),fluoride group (fluoride ion:10.0 mg/L) and chondroitin sulfate group (fluoride ion:10.0 mg/L,the rats were performed intraperitoneal injection with 0.66 mg/kg chondroitin sulfate for 5 days after intaking fluoride for 90 days) on the basis of random number table.Five rats were in each group,and the experiment was carried out for 95 days.The capability of learning and memory of rats were tested by Morris water maze test;the expression of phospho-Erk1/2,Erk1/2,MMP-2 and MMP-9 protein in brain tissue was detected by Western blotting;the expression of phospho-Erk1/2,MMP-2 and MMP-9 in hippocampus CA2 area of brain was detected by immunohistochemistry.Results More vesicles and swelling of mitochondria or endoplasmic reticulum were observed in SH-SY5Y cell treated with fluoride through electron microscope,but relatively less in chondroitin sulfate group.Survival rate and amount of SH-SY5Y treated with chondroitin sulfate [(92 ± 23)% and (7.83 ± 1.38) × 106/ml] were significantly higher than that of fluoride group [(55 ± 2)%,(2.19 ± 1.26) × 106/ml,P < 0.05].Animal experiment results showed that most rats in control group and chondroitin sulfate group used spatial direct search strategy,and the amount of this search strategy (2.20 ± 1.09,3.40 ± 1.34) was more than that in fluoride group (0.40 ± 0.54,P < 0.05).The expression of phospho-Erk1/2 in brain tissue of rats in fluoride group (3.26 ± 0.88) was significantly higher than that in control group (1.53 ± 0.28) and chondroitin sulfate group (2.36 ± 0.87,P < 0.05).Immunohistochemistry results showed that average gray value of phospho-Erk1/2 in chondroitin sulfate group (220.20 ± 3.09) was significantly higher than that of the control group and the fluoride group (100.00 ± 0.00,130.98 ± 1.27,P < 0.05).The average gray value of MMP-2 in the fluoride group (294.52 ± 5.18) was significantly higher than that in control group and chondroitin sulfate group (100.00 ± 0.00,117.95 ± 1.55,P < 0.05).The average gray value of MMP-9 protein of the fluoride group (993.64 ± 3.66) and the chondroitin sulfate group (1 167.30 ± 239) was significantly higher than that of control group (100.00 ± 0.00,P < 0.05).Conclusions Erk1/2 pathway possibly maintains the stability of cell survival by regulating the expression of MMP-2 and MMP-9.Chondroitin sulfate can protective nerve cells and reduce the nervous damage caused by fluorosis to some certain extent.

8.
Chinese Journal of Endemiology ; (12): 265-270, 2018.
Article in Chinese | WPRIM | ID: wpr-701312

ABSTRACT

Objective To study the expression of silent information regulator (SIRT) in brains of rats with chronic fluorosis and reveal the correlation between SIRT1 and the ability of learning and memory of rats.Methods Sixty SD rats were selected and their body weight was (100 ± 20) g,according to the body mass of the rats,random number table method was used to divide rats into control group,low and high fluoride groups,experimental period was 3 and 6 months (ten rats in each experimental period,half males and half females).In control group,the rats were fed with drinking water containing no more than 0.5 mg/L fluoride;the rats in low and high fluoride groups were fed drinking water containing 5.0 and 50.0 mg/L fluoride,respectively.All of rats were fed the same standard food containing no more than 0.6 mg/kg fluoride.Three degree method was used to check the formation of dental fluorosis.Rat urinary fluoride was determined via the fluoride electrode method;Morris water maze method was used to detect the ability of learning and memory of rats (the escape latency time,the number of crossing the platform and stay time in platform quadrant);the protein and mRNA expression levels of SIRT1 were detected by Western blotting and Real-time PCR,respectively.Results In the experimental period of 3 and 6 months,no dental fluorosis was observed of rats in control group,but there were different degrees of dental fluorosis in low and high fluoride groups,especially in high fluoride group.The urinary fluorine contents [(1.60 ± 0.09),(1.91 ± 0.16) mg/L;(1.94 ± 0.19),(2.31 ± 0.18) mg/L] of rats fed with low and high fluoride for 3 or 6 months were significantly higher than those in control group [(1.08 ± 0.15),(1.09 ± 0.17) mg/L,P < 0.05].The escape latency time [(18.36 ± 2.80) s] of rats in the high fluoride group at the end of 3 months was higher than that of control group [(6.68 ± 3.01) s,P < 0.05],the number of stay time in platform quadrant [(12.91 ± 3.25) s] was lower than that of control group [(19.97 ± 3.30) s,P < 0.05].The escape latency time [(15.46 ± 4.56),(28.16 ± 4.00) s] of rats in low and high fluoride groups at the end of 6 months were all higher than that of control group [(6.62 ± 2.31) s,P < 0.05];the number of crossing the platform and stay time in platform quadrant [(2.25 ± 1.71) times,(12.73 ± 3.55) s;(1.40 ± 1.15) times,(9.26 ± 1.72) s] of these rats were significantly lower than those of the control group [(4.00 ± 1.58) times,(19.53 ± 4.36) s,P < 0.05].The expression levels of protein [(73.84 ± 9.68)%,(73.23 ± 4.51)%;(53.30 ± 17.63)%,(54.69 ± 18.71)%] and mRNA [(70.33 ± 4.89)%,(66.27 ± 3.38)%;(37.72 ± 4.89)%,(44.15 ± 1.74)%] of SIRT1 in the hippocampus and cortex of rats fed with high fluoride for 3 or 6 months were significantly lower than those in control group [(100.00 ± 13.51)%,(100.00 ± 13.60)%;(100.00 ± 15.37)%,(100.00 ± 12.19)%;(100.00 ± 2.65)%,(100.00 ± 4.34)%;(100.00 ± 3.40)%,(100.00 ± 4.52)%,P < 0.05].Whereas,the decreased expression levels of protein [(77.65 ± 14.51)%,(71.51 ± 8.27)%] and mRNA [(57.78 ± 1.96)%,(63.76 ± 2.16)%] of SIRT1 in the hippocampus and cortex of rats in the low fluoride group were only observed at the end of 6 month of experiment (P < 0.05).The expression of protein of SIRT1 in the hippocampus and cortex of rats in 3 or 6 months was negatively correlated with the escape latency time of rats (r=-0.598 5,-0.493 2;-0.782 6,-0.777 3,P< 0.05),and it was positively correlated with the number of crossing the platform (r =0.547 7,0.523 3;0.720 5,0.715 4,P < 0.05).Conclusion The decrease of the ability of learning and memory in rats with chronic fluorosis may be related to the decreased expression of SIRT1 influenced by chronic fluorosis.

9.
Article in Chinese | WPRIM | ID: wpr-701260

ABSTRACT

Objective To evaluate the influence of fluoride on mitochondrial membrane potential of neuroblastoma SH-SY5Y cells,and on the expression levels of mitochondrial proteins mitofusion 1 (Mfn1) and fission 1 (Fis1).Methods A stable and feasible culture method of SH-SY5Y cells in vitro was established with different concentration of sodium fluoride [0.0 (control),0.4,2.0 and 4.0 mmol/L],and various periods exposure of 6,12,24,48 h;the mitochondrial membrane potential of SH-SY5Y cells was detected by mitochondrial membrane potential assay kit (JC-1);and the expression levels of Mfn1 and Fis1 proteins were detected by Western blotting.Results Compared with the control group (1.63 ± 0.18,1.13 ± 0.15,1.30 ± 0.02) for various periods exposure (6,12,48 h),the red/green fluorescence ratios of the mitochondrial membrane potential of SH-SY5Y cells exposed to 2.0 and 4.0 mmol/L of sodium fluoride were decreased significantly (1.01 ± 0.10,0.80 ± 0.04;0.75 ± 0.13,0.62 ± 0.10;0.82 ± 0.01,0.56 ± 0.04,P < 0.05);compared with the control group (0.93 ± 0.03,1.05 ± 0.07,1.17 ± 0.04) for various periods exposure,the expression levels of mitochondrial Mfn1 protein were decreased significantly in 0.4,2.0,4.0 mmol/L sodium fluoride groups (6,12,48 h:0.75 ± 0.02,0.65 ± 0.05,0.57 ± 0.06;0.83 ± 0.06,0.79 ± 0.06,0.69 ±0.06;0.98 ± 0.05,0.73 ± 0.07,0.62 ± 0.09,P < 0.05).Compared with the control group (0.90 ± 0.05) for exposure time 12 h,the expression levels of Fis1 protein were increased significantly in 2.0,4.0 mmol/L sodium fluoride groups (1.14 ± 0.06,1.23 ± 0.06,P < 0.05).Conclusions The mitochondrial membrane potential and the expression levels of mitofusion 1 and fission 1 of SH-SY5Y cells treated with fluoride are abnormal,which might be associated with the theory of nerve cell damage from high oxidative stress.

10.
Article in Chinese | WPRIM | ID: wpr-688184

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<p><b>OBJECTIVE</b>To study the distribution of haplotypes of Y-chromosomal short tandem repeats (Y-STR) loci among three ethnic minorities from Guizhou, China.</p><p><b>METHODS</b>Twenty four Y-STR loci of 174 unrelated males were amplified with a Microreader(TM)24Y Direct ID System kit. Capillary electrophoresis was carried out on an ABI 3100 Genetic Analyzer, and the data was analyzed with GeneMapper software.</p><p><b>RESULTS</b>Seventy six haplotypes were identified for the 24 Y-STR loci among the three ethnic minorities, including 13 from the Qiangs, 35 from the Manchurians, and 28 from the Shes, with the corresponding Haplotype Diversity (HD) being 0.7327, 0.9578, and 0.9344. Genetic distance between the Shes and Qiangs was relatively close, whilst that for Manchurians was relatively far.</p><p><b>CONCLUSION</b>Analysis of the genetic characteristics and relationship of the three ethnic minorities from Guizhou can provide a reference for the study of their origin, evolution and patrilineal fusion.</p>

11.
Chinese Journal of Pathology ; (12): 491-496, 2017.
Article in Chinese | WPRIM | ID: wpr-809009

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Objective@#To investigate the effect of lovastatin on oxidative stress and apoptosis in neurons induced by β-amyloid peptide (Aβ).@*Methods@#Primary culture of rat hippocampal neuron was treated with Aβ oligomers alone or combined with lovastatin. The levels of OH-, H2O2, O2·-, malondialdehyde, superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activities were measured by biochemical methods and protein expression of caspase-3 and bcl-2 was detected by Western blot.@*Results@#As compared with the control group, treatment of 0.5 μmol/L Aβ oligomers for 48 h led to significant increase of OH-, H2O2, O2·- and malondialdehyde content, inhibition of SOD and GSH-PX activities, enhanced caspase-3 expression and decreased bcl-2 expression. Interestingly, these neurotoxic modifications on the levels of OH-, H2O2, O2·- and malondialdehyde content, SOD and GSH-PX activities, and the protein expression of cleaved caspase-3 and bcl-2 were significantly attenuated when the cells were pretreated with 0.1 μmol/L lovastatin for 24 h before exposure of Aβ oligomers.@*Conclusion@#Lovastatin may play an important role in antagonizing the neurotoxicity of Aβ through a mechanism likely related to the inhibition of oxidative stress.

12.
Article in Chinese | WPRIM | ID: wpr-510306

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Objective To investigate the differences of X-ray findings of skeletal fluorosis between coal-burning type endemic fluo-rosis and industrial fluorosis.Methods The patients were randomly selected as research objects including 60 cases of coal-burning type endemic osteofluorosis and 60 cases of industrial osteofluorosis.The X-ray findings on the left forearm,crus and pelvic radio-graphs of these patients were analyzed retrospectively to find out the differences between skeletal fluorosis of coal-burning type endemic fluorosis and industrial fluorosis.Results X-ray features are no significant statistical differences between coal-burning type endemic fluorosis and industrial fluorosis,except these of interosseous membrane ossification of forearm and crus (forearmχ2=10.909,P<0.05;crusχ2=8.547,P<0.05),obturator membrane ossification of pelvis (χ2=36.554,P<0.05),periosteal proliferation outside bone of crus (χ2=4.937,P<0.05),and ossification of soleus (χ2=4.904,P<0.05).Conclusion The X-ray signs of endemic osteofluorosis and industrial skeletal fluorosis are almost similar,but there are some differences between them.

13.
Article in Chinese | WPRIM | ID: wpr-506144

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Objective To explore the influences of protein kinase Cβ (PKC3) inhibitor LY333531 on oxidative injury and apoptosis of SH-SY5Y cells induced by fluorosis.Methods The SH-SY5Y cell model of fluorosis was established,and the experiment was divided into three groups:control group [0.0 mmol/L sodium fluoride (NaF) and 0.0 μmol/L LY333531],the fluoride group (0.5 mmol/L NaF and 0.0 μmol/L LY333531),and the PKCβ inhibitor group (0.5 mmol/L NaF and 0.2 μmol/L LY333531),n =3.Flow cytometry was used to detect the changes of reactive oxygen species (ROS) and apoptosis rate,fluorescent probe technique was used to detect mitochondrial membrane potential after each group for 48 h.Results Compared with the control group [(3.32 ± 0.29) × 103,0.60 ± 0.09,(7.58 ± 1.20)%],the level of ROS [(5.99 ± 0.32) × 103] was increased,mitochondrial membrane potential (0.28 ± 0.06) was decreased,and the apoptosis rate [(18.00 ± 2.32)%] was increased in the fluoride group (all P < 0.05);compared with the fluoride group,the level of ROS [(5.12 ± 0.25) × 103] was decreased,mitochondrial membrane potential (0.42 ± 0.03) was increased,and the apoptosis rate [(11.79 ± 0.70)%] was decreased in the PKCβ inhibitor group (all P < 0.05).Conclusions Excess fluoride could cause oxidative damage and apoptosis in cells.PKC3 inhibitor LY333531 has a protective effect in oxidative damage and apoptosis by fluorosis.

14.
Chinese Journal of Endemiology ; (12): 327-332, 2017.
Article in Chinese | WPRIM | ID: wpr-614576

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Objective To investigate the possible mechanism of endonuclease G (Endo G)-mediated non-Caspase-dependent apoptotic pathway in brain neuronal apoptosis in chronic fluorosis rats.Methods Sixty Sprague Dawley (SD) rats (half male and half female) were randomly divided into two groups:control group fed with tap water with fluoride content < 0.5 mg/L and fluorine group in which sodium fluoride was added into drinking water with fluoride content of 50.0 mg/L.Both groups were fed with standard food with fluorine content < 0.5 mg/kg.The experiment period was 10 months.At the end of the experiment,all the animals were sacrificed,and brain tissue was taken.Flow cytometry was used to examine apoptosis rate,immune-histochemistry was employed to detect the distribution of Endo G in brain tissue;Western blotting was used to test the protein expression of Endo G.Results Compared to the apoptosis rate of control group [(1.3 ± 0.6)%,(1.9 ± 0.3)%],the apoptosis rate in hippocampus and cortex of rats with chronic fluorosis [(2.6 ± 0.6)%,(3.1-± 0.7)%] was significantly increased (t =3.1,3.4,all P < 0.05).The Endo G positive neurons and their degree of staining in CA1,CA2,CA3 and CA4 of hippocampus,frontal cortex as well as the upper layer of parietal cortex [(11.1 ± 2.2),(10.2 ± 1.9),(9.8 ± 3.1),(9.9 ± 1.6),(10.6 ± 2.9),(8.2 ± 2.4),(11.1 ± 2.8) scores] in rats with chronic fluorosis were significantly higher than those in the control group [(5.8 ± 1.8),(6.7 ± 2.6),(5.2 ± 2.4),(7.2 ± 2.1),(7.7 ± 2.6),(6.1 ± 1.9),(8.1 ± 2.6) scores,t =2.9,2.5,2.4,2.3,2.2,2.5,2.3,P < 0.01 or < 0.05].The protein level of Endo G in the mitochondria of rat brains with chronic fluorosis [(86.4 ± 7.2)%,(83.9 ± 6.8)%] was significantly lower than that of control group [(100.0 ± 6.1)%,(100.0 ± 5.5)%,t =2.6,2.3,all P < 0.05].Meanwhile,the protein level of Endo G in the nucleus of neurons from chronic fluorosis rats [(117.5 ± 6.4)%,(115.2 ± 6.2)%] was significantly higher than that of the control [(100.0 ± 5.2)%,(100.0 ± 5.5)%,t =2.5,2.2,all P < 0.05].Conclusion The high expression of Endo G and nuclear transfer are related to the neuron apoptosis in chronic fluorosis rat,which may be one of the mechanisms of brain injury of the disease.

15.
Chongqing Medicine ; (36): 4702-4704, 2017.
Article in Chinese | WPRIM | ID: wpr-668528

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Objective To study the frequency of 9 bp sequence deletion of mithochondrial DNA in three ethnic nationalities of Guizhou Province .Methods A total of 183 male blood samples were randomly extracted from Miao nationality (69 cases) in Leishan County ,Shui nationality (44 cases) and Buyi nationality (70 cases) in Libo County .Polymerase chain reaction(PCR) and di-rect sequencing were used to detect the mithochondrial DNA 9 bp sequence deletion .Results The standard type ,deleting type and 3 type were found in the samples of 3 ethnic nationalities in Guizhou Province ,the highest deletion frequency was Shui nationality (40 .91% ) in Libo County ,1 cases of 3 type was detected in Miao nationality of Leishan County ,and the mtDNA 9 bp deletion fre-quency had no statistical difference among 3 ethnic nationalities(P>0 .05) .Conclusion The frequency of mtDNA 9 bp deletion is different among three native minorities ,the genetic variation in Shui nationality is larger ,compared with Miao nationality genetic re-lationship ,Shui nationality has a relatively closer affinity with Buyi nationality .

16.
Chinese Journal of Epidemiology ; (12): 389-393, 2016.
Article in Chinese | WPRIM | ID: wpr-237536

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Objective To investigate the association between HLA-DP gene polymorphism and the susceptibility to hepatitis B virus (HBV) infection in people in Buyi,Miao and Shui ethnic groups in Guizhou.Methods A total of 256 patients with HBV infection,142 HBV self-cleared patients and 135 controls were recruited from 3 ethnic groups for this case-control study.Genotypes of rs3077 and rs9277535 were determined by using real-time PCR with a TaqMan-MGB probe.Results Compared with healthy subjects and self-cleared subjects,the allele distribution of rs9277535 was significantly associated with chronic HBV infection (P<0.05),no significant differences in rs3077 allele and genotype distributions were found among 3 groups (P>0.05).Compared with the rs9277535 GG genotype,the AA and AG genotypes were protective factors against HBV infection in dominant model after adjustment for age and sex (OR=0.645,95%CI:0.421-0.988),and no significant difference in rs3077 locus was found in the same analysis (P>0.05).For men,the rs3077 locus CC and CT genotypes were also protective factors against HBV infection (OR=0.493,95%CI:0.266-0.916),and there was no significant difference in rs9277535 locus compared the genotype distributions among 3 groups in dominant mode.Comparison of allele and genotype distribution in 3 ethnic groups showed that HLA-DP gene rs3077 genotype distributions had significant difference between the HBV infection group and the healthy control group in Buyi ethnic group (x2=6.726,P=0.036).Conclusions The presence of rs9277535A allele at HLA-DP gene polymorphisms might decrease the risk for HBV infection,the rs3077 C allele at HLA-DP gene polymorphisms might also confer protective effect against HBV infection in women.No significant correlation between HLA-DP gene rs3077 and rs9277535 locus polymorphism and HBV clearance was found in this study.The HLA-DP gene rs3077 genotype distribution of HBV-infected patients had significant differences among different ethnic groups.

17.
Chinese Journal of Endemiology ; (12): 333-337, 2016.
Article in Chinese | WPRIM | ID: wpr-498021

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Objective To investigate the influence of chronic fluorosis on protein kinase Cβ (PKCβ)/p66shc signal pathway in the brain of rats,and reveal the molecular mechanism of brain damage.Methods According to body weight by the random number table method thirty SD rats were divided into three groups of 10 each (half females and half males),the normal control group [less than 0.5 mg/L of fluorine (prepared with NaF) in drinking water],low fluoride exposure group (10.0 mg/L fluorine),and high fluoride exposure group (50.0 mg/L fluoride).The experiment period was 6 months.The protein level of PKCβ,p66shc,phospho-p66shc and preserved ammonia acyl isomerase (Pin1) in rat brain was detected by Western blotting.The level of neuron nuclear antigen (NeuN),p66shc and phospho-p66sh in brain of rats was detected by immunohistochemistry.Results By Western blotting,the levels of PKCβ,Pin1 and phospho-p66shc protein in brain tissue in high fluoride exposure group [(193.00 ± 57.53)%,(228.21 ± 71.14)%,(201.54 ±:50.86)%] were higher than those of the normal control groups [(100.00 ± 21.24)%,(100.00 ± 40.55)%,(100.00 ± 13.35)%,all P < 0.05].By immunohistochemistry,the numbers of NeuN staining in brain tissue of the rats in both high and low fluoride exposure groups [(49.50 ± 12.57)%,(65.66 ±14.58)%] were lower than that of the control group [(100.00 ± 18.32)%,all P < 0.01].The level of phospho-p66shc protein in brain tissue in high fluoride exposure group [(242.66 ± 93.01)%] was higher than those of the low fluoride exposure and the normal control groups [(152.53 ± 60.65)%,(100.00 ± 25.63)%,all P < 0.01].Conclusion Chronic fluorosis has increased the expressions of PKCβ,Pin1 and phospho-p66shc at protein level in brain of rats,which may be related to the molecular mechanism of brain damage resulted from chronic fluorosis.

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Chinese Journal of Endemiology ; (12): 547-551, 2016.
Article in Chinese | WPRIM | ID: wpr-496589

ABSTRACT

Objective To investigate the possible pathological role of mitochondrial apoptosis pathways and its factors in fluorosis-induced apoptosis of human hepatocellular carcinoma cell strain (HepG2).Methods Under the stimulation of 1,3,6 and 9 mmol/L concentrations of NaF in vitro for 24 h (n =5),while normal control group was cultured under normal condition,the cytotoxicity was measured with MTT.The mitochondrial apoptosis inducing factor (AIF) was measured at both mRNA (n =5) and protein levels (n =6),respectively,by real-time PCR and Western blotting.The mitochondrial apoptosis related factors,such as B-cells lymphoma-2 (Bcl-2),Bcl-associated X protein (Bax),cytochrome C,caspase-9 and caspase-3 were measured at protein levels (n =6).Results After treated with 0,1,3,6 and 9 mmol/L NaF for 24 h,the cell absorbance of HepG2 cells was 0.307 ± 0.031,0.333 ± 0.028,0.230 ± 0.011,0.178 ± 0.001 and 0.152 ± 0.003,respectively,and the differences were statistically significant among groups (F =82.224,P < 0.01).After treated with 3 mol/L NaF for 24 h,the mRNA level of AIF was [(153.14 ± 5.41)%] which was increased compared to the control group [(100.00 ± 4.70)%,t =-4.73,P <0.05].Under the same condition,the protein levels of AIF,Bcl-2,cytochrome C in cytoplasm,caspase-9 and caspase-3 were (152.16 ± 47.30)%,(171.90 ± 51.52)%,(458.00 ± 19.48)%,(527.17 ± 200.67)% and (432.70 ±64.27)%,which were increased compared to those of the control groups [(100.00 ± 48.86)%,(100.00 ± 34.44)%,(100.00 ± 116.59)%,(100.00 ± 19.58)% and (100.00 ± 137.16)%,t =-3.80,-3.96,-15.76,-4.64,-5.06,all P < 0.05],while the protein levels of Bax and cytochrome C in mitochondrion were (24.66 ± 26.04)%,(72.99 ±45.34)%,which were decreased compared to those of the control groups [(100.00 ± 44.01)%,(100.00 ± 34.14)%,t =6.35,0.68,all P < 0.05].Conclusion The mitochondrial apoptosis pathway and related factors may be involved in NaF-induced cell death in HepG2 cells.

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Chinese Journal of Endemiology ; (12): 178-181, 2016.
Article in Chinese | WPRIM | ID: wpr-489865

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Objective To detect the levels of oxidative stress in brain and serum of rats with chronic fluorosis and the antagonistic effects of vitamin E (VitE),and to reveal the role of oxidative stress in brain injury.Methods Thirty healthy SD rats were divided into three groups based on body weight by means of a random number table (10 rats in each group,half male and half female).In the control group,the rats were fed with drinking water containing less than 0.5 mg/L fluoride;in the fluoride group,the rats were fed with high doses of sodium fluoride in drinking water (50.0 mg/L) and the VitE antagonistic group were fed with the same content of fluoride in drinking water as the fluoride group,but adding VitE (50.0 mg/kg) by intragastric administration once a day.All rats were fed with normal diet (6.2 mg/kg).After exposure to fluoride for 10 months,all rats were put to death,dental fluorosis of the rats was examined and the fluoride content in bone was determined by fluoride-ion selective electrode;the activity of superoxide dismutase (SOD) was determined by the xanthine oxidase method and glutathione peroxidase (GSH-Px) by the colorimetric method,the level of malonaldehyde (MDA) by the glucosinolates barbituric acid fluorescence method and the levels of OH-,H2O2 and O-·2 in rat serum and/or brain were detected by the colorimetric method.Results In the rats of the fluoride group,fluoride content in bone was higher as compared to control [bone fluoride:(211.07 ± 48.52) vs.(33.40 ± 9.26) mg/kg,P < 0.01].The activities of SOD and GSH-Px in rat brains of the fluoride group [(20.10 ± 1.98) kU/g,(28.70 ± 19.35) kU/L] were significantly lower than those of controls [(37.05 ± 3.13) kU/g,(59.63 ± 12.83) kU/L,all P < 0.01],the activity of SOD in VitE antagonistic group [(26.27 ± 1.74) kU/g] was higher than the fluoride group (P < 0.01);the activities of SOD and GSH-Px in rat serum of the fluoride group were significantly decreased [(11.55 ± 1.75) kU/L,(79.50 ± 19.18) U/L] than those of controls [(20.79 ± 2.43) kU/L,(170.00 ± 14.68) U/L,all P < 0.01],the activity of SOD in VitE antagonistic group [(17.23 ± 0.68) kU/L] was higher than the fluoride group (P < 0.01).The levels of MDA in rat brain and serum of the fluoride group [(8.84 ± 0.69) μmol/L,(1.46 ± 0.11) nmol/L] were significantly higher than those of controls [(1.27 ± 0.74) μmol/L,(0.83 ± 0.10) nmol/L,all P< 0.01],VitE antagonistic groups [(4.51 ± 1.13) μmol/L,(1.29 ± 0.02) nmol/L] were lower than the fluoride groups (all P < 0.01).The levels of OH-,H2O2 and O-·2 in rat brains of the fluoride group [(24.24 ± 1.80) kU/g,(15.28 ± 2.97) mmol/L,(6.53 ± 0.96) U/g] were significantly higher than those of controls [(11.44 ± 1.63) kU/g,(5.28 ± 1.20) mmol/L、,(2.93 ± 0.42) U/g,all P < 0.01],VitE antagonistic groups [(14.43 ± 0.76) kU/g,(8.09 ± 0.55) mmol/L,(4.41 ± 0.49) U/g] were lower than the fluoride groups (all P < 0.01).Conclusions Elevated levels of oxidative stress are found in brain and serum of the rats with chronic fluorosis,which may be a main mechanism of brain injury.VitE may play an important antagonistic role in oxidative damage induced by fluoride toxicity.

20.
Article in Chinese | WPRIM | ID: wpr-489838

ABSTRACT

Objective Through observation of the expression and activity of extracellular regulated protein kinase 5 (ERK5) and its relationship with the learning and memory ability in rats with chronic fluorosis,to further study the pathogenesis of chronic fluorosis in nervous system.Methods Thirty SD rats were divided into 3 groups according to body weight by means of a random number table (10 rats in each group,half male and half female).The rats in control group were fed with free drinking tap water containing less than 0.5 mg/L fluoride (NaF);the rats in low fluoride group with 10.0 mg/L fluoride;the rat in high dose fluoride group with 50.0 mg/L fluoride.After 6months of experiment,rat brain tissue was took,mRNA expression level of ERK5 was detected by real-time fluorescence quantitative PCR (real-time PCR),protein expression level and activity of ERK5 were detected by Western blotting;the learning and memory ability of rats with chronic fluorosis were detected by Morris water maze test.Results The rat in groups exposed to fluoride exhibited different degrees of dental fluorosis and the fluoride content in urine of rats increased gradually with increase of fluoride doses (F =164.10,P < 0.05).The protein levels of phosphor-ERK5 in the control group,low fluoride group and high fluoride group were 0.13 ± 0.03,0.29 ± 0.10and 0.43 ±0.17,respectively,the difference was statistically significant (F=11.96,P< 0.05),and low fluoride group and high fluoride group were higher than control group (all P < 0.05).The total protein levels of ERK5 in control group,low fluoride group and high fluoride group were 0.32 ± 0.11,0.37 ± 0.13 and 0.49 ± 0.16,respectively,the difference was statistically significant (F =3.45,P < 0.05),and high fluoride group was higher than control group (P < 0.05).The expression of ERK5 mRNA in rat brains between groups was not significantly different (F =0.81,P > 0.05).The second,third,and forth days of directional navigation experiment,the time of escape latency and the number of crossing the platform between groups were statistically significant (H =28.20,29.90,26.47,27.23,35.34,27.62,all P < 0.01);the fifth day of space exploration experiment,the difference of the time of the first crossing platform and the number of crossing the platform between groups were statistically significant (H =31.41,30.80,all P < 0.01);the protein level of phosphor-ERK5 in brain tissue of rats was negatively correlated with the number of the first crossing platform (r =-0.470,P < 0.01),while positively related to escape latencies at the fifth day of the test (r =0.591,P < 0.01).Conclusion The changes of ERK5 signaling pathway in rat brain tissue caused by chronic fluorosis are found,which are related to the decrease of leaming and memory ability of animals with chronic fluorosis.

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