BACKGROUND:Previous studies have shown that N-methyl-D-aspartic acid receptor(NMDA)receptors are associated with fluorine,but the role in fluoride-induced endoplasmic reticulum stress remains unclear. OBJECTIVE:To observe the changes of excitatory neurotransmitter NMDA receptor and endoplasmic reticulum stress IRE1α-ASK1-JNK pathway protein expression in brain tissue of rats with experimental fluorosis,and to investigate the pathogenesis of neurological injury in fluorosis by giving NMDA receptor inhibitor to SH-SY5Y cells. METHODS:(1)Animal model:18 1-month-old SD rats were randomly divided into control group(drinking water fluoride content<0.5 mg/L),low fluoride group(drinking water fluoride content 10.0 mg/L)and high fluoride group(drinking water fluoride content 100.0 mg/L),with 6 rats in each group,half of each sex.After 6 months of fluoride intake,the rats were observed for the occurrence of dental fluorosis,and the 24-hour urinary fluoride content was measured.After anesthesia and euthanasia,the brain tissue of rats was taken to observe the pathological changes.Western blot assay was used to detect NMDA receptors and IRE1α,ASK1 and JNK protein expression in the brain tissue.(2)Cell model:SH-SY5Y cells were cultured in vitro and treated with sodium fluoride at final concentrations of 0.3 mmol/L and 3 mmol/L.The fluoride-stained cells were interfered with 10 μmol/L NMDA receptor antagonists Ifenprodil and MK-801 to observe the relevant protein changes. RESULTS AND CONCLUSION:(1)The incidence of dental fluorosis and urinary fluoride level in rats in the high fluoride group were significantly higher than that in the control and low fluoride groups(P<0.05).(2)Compared with the control group,the cytoplasm of neuronal cells in the CA3 area of the hippocampus in the low fluoride group was slightly more basophilic,while the neuronal cells in the CA3 area of the high fluoride group were disorganized,with increased basophilicity and some of the nuclei solidified.(3)In rat brain tissue,the expressions of NR2A in the high fluoride group and NR2B in the low fluoride group were significantly higher compared with the control group(P<0.05),and NR2B,IRE1,ASK1,and p-JNK protein expression levels were increased in the high fluoride group compared with the control and low fluoride groups(P<0.05).(4)In SH-SY5Y cells,NR1,NR2A and NR2B protein expressions were significantly increased in the high fluoride group compared to the control group(P<0.05).The protein levels of NR1 and NR2A were significantly reduced in the high fluorine + Ifenprodil group and high fluorine + MK-801 group compared with the high fluorine group(P<0.05).NR2B protein expression was significantly lower in the high fluorine + Ifenprodil group than that in the high fluorine group(P<0.05).(5)In SH-SY5Y cells,IRE1,ASK1,and p-JNK protein expression was significantly higher in the high fluoride group compared with the control group(P<0.05),while ASK1 and p-JNK protein expressions were significantly decreased in the high fluorine + Ifenprodil group and high fluorine + MK-801 group compared with the high fluorine group(P<0.05).IRE1 protein level was significantly lower in the high fluorine + Ifenprodil group than that in the high fluorine group(P<0.05).(6)It is concluded that excessive fluorine intake activates NMDA receptors in the central nervous system,causing increased expression of endoplasmic reticulum stress IRE1α,ASK1,and p-JNK proteins,and the use of NMDA receptor inhibitors has a mitigating effect on endoplasmic reticulum stress caused by fluorosis.

Flagella are the main motility structure of Clostridioides difficile that affects the adhesion, colonization, and virulence of C. difficile in the human gastrointestinal tract. The FliL protein is a single transmembrane protein bound to the flagellar matrix. This study aimed to investigate the effect of the FliL encoding gene flagellar basal body-associated FliL family protein (fliL) on the phenotype of C. difficile. The fliL gene deletion mutant (ΔfliL) and its corresponding complementary strains (: : fliL) were constructed using allele-coupled exchange (ACE) and the standard molecular clone method. The differences in physiological properties such as growth profile, antibiotic sensitivity, pH resistance, motility, and spore production ability between the mutant and wild-type strains (CD630) were investigated. The ΔfliL mutant and the : : fliL complementary strain were successfully constructed. After comparing the phenotypes of strains CD630, ΔfliL, and : : fliL, the results showed that the growth rate and maximum biomass of ΔfliL mutant decreased than that of CD630. The ΔfliL mutant showed increased sensitivity to amoxicillin, ampicillin, and norfloxacin. Its sensitivity to kanamycin and tetracycline antibiotics decreased, and the antibiotic sensitivity partially returned to the level of CD630 strain in the : : fliL strain. Moreover, the motility was significantly reduced in the ΔfliL mutant. Interestingly, the motility of the : : fliL strain significantly increased even when compared to that of the CD630 strain. Furthermore, the pH tolerance of the ΔfliL mutant significantly increased or decreased at pH 5 or 9, respectively. Finally, the sporulation ability of ΔfliL mutant reduced considerably compared to the CD630 strain and recovered in the : : fliL strain. We conclude that the deletion of the fliL gene significantly reduced the swimming motility of C. difficile, suggesting that the fliL gene is essential for the motility of C. difficile. The fliL gene deletion significantly reduced spore production, cell growth rate, tolerance to different antibiotics, acidity, and alkalinity environments of C. difficile. These physiological characteristics are closely related to the survival advantage in the host intestine, which is correlated with its pathogenicity. Thus, we suggested that the function of the fliL gene is closely related to its motility, colonization, environmental tolerance, and spore production ability, which consequently affects the pathogenicity of C. difficile.
Humans ; Clostridioides/metabolism* ; Clostridioides difficile/metabolism* ; Bacterial Proteins/metabolism* ; Virulence ; Anti-Bacterial Agents/metabolism*

Objective:To study the effects of fluoride exposure on skeletal development of zebrafish larvae and its possible molecular mechanisms.Methods:Six hours post fertilization(6 hpf) wild-type zebrafish embryos were selected and exposed to sodium fluoride [NaF, control group (0 mg/L NaF), low fluoride group (25 mg/L NaF) and high fluoride group (100 mg/L NaF)] for 9 days. Fluorine ion selective electrode was used to detect the overall fluorine content of zebrafish larvaes, and the death and development of zebrafish larvaes were observed and counted. Bone mineralization and chondrogenesis of the zebrafish larvaes were analyzed by alizarin red staining and alcin blue staining, respectively. The expression levels of sry-related-high-mobilty-group box 9a (Sox9a), osteprotegerin (OPG) and receptor-activator of nuclear factor kappa beta ligant (RANKL) were analyzed by real-time quantitative PCR.Results:Compared with control group [(0.12 ± 0.01) μg/140 larvaes], the overall fluorine contents of zebrafish larvaes in low fluoride group [(0.28 ± 0.03) μg/140 larvaes] and high fluoride group [(0.64 ± 0.10) μg/140 larvaes] were significantly higher, and the differences were statistically significant ( P < 0.05). Compared with control group, zebrafish larvaes in high fluoride group had shorter body length, higher swim bladder loss rate and higher spinal curvature rate ( P < 0.05). The alizarin red staining area, integrated optical density (IOD) and the number of mineralized vertebrae were higher in low fluoride group, while the alcin blue staining area of cartilage formation was lower ( P < 0.05). In the high fluoride group, alizarin red staining area, IOD and the number of mineralized vertebrae were lower, while the alcin blue staining area of cartilage formation was higher ( P < 0.05). Compared with control group, the expression levels of OPG mRNA and OPG/RANKL mRNA in low fluoride group were higher ( P < 0.05); the expression level of RANKL mRNA was higher in high fluoride group, while the expression level of OPG/RANKL mRNA was lower ( P < 0.05). Conclusion:A short period of fluoride exposure from zebrafish embryo to zebrafish larvae can cause abnormal bone development of zebrafish larvae, which may be related to endochondral osteogenesis and OPG/RANKL pathway.

Objective:To investigate the effects of excessive fluoride exposure on astrocytes and the expression of glial fibrillary acidic protein (GFAP), in vitro and in vivo. Methods:(1) In vivo experiment: 24 SPF SD rats, half male and half female, were randomly divided into control and fluoride exposed groups according to sex and body weight, 12 rats in each group. Rats were fed with < 1 mg/L and 50 mg/L sodium fluoride solution prepared by tap water for 6 months, respectively. The expression levels of GFAP protein in rat brain tissue were measured by immunofluorescence, immunohistochemistry and Western blotting. (2) In vitro experiment: adult (6-month-old) rat cortical astrocytes were extracted and cultured in primary culture (4 mmol/L sodium fluoride solution for 24 h), and the astrocytes were identified by immunofluorescence, and GFAP mRNA and protein expression levels were detected by real-time fluorescence quantitative PCR and Western blotting, respectively, and astrocytes apoptosis and calcium ion content were detected by flow cytometry. Results:(1) In vivo experiment: the results of immunofluorescence, immunohistochemistry and Western blotting showed that the GFAP protein expression level in brain tissue of rats exposed to fluoride was higher than that of control group (0.440 ± 0.200 vs 0.250 ± 0.120, t =-5.93, P = 0.027; 0.270 ± 0.020 vs 0.240 ± 0.050, t =-4.87, P = 0.040; 1.017 ± 0.001 vs 0.486 ± 0.006, t =-52.48, P = 0.001). (2) In vitro experiment: GFAP positive cells were identified as astrocytes by immunofluorescence; GFAP mRNA expression level was higher in fluoride exposed group than that of control group by real-time fluorescence quantitative PCR (2.780 ± 0.120 vs 0.134 ± 0.005, t =-37.84, P = 0.001). The Western blotting results showed that the GFAP protein expression level was higher in fluoride exposed group than that of control group (2.76 ± 0.10 vs 1.38 ± 0.05, t =-20.44, P = 0.002). Flow cytometry results showed that the apoptosis rate of astrocytes was higher in fluoride exposed group than that of control group (%: 55.0 ± 1.0 vs 3.5 ± 0.6, t =-10.28, P = 0.009) and the calcium ion content was lower than that of control group (%: 54 ± 9 vs 72 ± 13, t = 4.64, P = 0.043). Conclusion:Excessive fluoride exposure causes increased GFAP expression in astrocytes in vitro and in vivo, promotes apoptosis, and affects calcium signaling pathways.

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.

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.

【Objective】 To explore the protective effects of exogenous hydrogen sulfide （H2S） on oxygen glucose deprivation and re-oxygenation （OGD/R）-induced SH-SY5Y cell injury. 【Methods】 OGD/R model was established in SH-SY5Y cells. Based on interferences， SH-SY5Y cells were divided into control group （no interference）， OGD/R model group， and treatment groups （OGD/R model treated with different concentrations of NaHS （H2S donor）. The cells were cultured in low glucose medium without fetal bovine serum under 37 ℃， 93% N2 and hypoxia （2% O2） for 12 h， then in complete medium under normoxia （5% O2 ） for 24 h. CCK-8 was used to detect cell viability， and flow cytometry was used for assaying the level of cellular apoptosis. Western blot was used to detect the expression levels of endoplasmic reticulum stress-related proteins including glucose regulatory protein 78 （GRP78）， CCAAT/enhancer-binding homologous protein （CHOP）， NF-κB P65 and COX-2. 【Results】 The cell viability was lower in OGD/R model group than in the control group （P<0.05）. NaHS treatment at a concentration of 0-0.5 mmol/L increased cell viability in OGD/R model group in a dose-dependent manner （P<0.05）. In contrast， NaHS （C>0.5 mmol/L） decreased cell viability of OGD/R （P<0.05）. The apoptosis level of early stage in OGD/R model group was significantly higher than that in control group （P<0.05）. NaHS treatment at concentrations of 0.2 and 0.4 mmol/L lowered the apoptosis level of early stage in OGD/R model group （P<0.05）. Western blot showed that the protein expression levels of GRP78， CHOP， NF-κB p65， and COX-2 were significantly higher in OGD/R model group than those in control group （P<0.05）. When compared with OGD/R model group， the expression levels of GRP78， CHOP， NF-κBp65 and COX-2 were decreased by NaHS at concentrations of 0.2， 0.4 and 4 mmol/L （P<0.05）， but there was no statistically significant difference among the treatment groups （P>0.05）. 【Conclusion】 NaHS can rescue cell viability of OGD/R-induced cell injury. It may be achieved by inhibiting the activation of NF-κBp65 and COX-2 proteins induced by endoplasmic reticulum stress to reduce the level of cell apoptosis.

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.

OBJECTIVETo study the distribution of haplotypes of Y-chromosomal short tandem repeats (Y-STR) loci among three ethnic minorities from Guizhou, China.

METHODSTwenty 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.

RESULTSSeventy 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.

CONCLUSIONAnalysis 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.

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.