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Background Chronic excessive exposure to fluoride can cause damage to the central nervous system and a certain degree of learning and memory impairment. However, the associated mechanism is not yet clear and further exploration is needed. Objective Using 4D unlabelled quantitative proteomics techniques to explore differentially expressed proteins and their potential mechanisms of action in chronic excessive fluoride exposure induced brain injury. Methods Twenty-four SPF-grade adult SD rats, half male and half male, were selected and divided into a control group and a fluoride group by random number table method, with 12 rats in each group. Among them, the control group drank tap water (fluorine content<1 mg·L−1), the fluoride group drank sodium fluoride solution (fluorine content 10 mg·L−1), and both groups were fed with ordinary mouse feed (fluoride content<0.6 mg·kg−1). After 180 d of feeding, the SD rats were weighed, and then part of the brain tissue was sampled for pathological examination by hematoxylin-eosin (HE) staining and Nissl staining. The rest of the brain tissue was frozen and stored at −80 ℃. Three brain tissue samples from each group were randomly selected for proteomics detection. Differentially expressed proteins were screened and subcellular localization analysis was performed, followed by Gene Ontology (GO) function analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, cluster analysis, and protein-protein interaction analysis. Finally, Western blotting was used to detect the expression levels of key proteins extracted from the brain tissue samples. Results After 180 d of feeding, the average weight of the rats in the fluoride group was significantly lower than that in the control group (P<0.05). The brain tissue stained with HE showed no significant morphological changes in the cerebral cortex of the fluoride treated rats, and neuron loss, irregular arrangement of neurons, eosinophilic changes, and cell body pyknosis were observed in the hippocampus. The Nissl staining results showed that the staining of neurons in the cerebral cortex and hippocampus of rats exposed to fluoride decreased (Nissl bodies decreased). The proteomics results showed that a total of 6927 proteins were identified. After screening, 206 differentially expressed proteins were obtained between the control group and the fluoride group, including 96 up-regulated proteins and 110 down-regulated proteins. The differential proteins were mainly located in cytoplasm (30.6%), nucleus (27.2%), mitochondria (13.6%), plasma membrane (13.6%), and extracellular domain (11.7%). The GO analysis results showed that differentially expressed proteins mainly participated in biological processes such as iron ion transport, regulation of dopamine neuron differentiation, and negative regulation of respiratory burst in inflammatory response, exercised molecular functions such as ferrous binding, iron oxidase activity, and cytokine activity, and were located in the smooth endoplasmic reticulum membrane, fixed components of the membrane, chloride channel complexes, and other cellular components. The KEGG significantly enriched pathways included biosynthesis of secondary metabolites, carbon metabolism, and microbial metabolism in diverse environments. The results of differential protein-protein interaction analysis showed that the highest connectivity was found in glucose-6-phosphate isomerase (Gpi). The expression level of Gpi in the brain tissue of the rats in the fluoride group was lower than that in the control group by Western blotting (P<0.05). Conclusion Multiple differentially expressed proteins are present in the brain tissue of rats with chronic fluorosis, and their functions are related to biosynthesis of secondary metabolites, carbon metabolism, and microbial metabolism in diverse environments; Gpi may be involved in cerebral neurological damage caused by chronic overdose fluoride exposure.
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Objective To explore the relationship between body muscle mass and body mass index of school-age children and their growth differences in different seasons. Methods A total of 526 cases of preschool children who came to Hengshui People's Hospital for examination from June 2018 to June 2020 were selected as observation objects, including 300 males and 226 females, aged 6-11 years old, with an average age of (8.2 years ±0.2 years). According to the body mass index (BMI), children are divided into normal body weight, overweight weight and obese groups. According to the season, preschool children were divided into four groups, spring group, summer group, autumn group, and winter group, with 131 cases in each group. The physique of preschool children was tested, and the serum 25(OH)D was detected at the same time, the body muscle mass was measured by the bioelectrical impedance method, and the whole body muscle mass index was calculated. Multi-factor linear regression was used to analyze the relationship between vitamin D nutritional status and muscle mass index ; To study the average vitamin D content of children and the differences in different seasons. Results There were 396 children with normal BMI, 90 were overweight, and 40 were obese. The children's normal weight, overweight, and obesity were divided into groups. According to the increase in BMI, the normal vitamin level group, overweight group, and obesity group also decreased, and the difference was statistically significant (P0.05). Compared with the vitamin D deficiency and deficiency groups, the number of children with sufficient vitamin D is also increasing, and the children with sufficient vitamin D have a fixed-point visit to the MMI. According to the analysis results, it is observed that there is a statistically significant difference in vitamin D nutrition and body muscle mass levels (P<0.05). The serum 25(OH)D levels of children in summer and autumn were significantly higher than those in spring and winter, and the difference was statistically significant (P<0.05). Conclusion The body muscle mass and body mass index of preschool children have a significant relationship with 25(OH)D. When 25(OH)D is sufficient, higher body muscle mass can be obtained and the body mass index can be decreased. The growth difference in different seasons is manifested in the higher 25(OH)D in summer, which is more conducive to the growth and development of preschool children.
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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.
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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.
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OBJECTIVE@#To identify the pathogenesis in two patients of restrictive cardiomyopathy (RCM) using high-throughput sequencing.@*METHODS@#Peripheral blood samples from the two patients and their parents were collected and genomic DNAs were extracted to conduct targeted next generation sequencing or whole exome sequencing. Bioinformation analysis was performed to identify the pathogenic variants in genes associated with cardiomyopathy, which were further validated by Sanger sequencing.@*RESULTS@#By high throughput sequencing, we detected a de novo heterozygous variant c.549+1G>T in TNNI3 gene in patient 1. The variant has not been reported previously and was predicted to be pathogenic in line with American College of Medical Genetics and Genomics (ACMG) guidelines (PVS1+PS2+PM2). Another heterozygous variant c.433C>T (p.Arg145Trp) in TNNI3 gene was identified in patient 2 and his father. The variant had been reported as pathogenic variant in Clinvar and HGMD databases; based on ACMG guidelines, the variant was predicted to be likely pathogenic (PS3+PM1+PP3).@*CONCLUSION@#TNNI3 variants may be the causative gene responsible for restrictive cardiomyopathy in the two patients. High throughput sequencing results provide bases for the diagnosis of restrictive cardiomyopathy.
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Child , Humans , Cardiomyopathy, Restrictive/genetics , Genomics , Heterozygote , Mutation , Exome SequencingABSTRACT
Objective:To investigate the effects of cancer susceptibility candidate gene 19 (CASC19) regulating the expression of microRNA-449b-5p (miR-449b-5p) on the proliferation, apoptosis and radiation sensitivity of cervical cancer cells.Methods:(1) HeLa cells of cervical cancer cell line were cultured. HeLa cells were irradiated with X-ray at different doses (0, 2, 4, 6, 8 Gy, respectively), then the expression level of CASC19 mRNA and miR-449b-5p were detected by real-time quantitative PCR. (2) HeLa cell proliferation, apoptosis, radiation sensitivity (expressed as a survival fraction) and its related protein expression included cyclin D1, cleaved-caspase-3, and histone variant H2AX (γ-H2AX) were examined after different treatment including silencing CASC19 expression, over-expressing miR-449b-5p, down-regulating miR-449b-5p and silencing CASC19 expression. (3) The dual luciferase reporter gene experiment and real-time quantitative PCR technology were used to verify the targeting relationship between CASC19 and miR-449b-5p.Results:(1) With the increase of X-ray irradiation different dose (0, 2, 4, 6, 8 Gy), the expression level of CASC19 mRNA in HeLa cells gradually increased ( F=502.681, P=0.000), and the expression level of miR-449b-5p gradually decreased ( F=202.936, P=0.000).(2) After silencing CASC19 expression or over-expressing miR-449b-5p, the survival rate of HeLa cells was significantly reduced ( P<0.05), the apoptosis rate was significantly increased ( P<0.05), the survival fraction was significantly reduced ( P<0.05), the expression level of cyclin D1 protein was significantly reduced ( P<0.05), and the expression levels of cleaved-caspase-3 and γ-H2AX protein were significantly increased ( P<0.05). After down-regulating miR-449b-5p and silencing CASC19 expression, the survival rate of HeLa cells was significantly reduced ( P<0.05), the apoptosis rate was significantly increased ( P<0.05), the survival fraction was significantly reduced ( P<0.05), the expression levels of cyclin D1 and γ-H2AX protein were significantly increased ( P<0.05), and the expression level of cleaved-caspase-3 was significantly decreased ( P<0.05). (3) Over expression of miR-449b-5p could significantly reduce the luciferase activity of CASC19 wild type (1.00±0.09 versus 0.37±0.05, P<0.01), but there were no significant effect on the luciferase activity of CASC19 mutant type (0.92±0.07 versus 0.94±0.05, P>0.05). After the expression of CASC19 was silenced, the expression level of miR-449b-5p in HeLa cells increased significantly (1.00±0.12 versus 4.84±0.49, P<0.05). After overexpression of CASC19, the expression level of miR-449b-5p in HeLa cells was significantly reduced (1.00±0.09 versus 0.38±0.04, P<0.05). Conclusion:CASC19 in HeLa cells negatively regulates the expression of miR-449b-5p, and down-regulating the expression of miR-449b-5p could partially reverse the effects of silencing CASC19 on HeLa cell proliferation, apoptosis and radiation sensitivity.
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Objective@#To investigate the effects of cancer susceptibility candidate gene 19 (CASC19) regulating the expression of microRNA-449b-5p (miR-449b-5p) on the proliferation, apoptosis and radiation sensitivity of cervical cancer cells.@*Methods@#(1) HeLa cells of cervical cancer cell line were cultured. HeLa cells were irradiated with X-ray at different doses (0, 2, 4, 6, 8 Gy, respectively), then the expression level of CASC19 mRNA and miR-449b-5p were detected by real-time quantitative PCR. (2) HeLa cell proliferation, apoptosis, radiation sensitivity (expressed as a survival fraction) and its related protein expression included cyclin D1, cleaved-caspase-3, and histone variant H2AX (γ-H2AX) were examined after different treatment including silencing CASC19 expression, over-expressing miR-449b-5p, down-regulating miR-449b-5p and silencing CASC19 expression. (3) The dual luciferase reporter gene experiment and real-time quantitative PCR technology were used to verify the targeting relationship between CASC19 and miR-449b-5p.@*Results@#(1) With the increase of X-ray irradiation different dose (0, 2, 4, 6, 8 Gy), the expression level of CASC19 mRNA in HeLa cells gradually increased (F=502.681, P=0.000), and the expression level of miR-449b-5p gradually decreased (F=202.936, P=0.000).(2) After silencing CASC19 expression or over-expressing miR-449b-5p, the survival rate of HeLa cells was significantly reduced (P<0.05), the apoptosis rate was significantly increased (P<0.05), the survival fraction was significantly reduced (P<0.05), the expression level of cyclin D1 protein was significantly reduced (P<0.05), and the expression levels of cleaved-caspase-3 and γ-H2AX protein were significantly increased (P<0.05). After down-regulating miR-449b-5p and silencing CASC19 expression, the survival rate of HeLa cells was significantly reduced (P<0.05), the apoptosis rate was significantly increased (P<0.05), the survival fraction was significantly reduced (P<0.05), the expression levels of cyclin D1 and γ-H2AX protein were significantly increased (P<0.05), and the expression level of cleaved-caspase-3 was significantly decreased (P<0.05). (3) Over expression of miR-449b-5p could significantly reduce the luciferase activity of CASC19 wild type (1.00±0.09 versus 0.37±0.05, P<0.01), but there were no significant effect on the luciferase activity of CASC19 mutant type (0.92±0.07 versus 0.94±0.05, P>0.05). After the expression of CASC19 was silenced, the expression level of miR-449b-5p in HeLa cells increased significantly (1.00±0.12 versus 4.84±0.49, P<0.05). After overexpression of CASC19, the expression level of miR-449b-5p in HeLa cells was significantly reduced (1.00±0.09 versus 0.38±0.04, P<0.05).@*Conclusion@#CASC19 in HeLa cells negatively regulates the expression of miR-449b-5p, and down-regulating the expression of miR-449b-5p could partially reverse the effects of silencing CASC19 on HeLa cell proliferation, apoptosis and radiation sensitivity.
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Objective:To investigate the specific effects of combined use of dinoprostone and phloroglucinol in painless delivery in full-term pregnant patients.Methods:100 primiparas admitted from Oct. 2016 to Oct. 2018 in our hospital were enrolled in this study. The 100 primiparas all met the following conditions: full-term pregnancy induced labor, singletons, no local complications, no maternity contraindications, no prostaglandins contraindications, no anesthesia contraindications and bishop scores of cervical were six points or less. By using the random number table method, they were divided into observation group and control group in the two groups, 50 cases in each group. Patients in the control group were induced labor by intravenous oxytocin infusion, and labor analgesia was performed under anesthesia when the opening of uterine orifice was 3 cm. In the observation group, labor induction mode was changed on the basis of the control group. Dinoprostone was used to induce labor at the posterior vaginal vault of the patient, and intravenous infusion of phloroglucinol was performed when the opening of uterine orifice was 3 cm and labor analgesia was performed under anesthesia. The cervical maturation rate, labor induction rate, specific labor process, pain degree and pregnancy outcome were compared between the two groups.Results:The effective rate of cervical maturation in the observation group (94.00%) was significantly higher than that in the control group (76.00%) , and the difference was statistically significant ( P<0.05) . The comparison results of pain degree between the two groups showed that: 36 cases (72.00%) with grade I pain in the observation group, significantly higher than 24 cases (48.00%) of the control group, and 2 cases (4.00%) with grade III pain in the observation group, significantly lower than 8 cases (16.00%) in the control group, with statistically significant differences ( P<0.05) . There was no significant difference between the two groups in term of the number of patients with grade II pain patients ( P>0.05) . There were 46 cases (92.00%) of successful labor induction in the observation group and 38 cases (76.00%) in the control group. The success rate of labor induction in the observation group was significantly higher than that in the control group, with statistically significant difference ( P<0.05) . The incubation period, active period, first stage and total length of labor in the observation group were significantly shorter than those in the control group, with statistically significant differences ( P<0.05) . There was no significant difference in the length of the second and third stages of labor between the two groups ( P>0.05) . There was no significant difference in neonatal asphyxia rate, Apgar score and postpartum blood loss between the two groups ( P>0.05) . Conclusion:The application of dinoprostone in painless delivery can effectively shorten the length of labor, and has a good effect on pain relief and smooth delivery, which can further accelerate the labor process of patients, accelerate cervical maturation, soften the cervix of patients, reduce spasm, and improve the rate of labor induction by combining with phloroglucinol, with more accurate efficacy.
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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.
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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.
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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.
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OBJECTIVE To investigate the effect of Turkish galls extract (TGE) on the expression of IgA in serum,urine and renal tissue of IgA nephropathy (IgAN) model rats.METHODS Fifty healthy male Sprague Dawley rats were randomly divided into normal control group,IgAN model group,and TGE 75,150 and 300 mg· kg-1 groups,10 rats per group.The model of IgAN rats was established with bovine serum albumin (BSA)+lipopolysaccharide (LPS)+carbon tetrachlorid (CCl4)for 12 weeks.From the 13th week,TGE was ig administrated once a day for 4 weeks.At the end of the 12th and 16th weeks,24 h urine protein was measured by BCA method.At the end of the 16th week,serum and urinary IgA levels were measured by enzyme linked immunosorbent assay(ELISA),serum creatinine(SCR) and blood urea nitrogen (BUN) were detected by an automatic biochemical analyzer,and the renal pathological changes were evaluated with an Oxford classification scoring system.The deposition of IgA immune complex in the kidney was observed by immunofluorescence assay.RESULTS At the end of 12th week,24 h urine protein increased in all IgAN groups (P＜0.05),compared with normal control group.At the end of 16th week,24 h urine protein,IgA content in serum and urine,SCr and BUN content in serum,score in Oxford classification of renal tissue and deposition of IgA immune complex in the kidney in IgAN model group were all higher than in normal control group (P＜0.05).Compared with IgAN model group,24 h urine protein,IgA content in serum and urine and SCr content in serum were decreased in all TGE groups (P＜0.05),and BUN content in serum and deposition of IgA immune complex in the kidney decreased in TGE 150 and 300 mg·kg-1 groups (P＜0.05).The score in Oxford classification of renal tissue was decreased in TGE 300 mg· kg-1 group only.CONCLUSION TGE has curative effect on IgAN model rats by reducing serum and urinary IgA and decreasing IgA immune complex deposition.
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Objective To investigate the effect of berberine on the radiosensitivity of cervical cancer cells.Methods 5,10,15,20 μmol/L of berberine were used to treat cervical cancer cell lines of Siha,HeLa,Caski.DMSO was applied as control of drug treatment.Cell proliferation was detected by the CCK-8 method,and then the half inhibitory concentration of berberine was calculated.Cell apoptosis and cell cycle distribution were detected by flow cytometry.Protein expressions of Cleaved Caspase-3,Cyclin B1,CDK1,STAT3 and p-STAT3 were detected by Western blot.Cervical cancer cells of Siha were treated by berberine with a half inhibitory concentration for 24 h and then irradiated with 0,2,4,6,8 Gy of X-rays.Cell clone assay was used to detect cell survival.Results Berberine could inhibit the growth of cervical cancer cells with a half inhibition concentration of(16.84 ± 3.52),(23.54 ± 8.67),(21.86 ± 6.35)μmol/L for Siha,Caski,and HeLa cells,respectively.The berberine at 17 μmol/L could induce apoptosis (t =56.847,P ＜ 0.01) and G2/M phase arrest (t =47.251,P ＜ 0.01) in Siha cells,which also inhibited the expressions of Cyclin B1,CDK1 and p-STAT3 and promoted the expression of cleaved Caspase-3,but did not influence the expression of STAT3 in cervical cancer cells.Treatment of cells with 17 μmol/L berberine increased the radiosensitivity of cervical cancer cells with a sensitivity enhancement ratio of 1.55.Conclusions Berberine can inhibit cell proliferation,promote apoptosis,block cell cycle,and increase radiosensitivity of cervical cancer cells.
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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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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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Objective To study the imaging appearances of congenital splenorenal venous shunt,and to evaluate their clinical significances.Methods Of 127 283 patients who underwent upper abdominal CT scanning,6 patients were diagnosed to have congenital splenorenal venous shunt.The imagings were studied retrospectively.Plain scanning,enhanced CT and Doppler ultrasonography were performed on all these patients.Results The incidence was about 47/million.The six patients were all females,the age was between 32 to 67 years.The mean age was 48.8 years.Enhanced CT demonstrated that there were twisted and dilated shunting blood vessels between the splenic and renal veins.The diameters in four patients were larger than the splenic and portal veins.In the remaining two patients,they were smaller shunting blood vessels.One patient had an associated absence of right portal vein.Two patients had associated dysplasia of portal veins and splenic veins.MPR,CPR,MIP and VR could three-dimensionally depict the courses,the beginnings and the ends of the splenorenal shunts.Doppler ultrasonography showed counterflow between the portal and splenic veins,and showed the blood in the splenic vein to flow into the left renal vein.Conclusions A congenital splenorenal venous shunt is one of the rare form of congenital extrahepatic portosystemic venous shunts.Enhanced MSCT scan combining with its post-processing techniques could clearly demonstrate the shunt vessels and the associated lesions.Doppler ultrasonography could further demonstrate the shunt direction.
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Objective To observe the expression of neural nicotinic acetylcholine receptor subunit α3 (α3nAChR) and extracellular regulated protein kinases (ERK1/2),c-Jun N-terminal kinase (JNK),p38 kinases of mitogen-activated protein kinase (MAPK) pathway in human neuroblastoma cell line SH-SY5Y overexposed to fluoride,and try to investigate the molecular mechanism of cell damage caused by overexposure of fluoride.Methods The SH-SY5Y cell with low expression of α3nAChR suppressed by silence interference RNA served as α3nAChR silence group;the normal SH-SY5Y cell served as control group,and the effect of silencing of αt3nAChR gene in SHSY5Y was detected by Western blotting and real-time PCR;SH-SY5Y cell was treated with different concentrations of fluoride (0.000,0.005,0.050,0.500,1.000,2.500,5.000 mmol/L),the safe concentration of fluoride in SHSY5Y cell was detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay;the SH-SY5Y cell of control group and α3nAChR silence group were treated with 4.000 mmol/L fluoride for 0,4,8,12,24,36,48 h according to the results of MTT assay;the expression of ERK1/2,JNK,p38 kinases of MAPK pathway in SH-SY5Y at protein levels was measured by Western blotting.Results The expression of α3nAChR mRNA (0.04 ± 0.03) and protein (12.0 ± 2.5) in α3nAChR silence group was decreased significantly compared with those of control group (1.00 ± 0.11,100.0 ± 11.3,t =24.58,28.80,all P ＜ 0.05).The viability of SH-SY5Y cell treated with 5.000 mmol/L fluoride (0.53 ± 0.15) was decreased significantly compared with that of SH-SY5Y cell treated with 0.000 mmol/L fluoride (1.05 ± 0.05,P ＜ 0.05).The increased expression of phospho-ERK1/2 was found in α3nAChR silence group and control group incubated with fluoride with time prolonged,and the expression of phospho-ERK1/2 increased significantly at time points 24,36 and 48 h (188.33 ± 7.33,200.00 ± 10.01,213.33 ± 11.55;125.33 ± 5.69,136.00 ± 4.52,155.33 ± 6.51) compared to 0 h in the same groups (100.00 ± 0.00,100.00 ± 0.00,all P ＜ 0.05),and the expression of phospho-ERK1/2 was higher significantly in α3nAChR silence group than those of control group (t =9.26,7.63,5.72,all P ＜ 0.05);no change of expression of total-ERK1/2 in the two groups was found with the passage of time.The gradually increased expression of phospho-JNK was found in α3nAChR silence group and control group,among which,the expression of phospho-JNK in o3nAChR silence group at time points 12,24,36 and 48 h (154.00 ± 6.25,149.00 ± 5.57,156.00 ± 6.08,141.67 ± 2.52) and in control group at 8,12,24,36,48 h (133.33 ± 10.69,173.00 ± 4.00,175.00 ± 11.79,200.67 ± 11.93,200.33 ± 18.58) was compared to those at 0 h in the same groups (100.00 ± 0.00,100.00 ± 0.00),and the difference was significant (all P ＜ 0.05);the higher expression of phospho-JNK was found in α3nAChR silence group other than control group at 8,12,24,36,48 h (t =-4.28,-5.02,-2.89,-8.33,-6.18,all P ＜ 0.05);no change of expression of total-JNK was found in the two groups (P ＞ 0.05).The increased expression of phospho-p38 was detected in control group at time points 24,36 and 48 h (120.33 ± 4.51,122.00 ± 7.55,119.67 ± 7.57) compared to 0 h in the same groups (100.00 ± 0.00,all P ＜ 0.05),and the expression of phospho-p38 was significantly higher than that in α3nAChR silence group at the same time points (93.33 ± 9.61,94.00 ± 5.01,98.33 ± 5.69,t =-4.01,-6.73,-5.59,all P ＜ 0.05);no change of expression of total-p38 was found in the two types of SH-SY5Y cells treated with fluoride (P ＞ 0.05).Conclusion When SH-SY5Y cells are exposed to fluoride;activation of ERK1/2 may be not depend on α3nAChR;α3nAChR may have protected the cell from apoptotic injury caused by activation of JNK pathway,and the activation of p38 may be depend on nAChRα3.
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Objective To investigate the clinical significance of the combination detection of blood lactic acid,B-type brain natri-uretic peptide(BNP)and homocysteine(Hcy)in elderly acute cerebral infarction.Methods The arterial and venous blood during a-cute onset stage,at 24,48,72 h after thrombolysis and at 12 h after fasting in 85 cases of acute cerebral infarction was collected for detecting blood lactic acid,BNP and Hcy,performing the dynamic monitoring analysis and the comparative observation with the healthy control group.Results The blood lactic acid,BNP and Hcy levels in the acute onset stage group and the recovery group were significantly increased compared with the healthy control group(P <0.05).The blood lactic acid,BNP and Hcy levels during the onset stage in the acute cerebral infarction group were(6.47 ±3.92)mmol/L,(100.52 ±48.96 )pg/mL and(48.96 ±15.13 )μmol/L respectively,which were significantly higher than those at 48,72 h of postoperative recovery,the differences between them were statistically significant(P <0.05).The blood lactic acid,BNP and Hcy levels during postoperative convalescence in the cere-bral infarction group were gradually decreased and declined to the minimal level until postoperative 72 h,which were(2.16±1.83) mmol/L,(33.61±10.42)pg/mL and(18.87±8.27)μmol/L respectively.Conclusion Blood lactic acid,BNP and Hcy are closely related with the occurrence and development of cerebral infarction,the joint detection of these three indicators provide a reliable ba-sis for the early diagnosis of elderly acute cerebral infarction.The active and effective treatment on the patient should be timely a-dopted and the dynamic monitoring should be performed in order to correctly assess the prognosis.
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Objective To observe the effects of fluorosis on the levels of endogenous cystathionine beta-synthase (CBS) and hydrogen sulfide (H2S) in rats.Methods According to body weight,forty-eight Sprague-Dawley rats (body weight 105-180 g) were divided into three groups by a random number table(16 rats in each group,half male).Fluorine contents of the feed in control group,low-fluoride group and high-fluoride group were 9.80,15.40 and 23.80 mg/kg.After 6 months of fluorine exposure,the fluorine contents of urine and bone were determined by the method of fluorine ion-selective electrode ; H2S levels in serum and brain and the activity of CBS in brain were detected by methylene blue; and protein expression of CBS was detected by Western blotting.Results Compared with control group,dental fluorosis was found in rats of low-fluoride and high-fluoride groups.The differences of fluorine contents of urine and femur were statistically significant between groups(F =65.16,67.93,all P ＜ 0.05).The urinary and femoral fluorine in low-fluoride groups [(5.25 ± 0.45)mg/L,(1 196.54 ± 72.78)mg/kg] and high-fluoride groups[(13.17 ± 0.98)mg/L,(2 656.61 ± 170.12)mg/kg] were higher than those of control groups [(3.64 ± 0.20)mg/L,(870.71 ± 71.51)mg/kg,all P ＜ 0.05],and the increases were in a dose-dependent fashion(all P ＜ 0.01).The differences of H2S contents in serum and brain were statistically significant(F =4.83,1 456.13,all P ＜ 0.05).The H2S content in serum was higher in high-fluoride group [(17.64 ± 2.38) μ mol/L] than that of the control group [(10.29 ± 0.74) μ mol/L,P ＜ 0.01].The H2S contents in brain were higher in the low-fluoride [(364.74 ± 2.06)μmol/L] and high-fluoride groups [(513.43 ± 4.18) μmol/L] than those of the control group[(314.94 ± 0.72)μmol/L,all P ＜ 0.01],and the increase was in a dose-dependent fashion (P ＜ 0.01).The difference of CBS activity was statistically significant between groups (F =760.63,P ＜ 0.01).The CBS activities were lower in low-fluoride [(438.90 ± 2.83) mmol· kg-1· min-1] and high-fluoride groups [(529.83 ± 2.37)mmol· kg-1· min-1] than those of the control group [(596.33 ± 2.75) mmol · kg-1· min-1,all P ＜ 0.01],whereas the protein expression of CBS in brain in high-fluoride group (1.49 ± 0.08) was higher than that of the control group (1.19 ± 0.06,P ＜ 0.05).Conclusion Chronic fluorosis can affect the levels of endogenous CBS and H2,S,and the increases are in a dose-dependent fashion in addition to CBS activity.
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Objective To investigate the effect of LI-cadherin- SiRNA protein on the growth and metastatic potentials of transplanted human hepatocellular carcinoma cell lines (Hep3B) in nude mice.Methods We transfected LI-cadherin- SiRNA to Hep3B cells,Hep3B cell suspension (transfected or control ) was injected subcapsullaryly into the spleen of nude mice,hepatic metastasis was observed by naked eye and immunohistochemistry.In addition,Western-blot was used to detect the level of LI-cadherin in different metastatic site.Results (1) Hep3B was green after successful transfect interference vector under fluorescence inverted microscope,in this study,the transfect rate was 80％.(2) Hep3B liver metastasis model in nude mice was established.The metastasis rates in the empty plasmid carrying group,the control group and the SiRNA transfect group were 50％,60％ and 80％,respectively.The number of metastasis caner nodules in the SiRNA transfect group was 26,significantly higher than other two groups.(3) The level of protein expression for LI-cadherin in the SiRNA transfect group is significantly lower than the control group and the empty plasmid carrying group.Conclusions LI-cadherin is crucial and important for the adhension capability of HCC in its migration.SiRNA transfected LI-cadherin increases the metastasis in a nude mouse model inoculated with human hepatocellular carcinoma cell lines.