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Objective:To investigate whether the necroptosis pathway receptor interacting protein 1-receptor interacting protein 3-mixed lineage kinase domain-like protein (RIP1-RIP3-MLKL) is involved in fluoride-induced osteoblastic death.Methods:Human osteosarcoma cell line (Saos-2 cells) were cultured in vitro and divided into NC group, sodium fluoride (NaF) groups (5.0, 10.0, 20.0 and 40.0 mg/L NaF), necroptosis inhibitor Necrostatin-1 (Nec-1) group (50.0 μmol/L Nec-1) and NaF + Nec-1 groups (5.0, 10.0, 20.0 and 40.0 mg/L NaF + 50.0 μmol/L Nec-1). After cultured for 24 and 48 h, respectively, cell proliferation was determined via the CCK-8 method, and the activity of lactate dehydrogenase (LDH) was determined by chemical colorimetry. To further analyze the influence of NaF on RIP1-RIP3-MLKL pathway, Saos-2 cells were divided into NCⅡgroup and NaFⅡgroups (2.5, 5.0, 10.0, 20.0 and 40.0 mg/L NaF). After cultured for 24 and 48 h, respectively, the protein expression levels of RIP1, RIP3 and MLKL were determined by Western blotting. Results:The cell proliferation rates (%: 100.00 ± 0.59, 104.90 ± 0.44, 104.16 ± 0.41, 82.45 ± 1.91, 64.59 ± 1.83, 103.56 ± 0.41, 107.18 ± 0.87, 105.35 ± 1.28, 89.63 ± 1.20, 77.51 ± 1.30; 100.00 ± 0.33, 107.92 ± 0.44, 101.78 ± 1.06, 75.45 ± 0.39, 57.94 ± 1.17, 106.74 ± 0.21, 111.85 ± 0.21, 107.82 ± 0.68, 82.34 ± 0.56, 70.19 ± 0.99) among all groups were significantly different at both 24 and 48 h ( F = 77.13, 2 313.43, P < 0.05). Except the cell proliferation rate of the 10.0 mg/L NaF + Nec-1 group that was not significantly different with that of the 10.0 mg/L NaF group at 24 h ( P > 0.05), the cell proliferation rates of other NaF + Nec-1 groups were significantly higher than those of corresponding NaF groups at both 24 and 48 h ( P < 0.05). The proliferation rate was negatively correlated with fluoride concentration ( r24 h = - 0.976, r48 h = - 0.969, P < 0.001). The LDH activity in all concentrations of NaF groups was significantly higher than that in NC group and corresponding NaF + Nec-1 groups at both 24 and 48 h ( P < 0.05). The LDH activity was positively correlated with fluoride concentration ( r24 h = 0.985, r48 h = 0.988, P < 0.001). The protein expression levels of RIP1, RIP3 and MLKL of 5.0 mg/L NaFⅡ group at 24 h, RIP3 of 5.0 mg/L NaFⅡ group at 48 h, and RIP1, RIP3 and MLKL of 10.0, 20.0 and 40.0 mg/L NaFⅡ groups at both 24 and 48 h were higher than that in NCⅡ group ( P < 0.05). The protein expression levels of RIP1, RIP3 and MLKL were positively correlated with fluoride concentration ( r24 h-RIP1 = 0.881, r48 h-RIP1 = 0.952, r24 h-RIP3 = 0.867, r48 h-RIP3 = 0.938, r24 h-MLKL = 0.758, r48 h-MLKL = 0.907, P < 0.001). Conclusion:Fluoride can directly cause necroptosis of osteoblasts through the RIP1-RIP3-MLKL pathway, and the severity of cell damage is closely related to fluoride concentration, Nec-1 has partially reversed the effects of fluoride.
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Objective:To observe the effect of sodium fluoride (NaF) on the expression of phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) signal and apoptosis in human osteosarcoma Saos-2 cells.Methods:Saos-2 cells were divided into 0 (control), 5, 10, 20 and 40 mg/L fluoride groups ( n= 3) according to the dose of NaF. The cells were collected after 24 and 48 h of culture in vitro. Western blotting was used to detect the expressions of PI3K/Akt signal and Forkhead transcription factor (FoxO) 1, and the apoptosis level of Saos-2 cells was detected by flow cytometry. Results:There were no significants differences in the expressions of PI3K and Akt in Saos-2 cells at 24 and 48 h ( P > 0.05). At 24 h, the expressions of phosphorylated PI3K (p-PI3K) in 5, 10, 20 and 40 mg/L fluoride groups (0.40 ± 0.06, 0.45 ± 0.02, 0.37 ± 0.06, 0.32 ± 0.06) were higher than that in the control group (0.28 ± 0.04, P < 0.05); at 48 h, the expressions of p-PI3K in 5 and 10 mg/L fluoride groups (0.46 ± 0.06, 0.58 ± 0.03) were higher than that in the control group (0.29 ± 0.04, P < 0.05), and that in the 40 mg/L fluoride group (0.21 ± 0.03) was lower than that in the control group ( P < 0.05). At 24 h, the expressions of phosphorylated Akt (p-Akt) in 5, 10 and 20 mg/L fluoride groups (0.27 ± 0.01, 0.30 ± 0.03, 0.27 ± 0.03) were higher than that in the control group (0.20 ± 0.02, P < 0.05); at 48 h, the expressions of p-Akt in 5 and 10 mg/L fluoride groups (0.42 ± 0.04, 0.60 ± 0.02) were higher than that in the control group (0.27 ± 0.01, P < 0.05), and that in the 40 mg/L fluoride group (0.18 ± 0.02) was lower than that in the control group ( P < 0.05). The expressions of FoxO1 in 10, 20 and 40 mg/L fluoride groups at 24 h (0.38 ± 0.07, 0.41 ± 0.06, 0.47 ± 0.08) were higher than that in the control group (0.34 ± 0.04, P < 0.05). At 48 h, the expressions of FoxO1 in 5, 10, 20 and 40 mg/L fluoride groups (0.36 ± 0.08, 0.37 ± 0.10, 0.54 ± 0.04, 0.73 ± 0.04) were higher than that in the control group (0.28 ± 0.04, P < 0.05). At 24 and 48 h, the apoptosis rates of control group and 5, 10, 20 and 40 mg/L fluoride groups were (2.867 ± 0.583)%, (3.647 ± 0.035)%, (3.773 ± 0.095)%, (5.440 ± 0.325)%, (7.203 ± 0.476)%; (3.707 ± 0.286)%, (4.023 ± 0.241)%, (4.970 ± 0.368)%, (12.473 ± 0.949)%, (19.387 ± 1.892)%, respectively. The apoptosis level of 40 mg/L fluoride group was higher than that of control group at 24 h ( P < 0.05), and that of 20 and 40 mg/L fluoride groups were higher than that of control group at 48 h ( P < 0.05). Conclusion:Fluoride can directly activate the PI3K/Akt signaling pathway in osteoblasts, and then has anti-apoptotic effect.
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BACKGROUND:There are no systematic and coherent studies on the mitochondrial apoptotic pathway of fluoride-induced osteoblast apoptosis, and the specific pathways to induce apoptosis in osteoblasts by fluorine are stil unclear. OBJECTIVE:To explore the possible pathways of apoptosis in osteoblasts induced by fluoride and its molecular characteristics. METHODS:A fluorosis model of human osteosarcoma cel line Saos-2 was establishedin vitro. After in vitro culture, the cels were treated with sodium fluoride at different concentrations (0, 5, 10, 20, 40, 80 mg/L). Flow cytometry was used to inspect the mitochondrial membrane potential at 24 hours after intervention; 84 apoptosis-related genes were detected by PCR Array; parts of the differentialy expressed genes were verified by western blot method. RESULTS AND CONCLUSION: When the concentrations of sodium fluoride were 20, 40 and 80 mg/L, the mitochondrial membrane potentials in osteoblasts were 27.0%, 28.8%, 38.6%, respectively (alP < 0.05). PCR array found 13 genes upregulated and 15 genes down-regulated. Immunoblotting results showed Bim, Caspase 9, Caspase 14, BCL2, BAX expressions enhanced with increasing doses of sodium fluoride; Caspase 3 expression was decreased at the concentration of 5 mg/L, but increased gradualy at over 10 mg/L. Caspase 7 expression had no significant difference. The expression of Caspase 10 decreased with the increasing doses of sodium fluoride. These findings indicate that fluoride may induce apoptosis in osteoblasts through the mitochondrial pathway (including the endoplasmic reticulum stress pathway) and death receptor pathway.
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BACKGROUND:At present, there was lack of reports on the efficacy of thoracolumbar tuberculosis complicated with severe kyphosis (>90°). Choice of surgical treatment is necessary for patients with severe spinal tuberculosis kyphosis, affected heart and lung function and neurological disorders. OBJECTIVE:To retrospectively analyze the repair effect of I-stage posterior osteotomy orthopedic fixation and II-stage anterior debridement interbody bone grafting fusion in repair of patients with thoracolumbar tuberculosis complicated with severe kyphosis. METHODS:Total y 53 patients with spinal tuberculosis complicated with severe kyphosis were enrol ed. Patients underwent posterior osteotomy orthopedic fixation in the first stage, and underwent anterior debridement interbody bone grafting fusion in the second stage. X-ray, CT, MRI and other imaging examinations were conducted before and after the treatment. Erythrocyte sedimentation rate, C-reactive protein, pain visual analog scale scores, kyphosis and ASIA spinal cord injury classification before and after the treatment were compared and analyzed for clinical evaluation of efficacy. RESULTS AND CONCLUSION:Al patients had a successful surgery. The operative time was 290 (195-420) minutes, and the intra-operative amount of blood loss was 1800 (1 100-3 300) mL, the average number of fixed segments were 11.8 (9-16). Al these 53 patients were fol owed up for 26-28 months. The erythrocyte sedimentation rate and C-reactive protein of patients after treatment gradual y recovered to normal, and recovered to normal levels at the final fol ow-up. The mean correction of sagittal Cobb angle was 77.92°, the correction rate reached to 74.6%at the final fol ow-up. Til the final fol ow-up, the average loss of corrective angle was 1.35°. The lower back pain and limitation of function obtained varying degrees of al eviating after treatment. The visual analog scale scores in the final fol ow-up were significantly lower than those before treatment (t=19.219, P<0.001). ASIA spinal cord injury scores gradual y increased. Patients recovered the ability to live and work in varying degrees. These results suggest that I-stage posterior osteotomy orthopedic fixation combined with II-stage anterior debridement interbody bone graft fusion is an effective methods for repair of thoracolumbar tuberculosis complicated with severe kyphosis. The lesions of patients with thoracolumbar tuberculosis complicated with severe kyphosis who were enrol ed in this study involve multiple vertebral body, long bone defect, and often need long segmental al ograft bone grafting, with long-time of bone grafting fusion, therefore, zygapophyseal bone grafting fusion should be conducted to increase the stability of posterior bone grafting.
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Objective To observe gene different expression of unfolded protein response signaling pathway in human osteoblasts under the excessive fluoride ,and explore the role of endoplasmic reticulum stress in fluorosis .Methods Human osteoblasts were cultured with fluoride ,intervening for 24 h .Cell viability and apoptosis were inspected by MTS assay and flow cytometer respective‐ly .The UPR signaling pathway was examined by real time PCR array ,and protein expressions were detected by Western blot .Re‐sults T he cell survival rates w ere (100 .678 5 ± 2 .830 3 )% ,(105 .393 4 ± 2 .538 4 )% ,(106 .125 7 ± 2 .048 3 )% ,(77 .977 3 ± 2 .544 3)% (P<0 .05) ,(30 .237 7 ± 0 .632 73)% (P<0 .05) treated with sodium fluoride at the concentration 0 ,5 ,10 ,20 ,40 ,80 mg/L respectively .Apoptosis rate inspected by flow cytometer was 4 .8% in 5 mg/L group ,13 .8% in 10 mg/L group ,37 .0% in 20 mg/L group ,58 .9% in 40 mg/L group ,63 .2% in 80 mg/L group (P<0 .05) .Only 1 gene was down regulated and 14 genes were up regulated .Western blot analysis showed BIP ,ATF4 ,CHOP and IRE1 both showed their protein expression gradually up regula‐ted with fluorine dose .XBP1 expression gradually increased in NaF 5-20 mg/L ,and its expression decreased at 40 and 80 mg/L . Conclusion Sodium fluoride can cause osteoblasts endoplasmic reticulum stress pathway through PTEN and IRE1 pathway ,and at high concentrations can cause apoptosis of osteoblast .
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BACKGROUND:Previous studies have shown that minichromosome maintenance 3 is related with fluorosis, but the expression of minichromosome maintenance 3 in fluorosis patients is not clear yet. OBJECTIVE:To analyze the mRNA expression level of minichromosome maintenance 3 in peripheral blood from patients exposed to fluoride and normal controls. METHODS:Eleven patients with mild fluorosis by drinking water (exposure group) and 11 cases of control (non-exposure group) were selected for research. SYBRGreen1 real-time quantitative PCR was used to determine the mRNA expression of minichromosome maintenance 3 in peripheral blood mononuclear cel s, and the liver and renal function indicators were detected. RESULTS AND CONCLUSION:mRNA expressions of minichromosome maintenance 3 in the exposure group and non-exposure group were (0.573 60±0.102 59) and (0.550 0±0.171 81), respectively, and there was no significant difference between two groups (P>0.05). There were no significant differences in the liver and renal function indicators between two groups. The results indicate that mild fluorosis has no significant effect on mRNA expression of minichromosome maintenance 3 in the peripheral blood mononuclear cel s. More indicators are needed to compressively analyze the effect of fluoride on the liver and renal functions.
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Objective To investigate the expression and significance of Smad4 in peripheral hepatocytes of lesions in mice with hepatic alveolar echinococcosis.Methods Eight mice in the test group were inoculated with alveolar echinococcosis and 8 mice in the control group were injected with normal saline.The expression of Smad4 protein in the hepatic tissue of the mice was detected by immunohistochemistry method,and the data were analyzed by chi-square test.The effect of alveolar echinocoeeosis on the expression of Smad4 protein was investigated.Results Smad4 was detected in cell nuclei and partly in the cytoplasm.Six months after the establishment of the mice model for alveolar echinococeosis,the expression of Smad4 in the hepatic tissue in the test group was significantly higher than in the control group(x2=19.869,P＜0.05).The number of Smad4 with positive expression in the hepatocytes in the test group was slightly higher than in the control group,and the expression intensity of Smad4 in the test group was greater than in the control group(x2=58.3 10,P＜0.05).Conclusions The increase of the expression of Smad4 protein in the periphery hepatocytes and tissues of hepatic alveolar echinococcosis lesions may play a role in hepatic cirrhosis and immune evasion.
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Objective To explore the signal transduction pathway of cyst fluid of Echinococcus granalosus in anti-parasite mechanisms through investigating the effect of cyst fluid on the expression of Toll-like receptor(TLR) in cells. Methods Changes of TLR4 and TGF-β1 expression of 7404 liver cells were detected by quantitative PCR. Results After treatment with increasing concentration cyst fluid the expres-sion of TLR4 was reduced. TGF-β1 expression of liver cells increased with the dose. TLR2 expressions in each group were very low. Conclusion Cyst fluid can increase the expression of TLR4, suggesting that the TLR4 signal transduction pathway involve anti-cyst fluid of Echinococcus granulosus. High concentrations of cyst fluid contribute to TGF-β1 expression which plays a role in immune evasion.
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Objective: To investigate the effect of black currant extract on human esophageal squamous cancer cell Eca109 line to reveal its probable mechanism. Methods: The survival cells and protein synthesis of tumor cell lines treated with 10-1, 10 -2,10-3 g/ml of the water extracts of black currant for 24 h were determined by MTT and Bradford assays, and the cell-DNA ploidy distribution and apoptotic rate were measured by flow cytometry (FCM) and morphological observation. Results: There was significant inhibition of the cell survival and protein synthesis of cancer cells at dose of 10-1 g/ml of the extracts. In the normal rat liver cells, the cell survival was not affected. An apoptosis peak appeared before diploid peak in FCM and apoptotic rate was 49.6%, and morphological change of apoptosis was observed. Conclusion: Black currant could significantly inhibit growth of human esophageal squamous cancer cell Eca109 line through inducing apoptosis.
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0.05) was observed in the expression of PDI in fluoride treated groups, and the expression of XBP-1 in 10-80 mg/L groups was significantly higher with a dose-dependent manner, and in 2.5-80 mg/L groups, the expression of BIP/?-actin was up-regulated (P