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Objective To clarify the role of nuclear factor κB(NF-κB) signaling pathway in pathogenesis of Kashin-Beck disease(KBD) by observing the expression of NF-κB p65 in the whole blood samples of patients with KBD and controls,and the expression of NF-κB p65 in C28/I2 chondrocyte, and to analyze the role of NF-κB p65 molecule in chondrocyte apoptosis. Methods Through a case-control study, 161 patients with KBD (KBD group) were selected from Xunyi, Yongshou, Changwu, Linyou, Qianyang and Long counties in KBD endemic areas and 312 healthy people(control group) were matched by age and sex in Shaanxi Province. Venous blood samples were collected from patients and healthy controls, which were anticoagulated and used for determination of NF-κB p65 protein.According to the group design,the model of C28/I2 chondrocyte oxidative damage was established.The experiments were divided into 4 groups including control group(C), tBHP injury group (O, tBHP 300.00 μmol/L), low selenium pre-protection group (OS1, 0.05 mg/L Na2SeO3+ 300.00 μmol/L tBHP), and middle selenium pre-protection group(OS2, 0.10 mg/L Na2SeO3+ 300.00 μmol/L tBHP). Then, cell apoptosis was detected by Hoechst 33342 and reactive oxygen species (ROS) was detected by dichlorfluorescein(DCF) method. The protein was extracted by Trizol method, then protein expression level of NF-κB p65 molecule was detected by Western blotting in whole blood samples and C28/I2 chondrocyte. Results The differences in age and sex were not statistically significant between KBD group and control group (t = 0.336, P > 0.05; χ2= 0.407, P > 0.05). The protein expression level of NF-κB p65 in KBD group was 1.835 times as high as that of control group (KBD:0.167 ± 0.026, control: 0.091 ± 0.014, t = 5.147, P < 0.01). Under the fluorescence microscope, chondrocyte showed strong blue fluorescence in tBHP group and the level of ROS(1.219 ± 0.104) was higher than those of low and middle selenium pre-protection groups(0.832 ± 0.077, 0.635 ± 0.070, P < 0.05).The protein expression level of NF-κB p65 in tBHP group (1.563 ± 0.351) was higher than that of control group (0.451 ± 0.069, P < 0.05), and protein levels of NF-κB p65 had a decreasing tendency in low and middle selenium pre-protection groups compared to tBHP group. Conclusion The NF-κB signaling pathway is up-regulated in KBD patients, moreover, chondrocyte experiments show that cell apoptosis is mediated via upregulation of NF-κB p65,which suggests NF-κB signaling pathway may play an important role in pathogenesis of KBD.
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Objective To explore the effects of oxidative damage and selenium on the apoptosis of articular chondrocytes and the expression of selenoprotein genes.Methods C28/I2 chondrocytes were preincubated for 24 h,using sodium selenite (Na2SeO3) or t-butyl hydroperoxide (tBHP) for 24 h.The experiment was divided into six groups,including control group (C,0.00 mg/L Na2,SeO3 + 0.00 μmol/L tBHP),selenium beforehand protection group (S2,0.10 mg/L Na2SeO3),oxidative damage group (O,150.00 μmol/L tBHP),low dose selenium protection group (OS 1,0.05mg/L Na2SeO3 + 150.00 μmol/L tBHP),medium dose selenium protection group (OS2,0.10 mg/L Na2SeO3 + 150.00 μmol/L tBHP),and high dose selenium protection group (OS3,0.15 mg/L Na2SeO3 + 150.00 μmol/L tBHP).After 24 h,Hoechst 33342 staining method was used to observe apoptosis,mRNA expression of glutathione peroxidase 1 (GPX1),GPX4,deiodinase 2 (DIO2),DIO3,selenoprotein P (SEPP1),thioredoxin reductase 1 (TrxR-1) and selenoprotein W(Sel W) was detected by Real-time PCR,both experiments were done three times.Results Apoptotic rates of C,S2,O,OS1,OS2,OS3 groups [(0.78 ± 0.06)％,(13.61 ± 7.11)％,(92.27 ± 3.44)％,(71.38 ± 5.22)％,(44.31 ± 9.16)％,(72.46 ± 4.69)％] were compared between groups,the differences were statistically significant (F =120.10,P ＜ 0.01).The apoptotic rates of O group was significantly higher than that of C group (P ＜ 0.05);compared to O group,the apoptotic rates of OS1,OS2,OS3 groups decreased significantly (P＜ 0.05),OS2 group was the most obvious.DIO2,SEPP1,GPX1,GPX4,TrxR-1,Sel W mRNA levels were compared in the six groups,the differences were statistically significant (F =24.60,14.53,127.60,30.60,637.10,59.64,P ＜ 0.01).Compared to C group (1.00 ± 0.00),the mRNA levels of GPX1 (0.10 ± 0.05),GPX4 (0.43 ± 0.09),TrxR-1 (0.11 ± 0.05) and Sel W (0.72 ± 0.15) in O groups were decreased significantly (P ＜ 0.05);compared to 0 group,the mRNA levels of GPX1 in OS1 (0.20 ± 0.03),OS2 (0.74 ± 0.10),and OS3 (0.30 ± 0.07) were increased significantly (P ＜ 0.05).Conclusion Down-regulated expression of selenoprotein genes are involved in the regulation process of articular cartilage apoptosis caused by oxidative stress,selenium also has a regulatory role in selenoprotein gene expression in articular chondrocytes.
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Objective To explore the effects of oxidative damage and selenium on the apoptosis of articular chondrocytes and the expression of selenoprotein genes.Methods C28/I2 chondrocytes were preincubated for 24 h,using sodium selenite (Na2SeO3) or t-butyl hydroperoxide (tBHP) for 24 h.The experiment was divided into six groups,including control group (C,0.00 mg/L Na2,SeO3 + 0.00 μmol/L tBHP),selenium beforehand protection group (S2,0.10 mg/L Na2SeO3),oxidative damage group (O,150.00 μmol/L tBHP),low dose selenium protection group (OS 1,0.05mg/L Na2SeO3 + 150.00 μmol/L tBHP),medium dose selenium protection group (OS2,0.10 mg/L Na2SeO3 + 150.00 μmol/L tBHP),and high dose selenium protection group (OS3,0.15 mg/L Na2SeO3 + 150.00 μmol/L tBHP).After 24 h,Hoechst 33342 staining method was used to observe apoptosis,mRNA expression of glutathione peroxidase 1 (GPX1),GPX4,deiodinase 2 (DIO2),DIO3,selenoprotein P (SEPP1),thioredoxin reductase 1 (TrxR-1) and selenoprotein W(Sel W) was detected by Real-time PCR,both experiments were done three times.Results Apoptotic rates of C,S2,O,OS1,OS2,OS3 groups [(0.78 ± 0.06)％,(13.61 ± 7.11)％,(92.27 ± 3.44)％,(71.38 ± 5.22)％,(44.31 ± 9.16)％,(72.46 ± 4.69)％] were compared between groups,the differences were statistically significant (F =120.10,P ＜ 0.01).The apoptotic rates of O group was significantly higher than that of C group (P ＜ 0.05);compared to O group,the apoptotic rates of OS1,OS2,OS3 groups decreased significantly (P＜ 0.05),OS2 group was the most obvious.DIO2,SEPP1,GPX1,GPX4,TrxR-1,Sel W mRNA levels were compared in the six groups,the differences were statistically significant (F =24.60,14.53,127.60,30.60,637.10,59.64,P ＜ 0.01).Compared to C group (1.00 ± 0.00),the mRNA levels of GPX1 (0.10 ± 0.05),GPX4 (0.43 ± 0.09),TrxR-1 (0.11 ± 0.05) and Sel W (0.72 ± 0.15) in O groups were decreased significantly (P ＜ 0.05);compared to 0 group,the mRNA levels of GPX1 in OS1 (0.20 ± 0.03),OS2 (0.74 ± 0.10),and OS3 (0.30 ± 0.07) were increased significantly (P ＜ 0.05).Conclusion Down-regulated expression of selenoprotein genes are involved in the regulation process of articular cartilage apoptosis caused by oxidative stress,selenium also has a regulatory role in selenoprotein gene expression in articular chondrocytes.
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Objective To observe the protein expressions of PI3Kp110, pAkt, pGSK3β of phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt) signaling pathway in the whole blood of patients with Kashin-Beck disease and analyze the status of PI3K/Akt signaling pathway. Methods Patients with Kashin-Beck disease ( KBD group ) were from six counties ( Xunyi , Linyou , Yongshou , Qianyang , Changwu and Long County) of Shannxi Province in Kashin-Beck disease areas , and the healthy controls (control group) were matched by age and sex. Venous blood was collected from patients and healthy controls. Trizol method was applied to extract the whole blood protein; protein expression levels of PI3K/Akt signaling pathway in whole blood were detected by Western blotting; the gray values were observed and recorded by the sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) and GDS-8000 gel imaging analysis system. Differences between the two groups were assessed by Student’s t-test. Results Compared age and sex between KBD group and control group, differences were not statistically significant(t=0.701, P>0.05;χ2=0.400, P>0.05). The protein expression levels of PI3Kp110, pAkt and p-GSK3β in KBD group were higher than that in control group(156.1 ± 92.1 vs. 79.5 ± 21.5, 113.7 ± 15.2 vs. 43.3 ± 10.7 and 105.9 ± 17.5 vs. 37.3 ± 12.0, respectively) and the differences were statistically significant (t=2.563, 6.567, 7.916; all P < 0.05 or < 0.01). Conclusion The PI3Kp110, pAkt and p-GSK3β expressions of signaling pathway proteins in the whole blood of patiens with Kashin-Beck disease are up-regulated significantly and the status of PI3K/Akt signaling pathway is activated.
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Objective To investigate the relationship between erythrocyte immune function and selenium (Se)level. Methods Forty-nine Kashin-Beck patients in endemic area aged 13- 16 years were divided into two groups and were orally given either selenized yeast or sodium selenite to provide 200 μg selenium per day for 12 weeks. Erythrocyte selenium level, glutathione peroxidase activity, the rosette formation rates of red blood cells complement receptor type Ⅰ(CR1), the immune function of red blood cells, and circulating immune complexes(CIC) were determined. Results After supplementing with selenium for 12 weeks, erythrocyte selenium level, glutathione peroxidase activity, the rosette formation rates of red blood cells CR1 were significantly increased. But the difference in rosette formation rates of IC and CIC content was not significant between before and after Se supplementation. Conclusion The increase of the immune function of the erythrocyte by selenium-supplement may be one of the effective mechanisms for the prevention of Kashin-Beck disease.
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1.8.PCR was performed using the human Gpx-1 gene,and we obtained the needed target gene fragments.Conclusion The alkaline lysis extraction method is reliable for obtaining high quantities of DNA from blood clot suited for PCR amplification.
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Objective According to the distribution of low selenium areas, low nutrition state of the residents and the affecting cartilage growth and articular cartilage of Kashin-Beck Disease(KBD),the chondrocyte differentia- tion and differential expression of collagen types Ⅰ , Ⅱ and Ⅹ in articular cartilage from Chinese mini-pigs treated with low selenium were investigated in order to gain insight into the effects of these conditions on chondrocyte differ- entiation in KBD cartilage. Mothods Eleven male juvenile mini-pigs, aged from 4 weeks to 6 weeks after birth, were divided into 3 groups. The Se content in the diet of the “low Se” group was 0. 035mg/kg diet, and 0. 175 mg/kg diet in the control. For Se-supplemented group 0. 390mg /kg diet was added. The content of Se in blood was assayed at the beginning and at the end of each experiment. Samples of articular cartilage were taken from the right femur condylus, and collagen types Ⅰ , Ⅱ and Ⅹ in articular cartilage were analyzed by immunohistochemistry and in situ hybridization. Results①All cartilage samples from juvenile mini-pigs fed with low selenium diet revealed a re- duction in type Ⅹ collagen mRNA expression in the hypertrophic chondrocytes as shown by in situ hybridization, and reduced type Ⅹ collagen deposition in the lower hypertrophic zone as shown by immunohistochemistry. ②Addition of selenium to the diet restored the type Ⅹ collagen to normal level. ③Type Ⅱ collagen was evenly distributed over the entire articular cartilage in all experimental and control groups. Type Ⅱ collagen mRNA signals were most prominent in the upper articular layer as well as in the hypertrophic zone in all groups. Type Ⅱ collagen expression was restrict- ed to the zone of endochondral ossification in all experimental groups and the control. Conclusion Low selenium has an down-regulatory role on the synthesis and deposition of collagen type Ⅹ in hypertrophic chondrocytes in articular cartilage of mini-pigs. Supplement of the low Se diet with additional Se restored the signals of collagen type Ⅹ to nor- mal levels. These findings indicate that selenium deficiency may disturb chondrocyte differentiation to hypertrophic cells in the growth plate,and worthy to be investigated further.
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Objective Molecular detection of methicillin-resistant S. aureus(MRSA)in the Intensive Care Unit (ICU)will be helpful for the control of transmission among patients. Methods Both mecA and femB genes of 233 patients in ICU were examined ICU by polymerase chain reaction(PCR) for the presence of MRSA. Swabs were taken from various sites, such as axilla,nose , skin lesions and throat, and incubated over night in salt broth cultures. Results One hundred and five of patients(45.06％) were positive for mecA gene, twenty six(11. 16％)were femB positive and fifteen (6%) patients were positive for MRSA, and such screening data were available within 6～ 7 h fol- lowing admission. Conclusion Specific PCR approaches is helpful for routine conventional diagnosis of MRSA, mecA/femB PCR detection offers a rapid and specific alternative for screening MRSA from patients in high-risk ar- eas.