| Section 1 The study of TNF-? is upregulated in T2DMpatients with fractureObjective:Fracture detection in patients with type 2 diabetes after venous serum hs-CRP,TNF alpha,beta,IL-1 IL-6,IP-10 and the content of RANTES and the fracture and fracture patients without diabetes and diabetes patients were compared with a,for the study of inflammatory markers in patients with type 2 diabetes and chemokines,lay a foundation role in the fracture healing process.Methods:1.Serum samples from bone fracture patients with or without T2DMTotal of 28 T2 DM patients with bone fracture(6 cases of tibial fracture,11 cases of fracture of tibia and fibula and 11 case of femoral fracture)were implicated in this study.And there were 25 cases of bone fracture patients(4 cases of tibial fracture,12 cases of fracture of tibia and fibula and 9 case of femoral fracture)without T2 DM or T1 DM were taken as control.In another control group,there were 22 T2 DM patients without bone fracture and infectious diseases.All serum samples were isolated from the first venous blood specimens from subjects with bone fracture when registered at emergency department.Detailed characteristics of these subjects were described in Table 1.Written consent was obtained from each subject before the study.And the study was approved by the Ethics Committee of the Affiliated Hospital of Inner Mongolia Medical University.2.Cells medium and reagentsHuman MG-63 osteoblast-like cells were provided by the cell resource center of Chinese academy of medical sciences(Beijing,China)and were cultured in Eagle's minimal essential medium(EMEM)(GIBCO,Rockville,MD,USA),supplemented with 2m M Glutamine(Sigma-Aldrich Corporation,St.Louis,MO,USA),1% Non Essential Amino Acids(NEAA)(Thermo Scientific,Rockford,IL,USA)and 10% fetal bovine serum(FBS)(Gibco,Rockville,MD,USA).The cells were incubated at 37 °C,with 5% CO2.D-glucose and TNF-? were purchased from Sigma-Aldrich Corporation(St.Louis,MO,USA)and were dissolved in EMEM with 2% FBS.3.Enzyme-linked immunosorbent assay(ELISA)for cytokinesSerum or supernatant levels of hs CRP,IL-1?,TNF-?,IL-6,IP-10 and RANTES were quantitatively examined with the solidphase enzyme-linked immunosorbent assay(ELISA)kit(Abcam,Cambridge,UK)under the guidance of product's manual.Briefly,precoated microplates(with monoclonal mouse anti-human antibody against hs CRP,IL-1?,TNF-?,IL-6,IP-10 or RANTES)were successively subject to the blockage with 2% Bovine Serum Albumin(BSA;Sigma-Aldrich,St.Louis,MO,USA),to the inoculation with the serially-diluted standardsserum samples or cell supernatant samples and then to the inoculation with the polyclonal antibody solution against hs CRP,IL-1?,TNF-?,IL-6,IP-10 or RANTES conjugated with horseradish peroxidase).Finally,the optical density of each well was determined immediately at 450 nm.4.Quantitative analysis of IL-1? or TNF-? m RNA with RT-q PCRCellular m RNA samples in MG-63 cells were isolated with the TRIzol reagent(Invitrogen,Carlsbad,CA,USA)according to the manufacturer's manual,and were supplemented with the RNasin? Plus RNase Inhibitor(Promega,Madison,WI,USA),and were stored at-80 °C before use.RT-q PCR assay for each m RNA sample was performed with the SYBR green One Step RT-PCR Kit(Takara,Tokyo,Japan)accordingly.Primers for TNF-?(PF: 5'-TCT CAT CAG TTC TAT GGC CC-3',PF: 5'-GGG ATG AGA CAA GGT ACA AC-3'),IL-1?(PF: 5'-AAG CTG AGG AAG ATG CTG-3',PR: 5'-ATC TAC ACT CTC CAG CTG-3')or ?-actin(PF: 5'-TGT ACG CCT CTG GCC GTA CC-3',PR: 5'-GAT GGG CAC AGT GTG GGT GA-3')were synthesized by Sangon Biotech Company(Shanghai,China).Each reaction was performed in 20 ?L containing 4 ?L 5x RT-PCR Buffer,1 ?L d NTP Mix(containing 10 m M of each d NTP),2 ?L RNA,0.5 ?L forward/reverse primer(10 ?M),0.5 ?L RNase inhibitor(Promega,Madison,WI,USA),and was supplemented with RNase-free water to a total volume of 20 ?L.RT-q PCR reaction was performed as following: For the reverse transcription,the reaction was ran at 42 °C for 5 minutes and 95 °C for 10 seconds;for the application,the reaction was ran for 40 cycles of 95 °C for 5 seconds and 60 °C for 20 seconds.Relative level of TNF-? or IL-1? was calculated by ?? Ct method,with ?-actin as control [27].5.Western blotting analysisTotal cellular protein samples were prepared with a Cell Lysis Buffer(Cell Signaling Technology Inc.,Danvers,MA,USA)according to the manual and supplemented with a protease inhibitor cocktail(Pierce,Rockford,IL,USA).For the Cytochrome c release assay,the cytoplasmic protein was isolated by the Mitochondria/Cytosol Fractionation Kit(Abcam,Cambridge,UK)according to the manufacturer's manual.Each protein sample was separated by 12 % SDS-PAGE gel(with a loading amount of 30 ?g for each sample)and was transferred on nitrocellulose membrane(Millipore,Bedford,MA,USA).The membrame was successively blocked overnight with 3 % BSA in Tris-buffered saline containing 0.05% Tween 20(TBST),incubated with the first antibody(Cytochrome c,Caspase 3,Poly(ADP-ribose)polymerase(PARP),or ?-actin)for 1 hour at room temperature or overnight at 4 °C,and then followed by the incubation with HRP-conjugated goat-anti-rabbit secondary antibody(Pierce,Rockford,IL,USA).Three-time washing of the membrane was necessary before each inoculation,specific binding band was detected by ECL kit(Amersham Pharmacia Biotech,Amersham,UK).Results:1.Upregulation of pro-inflammatory cytokines in the T2 DM patients with bone fractureTo investigate the promotion to pro-inflammatory cytokines and chemokines in T2 DM patients with bone fracture,we examined the serum levels of hs-CRP,TNF-?,IL-1?,IL-6,IP-10 and RANTES in T2 DM patients with or without bone fracture(Tibial fracture,fracture of tibia and fibula,or femoral fracture).The non-T2 DM patients with bone fracture were also included.It was shown in table 1,that there was no significant difference in age and gender among the three groups of patients;and there was neither significant difference in Body Mass Index(BMI),diabetes mellitus(DM)duration,fasting glucose and other DM-associated characteristics.However,as shown in Figure 1A-D,the level of hs CRP)was significantly higher in the group of T2 DM patients with fracture,than in the T2 DM control patients(p < 0.0001)or than in fractured patients without T2DM(p < 0.01).Moreover,there was also significant difference in the serum proinflammatory cytokines such as TNF-?,IL-1? and IL-6 between T2 DM patients with fracture and T2 DM control patients,or between the T2 DM patients with fracture and fractured patients without T2 DM.The difference in IL-1?(p < 0.01 or p < 0.05),TNF-?(p < 0.001 or p < 0.01)or IL-6(p < 0.001 or p < 0.05)was also significant.In addition,the serum levels of IP-10 and RANTES were also significantly higher in the bone-fractured T2 DM patients(p < 0.01 or p < 0.001,compared with T2 DM control patients or with fractured patients without T2 DM,Figure 2E and 2F)Thus,we confirmed the high promotion of proinflammatory cytokines and chemokines in T2 DM patients with bone fracture.2.High glucose induces TNF-? and IL-1?in human osteoblast-like MG-63 cellsTo further determine the promotion to pro-inflammatory cytokines by high glucose in human osteoblast cells,we then examined the promotion to TNF-? and IL-1? in both m RNA and protein levels.MG-63 cells were treated with 5,10,20 or 40 m M D-glucose for 8(for m RNA assay)or 12(for protein assay)hours,and the m RNA or protein level of TNF-? and IL-1? was evaluated by RT-q PCR or by ELISA.Figure 2A demonstrated that the m RNA level of TNF-? was significantly upregulated to 1.680 ± 0.1589 or to 2.823 ± 0.2751 by the treatment with 20(Column 3 vs column 1,p=0.0203)or 40 m M(Column 4 vs column 1,p=0.0032)D-glucose,with a dose-dependence(Column 4 vs column 3,p=0.0228).And the IL-1? m RNA was also significantly promoted to 2.847 ± 0.1775 or to 4.574 ± 0.4457 by the D-glucose treatment with a concentration of 20(Column 3 vs column 1,Figure 2B,p=0.0007)or 40 m M(Column 4 vs column 1,Figure 2B,p=0.0004),also dose-dependently(Column 4 vs column 3,Figure 2B,p=0.0226).And the protein levels of both cytokines were also upregulated the high glucose.It was shown in Figure 2C and 2D that the TNF-? and IL-1? in the supernatant of MG-63 cells were markedly upregulated by either 20 or 40 m M D-glucose(p < 0.05,p < 0.01 or p < 0.001),with a dose-dependence(p < 0.05 or p < 0.01).Conclusion:1.In patients with type 2 diabetes before the changing rule of the inflammatory factors and chemokines for fractures in patients with type 2 diabetes in serum hs-CRP,TNF alpha,beta,IL-1 IL-6,IP-10 and the content of RANTES is greater than the type 2 diabetic patients without fracture,the former two is greater than the fracture patients without diabetes.2.TNF alpha and beta IL-1 in the m RNA and protein levels,as osteoblasts cultured glucose levels higher and higher,including 40 m M glucose group is highest,followed by 20 m M glucose group and comparison between different concentration groups,the difference is statistically significant.TNF alpha and beta IL-1 m RNA content and measuring dependencies to the concentration of glucose.Section 2 High sugar interfere with tumor necrosis factor alpha in vitromediated osteoblast apoptosis mechanism researchObjective:Test under different concentrations of glucose interferes with the tumor necrosis factor alpha osteoblast induced apoptosis of MG-63 rule,and by the methods of gene knock out the knockout osteoblast TNF alpha pieces,detecting the expression of osteoblast apoptosis and apoptosis related factor influence,for the study of patients with type 2 diabetes TNF alpha role in the fracture healing process,the molecular mechanism of fracture healing drink basis for prevention and cure.Methods:1.Cells medium and reagentsHuman MG-63 osteoblast-like cells were provided by the cell resource center of Chinese academy of medical sciences(Beijing,China)and were cultured in Eagle's minimal essential medium(EMEM)(GIBCO,Rockville,MD,USA),supplemented with 2m M Glutamine(Sigma-Aldrich Corporation,St.Louis,MO,USA),1% Non Essential Amino Acids(NEAA)(Thermo Scientific,Rockford,IL,USA)and 10% fetal bovine serum(FBS)(Gibco,Rockville,MD,USA).The cells were incubated at 37 °C,with 5% CO2.D-glucose and TNF-? were purchased from Sigma-Aldrich Corporation(St.Louis,MO,USA)and were dissolved in EMEM with 2% FBS.2.Enzyme-linked immunosorbent assay(ELISA)for cytokinesSerum or supernatant levels of hs CRP,IL-1?,TNF-?,IL-6,IP-10 and RANTES were quantitatively examined with the solidphase enzyme-linked immunosorbent assay(ELISA)kit(Abcam,Cambridge,UK)under the guidance of product's manual.Briefly,precoated microplates(with monoclonal mouse anti-human antibody against hs CRP,IL-1?,TNF-?,IL-6,IP-10 or RANTES)were successively subject to the blockage with 2% Bovine Serum Albumin(BSA;Sigma-Aldrich,St.Louis,MO,USA),to the inoculation with the serially-diluted standardsserum samples or cell supernatant samples and then to the inoculation with the polyclonal antibody solution against hs CRP,IL-1?,TNF-?,IL-6,IP-10 or RANTES conjugated with horseradish peroxidase).Finally,the optical density of each well was determined immediately at 450 nm.3.Western blotting analysisTotal cellular protein samples were prepared with a Cell Lysis Buffer(Cell Signaling Technology Inc.,Danvers,MA,USA)according to the manual and supplemented with a protease inhibitor cocktail(Pierce,Rockford,IL,USA).For the Cytochrome c release assay,the cytoplasmic protein was isolated by the Mitochondria/Cytosol Fractionation Kit(Abcam,Cambridge,UK)according to the manufacturer's manual.Each protein sample was separated by 12 % SDS-PAGE gel(with a loading amount of 30 ?g for each sample)and was transferred on nitrocellulose membrane(Millipore,Bedford,MA,USA).The membrame was successively blocked overnight with 3 % BSA in Tris-buffered saline containing 0.05% Tween 20(TBST),incubated with the first antibody(Cytochrome c,Caspase 3,Poly(ADP-ribose)polymerase(PARP),or ?-actin)for 1 hour at room temperature or overnight at 4 °C,and then followed by the incubation with HRP-conjugated goat-anti-rabbit secondary antibody(Pierce,Rockford,IL,USA).Three-time washing of the membrane was necessary before each inoculation,specific binding band was detected by ECL kit(Amersham Pharmacia Biotech,Amersham,UK).4.MTT assay and Cell apoptosis assayMTT assay(Invitrogen,Carlsbad,CA,USA)was performed for the cellular viability analysis.MG-63 cells seeded in 96-well plates with approximately 85% confluence were treated with 5,10,20 or 40 m M D-glucose,or(and)with 5,10,20 or 40 ng/m L TNF-? for 24,48 or 72 hours.Then each cell well was added with 10 ?L MTT solution and the plate was incubated at 37 °C for 4 hours.Post adding 100 ?L DMSO into each well to dissolve the formazan,the absorbance was measured on an ELISA plate reader with a test wavelength of 570 nm and a reference wavelength of 630 nm to obtain sample signal(OD570-OD630).The apoptosis of MG-63 cells was assayed with an annexin V-FITC apoptosis detection kit(Sigma-Aldrich,St.Louis,MO,USA).In brief,approximate 1 ×106 cells post treatment were stained with annexin V-FITC and propidium iodide,and then apoptotic cells were detected by a FACScan flow cytometer(BD Biosciences,San Jose,CA,USA).The apoptosis level was evaluated by a percentage of apoptotic cells to total cells.5.Statistical analysisStatistical analysis was performed using Graph Pad Prism 6(Graph Pad Software,La Jolla,CA,USA).Statistical evaluations are presented as mean ± SE.Data were analyzed using the Student's t test or by one-way ANOVA test,post confirming the normally-distributed data with Shapiro-Wilk test.A p value <0.05 or less was considered statistically significant.Results:1.TNF-? reduces viability in MG-63 cells in the presence of high D-glucoseWe supposed a possible influence on the viability of osteoblasts by TNF-? and high glucose.The viability of MG-63 cells which were treated with TNF-? and D-glucose with a range of concentrations were examined by MTT assay.It was indicated in Figure 3A that the viability of MG-63 was significantly reduced by the D-glucose treatment with 40 m M for 24 hours.And the viability reduction was also induced by the TNF-? treatment with 20 or 40 ng/m L(p < 0.05 respectively).Furthermore,the viability reduction of MG-63 cells was more significant by the TNF-? treatment,in the presence of D-glucose.Figure 3B demonstrated that the TNF-? treatment with 10 ng/m L significantly reduced the viability of MG-63 cells,in the presence of 20 m M D-glucose(p < 0.05).And the viability reduction was more significant by the TNF-? treatment with 20 or 40 ng/m L,in the presence of 20 m M D-glucose(p < 0.001 respectively),dose-dependently.To further determine the cell viability reduction by the treatment with TNF-? and D-glucose,we curved the viability of MG-63 cells,which were treated with 5 m M D-glucose(as control),20 m M D-glucose,20 ng/m L TNF-?,or both with 20 m M D-glucose and 20 ng/m L TNF-?.Figure 3C indicated that there was no significant difference in the viability curve between the MG-63 cells treated with 20 m M D-glucose and the MG-63 cells treated with 20 ng/m L TNF-?.However,the viability of TNF-?-treated MG-63 cells was significantly lower at 48 or 72 hour post treatment.Furthermore,the cellular viability decreased more significantly in the combination treatment group(p < 0.01 or p < 0.001)2.TNF-? promotes apoptosis and upregulates apoptosis-associated markers in MG-63 cells in the presence of high D-glucoseGiven the significant apoptosis induction by TNF-? in osteoblast cells,we then determined the apoptosis induction in MG-63 cells by TNF-?,in the presence of D-glucose.The apoptosis induction by the singular treatment with D-glucose or TNF-?,or by the combined treatment with both agents was assayed.Figure 4 demonstrated that the 20 m M D-glucose did not induce significant apoptosis in MG-63 cells,compared to the control MG-63 cells(5 m M D-glucose),whereas the treatment with 20 ng/m L TNF-? promoted significantly higher level of apoptosis at 24 or 48 hour post treatment(p < 0.05 or p < 0.01).Furthermore,there were more apoptotic cells in the combined treatment group(p < 0.05 respectively,compared to the TNF-? treatment).Then we analyzed in the four groups of cells,the apoptosis-associated markers,such as released cytochrome c,cleaved caspase 3 and lyzed PARP,by activated caspase 3 by western blot analysis.It was shown that D-glucose with 20 m M had no regulation on the cytochrome c release,caspase 3 activationand PARP lysis,whereas all of which were promoted by 20 ng/m L TNF-?(p < 0.05 or p < 0.01).Furthermore,the treatment with 20 m M D-glucose could aggravated the apoptosis induction by 20 ng/m L TNF-?(p<0.05 or p<0.01,compared to the TNF-? group,).Therefore,the high glucose synergizes the apoptosis promotion by TNF-? in MG-63 cells.Conclusion:1.The glucose and tumor necrosis factor alpha and osteoblast MG-6 energy metering dependence.2.D-glucose compound TNF alpha interference osteoblast after 48 and 72,vigor significantly decreased.3.The tumor necrosis factor alpha + glucose group of osteoblast apoptosis rate than with simple interference group.4.The tumor necrosis factor alpha + glucose group lead to osteoblast apoptosis related proteins cytochrome c,cleaved caspase 3 protein and lyzed PARP protein content is significantly greater than with simple interference group. |
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