| Background:Fracture is the most common clinical orthopedic disease, fracture healing is mainly divided into cartilage callus induction period, inflammation period, period, bony callus period and the reconstruction period, fracture healing in the hinder any link will affect the healing of fracture.Fracture healing in the cell is more, different stages are involved in different cells, fracture hematoma and inflammation period, local tissue under the action of various chemokines, fracture was between undifferentiated mesenchymal stem cells, lymphocytes and macrophages, osteoblast activation of fracture end, granulation tissue degradation and osteogenesis precursor cells, osteoblast precursor cells were proliferation and differentiation, extracellular matrix was generated, participated in fracture healing;In callus reaction period, by inside periosteum germinal layer and generated within the bone marrow stromal cells and inducing bone original cells internalization bone, cartilage and bone cell differentiated.In cartilage formation, fibroblast synthesis and secretion of collagen secretion calcium salt and so on, and cartilage, bone and cartilage cell proliferated differentiation gradually mature.In the callus formation, osteoblasts produce new bone matrix, osteoclasts in bone remodelling.Fracture healing in addition to the cells involved in, there are all kinds of collagen and extracellular matrix proteins involved in any cell and protein function, were likely to influence the fracture healing.Open fractures often accompanied by bacterial infection and bacterial infection release toxins,it will interfere with or damage the normal metabolism and function of fracture healing relevant cells, thereby blocking the process of fracture healing, so the bacterial infection was the main fracture nonunion or delayed union, one of the reasons is the osteoblast formation of bone fracture healing process and calcification (S2), bacteria cause local inflammation media release, caused an immune reaction eventually lead to osteoblast necrosis or apoptosis, osteogenesis function obstacle, eventually leading to not heal.Bacterial Lipopolysaccharide (LPS) Lipopolysaccharide is a major component of bacterial endotoxin, including specific polysaccharides, nonspecific polysaccharides and lipids.The toxicity of lipid A is the main part, and bacterial endotoxin is the unique structure of gram-negative bacteria cell walls, which are the main cause of heat source, bacteria have relevant research has shown that bacteria release of Lipopolysaccharide is a major cause of lead to don’t open fractures healed and Lipopolysaccharide inflammatory process how to cause local inflammation, the reaction pathways by which influence the formation of osteoblasts bone of the specific mechanism is unclear, the lack of relevant research both at home and abroad, and the problem for the clinical treatment of fracture nonunion or delayed union of its great are significance.Inflammation is the body to the outside world infringement and damage of a normal defense, it is good for normal fracture healing process, but the fracture after the release of a large number of inflammatory factors will lead to osteoblasts can’t or osteoclast activation, a study reported that don’t heal inflammation caused by fractures, is mainly caused by bacteria lipopolysaccharide osteoblast inflammation, such as release?Interleukin 1, interleukin 6 and activation factor ligands (RANKL/S3), or inducing osteoblast secrete osteoclast activation factor, which can induce osteoclast maturity and activation.But how bacteria lipopolysaccharide mediated osteogenesis of intracellular signal transduction mechanism is unclear.Toll-like receptors (TLR) are a kind of important proteins involved in nonspecific immune molecules, TLR are the body’s natural monitor, can monitor a variety of different disease molecular model, is the first channel of the body fight infection. TLR can identify bacteria lipopolysaccharide, so as to induce the secretion of pro-inflammatory cytokines. TLR to identify bacteria lipopolysaccharide play a role of inflammation mainly through toll-like receptors signaling myeloid differentiation factor (MyD88) depend on the mechanism, the main process is that the ligand binding to the Toll receptor MyD88 gathered in Toll receptor TIR domain structure, gathered with IRAK myeloid differentiation factor (MyD88) after (S1) interaction, make its phosphorylation, phosphorylation of IRAK factor binding associated with tumor necrosis factor receptor mediated inflammatory response in the end.That is bacterial lipopolysaccharide (MyD88) generated by myeloid differentiation factor inflammatory factors play an inhibition of osteoblast osteogenetic process, but the specific mechanism is unclear.Fracture happens, in under the action of all kinds of inflammatory factors and other cytokines, near the fracture between,Bone marrow mesenchymal stem cells into osteoblast differentiation development, its transformation and differentiation process of adjustment is very sophisticated, its action mechanism, the relevant reports in recent years, some scholars found that the closed fracture end runt related transcription factors-2 (Runx2) and osteocalcin (OCN osteocalcin,) leels, and studies have found that if the experimental animals to knock the runt related transcription factors-2, lower the osteoblast, fracture healing is difficult, so we can preliminary thought runt related transcription factors-2 as the key of start between Bone marrow mesenchymal stem cells, studies have found that runt related transcription factors the activation of signaling pathways and BMP 2-7 (ipads morphogenetic protein, BMP) caused by body activation associated phosphorylation in cells, which form the osteocalcin, stimulate the osteogenesis of the key factors.Therefore this research in view of the infection, bone nonunion, fracture healing, on mouse osteoblast as the research object, and speculated that bacteria lipopolysaccharide by TLR influence on osteoblast differentiation, by measuring the activity of alkaline phosphatase and bone matrix ossification, the expression of alkaline phosphatase and osteocalcin and Runx2, clear lipopolysaccharide effects on osteoblast differentiation and matrix mineralization;At the same time, we further defined through RNA interference technology lipopolysaccharide TLR-4 dependent pathway effects on osteoblast differentiation.This article research results can provide bacteria infection, leading to fracture healing with molecular biology foundation, also in the treatment of infection caused by fracture healing and provide new ideas for the selection of drug targets, as a result of this article can guide the clinical prevention of fractures healed, is of great significance.Objective:(1) To cultivate mice ossification cell line MC3T3 E1, and remove the osteoblast siRNA TLR-4 express (TLR-4 specific siRNA transfection MC3T3 El cells).(2) With different concentrations of lipopolysaccharide cultivated ossification cell line MC3T3 El, using immune protein imprinting measuring TLR-4 protein levels.(3) The RT-PCR method was used to measure dose lipopolysaccharide treatment with different concentration of ossification cell line MC3T3 E1-osteogenesis markers in ALP, OCN and Runx2 mRNA level change, thus the analysis of the influence of lipopolysaccharides for osteoblasts osteogenesis function measurement. Dose lipopolysaccharide(4) With different concentration on osteogenesis cell line MC3T3 El, To detect of osteoblast alkaline phosphatase activity and substrate ossification measuring dependency, and the analysis of different concentrations of lipopolysaccharide osteoblast osteogenetic effects.(5) With different concentrations of lipopolysaccharide effect on removal of siRNA TLR-4 express (TLR-4 specific siRNA transfection MC3T3 E1 cells) osteoblast cultivated in mice, using the method of the Von Kossa measurement matrix ossification, detection of alkaline phosphatase activity, and to measure different doses of lipopolysaccharide knockout osteoblast cell activity and apoptosis.MethodsReagents, Cell culture and treatmentMurine osteoblastic MC3T3-E1 cell line was purchased from American Type Culture Collection (ATCC) (Rockville, MD, USA), and was cultured in a-Minimal essential medium (a-MEM) (GIBCO, Rockville, MD, USA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA, USA) and 1% penicillin/streptomycin (CSPC Pharmaceutical Group Limited, Shijiazhuang, China) at 37℃ under 5% CO2. Lipopolysaccharides (LPS) from Escherichia coli 0111:B4, which was purified by phenol extraction was purchased from Sigma-Aldrich (St. Louis, MO, USA), and was dissolved in a-MEM supplemented with 2% FBS. For the LPS treatment, MC3T3-E1 cells with more than 85% confluence were treated with 0,50,100,200, or 400 ng/mL LPS for 0,4,8,12, or 24 hours. To abrogate the TLR-4 expression in MC3T3-E1 cells, siRNA-TLR-4 or control siRNA (GenePharma Technology, Shanghai, China) was transfected into MC3T3-E1 cells with 30 or 60 nM by lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).Quantitative analysis of the mRNA levels of TLR-4, ALP, OCN and Runx2Cellular mRNA from MC3T3-E1 cells was isolated with the Kit for mRNA Isolation and Purification (Takara&Clontech, Tokyo, Japan), and was quantified by real-time quantitative PCR (RT-qPCR) with a one-step SYBR Green PCR Kits (TaKaRa, Tokyo, Japan) and with paired primers for TLR-4, ALP, OCN or Runx2. The RT-qPCR reaction was performed on the (Roche Diagnostics, Mannheim, Germany). The paired primers for each marker were synthesized by Shanghai Sangon Company (Sangon, Shanghai, China). All data were presented as the fold change over the internal control Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and were calculated with the △△Ct methodWestern blotting assay for TLR-4Cytosolic protein was isolated with the Cell Lysis Buffer (Cell Signaling Technology Inc., Danvers, MA, USA), and was supplemented with a protease inhibitor cocktail kit (Roche Biochemicals, Basel, Switzerland). Protein samples were separated by the 12% SDS-PAGE gel, and were transferred to polyvinylidene fluoridehydrophobic membrane (Millipore, Bedford, MA, USA). Then non-specific binding sites on the membrane was blocked with 5% skimmed milk (Solarbio, Beijing, China) overnight at 4℃, and the membrane was inoculated with the rabbit polyclonal antibody against TLR-4 (Abcam, Cambridge, UK) or against GAPDH (Sinobio, Beijing, China) at room temperature for 2 hours, and then was inoculated with the peroxidase-conjugated secondary antibody against rabbit IgG (Pierce, Rockford, IL, USA) at room temperature for 1 hour. Finally, the electrochemoluminescence (ECL) detection system (Amersham, Uppsala, Sweden) was used to visualize the specific binding. The TLR-4 level was presented as a relative gray value to GAPDH.Von Kossa staining and ALP activity assayTo examine the effect of LPS on osteoblastic differentiation, the MC3T3-E1 cells were cultured in a-MEM with 10% FBS, with 0,50,200 or 400 ng/mL LPS. Matrix mineralization in MC3T3-E1 cells was assayed by Von Kossa staining as previously described (Chan et al.,2008). For the ALP assay, MC3T3-E1 cells were lyzed post treatment, and the cell lysate with a protein concentration of 2 mg was added to the assay buffer (1 mM MgCl2,50 mM Tris-HC1/pH 9.2) containing 2 mM p-nitrophenol phosphate. After incubation for 10 minutes at 37℃, the reaction was terminated by 0.45 M NaOH, and the absorbance of p-nitrophenol liberated in the reactive solution was read at 420 nm. The ALP activity was defined as a relative level to control group.MTT cell viability assayCell viability was measured using the MTT method. Briefly, Cells were incubated for 24 hours with 0,50,100,200,400 ng/ml LPS.20 μL MTT in the final concentration of 5 mg/mL was added and the cells were incubated for 4 hours at 37℃. Then the supernatants were carefully removed.100 μL DMSO was added to each well and pipetted up and down to dissolve crystals. The plate was put into the 37℃ incubator to dissolve air bubbles and measured at 570 nm wavelength using a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). The results were expressed as (A570 of experimental wells)/(A570 of control wells) x 100%.Cells apoptosis-related analysisAnalysis of cell apoptotic percentage was performed by Annexin V-FITC Apoptosis Detection Kit. Briefly, the cells were centrifuged at 2000 RPM for 5 minutes and washed twice with PBS, then the cells were suspended in the 400 μl 1×Binding Buffer at a concentration of 1×105 cells/mL, then 5 μL of Annexin V-FITC and Propidium Iodide was added in turn and mixed. The treated cells were placed in the dark at RT for 5-15 minutes to perform flow cytometry analysis.Statistical AnalysisStatistical difference was analyzed with Student’s t test between two groups with SPSS 18.0 software (IBM SPSS, Armonk, NY, USA). A p value less than 0.05 was considered to be significant.ResultsLPS promotes TLR-4 in mouse osteoblast MC3T3-E1 cellsTo recognize the LPS signaling in osteoblast cells, we firstly examined the TLR-4 expression in the LPS-treated MC3T3-E1 cells. It was demonstrated in Figure 1A that the LPS treatment with 100,200 or 400 ng/mL significantly upregulated the mRNA level of TLR-4 in the MC3T3-E1 cells (P<0.05, P<0.01, or P<0.001), with a marked dose-dependence (P<0.05). Then we evaluated the time-dependence of the TLR-4 upregulation by LPS. Figure 1B indicated that the TLR-4 mRNA upregulation was significant since 4 hours post treatment (H.P.T.) with 200 ng/mL LPS, and lasted until 8 H.P.T, with a significant difference between 4 and 6 H.P.T. (P<0.05). To reconfirm the TLR-4 upregulation by LPS, we analyzed the TLR-4 in protein level in the LPS-treated MC3T3-E1 cells. As shown in Figure 1C, the TLR-4 upregulation was also significant by the LPS treatment with 100,200 or 400 ng/mL in protein level (P <0.01 or P<0.001), also with a dose-dependence (P<0.01). And the time-dependence (P<0.01) of the TLR-4 upregulation by LPS treatment was also reconfirmed in protein level (P<0.01 or P<0.001, Figure 1D). Taken together, we confirmed the TLR-4 upregulation by LPS in mouse osteoblast MC3T3-E1 cells.LPS inhibits matrix mineralization and ALP activity in MC3T3-E1 cellsIn order to investigate the influence of LPS treatment on the osteoblast differentiation, we then examined the matrix mineralization and ALP activity in the LPS-treated MC3T3-E1 cells. Figure 2A demonstrated that the ALP activity markedly decreased in the groups of MC3T3-E1 cells with 100,200 or 400 ng/mL LPS treatment (P<0.05, P<0.01, or P<0.001), with a dose-dependence (P<0.05). And the matrix mineralization was also measured in above-mentioned groups. It was indicated in Figure 2B that when LPS with 200 or 400 ng/mL was added to the cell culture, the MC3T3-E1 cells formed significantly less mineralization dots (P<0.01 for 200 ng/mL LPS or P<0.001 for 400 ng/mL LPS, Figure 2C), dose-dependently (P<0.01).LPS suppresses the osteogenic marker expression in MC3T3-E1 cellsOsteoblast differentiation is characterized by the promotion to the expression of multiple well-known "osteogenic markers", such as ALP, OCN and Runx2 (Bandow et al.,2010). We thus analyzed the effects of LPS on the expression of ALP, OCN and Runx2 in mRNA levels by real-time quantitative PCR in osteoblasts in the presence or absence of LPS. As shown in Figure 3 A, the mRNA level of ALP was significantly reduced by the treatment with 100,200 or 400 ng/mL (P<0.05 for 100 ng/mL, P <0.01 for 200 or 400 ng/mL), with a significant difference between the groups of 100 and 400 ng/mL. And the reduction in mRNA levels was also confirmed in OCN and Runx2. The OCN (Figure 3B) and Runx2 (Figure 3C) mRNA levels were also markedly downregulated by the LPS treatment with more than 100 ng/mL (P<0.05 for 100 ng/mL for OCN or Runx2, P<0.01 for 200 or 400 ng/mL for the both markers), and there was also a significant dose-dependence for the LPS treatment for either OCN or Runx2. Thus, we confirmed the inhibition of osteoblast differentiation by LPS treatment.RNAi-mediated TLR-4 knockdown abrogates the inhibition by LPS to osteoblast differentiationTo further investigate the role of TLR-4 in the LPS-medicated inhibition of osteoblast differentiation, we then abrogated the TLR-4 expression in the LPS-treated MC3T3-E1 cells, and then re-examined the matrix mineralization, the ALP activity and the expression of ALP, OCN and Runx2. Figure 4A indicated a significant downregulated TLR-4 mRNA in the MC3T3-E1 cells which were transfected with TLR-4-specific siRNAs (siRNA-TLR-4) in the MC3T3-E1 cells (p< 0.001 for either 30 or 60 nM). And the western blot analysis results also confirmed a significant downregulation of TLR-4 in protein level in the MC3T3-E1 cells (p< 0.001, Figure 4B). Moreover, the TLR-4 abrogation significantly ameliorated the inhibited matrix mineralization (p< 0.05, Figure 4C and 4D). And the mRNA levels of ALP, OCN and Runx2 were also significantly higher in the siRNA-TLR-4 group than in the siRNA-Con group (P<0.05 or P<0.01) with a concentration of either 30 or 60 nM. Thus, we confirmed the TLR-dependence of the LPS-mediated inhibition of osteoblast differentiation in MC3T3-E1 cells.Conclutions:(1) we successfully cultivate ossification cell line MC3T3 E1, and remove the osteoblast siRNA TLR-4 express (TLR-4 specific siRNA transfection MC3T3 E1 cells).(S1)(2)Lipopolysaccharide inhibits osteoblast MC3T3 E1 TLR-4 protein expression, and the measurement and time correlation.(3)The RT-PCR method was used to measure concentration with different doses of lipopolysaccharide treatment of ossification cell line MC3T3 E1-osteogenesis markers in the change of alkaline phosphatase and osteocalcin, Runx2,lipopolysaccharide effects on osteoblast of above three kinds of osteogenesis function marks the existence of the metering dependency, a negative correlation.Dose lipopolysaccharide.(4) With different concentration on osteogenesis cell line MC3T3 E1, detected osteoblast alkaline phosphatase activity and measuring dependency matrix ossification, at the same time, we measured the the influence of different doses on osteoblast apoptosis and activity, lipopolysaccharide has inhibitory effect on osteoblast function, at the same time can reduce the proportion of osteoblast activity increase osteoblast apoptosis.(5) Knockout TLR-4 osteoblast MC3T3 E1 its matrix ossification degree and alkaline phosphatase, OCN, Runx2 mRNA level and higher levels of osteoblast MC3T3 knockout TLR-4-E1 cell activity, cell apoptosis rate reduced.(S4)Key wordslipopolysaccharide;... |
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