| Background:Bone fracture is a prevalent medical condition, in which the urgent alteration of the bone microenvironment causes a reduced blood supply and subsequent hypoxia. And the healing process of bone fracture starts with the hematoma and inflammation surrounding the injured bone and ends with the re-structure of the broken bone (McKibbin 1978, Remedios 1999). However, the healing process is promoted or hindered by several factors mainly via affecting the growth and differentiation of osteoblasts and the mineralization of the collagen matrix (Harada & Rodan 2003). Serious hypoxia is one of the most prominent outcomes following fractures, significantly influencing their healing process (Heppenstall et al.1976). Hypoxia promotes the expression of such transcriptional factors and cytokines (Warren et al. 2001) as Hypoxia-inducible factors (HIFs), vascular endothelial growth factor (VEGF) (Akeno et al.2001), bone morphogenetic protein-2 (BMP-2) (Bouletreau et al.2002, Wang & Han 2014).Studies have shown a significant interaction between the local and systemic inflammatory response after severe trauma. However, the involvement of the inflammatory responses in mechanisms of bone fracture healing is still poorly comprehended. Various types of immune cells and cytokines have been indicated to be involved in the wound healing or bone regeneration (Naik et al.2009, Gerstenfeld et al.2003, Zhang et al.2002, Kolar et al.2010). Studies have indicated the infiltration of immune cells into already existing fracture hematomas on the early inflammatory phase of fracture healing (Andrew et al.1994, Hauser et al.1997). And such cytokines as interleukin-8 (IL-8)(Hoff et al.2013), transforming growth factor beta (TGF-β) (Westhauser et al.2015) and IL-6 (Herlin et al.2015) are confirmed to be upregulated by hypoxia in an association with the bone fracture.High-mobility group protein B1 was initially described as a cytokine-like factor in models of sepsis (Wang et al.1999), and now is well-known to be a key mediator of inflammation in multiple injury models, such as hemorrhagic lung injury (Kim et al. 2005), hepatic ischemia-reperfusion (Tsung et al.2005), hemorrhagic shock (Yang et al.2006). HMGB1 interacts with Toll-like receptor (TLR)-4 (Yu et al.2006, Park et al.2006, Park et al.2004) and initiates initial inflammatory response to injury. However, the role of HMGB1 in the inflammatory response following bone fractures is unknown.The oxygen supply/blood flow deprivation or interruption during bone fracture results in an ischemic-hypoxic condition for local osteoblasts and bone mesenchymal stem cells (MSCs) and inhibits the bone repairing. Particularly, hypoxia exerts a significant influence, via regulating the expression of transcriptional factors and cytokines such as Hypoxia-inducible factors (HIFs), vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP-2). And such factors/cytokines then pose regulatory roles on the wound healing or bone regeneration. Bone morphogenetic proteins (BMPs), such as BMP-2, alkaline phosphatase (ALP), osteopontin (OPN) and osteocalcin (OCN), have been indicated to be deregulated by hypoxia in the differentiation of MSCs during bone repair. Moreover, sustained hypoxia even leads to the apoptosis and even necrosis of osteoblasts and MCSs.N-methyl pyrrolidone (NMP) has been approved as a safe and biologically inactive small chemical constituent in medical devices. Recently, NMP has been reported to have the pharmaceutical property in enhancing bone regeneration in a rabbit calvarial defect model in vivo. Therefore, NMP might be useful for the treatment of osteoporosis or other bone diseases associated with excessive bone resorption. In addition, it exerts an anti-inflammatory potential on the lipopolysaccharide (LPS)-induced inflammatory process. The LPS-promoted levels of TNF-α, IL-1β, IL-6, iNOS and COX-2 were inhibited by NMP in a dose-dependent manner. And the effect of NMP is mediated through the downregulation of NF-κB pathway.The purpose of this study was to examine the promotion to HMGB1 in the hematoma specimens and in macrophages under hypoxia. Then we investigated the regulation of HMGB1 on the proliferation of osteoblast cells, and on the activation of TLR-4 and RAGE under normoxia or hypoxia. We also examined the phosphorylation of extracellular signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK) in the HMGB1-treated osteoblast cells under normoxia or hypoxia. In addition, the xx-specific siRNA was transfected into the HMGB1-treated osteoblast cells under hypoxia and re-examined the cell proliferation and the ERK/JNK phosphorylation. Our study recognized the key regulatory role of hypoxia-induced HMGB1 on the proliferation of osteoblast cells via regulating the ERK/JNK phosphorylation, TLR-4-dependently.In the present study, we investigated the effect of NMP on the hypoxia-induced apoptosis and differentiation of osteoblasts, and then we examined the molecular mechanism underlining such effect in osteoblasts. Our study implies the protective role of NMP in the hypoxia-induced impairment in osteoblast differentiation.Objective:1. To study the mechanism of Hypoxia-induced HMGB1 in would tissues promotes the osteoblast cell proliferation via activating TLR signaling2. To study the mechanism of N-methyl the hypoxia-reduced osteoblast differentiation via inhibiting the NF-κB signaling by pyrrolidone (NMP).Materials and Methods:The part one:Hematoma samples, Cell culture and treatmentTotal of 31 patients with open femur fracture were enrolled from March 2014 to March 2015 in the Department of Orthopedics and Traumatology, Nanfang Hospital, Southern Medical University, and were involved in this study. The accumulated hematoma samples in the wound and the fresh bleeding sample during surgery were collected for the HMGB1 assay. Written consent was obtained from each patient before the study, which was also approved by the Ethics Committee of Nanfang Hospital, Southern Medical University.U937 cells were cultured and maintained in RPMI-1640 medium (HyClone, Logan, UT, USA) which was supplemented with 10% or 2% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA), with 50μg/ml penicillin and with 50μg/ml streptomycin (CSPC Pharmaceutical Group Limited, Shijiazhuang, China). Cultures can be established by centrifugation with subsequent resuspension of 1-2 x 105 viable cells/mL. Fresh medium was updated every 3 to 4 days (depending on cell density). For hypoxia treatment, cells were placed in a hypoxia incubator infused with a gas mixture of 5% CO2 and nitrogen to obtain 2% oxygen concentration. For the HMGB1 assay, U937 cells were incubated under normoxia or under hypoxia for 8,12, 24 or 48 hours, then the supernatant of U937 cells and cells in each group were collected for HMGB1 assay.MG-63 cells were also grown in RPMI-1640 medium supplemented with 10% FBS,50μg/ml penicillin and 50μg/ml streptomycin. The two types of cells were incubated at 37℃, with 5% CO2 in a humid incubator. For hypoxia treatment, cells were placed in a hypoxia incubator infused with a gas mixture of 5% CO2 and nitrogen to obtain 2% oxygen concentration. For the HMGB1 treatment, MG-63 cells with 85-95% confluence were incubated with RPMI-1640 (2% FBS) supplemented with HMGB1 for 0,0.2 or 1μg/mL for 24,48 or 72 hours under normoxia or under hypoxia. To knockout the TLR-4 in MG-63 cells, siRNA-TLR-4 and control siRNA were synthesized by GenePharma Technology (Shanghai, China) and were transfected into MG-63 cells with a concentraction of 30 or 60 nM by lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).ELISA for HMGBlHMGB1 in the hematoma or fresh bleeding samples, or in the supernatant of macrophage U937 cells were examined with the enzyme-linked immunosorbent assay (ELISA) kit (Westang Bio, Shanghai, China) under the guidance of the product’s manual. The microplate was added with 100μL standard samples or with the serially-diluted samples, and was incubated at 37℃ for two hours. Then samples were aspirated, and the plate was washed for four times with 100分l phosphate buffered saline with Tween 20 (PBST) in each well. Then the plate was added with 100分L antibody against HMGB1 in each well, and was incubated 37℃ for one hour. Post four-time washing, the plate was added with 100μL secondary antibody conjugated with horseradish peroxidase and was incubated for 30 minutes at 37℃. Post the inoculation with 100μL substrate at dark for 15 minutes, the specific binding optical density of each well was determined immediately at 450 nm. mRNA preparation and quantitative analysismRNA samples from MG-63 cells were prepared with the TRizol reagent (Life Technologies, Grand Island, NY, USA), and were quantified by real-time quantitative PCR (RT-qPCR). SYBR Green PCR Kits (TaKaRa, Tokyo, Japan) was utilized to quantify the mRNA level of ERK, JNK, TLR-4, or Tubulin in MG-63 cells. The qPCR was performed on the ABI PRISM 7300 detection system. The primers for each molecule were synthesized by Shanghai Sangon Company (Sangon, Shanghai, China). All data were presented as the fold change over the internal control Tubulin and were calculated with the ΔΔCt method (Livak & Schmittgen 2001).Western blotting assayCytosolic or nuclear protein samples were isolated with the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, Waltham, MA, USA), and were supplemented with a Protease Inhibitor Cocktail (Abcam, Cambridge, UK). Protein samples were separated by the 10% or 12% SDS-PAGE gel, and were transferred to nitrocellulose membrane (Millipore, Bedford, MA, USA). Then the membrane was blocked with 2% BSA (dissolved in PBST) overnight at 4℃ to cover the non-specific binding on the membrane, and the HMGB1, ERK with or without the phosphorylated Thr202/Thr204, JNK with or without the phosphorylated Thr183, TLR-4 or Tubulin was detected with rabbit polyclonal IgG against each marker (all from Cell Signaling Technology Inc. (Danvers, MA, USA) or Tubulin (Sinobio, Beijing, China). A peroxidase-conjugated secondary antibody against rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA) and the electrochemoluminescence (ECL) detection system (Amersham, Uppsala, Sweden) were used to present the specific binding. The level of HMGB1 or TLR-4 was presented as a relative gray value to Tubulin, whereas the level of p-ERK or p-JNK was presented as the relative gray value to ERK or to JNK.Cell proliferation assay with CCK-8Proliferation of MG-63 cells under normoxia or hypoxia, with or without the HMGB1 treatment, with the transfection with siRNA-TLR-4 or siRNA-Con was performed with CCK-8 assay (DOJINDO, Kumamoto, Japan). Briefly, MG-63 cells in each group were incubated with CCK-8. The 490 nm absorbance of cells was detected after visual color occurrence.Statistical AnalysisData was presented as mean±SD. And statistical difference was analyzed with SPSS 18.0 software (IBM SPSS, Armonk, NY, USA). The difference between two groups was analyzed by Student’s t test. A p value less than 0.05 was considered to be significant.The part two:Cells and cell culture/treatmentHuman osteoblastic hFOB 1.19 cells purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), and were cultured in the 1:1 mixture of Ham’s F12 medium and Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Rockville, MD, USA), which was supplemented with 2.5 mM L-glutamine (Sigma-Aldrich, St. Louis, MO, USA), with 0.3 mg/ml G418 (Thermo Scientific, Rockford, IL, USA) and with 10% fetal bovine serum (FBS, Sijiqing, Hangzhou, China). hFOB 1.19 cells were incubated in a humidified incubator with 5% CO2 at 37℃. For the hypoxia treatment, hFOB 1.19 cells were incubated in a hypoxia incubator with 5% CO2 and 2% oxygen. And the oxygen concentration was monitored continuously (Forma 3130, Thermo Fisher Scientific, Inc., Waltman, MA, USA). N-methyl pyrrolidone (NMP) (Sigma-Aldrich, St. Louis, MO, USA) treatment was performed with a concentration of 0,3,10 or 30 mM. Enzyme-linked immunosorbent assay (ELISA) for BMP-2, PINP, ALP or RUNX-2The intracellular levels of bone morphogenetic protein-2 (BMP-2), propeptide of type I procollagen I (PINP), alkaline phosphatase (ALP) or Runt-related transcription factor -2 (RUNX2) in hFOB 1.19 cells were quantified with the ELISA kit for BMP2, PINP, ALP or RUNX2 (all from Abcam, Cambridge, UK) according to the product’s manual. The microplate for each marker was incubated with 100μL serially-diluted standard samples or hFOB 1.19 cells-lyzed cellular samples overnight at 4℃. Then the plate was incubated with 100μL antibody solution (against BMP2,PINP, ALP or RUNX2) at 37℃ for one hour. Hereafter,100μL horseradish peroxidase-conjugated secondary antibody was added for an incubation at 37℃ for 30 minutes. Four-time washing with phosphate buffered saline adding Tween 20 (PBST) was performed before each incubation. Finally, the plate was incubated with 100μL substrate at dark for 15 minutes, and the plate was read at 450 nm with a spectrophotometer (Bio-Rad, Hercules, CA, USA).RT-qPCR analysis of p65 mRNA and NF-κB luciferase reporter assayTotal cellular mRNA samples from hFOB 1.19 cells were prepared with the Recover All Total Nucleic Acid Isolation Kit (Ambion, Austin, TX, USA) according to the kit’s manual, and were supplemented with 1μl RNasin(?) Plus RNase Inhibitor (Promega, Madison, WI, USA). The p65 mRNA level was quantified with the Takara One Step RT-PCT kit (Takara, Tokyo, Japan), with the p65-specific primers (Forward primer:5’-tgatcactaagcaggaagatgtg-3’, Reverse primer:5’-gaaggctcaggtcggccccag-3’). The quantification of p65 was relatively presented with P-actin as reference gene, via the ΔΔCt method.For the NF-κB luciferase reporter assay, hFOB 1.19 cells were plated into 96-well plates and cultured to approximately 85%-confluent. Then hFOB 1.19 cells were transfected with the NF-κB luciferase reporter plasmid (Genomeditech, Shanghai, China) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.Protein isolation and western blotting5×105 hFOB 1.19 cells post treatment were subject to the protein isolation with the Nuclear/Cytosol Fractionation Kit (BioVision, San Diego, CA, USA), the cytosolic and the nucleur fraction were collected respectively and were added with Protease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, MO, USA). Then each fraction was quantified with BCA Protein Assay Reagent Kit (Pierce, Rockford, IL, USA). To analyze the p65 and inhibitory kappa B (IκB) levels, each protein sample was separated by electrophoresis with 12% SDS-PAGE gradient gel, and then was transferred to nitrocellulose membrane (Millipore, Bedford, MA, USA). The membrane was then incubated with 2% BSA (4℃ overnight) to block out non-specific binding sites, was incubated (4℃ overnight) with the rabbit polyclonal antibody to p65 (Abcam, Cambridge, MA, USA), to IκB (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or to P-actin (Sinobio, Beijing, China), and then was incubated (room temperature for 1 h) with the horseradish peroxidase-1 inked secondary goat-anti-rabbit antibody (Bio-Rad Laboratories, Hercules, CA, USA). Three-time washing with Ix Phosphate Buffered Saline Tween-20 (PBST) was performed before each inoculation. And the specific p65, IκB or β-actin band was then quantified with Enhanced chemiluminescence (ECL) (Thermo Scientific, Rockford, IL, USA).MTT assay for cellular viabilityViability of hFOB 1.19 cells was examined with 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay kit (Invitrogen, Carlsbad, CA, USA). Briefly, hFOB 1.19 cells, post normoxia, hypoxia or (and) NMP treatment, were incubated with the MTT reagent at 37℃ for 3 hours. Optical densities (OD) at 450 nm were measured by spectrophotometer (Crystaleye, Olympus, Tokyo, Japan). Cellular viability was presented as average OD450 value.Statistical AnalysisStatistical difference was analyzed with SPSS 13.0 software (IBM SPSS, Armonk, NY, USA). The difference between two groups was analyzed by Student’s t test. A p value less than 0.05 was considered to be significant.Results:The part one:1.HMGB1 was upregulated in the hematoma of fractured bones and in macrophage U937 cells under hypoxiaTo recognize the role of HMGB1 in bone fracture, we firstly examined the level of HMGB1 in the hematoma specimens (n=31) which were produced during the bone fracture, with the fresh bleeding samples (n=31) as control. It was demonstrated in Figure 1A that the average HMGB1 level in the hematoma samples from bone fracture was 31.730±3.197 ng/mL, significantly higher than the level of 9.845± 0.967 ng/mL in the fresh bleeding samples from bone fracture (p<0.0001). Then we investigated the regulation of HMGB1 by hypoxia in the human macrophage, which is the main type of HMGB1 producers in blood. As shown in Figure 1B, from 12 to 48 hours post treatment, the HMGB1 level in the supernatant of macrophage U937 cells was significantly upregulated by the hypoxia treatment (p<0.01 or p<0.001), with the U937 cells under normoxia as control. In addition, we analyzed the nucleus and cytosol distribution of HMGB1 in U937 cells under hypoxia or normoxia. The western blotting results indicated that the HMGB1 in nucleus was not markedly different between the normoxia and hypoxia groups. However, the cytosolic HMGB1 was promoted by hypoxia, the relative cytosol/nucleus level of HMGB1 was significantly upregulated by hypoxia at 24 or 48 hours post treatment (p<0.01 respectively). Thus, we confirmed the upregulation of HMGB1 in the hematoma of fractured bones and in macrophage U937 cells under hypoxia.2.HMGB1 promotes the proliferation of osteoblast MG-63 cells and upregulates TLR-4We then investigated the regulation of the hypoxia-promoted HMGB1 on the proliferation of MG-63 cells. The growth curve of MG-63 cells which were treated with 0,0.2 or 1μg/mL HMGB1 was assessed. It was demonstrated that the MG-63 cells which were treated with 0.2 μg/mL HMGB1 grew to higher levels at 24,48 or 72 hour post treatment (p<0.05 or p<0.01) under normoxia. And the treatment with 1 μg/mL HMGB1 promoted even a higher level of cell proliferation than the treatment with 0.2 μg/mL HMGB1 (p<0.05 respectively). Such promotion to the cell proliferation was also confirmed in the MG-63 cells under hypoxia.That activity reduction of MG-63 cells was markedly inhibited by the treatment with 0.2μg/mL HMGB1 at 24,48 or 72 hour post treatment (p<0.05 or p<0.01).The inflammatory response exerted by HMGB1 is usually initiated by the interaction of HMGB1 with TLR-4 (Yu et al.2006, Park et al.2006, Park et al.2004). To recognize the mechanism underlining the proliferation promotion by HMGB1 in osteoblast MG-63 cells, we then examined the level of TLR-4 in MG-63 cells under normoxia or hypoxia, with or without the HMGB1 treatment. The western blot analysis indicated that hypoxia markedly upregulated TLR-4 in MG-63 cells under hypoxia rather than under normoxia, particularly in the presence of 0.2μg/mL HMGB1.3.HMGB1 induces the phosphorylation of ERK and JNK in MG-63 cellsHMGB1 initiates inflammatory reactions through the activation of ERK (van Beijnum et al.2008) and JNK (Wu et al.2013) signaling. To clarify the mechanisms by which the hypoxia-induced HMGB1 promotes the proliferation of MG-63 cells, we then determined the expression and the phosphorylation of ERK and JNK in the HMGB1-treated MG-63 cells under normoxia or hypoxia. It was indicated in Figure 3A that the mRNA level of ERK was significantly unregulated by hypoxia at 12 hour post treatment (p<0.05). Particularly, such upregulation was aggravated by additional HMGB1 treatment (0.2μg/mL) (p<0.05). And such aggravation lasted to the 24 hour post treatment (p<0.05). And the mRNA level of JNK was also upregulated by the HMGB1 treatment under hypoxia in MG-63 cells under hypoxia at 12 hour post treatment (p<0.05,). Then we examined the phosphorylation of both signaling markers in the HMGB1-treated MG-63 cells under normoxia or hypoxia with western blot analysis. Results demonstrated that the phosphorylation levels of both markers were markedly promoted by the hypoxia treatment (p<0.05 or p<0.01), and the hypoxia-promoted phosphorylation was also aggravated by the HMGB1 treatment (p<0.01). Taken together, the hypoxia upregulated the phosphorylation of both ERK and JNK, and the upregulation was aggravated by HMGB1.4.TLR-4 knockout inhibits the HMGBl-promoted proliferation of MG-63 cellsWe next elucidated the role of the main cell membrane HMGB1 receptors TLR4, in HMGB1-induced proliferation of MG-63 cells. TLR-4-specific siRNA was utilized to knockout the TLR-4 expression in the MG-63 cells, and results demonstrated in Figure 4A that siRNA-TLR-4 with 30 or 60 nM reduced the relative TLR-4 mRNA level from 1.00±0.12 or 1.00±0.13 to 0.53±0.058 or to 0.32±0.037 (p<0.01 respectively). And the siRNA-TLR-4 transfection also significantly blocked the hypoxia-induced TLR-4 in MG-63 cells in protein level at either 24 or 48 hour post transfection (p<0.01). Then we re-curved the growth of MG-63 cells under hypoxia or normoxia, post the transfection with 60 nM siRNA-TLR-4 or siRNA-Con. As shown in Figure 4C, the siRNA-TLR-4 significantly reduced the proliferation level of MG-63 cells under normoxia at 24,48 or 72 hour post transfection, compared with the siRNA-Con (p<0.05 or p<0.01). Moreover, the knockout of TLR-4 with siRNA-TLR-4 markedly reduced the cell viability than the siRNA-Con transfection (p<0.05 or p<0.01). Thus, we confirmed the TLR-4-dependence of the HMGB1-induced MG-63 cell proliferation under normoxia, or of the HMGB1-mediated cellular viability amelioration of MG-63 cells under hypoxia.5.TLR-4 knockout reduces the HMGB1-induced phosphorylation of ERK and JNK in MG-63 cellsWe also re-evaluated the phosphorylation levels of ERK and JNK in the hypoxia-treated MG-63 cells post the knockout of TLR-4. The level of ERK and JNK with or without phosphorylation were analyzed with western blotting in the hypoxia-treated MG-63 cells which were transfected with 60 nM siRNA-TLR-4 or with siRNA-Con. Results demonstrated that compared to the siRNA-Con, siRNA-TLR-4 significantly blocked the hypoxia-induced phosphorylation of ERK at either 24 or 48 hour post transfection (p<0.01). And the hypoxia-induced phosphorylation level of JNK was also markedly inhibited by siRNA-TLR-4 with 60 nM (p<0.01). Therefore, the hypoxia- and following promoted HMGB1-induced phosphorylation of ERK and JNK was TLR-4-dependent.The part two:1.Hypoxia reduces the expression of osteoblast differentiation-associated markersOsteoblast differentiation is characterized by the high expression of such markers as bone morphogenetic protein 2 (BMP-2), propeptide of type I procollagen I (PINP), alkaline phosphatase (ALP) and Runt-related transcription factor 2 (RUNX2). We firstly examined the expression of BMP-2, PINP, ALP and RUNX-2 with ELISA in the hFOB 1.19 cells under normoxia or hypoxia. The BMP-2 level was markedly reduced in the hFOB 1.19 cells under hypoxia than under normoxia (p<0.05 or p<0.01 for 6,12 or 24 hours post treatment). And such downregulation by hypoxia was also found in the expression of PINP (p<0.05 or p<0.01), of ALP (p<0.05, p<0.01 or p<0.001,) and of RUNX2 (p<0.05 or p<0.01).2.NF-κB signaling was promoted in the hypoxia-mediated reduction of osteoblast differentiationNF-κB pathway is induced by hypoxia in neurons, in T lymphocytes, in microglia cells, and also in various types of tumor cells. In order to identify the hypoxia-mediated reduction of osteoblast differentiation-associated markers, we then explored the activation of NF-κB pathway in the hFOB 1.19 cells under normoxia or hypoxia. We found that there was a high p65 mRNA level in the hypoxia-treated hFOB 1.19 cells (p<0.01 or p<0.001 for 6,12 or 24 hours post treatment). Western blotting assay also indicated an upregulation of p65 in protein level in the hypoxia-treated hFOB 1.19 cells (p<0.01 respectively for 12 or 24 hours post treatment). However, IκB, an inhibitor of NF-κB pathway, was markedly downregulated by hypoxia in hFOB 1.19 cells (p<0.05 or p<0.01). To substantiate the activation of NF-κB pathway by hypoxia, we exposed hFOB 1.19 cells to hypoxia, and then assessed the NF-κB activation using an NF-κB luciferase reporter, which contains four copies of NF-kB binding site.There was approximately 2-fold upregulation of luciferase activity in the hypoxic hFOB 1.19 cells, compared to the normoxia-treated hFOB 1.19 cells (p<0.01 or p<0.001). These data indicated that hypoxia induced p65 expression and its activation.3.NMP ameliorates the hypoxia-reduced osteoblast differentiationIn order to examine the possibility that NMP might modulate the hypoxia-mediated reduction of osteoblast differentiation, the osteoblast differentiation-characterized markers, such as BMP-2, PINP, ALP and RUNX-2 were re-evaluated in the NMP-treated hFOB 1.19 cells under hypoxia. We first investigated the effect of NMP on the viability of hFOB 1.19 cells under hypoxia. NMP exerted no influence on the cellular viability of hFOB 1.19 cells under normoxia. However, the NMP treatment markedly ameliorated the hypoxia-mediated viability reduction of hFOB 1.19 cells (p<0.05 or p<0.01). And such amelioration was dose-dependent (p<0.05).We then examined the BMP-2, PINP, ALP and RUNX2 levels with ELISA in the NMP-treated hFOB 1.19 cells under hypoxia. The BMP-2 reduction was significantly ameliorated by 10 mM NMP at 12 or 24 hours post treatment (p<0.05 or p<0.01). And such amelioration by the NMP treatment was also found in the PINP reduction at 12 or 24 hours post treatment (p<0.05 or p<0.01). In addition the NMP-mediated amelioration was also found from 6 or at 24 hours post treatment for the ALP reduction or for the RUNX2 reduction (p<0.05 or p<0.01). Taken together, NMP ameliorates the hypoxia-induced donwregulation of cellular viability and osteoblast differentiation.4.NMP inhibited the hypoxia-promoted NF-κB signaling in hFOB 1.19 cellsIn order to investigate the regulation by NMP on the hypoxia-mediated reduction of osteoblast differentiation, we then re-evaluated the activation of NF-κB pathway in the hFOB 1.19 cells under hypoxia, with or without the treatment with 10 mM NMP. The hypoxia-promoted p65 mRNA level was markedly inhibited at 12 or 24 hours post treatment (p<0.05 or p<0.01). And the western blotting results demonstrated that the protein level of p65 was inhibited, whereas the IκB level was upregulated by 10 mM NMP in the hypoxia-treated hFOB 1.19 cells (p<0.01 or p<0.001). Moreover, the NF-κB luciferase reporter assay indicated that the promoted luciferase activity was inhibited by the 10 Mm NMP in the hypoxic hFOB 1.19 cells (p<0.05 or p<0.01). Therefore, NMP inhibited the hypoxia-promoted NF-κB signaling in hFOB 1.19 cells.Conclusion:1.Hypoxia reduces the expression of osteoblast differentiation-associated markers2.HMGB1 promotes the proliferation of osteoblast MG-63 cells and upregulates TLR-43.HMGB1 induces the phosphorylation of ERK and JNK in MG-63 cells4.TLR-4 knockout inhibits the HMGB1-promoted proliferation of MG-63 cells5.TLR-4 knockout reduces the HMGB1-induced phosphorylation of ERK and JNK in MG-63 cells6.Hypoxia reduces the expression of osteoblast differentiation-associated markers7.NF-κB signaling was promoted in the hypoxia-mediated reduction of osteoblast differentiation8.NMP ameliorates the hypoxia-reduced osteoblast differentiation9.NMP inhibited the hypoxia-promoted NF-κB signaling in hFOB 1.19 cells... |
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