Osteopontin mRNA Expression Research Articles

Expression of Osteopontin mRNA During Periodontal Healing Following Scaling and Root Planing

Objective: To analyze osteopontin mRNA expression levels in subjects with periodontitis prior to (baseline) and 7, 14, and 28 days following scaling and root planing (SRP). Material and Methods: Gingival crevicular fluid was collected as clinical samples from four subjects with periodontitis (pocket depth, 4 - 5 mm) aged 35 - 54 years old as well as from three healthy subjects (controls). The osteopontin mRNA expression levels were measured by quantitative real-time polymerase chain reaction. Spearman’s rank correlation between osteopontin levels in gingival crevicular fluid and the modified gingival index (MGI) was also performed. Results: The Wilcoxon signed-rank test showed no significant difference in osteopontin mRNA expression levels between baseline and 28 days following SRP (p=0.068). The Friedman test showed no significant difference in osteopontin mRNA expression levels between baseline and following SRP (7, 14, or 28 days) (p>0.05). Spearman’s rank correlation showed no significant correlation between osteopontin mRNA expression levels and MGI (r=0.087; p=0.749). Conclusion: Following SRP of periodontal tissue, there was a decreasing trend in osteopontin mRNA expression; however, this finding was not statistically significant. Nevertheless, osteopontin can be used as a biomarker to monitor the healing process; however, further studies are required to clarify our results.

Comparative study of hydroxyapatite, fluor-hydroxyapatite and Si-substituted hydroxyapatite nanoparticles on osteogenic, osteoclastic and antibacterial ability.

This study compared the effects of hydroxyapatite (HA), fluor-hydroxyapatite (FHA) and Si-substituted hydroxyapatite (SiHA) on osteogenic differentiation, osteoclastic activity and antibacterial properties. HA, FHA and SiHA were prepared via a sol–gel reaction and characterized by scanning electron microscopic analysis (SEM), transmission electron microscopic analysis (TEM), and X-ray photoelectron spectrometry. Cell proliferation was evaluated using an MTT assay and cytoskeletal morphology was observed by fluorescence microscopy. Osteogenic differentiation was evaluated using alkaline phosphatase activity and Alizarin red staining. Quantitative real-time PCR was used to evaluate the mRNA expression of runt-related transcription factor 2 (Runx2) and osteopontin (OPN). New bone formation was tested using μCT, haematoxylin and eosin staining and TRAP staining. The antibacterial actions against Porphyromonas gingivalis (P. g) were evaluated through plate counting and live-dead bacterial staining. The results demonstrated that HA, FHA and SiHA can promote proliferation of bone mesenchymal stem cells (BMSCs). ALP activity in FHA extract with a concentration of 625 μg mL−1 was the highest after 14 days osteogenic induction; similar results were observed for Runx2 and OPN mRNA expression. HA, FHA and SiHA decreased trabecular space in bone defects, but FHA reduced osteoclastic activity and inhibited P. g growth. In conclusion, FHA can promote osteogenic activity, reduce osteoclastic activity and enhance antibacterial effects.

Coexistence of Anticoagulant and Anti-vascular Calcification Activities in Kribbella sp. UTMC 267 Metabolites.

Thrombotic disorders increase the risk of cardiovascular/cerebrovascular complications and represent a major health problem worldwide. Anticoagulants are extensively used in treatment of these disorders. Vitamin K antagonists, like Warfarin, are frequently used in medication. Vascular calcification (VC) is a significant side-effect of vitamin K antagonists especially Warfarin. There is an urgent need to find natural, efficient, non-toxic, and cost effective anticoagulants without dangerous side-effect like VC. In the present study, we evaluated the potential of thirteen fermentation broth extracts of actinobacteria (FBEA) (200 µg mL-1) to prolong whole blood prothrombin time (PT)/international normalized ratio (INR) and activated partial thromboplastin time (APTT). The fractions of the most effective FBEA were further investigated for their inhibitory effect on VC. The results showed PT/INR of the healthy blood samples was sensitive to the presence of five FBEA. Their INR index fell in the 1.2 to 8.6 range and six FBEA prolonged both PT/INR and APTT parameters (1.7-5 INR, and 46-59 s for APTT). The fractions of Kribbella sp. UTMC 267 FBE (200 µg mL-1), as the most efficient FBE, only inhibited intrinsic and common pathways of coagulation (APTT). Under calcification condition, Kribbella sp. UTMC 267 fractions (20 µg mL-1) showed significant inhibitory effect on VC in alizarin red staining (13.3-76 %) and alkaline phosphatase activity of VSMCs (33-62%). They also inhibited osteopontin mRNA expression in treated vascular smooth muscle cells (VSMCs) under that situation. So, we can introduce Kribbella sp. UTMC 267 FBE as a good candidate for more investigation on thrombotic medication.

The impact of photofunctionalized gold nanoparticles on osseointegration

ObjectivesThe aims of this study were to create a new surface topography using simulated body fluids (SBF) and Gold Nanoparticles (GNPs) and then to assess the influence of UV Photofunctionalization (PhF) on the osteogenic capacity of these surfaces. Materials and methodsTitanium plates were divided into six groups All were acid etched with 67% Sulfuric acid, 4 were immersed in SBF and 2 of these were treated with 10 nm GNPs. Half of the TiO2 plates were photofunctionalized to be compared with the non-PhF ones. Rat's bone marrow stem cells were seeded into the plates and then CCK8 assay, cell viability assay, immunofluorescence, and Scanning electron microscopy (SEM) were done after 24 hours. Gene expression analysis was done using real time quantitative PCR (qPCR) one week later to check for the mRNA expression of Collagen-1, Osteopontin and Osteocalcin. Alkaline phosphatase (ALP) activity was assessed after 2 weeks of cell seeding. ResultsOur new topography has shown remarkable osteogenic potential. The new surface was the most biocompatible, and the 10 nm GNPs did not show any cytotoxicity. There was a significant increase in bioactivity, enhanced gene expressions and ALP activity. ConclusionsGNPs enhances osteogenic differentiation of stem cells and Photofunctionalizing GNPs highly increases this. We have further created a novel highly efficient topography which highly enhances the speed and extent of osseointegration. This may have great potential for improving treatment outcomes for implant, maxillofacial as well as orthopedic patients.

The expression of Akt/mTOR in VSMC calcification induced by high phosphate and its regulation of Cbfα1

Objective: To observe the expression of protein kinase B (Akt) / mammalian target of rapamycin (mTOR) induced by high phosphorus in rat vascular smooth muscle cells (VSMC) calcification model, and its modulation on the expression of core binding factor alpha 1 (Cbfα1). Methods: Rat VSMC cells were cultured in vitro, and then divided into two groups: normal phosphorus group (Pi 1.3 mmol/L) and high phosphorus group (Pi 2.6 mmol/L). At day 7, calcium deposition was detected by Alizarin stain. The mRNA levels of Cbfα1 and osteopontin (OPN) were determined by real-time PCR. The protein expressions of p-Akt (ser473), p-mTOR (S2448), Cbfα1 and OPN were quantified by Western blot. Then, VSMC cultured with high phosphorus were treated with Akt inhibitor (Wortmannin) and mTOR inhibitor (Rapamycin) with different concentrations. After 24 h, the mRNA levels of Cbfα1 and OPN were determined and after 48 h, the protein expressions of p-Akt, p-mTOR, Cbfα1 and OPN were quantified. Also, at day 7, calcium deposition was also visualized by Alizarin stain. Results: After 7 days, compared with normal phosphorus group, calcium deposition was more obvious in high phosphorus group. The mRNA expressions of Cbfα1 and OPN increased significantly and the protein expressions of p-Akt, p-mTOR, Cbfα1 and OPN up-regulated significantly in high phosphorus group (all P<0.05). After treated with Wortmannin or Rapamycin for 24 h, compared with high phosphorus group, the mRNA expressions of Cbfα1 and OPN decreased significantly in high phosphorus + Wortmannin (30, 50 and 100 nmol/L) groups (all P<0.05) and high phosphorus + Rapamycin (1, 10 and 100 ng/ml) groups (all P<0.05). After treated with Wortmannin or Rapamycin for 48 h, compared with high phosphorus group, the protein expressions of p-Akt, Cbfα1 and OPN down-regulated significantly in high phosphorus + Wortmannin (30, 50 and 100 nmol/L) groups (all P<0.05). It showed a dose-dependent down-regulation of p-mTOR, Cbfα1 and OPN in high phosphorus + Rapamycin (1, 10, 100 ng/ml) groups (all P<0.05). After 7 days, compared with high phosphorus group, calcium deposition decresased significantly in high phosphorus + Wortmannin and high phosphorus + Rapamycin groups. Conclusions: High phosphorus can induce VSMC calcification. Akt and mTOR are involved in VSMC calcification induced by high phosphorus through the activation of Cbfα1.

Upregulation of osteopontin expression via the interaction of macrophages and fibroblasts under IL-1b stimulation

BackgroundFibrosis is attributed to dysregulation of tissue-remodeling. In remodeling areas, fibroblasts and macrophages actively make contact with each other. Osteopontin (OPN) is a pro-fibrotic molecule, whose expression is upregulated by interleukin (IL)-1β via secretion of its downstream cytokines, such as IL-6. Here, we investigated the effect of interaction between fibroblasts and macrophages under IL-1β stimulation on the expression of OPN. MethodsWe used human lung fibroblasts and THP-1 macrophages differentiated from THP-1 cells using phorbol 12-myristate 13-acetate. These cells were either cultured alone or co-cultured under IL-1β stimulation. Secretion of OPN and IL-6 were examined by enzyme-linked immunosorbent assay, and mRNA expression was assessed by quantitative real-time PCR. The effects of siRNA against IL-6 or OPN on OPN expression were evaluated. ResultsOPN expression increased when fibroblasts and THP-1 macrophages were co-cultured under IL-1β stimulation. The siRNA against IL-6 in fibroblasts suppressed the upregulation of OPN expression during co-culture, whereas siRNA against IL-6 in THP-1 macrophages did not. The upregulation of expression of OPN mRNA in fibroblasts or THP-1 macrophages when co-cultured under IL-1β stimulation was mediated by IL-6 from fibroblasts. OPN from THP-1 macrophages was involved in the increase of OPN expression in fibroblasts. ConclusionsThe present study revealed the crosstalk between fibroblasts and THP-1 macrophages under IL-1β stimulation, where IL-6 from fibroblasts, stimulated by IL-1β, upregulated OPN expression in fibroblasts themselves via increase in OPN from THP-1 macrophages. The fibroblasts/macrophages network may induce activation or qualitative changes in both cells, which contributes to inflammation-associated fibrosis.

Effects of tumor necrosis factor-α on osteogenic differentiation and Notch signaling pathway in human periodontal ligament stem cells

To evaluate the effects of tumor necrosis factor-α (TNF-α) on osteogenic differentiation and Notch signaling pathway of periodontal ligament stem cells (PDLSCs) and to investigate the regulatory role of Notch signaling pathway on the osteogenic differentiation of PDLSCs under the influence of TNF-α. PDLSCs were obtained through enzyme digestion and tissue block method. The expression levels of stem cell surface markers CD105, CD90, CD146, CD45, and CD31 were detected by fluorescence activated cell sorter (FACS). PDLSCs were divided into experimental (10 ng·mL⁻¹ TNF-α) and control groups (0 ng·mL⁻¹ TNF-α). The proliferation ability of PDLSCs was detected using cell counting kit-8 (CCK-8). The effect of TNF-α on the osteogenic ability of PDLSCs were tested by measuring alkaline phosphatase (ALP) activity and conducting alizarin red staining and quantitative real-time polymerase chain reaction (PCR). We tested Notch signal pathway receptors Notch1, Notch2, ligand JAG1, JGA2, and downstream gene Hes-1. Changes in DLL1 expression were detected by quantitative real-time PCR. FACS profiling showed that PDLSCs were strongly positive for CD105, CD90, and CD146 but negative for CD45 and CD31. CCK-8 results showed that TNF-α could promote the proliferation of PDLSCs (P<0.05). ALP activity in the experimental group was lower than that in the control group (P<0.05). Alizarin red staining showed that the experimental group had decreased mineralized nodules as compared with the control group. Quantitative real-time PCR results showed that the mRNA expression of osteogenic marker genes cementum attachment protein (CAP), osteopontin (OPN), and Runt-related transcription factor 2 (Runx2) significantly decreased in the experimental group as compared with those in the control group (P<0.05). The expression levels of Notch1, Notch2, JAG1, JGA2 and Hes-1 were significantly decreased (P<0.05), whereas those of Notch3 and DLL1 were increased in Notch signaling pathway-related molecules (P<0.05). TNF-α can promote PDLSCs proliferation and inhibit bone differentiation and Notch signaling pathway expression, indicating that the Notch signaling pathway regulates PDLSCs osteogenic differentiation.

Fate of adipose derived stem cells is correalted with expression of specific set of genes

Purpose: Adipose tissue represents an abundant, accessible and rich source of adult stem cells with the ability to differentiate into multiple lineages. Our understanding of the dynamic process requiring indispensable differentiation of adipose-derived stem cells (ASCs) remains limited. Here, we describe that differentiation of ASCs into adipogenic, osteogenic, and chondrogenic lineages is dependent on expression of certain specific genes. Methods: ASCs were isolated from human adipose tissue by enzymatic digestion using collagenase. Isolated ASCs were cultured as monolayer in DMEM low-glucose medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) at 37°C in a 5% CO2 incubator. Adipogenic differentiation of ASCs was done for 14 days using adipogenic medium. Osteogenic and chondrogenic differentiation of ASCs was done for 21 days using osteogenic and chondrogenic differentiation medium. Adipogenic differentiation of ASCs was accessed by Oil Red O staining, and gene expression of adipogenic markers. Osteogenic differentiation was accessed by Von Kossa staining, calcium estimation and gene expression of osteogenic markers. Chondrogenic differentiation was accessed by Alcian blue staining, and gene expression of chondrogenic markers. Results: Normal differentiation of ASCs into adipogenic lineage resulted in higher level of oil red o staining which represents higher adipose depots. The mRNA expression of adipogenic markers Adiponectin, FABP4, and PPAR-γ significantly increases with adipogenic differentiation. Interestingly, expression of TAZ decreases with adipogenic differentiation of ASCs. Differentiation of ASCs into osteogenic lineage resulted in higher amount of von Kossa staining along with significantly higher level of calcium concentrations. The mRNA expression of osteogenic markers RUNX2, Osteonectin, Osteopontin, ALP, and TAZ significantly increases during osteogenic differentiation. Differentiation of ASCs into chondrogenic lineage resulted in higher level of Alcian blue staining. The mRNA expression of chondrogenic markers Aggrecan, COL2A1, SOX9, and TAZ significantly increases during chondrogenic differentiation. Conclusions: We demonstrate that differential gene expression has critical role in ASCs lineage determination. By identifying and modulating expression of such genes, we may direct the differentiation fate of ASCs that will be useful as a cell therapy for regenerative medicine.

Effect of miR-21 down-regulated by H 2O 2 on osteogenic differentiation of MC3T3-E1 cells

To explore the effect and mechanism of miR-21 down-regulated which was induced by H 2O 2 on osteogenic differentiation of MC3T3-E1 cells. MC3T3-E1 cells were cultured and passaged, and the 7th generation cells were harvested to use in experiment. The MC3T3-E1 cells were treated with different concentrations (0, 40, 80, 160, and 320 μmol/L) of H 2O 2. The expression of miR-21 was detected by real-time quantitative PCR (RT-PCR) and the cell viability was determined by MTS. Then the appropriate concentration of H 2O 2 was obtained. To analyze the effect of H 2O 2 on osteogenic differentiation of MC3T3-E1 cells, the MC3T3-E1 cells were divided into blank control group (group A), H 2O 2 group (group B), osteogenic induction group (group C), and H 2O 2+osteogenic induction group (group D). The expression of miR-21 and the osteogenesis related genes expressions of Runx2, osteopontin (OPN), and collagen type Ⅰ alpha 1 (Col1a1) were detected by RT-PCR. The expression of phosphatase and tensin homolog (PTEN) was detected by Western blot. The extracellular calcium deposition was detected by alizarin red staining. To analyze the effect on osteogenic differentiation of MC3T3-E1 cells after the transfection of miR-21 inhibitor and siRNA-PTEN, the MC3T3-E1 cells were divided into H 2O 2 group (group A1), H 2O 2+osteogenic induction group (group B1), H 2O 2+osteogenic induction+miR-21 inhibitor group (group C1), and H 2O 2+osteogenic induction+miR-21 inhibitor negative control group (group D1); and H 2O 2 group (group A2), H 2O 2+osteogenic induction group (group B2), H 2O 2+osteogenic induction+siRNA-PTEN negative control group (group C2), and H 2O 2+osteogenic induction+siRNA-PTEN group (group D2). The osteogenesis related genes were detected by RT-PCR and the extracellular calcium deposition was detected by alizarin red staining. The results of MTS and RT-PCR showed that the appropriate concentration of H 2O 2 was 160 μmol/L. The expression of miR-21 was significantly lower in group B than in group A at 1 and 2 weeks ( P<0.05). The expression of miR-21 was significantly lower in group D than in group C at 1 and 2 weeks ( P<0.05). The expression of PTEN protein was significantly lower in group C than in groups A and D ( P<0.05). The mRNA expressions of Runx2, OPN, and Col1a1 were significantly lower in group D than in group C at 1 and 2 weeks ( P<0.05). The extracellular calcium deposition in group D was obviously less than that in group C. The expression of PTEN protein was significantly higher in group C1 than in group D1 ( P<0.05). The mRNA expressions of Runx2 and OPN were significantly lower in group C1 than in groups B1 and D1 at 1 and 2 weeks ( P<0.05). The mRNA expression of Col1a1 was significantly lower in group C1 than in groups B1 and D1 at 2 weeks ( P<0.05). The extracellular calcium deposition in group C1 was obviously less than those in groups B1 and D1. The mRNA expressions of OPN and Col1a1 were significantly higher in group D2 than in groups B2 and C2 at 1 week ( P<0.05). The extracellular calcium deposition in group D2 was obviously more than those in groups B2 and C2. H 2O 2 inhibits the osteogenic differentiation of MC3T3-E1 cells, which may be induced by down-regulating the expression of miR-21.

Fibroblast growth factor 23 is upregulated in the kidney in a chronic kidney disease rat model.

The hormone fibroblast growth factor 23 (FGF23) is secreted from bone and is involved in phosphorus (P) metabolism. FGF23 mainly binds the FGF receptor, which interacts with αKlotho in the kidney or parathyroid and regulates Na-dependent phosphate co-transporter type IIa (NaPi-IIa) and type IIc (NaPi-IIc) expression, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) activity, and parathyroid hormone (PTH) secretion. In this study, we utilized hemi-nephrectomized rats fed a high-P diet (HP Nx), rats subjected to a partial nephrectomy (PN) and rats with doxorubicin-induced renal failure (DXR) as chronic kidney disease (CKD) animal models and analyzed the P metabolism and FGF23 expression in the kidneys in each CKD model. We cultured HK2 cells with a high level of P, 1,25(OH)2D3 or transforming growth factor-β1 (TGFβ1) to investigate the FGF23 expression mechanism. In both the HP Nx and PN rats, the blood FGF23 and PTH levels were increased. However, the 1,25(OH)2D3 level was increased in the HP Nx rats and decreased in the PN rats. In all three animal models, the mRNA expression of αKlotho, NaPi-IIa and NaPi-IIc was decreased, and the mRNA expression of TGFβ1, collagen1a1, osteopontin and FGF23 was elevated in the kidney. FGF23 protein and mRNA were expressed at high levels in the extended tubule epithelium, which was an osteopontin-positive region in the HP and PN rats. FGF23 and osteopontin mRNAs were expressed in HK2 cells incubated with TGFβ1; however, these levels were not altered in HK2 cells incubated with 1,25(OH)2D3 and high P levels in vitro. Altogether, FGF23 is expressed in the kidneys in CKD model rats. Following stimulation with TGFβ1, the injured renal tubular epithelial cells are strongly suspected to express both FGF23 and osteopontin. FGF23 produced in the kidney might contribute to P metabolism in subjects with CKD.

The expression change of osteopontin after subarachnoid hemorrhage in brain cortex of rats

Objective To investigate the expression change of osteopontin (OPN) after subarachnoid hemorrhage (SAH) in brain cortex of rats. Methods 76 rats were randomly assigned to 3 groups: 6 rats in normal control group, 28 rats in sham group (4 rats in each group that corresponded with the time points of 6, 12, 24, 48 h, 3, 5 and 7 d). 42 rats with SAH were correspondingly assigned with the previous time spots with 6 rats in each group. The mRNA expression of OPN was measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and OPN expression by Western blotting analysis. And neurological scores were assessed for each group. Results The mortality and morbidity in the prechiasmatic injection model were close to the clinical incidence and the neurological scale was severe at 24 h (7.3 ±2.2, t=7.45, P=0.002). The whole mortality of the first part was 7.3%. The expressions of the total and active protein and mRNA of OPN increased dramatically at 12 h and reached the peak at 3 day (mRNA: 30.5±8.6, t=5.891, P=0.004; OPN: 16.2±4.1, t=6.320, P=0.003), then decreased gradually after 72 h which were still higher than that of sham group. Conclusion The prechiasmatic SAH model is suitable for study of the pathophysiological mechanisms of SAH due to the high reproducibility and stability. OPN may be involved in the pathological process of SAH, and was up-regulated significantly to the peak at 3 day after SAH. Key words: Subarachnoid hemorrhage; Osteopontin; Model, animal; Cerebral vasospasm

Carbon monoxide releasing molecule‑3 promotes the osteogenic differentiation of rat bone marrow mesenchymal stem cells by releasing carbon monoxide.

Stem cell‑based therapies are promising strategies to stimulate bone regeneration.Carbon monoxide releasing molecule‑3 (CORM‑3) exhibits multiple regulatory effects in a number of cells by releasing carbon monoxide (CO). The present study aimed to investigate the influence of CORM‑3 on the osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs). BMSCs were divided into five groups: A CORM‑3‑osteogenic group, in which cells were pretreated with CORM‑3 and subjected to osteogenic differentiation induction using osteogenic medium; an osteogenic group, in which cells were cultured in osteogenic medium; a degassed CORM‑3‑osteogenic group, in which cells were pretreated with degassed CORM‑3 and subjected to osteogenic differentiation induction; a CORM‑3 group, in which cells were cultured in control medium containing CORM‑3; and a control group, in which cells were cultured in control medium alone. The osteo‑specific mRNA and protein expression of runt‑related transcription factor2 (Runx2), osteocalcin (OCN) and osteopontin (OPN) were assessed using reverse transcription‑quantitative polymerase chain reaction and western blot analysis. Alkaline phosphatase (ALP) activity was also examined and mineralization was detected using alizarin red staining. Levels of Runx2, OCN and OPN mRNA and protein in the CORM‑3‑osteogenic group were significantly increased compared with the osteogenic group (P<0.05), with the exception of OCN protein levels on day3. The mRNA and protein expression of Runx2, OCN and OPN in the degassed CORM‑3‑osteogenic and osteogenic groups were similar. In addition, the mRNA and protein expression of Runx2, OCN and OPN in the CORM‑3 and control group were similar. ALP activity in the CORM‑3‑osteogenic group was increased from day3 and remained significantly higher compared with all other groups on days3,5and7 (P<0.05). Additionally, the results indicated that the optical density value of alizarin red staining in the CORM‑3‑osteogenic group was significantly increased compared with the other groups (P<0.05). Therefore, the present study demonstrated that CORM‑3 may promote the osteogenic differentiation of BMSCs by releasing CO.

Klf10 silencing inhibited mechanical force induced osteogenic differentiation of human periodontal ligament cells

To investigate the effect of Klf10 silence on human periodontal ligament cells (HPDLCs) under mechanical force. HPDLCs were isolated and transfected with Klf10 siRNA, and then exposed to centrifugal force for 6 h at 631 r/min. Purmorphamine, an hedgehog signaling pathway agonist, was used for intervention. The activity of alkaline phosphatase (ALP) was detected by ELISA. RT-PCR and Western blot were performed to detect the mRNA and protein expression of Klf10, Runt related transcription factor 2 (Runx2), osteopontin (OPN) and osteocalcin (OCN). The protein expression of glioma associated oncogene homolog 1 (Gli1) and patched-1 (PTCH1) was detected by Western blot. SPSS 20.0 software package was used to analyze the data. Mechanical force increased mRNA and protein level of Klf10, Runx2, OPN and OCN, and elevated ALP activity significantly (P<0.05). Mechanical force also upregulated the protein expression of GLI1 and Ptch1 significantly (P<0.05). Compared with the control group, Klf10 siRNA transfection significantly decreased mRNA and protein level of Klf10 (P<0.05). Klf10 siRNA significantly inhibited the activity of ALP, and downregulated mRNA and protein expression of Runx2, OPN and OCN (P<0.05). Moreover, Klf10 siRNA significantly inhibited protein expression of GLI1 and Ptch1 (P<0.05), and purmorphamine obviously inhibited the effect of Klf10 siRNA (P<0.05). Klf10 silencing could inhibit bone differentiation of human periodontal ligament cells under mechanical force, which may be through regulation of hedgehog signaling pathway.

Effect of total sonicated Aggregatibacter actinomycetemcomitans fragments on gingival stem/progenitor cells.

BackgroundAggregatibacter-actinomycetemcomitans (A.actinomycetemcomitans) are strongly associated with localized-aggressive-periodontitis (LAgP). The study’s aim was to test for the first time the effect of total sonicated A.actinomycetemcomitans-bacterial-fragments on gingival mesenchymal stem/progenitor cells’ (G-MSCs) proliferation and regenerative gene expression in-vitro.Material and MethodsG-MSCs were isolated, characterized, expanded and stimulated by total sonicated A.actinomycetemcomitans-bacterial-fragments (0 (negative-control), 15, 60, 120 and 240µg/ml; serovar-b; n=6/group). Cellular proliferation and NF-κβ (NFKB1), Alkaline Phosphatase (ALPL), Collagen-I (COL1A1), Collagen-III (COL3A1), Osteonectin (SPARC) and Osteopontin (SPP1) m-RNA expression were assessed via reverse-transcription-polymerase-chain-reaction (RT-PCR) at 24, 48 and 72 hours and CFUs-ability evaluated at twelve days.ResultsG-MSCs demonstrated stem/progenitor cells’ characteristics. A.actinomycetemcomitans-bacterial-fragments (up to 72 hours) resulted in marked G-MSCs’ proliferation over-time (p<0.001) and elevated NFKB1 (p=0.017), COL1A1 (p=0.025), SPARC (p=0.025), decreased ALPL (p=0.017), with no significant differences for COL3A1 and SPP1 expression or stimulation times (p>0.05; Friedman-test). Longer-term stimulation for twelve days reduced G-MSCs’ CFUs.ConclusionsSonicated A.actinomycetemcomitans-bacterial-fragments’ exert beneficial short-term effects on G-MSCs’ proliferative and non-mineralized tissue forming aptitude. Results shed new light on the importance of periodontal treatment for LAgP patients, using power driven sonic/ultrasonic devices, which, in addition to reducing the subgingival microbial load, produces cell-stimulatory A.actinomycetemcomitans-bacterial-fragments, with positive attributes on tissue reparative/regenerative responses of tissue resident stem/progenitor cells in their niche. Key words:Ultrasonic, Aggregatibacter actinomycetemcomitans, stem cells, gingiva, Aggressive periodontitis.

Roles of microRNA-539 and osteopontin in rheumatoid arthritis.

The present study aimed to investigate the role and mechanism of microRNA-539 (miR-539) in rheumatoid arthritis (RA). A total of 68 RA patients and 46 osteoarthritis patients were enrolled into the current study. Peripheral blood and joint fluid were collected prior to treatment. Reverse transcription-quantitative polymerase chain reaction was performed to detect osteopontin (OPN) mRNA and miR-539 expression levels, while ELISA and western blot analysis were applied to detect OPN protein expression. In addition, bioinformatics analysis predicted that miR-539 directly targeted OPN, while dual-luciferase assay was used to validate this finding. Furthermore, agomiR-539 transfection and OPN knockdown by siRNA were conducted in MH7A cells, and MTT assay was used to detect MH7A cell proliferation. The results indicated that OPN was significantly increased in the blood and joint fluid of RA patients, while miR-539 expression was significantly decreased in the two types of specimens (P<0.05). Subsequent to silencing OPN by siRNA, the proliferation of MH7A cells was decreased (P<0.05). Following upregulation of miR-539, OPN expression was significantly decreased and cell proliferation was inhibited. Dual-luciferase assay revealed that miR-539 regulated OPN expression through complementary binding to 3'-untranslated region. OPN was also significantly increased in the blood and joint fluid of RA patients, which may be associated with the downregulation of miR-539. Thus, miR-539 may promote the development and progression of RA through regulating OPN.

Isolation and screening of rare Actinobacteria, a new insight for finding natural products with antivascular calcification activity.

Vascular calcification (VC) is a significant pathological process in some life-threatening diseases. Several pathological mechanisms, including transdifferentiation of vascular smooth muscle cells to osteoblast-like cells and apoptosis are involved in VC. Compounds with an inhibitory effect on these processes are potentially efficient medications. In consideration of the multiple biological activities of Actinobacteria, this research was aimed at finding anti-VC metabolite-producing Actinobacteria. After the isolation and identification of Actinobacteria, the effect of their fermentation broth extracts on the apoptosis rate was measured using various methods, for example, ethidium bromide/acridine orange staining, DNA laddering and diphenylamine assays. The effect of the most effective fermentation broth extract of Actinobacteria (FBEA) on the mRNA expression of runt-related transcription factor 2 (Runx2) and osteopontin (OPN) was examined. Finally, the most effective FBEA was fractionated and the chemical composition of anti-VC fractions was analysed using GC-MS. Various VC inhibition rates were observed in the tested FBEA (20μgml-1 ; 17·9-60·15%). The inhibition of DNA fragmentation was 7-48%. The FBE with the greatest anticalcification activity belonged to Kribbella sp. UTMC 267 and, according to 16S rRNA analysis, Kribbella sancticallisti with a similarity of 98·53% is its nearest neighbour. The FBE of Kribbella sp. UTMC 267 reduced Runx2 mRNA expression by 2·95-fold and OPN mRNA expression by 28·57-fold, both of which are considered significant (P<0·05). Finally, GC-MS analysis showed the existence of potent anti-oxidative and anti-inflammation agents in FBE of Kribbella sp. UTMC 267. Actinobacterial metabolites can provide a new strategy for treating VC diseases by reducing the expression of osteogenic genes, the apoptosis rate and oxidative stress. This study highlights the therapeutic potential of Kribbella sp. metabolites and Actinobacteria as a new natural source for drug discovery programs in the nonantibiotic bioactivity field.

Effects of small molecules water that may retard kidney stone formation.

Water intake is important for preventing kidney stones. Small molecules water is a more active restructured water under magnetic field. Here, we studied the relation between small molecules water and crystal formation in the rat kidney. The small molecules water was prepared by a water machine providing a 0.8-T magnetic field. Calcium oxalate crystals were induced by 0.75% EG (ethylene glycol) in male Sprague-Dawley rats by drinking small molecules water and plain water for up to 6weeks, respectively. Urinary ions were assayed. Osteopontin mRNA expression and urinary LDH were detected. Crystals were observed using a light microscope and a polarizing microscope. A significantly reduced urinary calcium and phosphorus excretion occurred in the 2nd and the 4th week after treatment of small molecules water. Crystals were initially detected in 40% of the experimental rats in the small molecules water group at the 6th week, later than the control group in which crystals were detected in 60% of rats at the 4th week. After 6weeks of treatment, crystals were observed to form in renal cortex, medulla and papilla in the control group, whereas only to form in renal medulla and papilla in the small molecules water group. OPN mRNA expression significantly increased earlier in the 2nd week after treatment of the small molecules water compared to the control (P=0.016). Small molecules water may retard crystal formation, reduce urinary calcium and phosphorus excretion and promote earlier OPN mRNA expression in the rat kidney.

Reduced miRNA‑214 expression in oral mucosa contributes to the pathogenesis of oral lichen planus by targeting CD44.

The present study aimed to investigate the role of miRNAs during the pathogenesis of oral lichen planus (OLP). OLP is a chronic inflammatory disorder, which involves T‑cell mediated autoimmunity and affects the skin, scalp, nails and mucosa. Abundant T lymphocytes have been demonstrated to infiltrate the oral mucosa, in which the activated T cells trigger apoptosis of oral epithelial cells. Overexpression of osteopontin (OPN) and CD44 has been observed in the mucosa of patients with OLP, and it has been confirmed that OPN suppresses the apoptosis of activated CD8+ T cells via CD44. The present study initially detected the protein and mRNA expression of CD44 and OPN in the mucosa of patients with OLP by western blot analysis and reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR), and confirmed the previously reported overexpression of CD44 and OPN in patients with OLP. The current study demonstrated, by RT‑qPCR, that the expression of microRNA‑214 (miR‑214), miR‑216a and miR‑216b was significantly reduced in patients with OLP. By analyzing the association between the protein level of CD44 and the expression of microRNAs (miRNAs), the present study identified a negative correlation between the expression of miR‑214 and CD44 in the mucosa samples of patients with OLP. Subsequently, the present study confirmed that miR‑214 represses endogenous CD44 expression by targeting the 3'untranslated region in HeLa, Raji and Jurkat cells. The current study indicates that reduced miR‑214 may be associated with OLP and, therefore, may be a candidate for drug development.

Abstract P432: The Effect of Carbonyl Stress on Renal Injury Induced by Renal Congestion

Background: Due to the functional linkage between cardiac function and renal function, a decline in renal function exacerbates cardiac function and a decline in cardiac function exacerbates renal function. Renal congestion is reported to contribute to the decline of renal function in heart failure patients. Regarding the mechanism of kidney failure caused by congestion, although it is suggested that ischemia is involved due to an increase in venous pressure and a decrease in blood flow, it has not been sufficiently elucidated. Since overexpression of glyoxylose-1 (GLO1), a methylglyoxal (MG) metabolizing enzyme, which is a type of carbonyl stress, has been reported to attenuate impairment due to renal ischemia reperfusion (I/R). In this study, we aimed to investigate the effect of carbonyl stress on kidney damage due to renal congestion using GLO1 overexpressing rats. Methods: Male Wistar wild type (WT) rat and GLO1 transgenic (GLO1) rat were used for experiment. Left kidney was congested by placing silver clip on left renal vein under anesthesia. Two weeks after surgery, renal cortical and outer medullary mRNA markers of fibrosis [transforming growth factor-β (TGF-β) and fibronectin (FN)], tubular injury [osteopontin (OPN) and kidney injury molecule 1 (KIM-1)] and inflammations [monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α)] were measured. Results; Relative mRNA expression of cortical TGF-β (contralateral kidney; 1.00 + 0.09, congested kidney; 2.44 + 0.33, respectively), FN (1.00 + 0.11, 2.71 + 0.46), OPN (1.00 + 0.18, 6.41 + 1.47), KIM-1 (1.00 + 0.59, 21.20 + 6.31), MCP-1 (1.00 + 0.24, 2.93 + 0.56) and TNF-α (1.00 + 0.14, 1.62 + 0.18) were significantly increased in congested kidney compared to contralateral kidney. Relative mRNA expression of cortical TGF-β (GLO1tg; 1.69 + 0.20, WT; 2.44 + 0.33, respectively), FN (1.53 + 0.33, 2.71 + 0.46), TNF-α (1.00 + 0.14, 1.62 + 0.18) in GLO1tg rat were significantly decreased compared to WT rat. No significant change was observed between renal medullary area of WT rat and GLO1tg rat. Conclusion: Renal congestion induces renal fibrosis, tubular injury and inflammation, and carbonyl stress which could contribute to the pathophysiological mechanism on renal injury in cardiac failure.

Abstract P544: Osteopontin Deficiency Decreases Autophagy and Exacerbates Tubular Injury in Acute Kidney Injury Induced by Folic Acid

Osteopontin (OPN), a secreted glycosylated phosphoprotein and pro-inflammatory cytokine, has been implicated in the pathology of several renal conditions, especially renal fibrosis in chronic kidney disease. OPN is slightly expressed in renal tubular cells in normal condition, but after acute tubular injury, OPN is highly induced in these cells. However, the role of induced OPN is still unclear. The aim of this study was to clarify the roles of OPN in acute kidney injury (AKI). AKI was induced in wild type (WT) and OPN knockout (KO) mice by using folic acid (FA) injection (0.35mg/kg). After 2days of injection, 34% of WT mice died, whereas 54% of KO died from renal failure. Kidneys from survived mice were removed and the renal histological changes, protein expression were examined. BUN and Creatinine levels were markedly elevated in WT-AKI and KO-AKI mice (BUN: WT-sham; 25.7±4.7mg/dl, WT-AKI; 315.0±173.2mg/dl, KO-AKI; 337.7±163.7mg/dl, Creatinine: WT-sham; 0.08±0.03 mg/dl, WT-AKI; 1.60±0.87 mg/dl, KO-AKI; 1.80±0.94 mg/dl). Renal OPN mRNA expression was increased in WT-AKI mice compared to WT-sham mice (p&lt;0.05). High levels of OPN expression in renal tubular cells were induced in WT-AKI mice. TUNEL positive tubular cells were increased in KO-AKI mice compared to WT-AKI mice. In immunohistochemical analysis, Kidney injury molecules 1 (Kim-1) positive tubular cells were also highly increased in KO-AKI mice compared to WT-AKI mice. In contrast, LC3B (autophagy related protein) positive tubular cells were decreased in KO-AKI mice compared to WT-AKI mice. These results indicate that OPN deficiency exacerbates tubular injury via through the inhibiting autophagy in folic acid induced AKI mice.