Effect of sonicated extracts of Porphyromonas gingivalis on osteogenic differentiation of mouse osteoblasts / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the effects of sonicated extracts of Porphyromonas gingivalis (Pg) on osteogenic differentiation of mouse osteoblast cell line MC3T3-E1.</p><p><b>METHODS</b>PgW83 was cultured under standard anaerobic conditions and extracted by sonication. Mouse osteoblast cell line MC3T3-E1 was cultured with various concentrations of the extraction (0, 10, 100, 1000 mg/L). Western blotting was applied to investigate the expression of osteocalcin (OC), bone sialoprotein (BSP), osteopontin (OPN) and osteonectin (ON). The activity of alkaline phosphatase (ALP) was detected by microplate reader after 14 days. Mineralization nodule formation was measured by alizarin red staining after 21 days.</p><p><b>RESULTS</b>Compared with the control group, the extracts of Pg decreased OC and ON expression in a dose-dependent manner (OC relative expression:1.000 ± 0.000,0.852 ± 0.110,0.625 ± 0.451,0.213 ± 0.053), (ON relative expression: 1.000 ± 0.000, 1.035 ± 0.133,0.141 ± 0.023,0.020 ± 0.003) (P < 0.05). The expression of OPN was down-regulated significantly in MC3T3-E1 treated with 1000 mg/L extraction (0.572 ± 0.162) compared with control group, 10 and 100 mg/L (1.000 ± 0.000, 1.029 ± 0.135, 1.199 ± 0.337) (P < 0.05). The expression of BSP remained unchanged when the cells were cultured with or without extraction (BSP relative expression:1.000 ± 0.000,0.831 ± 0.182,0.897 ± 0.115,0.778 ± 0.235) (P > 0.05). Meanwhile, the extracts of Pg decreased ALP activity [control group:(0.0275 ± 0.0014) U/gprot, 10 mg/L: (0.0140 ± 0.0011) U/gprot, 100 mg/L: (0.0057 ± 0.0013) U/gprot, 1000 mg/L: (0.0020 ± 0.0008) U/gprot] (P < 0.05) and reduced mineralization nodule formation.</p><p><b>CONCLUSIONS</b>The results suggest that Pg may inhibit osteoblasts'osteogenic function by down-regulation of osteogenic differentiation related proteins.</p>

Effect of caspase-dependent pathway on apoptosis of human gingival fibroblasts induced by advanced glycation end products / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the effect of synthesized advanced glycation end products (AGE) on apoptosis of human gingival fibroblasts (HGF) and the possible role of caspase-dependent pathway in the process of AGE-induced apoptosis.</p><p><b>METHODS</b>HGF were incubated with AGE-human serum albumin (AGE-HSA). The activity of caspase-8, caspase-9 and caspase-3 were detected by microplate reader after 12 and 24 hours. HGF were incubated with caspase inhibitors for 1 hour, and then incubated with AGE-HSA for 24 hours, HGF was first stained by Hoechst33258 and observed under inverted microscope, and then double stained by annexin V and propidine iodide (PI) and observed by flow cytometry (FCM). The activity of caspase-3 was determined by caspase-3 assay kit and observed by microplate reader.</p><p><b>RESULTS</b>Caspases activity of caspase-8, -9, -3 was 0.1097 ± 0.0051, 0.0965 ± 0.0051 and 0.1280 ± 0.0103 after 12 h of incubation with AGE-HSA and HGF, respectively, and 0.1558 ± 0.0053, 0.1308 ± 0.0035 and 0.1954 ± 0.0051 after 24 h of incubation with AGE-HSA and HGF, respectively (P < 0.05). Positive cells number was 247.7 ± 32.4, 200.1 ± 14.6, 154.1 ± 14.4 and 131.3 ± 14.6 in caspase inhibitor groups by Hoechst33258 staining, respectively. Apoptotic rate was (25.57 ± 2.20)%, (38.87 ± 3.31)%, (17.17 ± 2.24)% and (14.73 ± 2.48)% in caspase inhibitor groups by annexin V-PI staining, respectively. The difference between different groups was significant (P < 0.05). Caspase-3 activity was reduced to 0.1274 ± 0.0076, 0.1465 ± 0.0062, 0.1044 ± 0.0051 in caspase inhibitor groups, respectively. The difference between different groups was significant (P < 0.05).</p><p><b>CONCLUSIONS</b>Apoptosis of HGF induced by AGE-HSA may be mainly through activation of caspase-dependant pathway in which cytoplasmic pathway may play a predominant role.</p>

Influence of environmental factors on synthesis rate of hydrogen peroxide by Streptococcus oralis / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the influence of a broad range of environmental conditions on initial rates of hydrogen peroxide produced by Streptococcus oralis (S. oralis).</p><p><b>METHODS</b>For each rate measurement, 1 ml aliquots of 10(12) cells/L mid-logarithmic phase S. oralis in TSBY were centrifuged and respectively washed by phosphate buffer containing 0.01-10 mmol/L glucose or sucrose, phosphate buffer with 5.0-7.5 pH or Bis-Tris buffer containing 0.01-100 mmol/L Ca(2+), 0.01-100 mmol/L F(-) or 0.01-100 mmol/L HFPO(3)(-). After S. oralis was cultured in respective buffer for 10, 20 and 30 min at 37 degrees C, the concentration of hydrogen peroxide in supernatant was assayed spectrophotometrically in 96-well micro-plate by ABTS-HRP at A(405).</p><p><b>RESULTS</b>Synthesis rate of hydrogen peroxide by S. oralis was 7.48 micromol/L per minute without carbohydrate, the synthesis rate of hydrogen peroxide by S. oralis increased with 0.01-10 mmol/L glucose and 0.01-10 mmol/L sucrose, but there was no statistically significant difference in synthesis rate among the carbohydrate groups. The rates of H2O2 synthesis were inhibited in the buffer at pH 5.0-6.0, compared with pH 7.0 (P < 0.05). Ca(2+) had little influence on the rate of H2O2 synthesis. IC(50) of H2O2 synthesis rates by S. oralis responded to FHPO(3)(-) and F(-) were 12.65 mmol/L and 1.90 mmol/L respectively.</p><p><b>CONCLUSIONS</b>Environmental conditions may influence the synthesis rate of H2O2 by S. oralis.</p>