[Regulation mechanism of resveratrol on odontogenic differentiation of human dental pulp stem cells].

PURPOSE: To investigate whether resveratrol promotes odontogenic differentiation of human dental pulp stem cells(DPSCs) by up-regulating the expression of silent information regulator 1 (SIRT1) and activating ß-catenin signaling pathway. METHODS: Different concentrations of resveratrol(0, 10, 15, 20 and 50 µmol/L) were used to treat DPSCs for 7 days and 14 days, and cell proliferative activity was detected by CCK-8. After odontogenic differentiation induced by 15 µmol/L resveratrol for 7 days, alkaline phosphatase(ALP) staining was performed and real-time quantitative reverse transcription PCR(qRT-PCR) was used to detect the mRNA expression of Runt-related transcription factor 2 (Runx2), dentin sialophosphoprotein(DSPP) and dentin matrix protein-1(DMP-1) in DPSCs. Western blot was used to detect the expression of SIRT1 in DPSCs on a specific day (0, 3rd, 5th, 7th and 14th) after differentiation induction. Western blot was also used to detect the expression of SIRT1 and activated ß-catenin during odontogenic differentiation of DPSCs treated by 15 µmol/L resveratrol for 7 days. The experimental data was analyzed with GraphPad Prism 9 software package. RESULTS: 15 µmol/L resveratrol had no significant effect on proliferation of DPSCs on the 7th and 14th day; 15 µmol/L resveratrol promoted odontogenic differentiation of DPSCs and up-regulated mRNA expression of RUNX2, DSPP, and DMP-1 in DPSCs; the expression of SIRT1 was the highest on the 7th day during odontogenic differentiation induction. Resveratrol resulted in the increasing protein expressions of SIRT1 and activated ß-catenin when DPSCs was induced to odontogenic differentiation for 7 days. CONCLUSIONS: Resveratrol promotes odontogenic differentiation of human DPSCs by up-regulating the expression of SIRT1 protein and activating ß-catenin signaling pathway.

Histone demethylase Jmjd3 modulates osteoblast apoptosis induced by tumor necrosis factor-alpha through directly targeting RASSF5.

Purpose: Regulation of gene expression is fine-tuned by a dynamic equilibrium between repressive modifications and transcriptional activation of histone tails. Jumonji domain-containing 3 (Jmjd3), also known as KDM6B, is a specific histone demethylase for trimethylation on histone H3 lysine 27 (H3K27me3) that specifically removes the methylation of H3K27me3 and promotes gene expression. Our previous study showed that Jmjd3 inhibits serum deprivation-induced osteoblast apoptosis. In this study, we clarified the role of Jmjd3 in tumor necrosis factor-alpha (TNF-α)-induced osteoblast apoptosis. Materials and Methods: Jmjd3 activity was inhibited by GSK-J4. Transfection of osteoblastic murine MC3T3-E1 cells with short hairpin RNA (shRNA) was used to establish stable Jmjd3 knockdown cells. Osteoblast apoptosis was detected using Annexin V-APC/PI staining, cysteinyl aspartate specific protease-3 (caspase-3) activity assays, and Western blot. Real-time polymerase chain reaction (PCR) and chromatin immunoprecipitation (ChIP) assays were performed to clarify the mechanism responsible for Jmjd3-regulated osteoblast apoptosis induced by TNF-α. Results: Based on Annexin V-APC/PI staining, caspase-3 activation, and poly ADP-ribose polymerase (PARP) cleavage, pretreatment with GSK-J4 and knockdown of Jmjd3 by shRNA transfection each inhibited osteoblast apoptosis. Furthermore, knockdown of Jmjd3 decreased the expression of Ras association domain family 5 (RASSF5), which is a pro-apoptotic gene of the Ras associated domain family. H3K27me3 levels in the promoter region of RASSF5 were up-regulated in the Jmjd3 knockdown cells. Conclusions: Jmjd3 regulated TNF-α-induced osteoblast apoptosis by targeting RASSF5.

[Inhibiting effect of transforming growth factor ß3 on IL-6 expression in MG63 induced by lipopolysaccharide].

PURPOSE: To explore the effect of transforming growth factor ß3 (TGF-ß3) on IL-6 expression in inflammatory MG63, and the mechanism by which TGF-ß3 exert its anti-inflammatory effect. METHODS: Cell line MG63 was stimulated by 20 µg/mL lipopolysaccharide of Porphyromonas endodontalis (P.e-LPS) to establish the inflammatory model of osteoblast. TGF-ß3 or TGFß1 varying from 5 to 20 ng/mL was added together with P.e-LPS for 24 h, then the mRNA expression of IL-6 was detected by real-time PCR, the role of TGF-ß3 on IL-6 protein was further verified by ELISA. MG63 was pretreated with 10 ng/mL TGF-ß3 for 30 min in RPMI 1640 medium without fetal bovine serum (FBS), then the cells were cultured for another 20 min with 20 µg/mL P.e-LPS, the phosphorylation level of ERK1/2 was measured by Western blot. Statistical analysis was performed using one-way ANOVA with SPSS13.0 software package. RESULTS: The results of real-time PCR revealed that, when MG63 was treated with 20 µg/mL P.e-LPS alone, the mRNA expression of IL-6 increased significantly(P<0.01). When TGF-ß1 was added with P.e-LPS, it could barely decrease IL-6 prominently at the highest concentration (P<0.05).Whereas, the inhibition effect of TGF-ß3 on IL-6 was dramatic (P<0.01), ELISA results showed that 10-20 ng/mL TGF-ß3 blocked the IL-6 expression at protein level (P<0.05). 20 µg/mL P.e-LPS promoted the phosphorylation level of ERK1/2 in MG63(P<0.01), while with 10 ng/mL TGF-ß3, the effect of P.e-LPS on ERK1/2 was blocked(P<0.05). CONCLUSIONS: TGF-ß3 is more potent than TGF-ß1 in inhibiting MG63, and ERK1/2 is involved in its anti-inflammatory effect.

[Effect of lipopolysaccharides from Porphyromonas endodontalis on the expression of interleukin-34 in mouse osteoblasts].

PURPOSE: To investigate the effects of lipopolysaccharides (LPS) extracted from Porphyromonas endodontalis (P.e) on the expression of interleukin-34 (IL-34) mRNA in MC3T3-E1 cells and the role of p38MAPK, ERK1/2, NF-κB and SIRT1 in the process. METHODS: MC3T3-E1 cells were treated with different concentrations of P.e-LPS(0-50 mg/L) and 20 mg/L P.e-LPS for different time (0-24 h). The expression of IL-34 mRNA was detected by real-time reverse transcription-polymerase chain reaction (real time RT-PCR). MC3T3-E1 cells were pretreated with inhibitor of NF-κB(BAY 11-7082),inhibitor of p38MAPK (SB203580), inhibitor of ERK1/2 (PD98059), agonist of sirtuin1 (SIRT1) [resveratrol (RES)] and inhibitor of SIRT1 (EX-527) for 1 h, and then were treated with 20 mg/L P.e-LPS. The expression of IL-34 mRNA was detected by real time RT-PCR. Statistical analysis was performed using one-way ANOVA and Dunnett t test with SPSS 13.0 software package. RESULTS: The level of IL-34 mRNA increased significantly after treatment with different concentrations of P.e-LPS(0-50 mg/L)，which indicated that P.e-LPS induced osteoblasts to express IL-34 mRNA in a dose-dependent manner. Maximal induction of IL-34 mRNA expression was observed in MC3T3-E1 cells treated with 20 mg/L P.e-LPS for 24 h.At 48 h, the expression of IL-34 mRNA decreased gradually. The mRNA of IL-34 decreased significantly after pretreatment with 10 µmol/L BAY-117082, SB203580 and PD98059 for 1 h. P.e-LPS-induced IL-34 upregulation was attenuated by pretreatment with RES, but increased by EX-527. CONCLUSIONS: These results suggest that P.e-LPS may mediate IL-34 mRNA expression in MC3T3-E1 cells. This process is dependent, at least in part, on p38MAPK, ERK1/2, NF-κB and SIRT1 signaling pathways.

[Mechanism of TNF-α in bone defect of chronic apical periodontitis].

PURPOSE: To investigate the effect of lipopolysaccharides(LPS) extracted from Porphyromonas endodontalis(P.e) on the expression of tumor necrosis factor-α(TNF-α) mRNA in MC3T3-E1 cells and the role of NF-κB signaling on the expression of macrophage colony stimulating factor (M-CSF) induced by TNF-α in MC3T3-El cells． METHODS: MC3T3-E1 cells were treated with different concentrations of P.e-LPS(0-50 mg/L) and 10 mg/L P.e-LPS for different time (0-24 h). The expression of TNF-α mRNA was detected by reverse transcription polymerase chain reaction(RT-PCR). MC3T3-E1 cells were treated with different concentrations of TNF-α(0-10 ng/L) for 6 h. The expression of M-CSF mRNA and protein was detected by RT-PCR and enzyme-linked immunoadsordent assay(ELISA).The expression of M-CSF protein was also detected in 10 ng/L TNF-α treated MC3T3-E1 cells after pretreated with BAY 11-7082 for 1 h, a special NF-κB inhibitor . Statistical analysis was performed using Multi-way ANOVA and Dunnett t test with SPSS 13.0 software package. RESULTS: The level of TNF-α mRNA increased significantly after treatment with different concentrations of P.e-LPS(0-50 mg/L)，which indicated that P.e-LPS induced osteoblasts to express TNF-α mRNA in dose dependent manners. Maximal induction of TNF-α mRNA expression was seen in the MC3T3-E1 cells treated with 10 mg/L P.e-LPS for 6 h. After 6 h, the expression of TNF-α mRNA decreased gradually .The expression of M-CSF mRNA and protein was increased in a does- dependent manner by different concentrations of TNF-α treatment（0-10 ng/L）. The expression of M-CSF protein increased from (37±2) ng/L(control group) to (301±8) ng/L(10 ng/L group).The protein of M-CSF decreased significantly after pretreatment with 10 µmol/L BAY 11-7082 for 1 h, and the expression of M-CSF proteins was reduced from (253±14) ng/L to (154±2) ng/L .BAY group had no significant difference from the control group. CONCLUSIONS: The expression of TNF-α mRNA was increased by P. endodontalis LPS treatment in osteoblast. TNF-α may induce the expression of M-CSF in MC3T3-E1 cells through the signaling of NF-κB. It suggests that TNF-α affect osteoblasts through autocrine way for bone destruction in chronic apical periodontitis induced by P.e-LPS.

[The effect of calcium hydroxide on IL-6 and TNF-α expression of osteoblast in periapical tissues].

PURPOSE: To compare the effect of calcium hydroxide in different position on pH and inflammation factor expression of periapical osteoblasts. METHODS: 140 sterilized single-rooted human teeth models were randomly divided into 6 experiment groups and one control group: Group 1-3:calcium hydroxide paste was placed in the apical half of root canal, the upper half of root canal and the pulp champer; Group 4-6:Apexcal was placed in the apical half of root canal, the upper half of root canal and the pulp champer; Group 7: the control group without medication. 10 teeth of each group were placed in P.e suspension, the IL-6 and TNF-α expression of MC3T3-E1 was tested at 3 d and 7 d. The other teeth of each group were placed in distilled water, and the pH in periapical region was tested at 3, 7, 14 and 21 d. SPSS 13.0 software package was used for statistical analysis. RESULTS: Calcium hydroxide placed in different position of the root canal increased periapical pH value and reached its peak at 14 d. The group in which calcium hydroxide paste was placed in pulp chamber gained lower pH level than other experimental groups. IL-6, TNF-α expression of MC3T3-E1 pretreated by P.e suspension of experimental groups was significantly reduced compared with control group, and there was no significant difference between the experimental groups. CONCLUSIONS: Calcium hydroxide placed in different position of the root canal could increase periapical pH value and reduce IL- 6, TNF-α expression of periapical osteoblasts.

[Expression of IL-34 in chronic periapical lesions and its clinical significance].

PURPOSE: To observe the expression of IL-34 mRNA in chronic periapical lesions and healthy periodontal ligaments and discuss the role of IL-34 in the etiology of chronic apical periodontitis. METHODS: A total of 25 periapical tissues from chronic apical periodontitis and 22 normal periodontal ligament tissue from extracted healthy teeth for orthodontic reason were selected. The expression of IL-34mRNA was detected by real-time PCR; the expression of IL-34 protein was detected by immunohistochemical analysis. Statistical analysis was performed using SPSS 13.0 software package. RESULTS: The level of IL-34 mRNA expression in periapical lesions (3.53±3.07) was significantly higher than that of the normal control (1.07±0.76); IL-34 was positively expressed in lymphocytes, plasma cells and macrophages. Image analysis software indicated that the level of IL-34 protein was significantly higher in periapical lesions than that in normal control (P<0.01). CONCLUSIONS: IL-34 may be closely related to inflammation of chronic apical periodontitis.

[Expression of Wnt5a in chronic apical periodontitis and its clinical significance].

PURPOSE: To detect the expression of Wnt5a in lesions of chronic apical periodontitis and determine the relationship between expression of Wnt5a and inflammation degree. METHODS: Ten patients with chronic apical periodontitis and 5 healthy controls were enrolled in this study. According to the inflammatory cell infiltration, the specimens were divided into 2 groups: severe inflammation group and mild inflammation group. The expression of Wnt5a was measured by real-time PCR (RT-PCR) and immunohistochemical analysis in the lesions of chronic apical periodontitis. The amount of Wnt5 expression was assayed and compared in different inflammation levels. Statistical analysis was performed using SPSS 13.0 software package. RESULTS: Wnt5a was detected in both groups. Expression of Wnt5a mRNA in patients were significantly higher than the controls (P<0.05). According to inflammation level, the positive expression rate of Wnt5a in severe inflammation group was significantly higher than the controls (P<0.01), and Wnt5a positive expression in mild inflammation group was also significantly higher than the controls (P<0.05). The expression of Wnt5a was significantly different between severe inflammation group and mild inflammation group (P<0.05). CONCLUSIONS: The expression of Wnt5a increases as the severity of tissue inflammation increases, which indicates that Wnt5a plays an important role in the development of chronic apical periodontitis.

[Effect of NF-κB on the expression of interleukin-6 induced by lipopolysaccharides of Porphyromonas endodontalis in MC3T3-E1 cells].

PURPOSE: To investigate the effect of NF-κB signaling on the expression of interleukin-6(IL-6) induced by lipopolysaccharides(LPS) extracted from Porphyromonas endodontalis(P.e) in MC3T3-El cells. METHODS: MC3T3-E1 cells were pretreated with BAY-117082 for 1 h, and then were treated with 10 mg/L P.e-LPS for different times. The translocation of NF-κB was observed by immunofluorescence. The expression of IL-6 was detected by reverse transcription polymerse chain reaction (RT-PCR) and enzyme-linked immuno sorbent assay (ELISA). Statistical analysis was performed using multi-way ANOVA and Dunnett's t test with SPSS 13.0 software package. RESULTS: The staining of NF-κB was mostly in cytoplasm in untreated cells. Rapid translocation of NF-κB into nucleus was observed in the cells stimulated for 30 min and mostly relocalization of NF-κB from nucleus to cytoplasm was observed after 60 min. Pretreatment with 10 µmol/L BAY-117082 for 1h significantly inhibited P.e-LPS-induced translocation of NF-κB .The mRNA and proteins of IL-6 decreased significantly after pretreatment with 10 µmol/L BAY-117082 and the expression of IL-6 proteins was reduced from (774.983±6.585) ng/L to (377.384±14.620) ng/L (P<0.01). The group of treatment with BAY-117082 alone had no significant difference from the blank control group. CONCLUSIONS: P.e-LPS can induce translocation of NF-κB in mouse osteoblast MC3T3-El, and P.e-LPS may induce the expression of IL-6 in mouse osteoblast through the signaling of NF-κB.

[Effect of CD-14 and toll like receptors on the expression of interleukin-6 induced by lipopolysaccharides of Porphyromonas endodontalis].

OBJECTIVE: To evaluate the effect of cluster of differentiation 14 (CD-14) and Toll like receptors (TLR) on the expression of interleukin-6 (IL-6) mRNA induced by Porphyromonas endodontalis (Pe) lipopolysaccharides (LPS). METHODS: MC3T3-E1 cells were treated with 10 mg/L Pe-LPS for different hours, and the cells uninvolved by anything as the blank group. The expression of IL-6 was detected by reverse transcription polymerse chain reaction (RT-PCR) and enzyme-liked immunosorbent assay (ELISA). The expression of CD-14, TLR-2 and TLR-4 mRNA was observed at different time point (0 - 24 h) by RT-PCR. The protein of CD-14, TLR-2 and TLR-4 was analyzed with a flow cytometer. MC3T3-E1 cells were pretreated with anti-CD-14, anti-TLR-2 and anti-TLR-4 antibody for 1 h, and then cells were stimulated with 10 mg/L Pe-LPS for 6 h. The expression of IL-6 mRNA was examined by RT-PCR. Statistical analysis was performed using one-way ANOVA Dunnett-t test with SPSS 11.0 software package. RESULTS: The IL-6 mRNA and proteins increased significantly after treatment with Pe-LPS. When MC3T3-E1 cells treated by Pe-LPS for 6 h, the expression of proteins soared from (11.696 ± 0.672) ng/L to (36.534 ± 0.574) ng/L (P < 0.01); In the control group, the CD-14 and TLR-4 mRNA are ambly-expression, and the ratios of CD-14 and TLR-4 positive cells were (39.038 ± 3.131)% and (11.438 ± 0.385)% respectively in MC3T3-E1. After treatment by Pe-LPS, the expression of CD-14 and TLR-4 mRNA increased significantly, and the ratios of CD-14 and TLR-4 positive cells markedly increased to (62.407 ± 1.800)% and (21.367 ± 2.271)%. TLR-2 expression did not change apparently after Pe-LPS treatment. The expression of IL-6 mRNA was partly inhibited by anti-CD-14 or anti-TLR-4 antibody, but not by TLR-2. CONCLUSIONS: Pe-LPS can induce the expression of IL-6 in osteoblast MC3T3-E1 through CD-14 and TLR-4, but not TLR-2.