Effects of Foxp3 gene silencing on the expression of inflammatory cytokines and the proliferation and migration of human periodontal ligament fibroblasts in an inflammatory environment / 华西口腔医学杂志

OBJECTIVES@#This study aimed to clarify the effects of Foxp3 silencing on the expression of inflammatory cytokines in human periodontal ligament cells (hPDLFs) in an inflammatory environment and on cell proliferation and invasiveness, as well as to explore the role of Foxp3 gene in the development of periodontitis.@*METHODS@#An small interfering RNA (siRNA) construct specific for Foxp3 was transfected into hPDLFs. Foxp3 silencing efficiency was verified by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, and the siRNA with the optimum silencing effect of Foxp3 gene was screened. Using lipopolysaccharide to simulate an inflammatory environment in vitro, CCK-8 detected the effect of silencing Foxp3 on hPDLFs proliferation under inflammatory conditions. Wound-healing experiments and transwell assays were conducted to detect the effect of silencing Foxp3 on hPDLF migration under inflammatory conditions. The expression of the inflammatory cytokines interleukin (IL)-6 and IL-8 was detected by RT-PCR and Western blotting under inflammatory conditions.@*RESULTS@#After siRNA transfection, RT-PCR and Western blotting analyses showed that the expression of Foxp3 mRNA in the Foxp3-si3 group decreased significantly (t=21.03, P<0.000 1), and the protein expression of Foxp3 also decreased significantly (t=12.8, P<0.001). In the inflammatory environment, Foxp3 gene silencing had no significant effect on hPDLFs proliferation (P>0.05), and Foxp3 gene silencing promoted hPDLFs migration (P<0.05). Moreover, the expression of IL-6 and IL-8 increased (P<0.05).@*CONCLUSIONS@#In an inflammatory environment, Foxp3 gene silencing promoted hPDLFs migration but had no significant effect on hPDLFs proliferation. The expression of inflammatory factors expressed in hPDLFs increased after Foxp3 gene silencing, indicating that Foxp3 gene inhibited inflammation in periodontitis.

Protective effect of low-level laser irradiation on lipopolysaccharide-mediated inflammatory injury of human periodontal ligament fibroblasts / 华西口腔医学杂志

OBJECTIVES@#To study the effect and mechanism of low-level laser irradiation (LLLI) on lipopolysaccharide (LPS)-induced inflammatory injury of human periodontal ligament fibroblasts (hPDLFs).@*METHODS@#hPDLFs were inoculated into well plates and randomly divided into the normal group, LPS group, and LPS+LLLI group. The cells in the normal group were cultured in conventional medium. The hPDLFs in the LPS and LPS+LLLI groups were cultured in RPMI1640 medium containing 1 mg·L@*RESULTS@#Compared with the normal group, the LPS group showed increased apoptosis rate of hPDLFs and intracellular free Ca@*CONCLUSIONS@#LLLI has a protective effect on the inflammatory injury of hPDLFs induced by LPS, and the effect is most obvious when the irradiation intensity is 4 J·cm

Periostin inhibits hypoxia-induced oxidative stress and apoptosis in human periodontal ligament fibroblasts p38 MAPK signaling pathway / 南方医科大学学报

OBJECTIVE@#To investigate the effect of periostin on hypoxia-induced oxidative stress and apoptosis in human periodontal ligament fibroblasts and the molecular mechanism involved.@*METHODS@# cultured human periodontal ligament fibroblasts were placed in an anaerobic gas-producing bag for hypoxia treatment for 48 h followed by treatment with periostin at low (25 ng/mL), moderate (50 ng/mL) or high (100 ng/mL) doses. MTT assay was used to measure the cell viability, and the cell apoptosis rate was determined using flow cytometry. The contents of IL-1β, IL-6 and TNF-α in the cells were determined with ELISA, and ROS levels were measured using a fluorescent plate reader. The intracellular SOD activity was detected using ELISA. The expressions of HIF-1α, P21, cyclin D1, Bax, cleaved caspase-3, Bcl-2, P38MAPK and p-p38 MAPK proteins in the cells were detected with Western blotting.@*RESULTS@#Hypoxia treatment significantly reduced the cell viability ( < 0.05), increased P21, Bax, and cleaved caspase-3 protein levels ( < 0.05), promoted cell apoptosis ( < 0.05), and decreased cyclin D1 and Bcl-2 protein levels ( < 0.05) in the cells. Compared with the hypoxic group, the cells treated with periostin at different concentrations showed significantly increased cell viability ( < 0.05) with significantly lowered apoptotic rates ( < 0.05) and decreased expression levels of Bax and cleaved caspase-3 ( < 0.05) but significantly increased expression levels of cyclin D1 and Bcl-2 ( < 0.05). Hypoxic exposure of the cells resulted in significantly increased expression levels of HIF-1α and p-p38 MAPK ( < 0.05) and increased levels of IL-1β, IL-6, TNF-α and ROS ( < 0.05) but decreased SOD activity ( < 0.05). Periostin treatment at different concentrations significantly lowered the expression levels of HIF-1α and p-p38 MAPK ( < 0.05) and the levels of IL-1β, IL-6, TNF-α and ROS ( < 0.05) and significantly increased SOD activity in the hypoxic cells ( < 0.05).@*CONCLUSIONS@#Periostin promotes the proliferation, inhibits apoptosis, enhances cellular antioxidant capacity, and reduces inflammatory damage in human periodontal ligament fibroblasts exposed to hypoxia possibly by inhibiting the activation of the p38 MAPK signaling pathway.

Effects of arecoline on apoptosis of human periodontal ligament fibroblasts / 口腔疾病防治

Objective @#To observe the effects of arecoline on the related protein expressions in the apoptosis of cultured human periodontal ligament fibroblasts (hPDLFs), such as p-JNK, p-p53, and Bcl-2.@*Methods @#hPDLFs were isolated from human periodontal ligament tissues and expanded in vitro. hPDLFs were treated with different concentrations of arecoline (0, 20, 40 and 80 μg/mL) for 12 h. The proliferative activities were evaluated by MTT. The expressions of p-JNK, p-p53, and Bcl-2 were determined by Western blot. @*Results@#Arecoline inhibited cell proliferation and induced apoptosis protein. The protein level of Bcl-2 was decreased, while p-JNK and p-p53 were increased (P < 0.05). The protein expressions were in a concentration-dependent manner with arecoline. @*Conclusion @#It demonstrates arecoline might contribute to the apoptosis of hPDLFs, and could destroy periodontal tissues.

Effects of IL-10 and glucose on expression of OPG and RANKL in human periodontal ligament fibroblasts

The effects of interleukin-10 (IL-10) and glucose on mRNA and protein expression of osteoprotegerin (OPG), and its ligand, receptor activator of nuclear factor-κB ligand (RANKL), were investigated in human periodontal ligament fibroblasts (HPDLFs). Primary HPDLFs were treated with different concentrations of IL-10 (0, 1, 10, 25, 50, and 100 ng/mL) or glucose (0, 5.5, 10, 20, 30, and 40 mmol/L). Changes in mRNA and protein expression were examined using the reverse-transcription polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. After IL-10 treatment, mRNA and protein levels of OPG were increased, while mRNA and protein levels of RANKL were decreased (P<0.05), both in a concentration-dependent manner. Glucose stimulation had the opposite concentration-dependent effect to that of IL-10 on OPG and RANKL expression. IL-10 upregulated OPG expression and downregulated RANKL expression, whereas high glucose upregulated RANKL and downregulated OPG in HDPLFs. Abnormal levels of IL-10 and glucose may contribute to the pathogenesis of periodontal disease.

Glucosamine Inhibits Lipopolysaccharide-induced Inflammatory Responses in Human Periodontal Ligament Fibroblasts

Glucosamine is commonly taken by the elderly without prescription as a nutritional supplement to attenuate the progression or symptoms of osteoarthritis. Previous studies demonstrated that glucosamine shows anti-inflammatory effects in tissues such as blood vessels and the heart. However, there have been few reports about the effects of glucosamine on oral inflammatory diseases. Therefore, in this study, the effects of glucosamine on lipopolysaccharide (LPS)-induced inflammatory responses were investigated using human periodontal ligament fibroblasts (HPDLFs). HPDLFs were incubated in the presence and absence of glucosamine (10 mM) for 24 h, followed by treatment with E. coli LPS (100 ng/ml) or vehicle. Quantitative RT-PCR and ELISA results showed that LPS exposure significantly increased the levels of IL-6 and IL-8 mRNA and protein, while the effect was significantly suppressed by glucosamine treatment. Glucosamine did not attenuate, but slightly increased, the LPS-induced activation of mitogen activated kinases (ERK, p38, JNK). However, it suppressed the LPS-induced increase in the DNA binding affinity and transcriptional activity of NF-kappaB. These results suggest that glucosamine exerts anti-inflammatory effects on HPDLFs exposed to LPS via inhibition of NF-kappaB activity, necessitating further studies using animal periodontitis models.

Experimental studies of hypoxia on the proliferation ability and ultrastructure of cultured human periodontal ligament fibroblasts / 实用口腔医学杂志

Objective: To observe the effect of hypoxia on changes of proliferation ability of cultured human periodontal ligament fibroblasts (HPLFS) in vitro. Methods: HPLFS were randomly divided into two groups (experimental group and control group) by different oxygen concentrations. The oxygen concentration of control group was 21%. The oxygen concentrations of experiment group were 10%, 5% and 2%. The proliferation ability of HPLFS was detected by MTT colorimetric assay. The cell ultrastructure was observed by transmission electron microscope (TEM). Results: MTT assay results showed that compared with the control group, at the 12 h and 24 h, cell proliferation was enhanced with the hypoxia degree. At 24 h, cell proliferation showed significant differences. At 48 h and 72 h, proliferation of the cultured HPLFS in severe hypoxia group reduced significantly. Observed by TEM, at 24 h, not only the number of mitochondria, rough endoplasmic reticulum but also cell process increased in the cultured HPLFS in severe hypoxia group. At 72 h, the number of lysosome increased and the cell structure degenerated. Conclusion: Long-time severe hypoxia may lower the repair and remodeling abilities of periodontium, which might be one of the important etiological factors of periodontal disease under condition of high altitude.

Effects of H2O2 and chlorhexidine on MMP-1, TIMP-1,2, Type 1 collagen, fibronectin and UNCL expressions in human periodontal ligament fibroblasts / 대한치주과학회지

PURPOSE: To evaulate the effects of chlorhexidine and H2O2 on matrix metalloproteinase-1 (MMP-1), tissue inhibitor of metalloproteinase(TIMP-1, TIMP-2), Type 1 collagen, fibronectin and UNCL expressions in human periodontal ligament fibroblasts (hPDLF). MATERIALS AND METHODS: 1.2x10(-1)%, 1.2x10(-2)% and 1.2x10(-3)% CHX and 3x10(-3)%, 3x10(-4)% and 3x10(-5)% H2O2 and mixture of CHX and H2O2 were applied to hPDLF for 1 min and 30 min. The mRNA expressions of MMP-1, TIMP-1 and 2, Type 1 collagen, fibronectin and UNCL in hPDLF were analysed by RT-PCR. RESULTS: The result were as follows: 1. The expression of UNCL mRNA was higher than that of other mRNAs. 2. 1.2x10(-3) % CHX increased mRNA expressions of hPDLF as application time increased. 3. H2O2 lower than 3x10(-3) % increased expression of UNCL mRNA, and did not decrease mRNA expression of hPDLF. 4. hPDLF treatment with 1.2x10(-1) % CHX (with or without H2O2) resulted in no gene expression. 5. hPDLF treatment with 1.2x10(-2) % CHX (with or without H2O2) for 30 minutes resulted in no gene expression. CONCLUSION: Because low concentration of CHX and H2O2 increased UNCL mRNA expression of hPDLF, low concentraction of CHX and H2O2 may have an antioxidative effect.

Effects of H2O2 and ascorbic acid on TIMP-2, Type 1 collagen, and PDLs22 levels in human periodontal ligament fibroblasts / 대한치주과학회지

Reactive oxygen species (ROS) have been implicated in the pathogenesis of various diseases. And vitamin C has shown a protective effect for the tissues. The aim of this study was to evaluate the effects of H2O2 and ascorbic acid on matrix metalloproteinase-1 (MMP-1), tissue inhibitor of metalloproteinase (TIMP: TIMP-1, TIMP-2), Type 1 collagen, fibronectin, and PDLs22 level in human periodontal ligament fibroblasts (hPDLF) via reverse transcription-polymerase chain reaction (RT-PCR). hPDLF was obtained from a healthy periodontium and cultured in Dulbecco's modified Eagles's medium plus 10% fetal bone serum. The concentration of ascorbic acid in hPDLF was 50 microgram/ml, and that of H2O2 in hPDLF was 0.03% and 0.00003%. Ascorbic acid only, H2O2 only and mixture of ascorbic acid and H2O2 were applied with hPDLF for 1-, 3-, and 30-min., respectively. The gene expression of MMP-1-, TIMP-1-, TIMP-2-, Type 1 collagen-, fibronectin-, and PDLs22-mRNA in hPDLF was analysed via RT-PCR. The results were as follows; 1. hPDLF in response to 30-min. incubation with 0.03% H2O2 did not show any gene expression. 2. In all the experimental groups, the gene expression of fibronectin mRNA showed the decreased tendency compared to control. 3. In all the experimental groups, the gene expression of TIMP-1 mRNA showed the tendency similar to control. 4. hPDLF in response to 30-min. incubation with 0.03% H2O2 and ascorbic acid increased mRNA induction for MMP-1. 5. In all the experimental groups, hPDLF increased mRNA induction for PDLs22, collagen type I, and TIMP-2 compared to control. Within the limited experiments, H2O2 and ascorbic acid increased mRNA induction for PDLs22, collagen type I, and TIMP-2 in hPDLF. More research will be needed in order to confirm the relative importance of the different roles of ROS and antioxidants in hPDLF from a periodontal regeneration or repair standpoint.

Effect of dexamethasone on proliferation of human periodontal ligament fibroblasts cultivated in vitro / 吉林大学学报(医学版)

Objective To investigate the effect of dexamethasone on proliferation of human periodontal ligament fibroblasts(hPDLF) cultivated in vitro,and search for optimal culture condition for hPDLF in vitro,and provide basis for further study on regeneration of periodontinum.Methods The hPDLF cultivated in vitro were divided into 5 groups and cultivated in 5 different culture media separately which all included 10% fetal bovine serium: ①control group(no DEX);②5 mg?L-1DEX;③10 mg?L-1 DEX;④20 mg?L-1 DEX;⑤50 mg?L-1 DEX.The proliferation of hPDLF was assyed by MTT method.The phase contrast microscope was used to observe the morphological changes of the cells.Results The PDLF appeared aggregation and cells on bottom of culture bottle showed squamae-shape after inoculated on plate with 24 hole for 3 d.There were more layers of smaller hPDLF with blunt prominency came forth after cultivated in DEX culture media.MTT method showed that DEX promoted the proliferation of hPDLF obviously compared with control group(P

Expression of mRNA for matrix metalloproteinases and tissue inhibitors of metalloproteinases in human gingival and periodontal ligament fibroblasts treated with lipopolysaccharide from Prevotella intermedia / 대한치주과학회지

Matrix metalloproteinases (MMPs) are a family of host-derived proteolytic enzymes and implicated in the remodeling and degradation of extracellular matrix under both physiological and pathological conditions. Connective tissue degradation in periodontal diseases is thought to be due to excessive MMP activities over their specific inhibitors. The effects of lipopolysaccharide (LPS) from Prevotella intermedia, one of the major putative pathogens of periodontitis, on the expression of mRNA for MMPs and tissue inhibitors of metalloproteinases (TIMPs) in human gingival and periodontal ligament fibroblasts were examined by reverse transcriptase-polymerase chain reaction (RT-PCR). The expression of mRNAs encoding MMP-1, -2, -3, -10, and -14 was increased in human gingival fibroblasts treated with P. intermedia LPS, whereas MMP-11 and TIMP-2 mRNA expression was decreased in these cells stimulated with LPS. P. intermedia LPS increased the MMP-1, -2, -10, -11, and -14 mRNA expression and decreased TIMP-1 and -2 mRNA expression in human periodontal ligament fibroblasts. These findings imply that P. intermedia LPS may play an important role in the connective tissue degradation in periodontitis.

A study of mutagenic effects of simulating static magnetic field produced by magnetic attachment on human periodontal ligament fibroblasts / 实用口腔医学杂志

0.05).Conclusion:It suggests that HPDLFs exposured to static magnetic field produced by magnetic attachment have little mutagenic effects on chromosomes and DNA.