Effects of low-level laser on the expression of interleukin-6, tumor necrosis factor‑α, osteoprotegerin, and receptor activator of nuclear factor-κB ligand in human periodontal ligament cells / 华西口腔医学杂志

OBJECTIVES@#This study aims to determine the effects of low-level laser (LLL) on the expression of interleukin-6 (IL-6), tumor necrosis factor (TNF)-α, osteoprotegerin (OPG), and receptor activator of nuclear factor-κB ligand (RANKL) in human periodontal ligament cells (HPDLCs) stimulated by high glucose; and identify the molecular mechanism of LLL therapy in the regulation of periodontal inflammation and bone remodeling during orthodontic treatment in diabetic patients.@*METHODS@#HPDLCs were cultured in vitro to simulate orthodontic after loading and irradiated with LLL therapy. The cultured cells were randomly divided into four groups: low glucose Dulbecco's modification of Eagle's medium (DMEM)+stress stimulation (group A), high glucose DMEM+stress stimulation (group B), hypoglycemic DMEM+LLL therapy+stress stimulation (group C), and hyperglycemic DMEM+LLL therapy+stress stimulation (group D). Groups C and D were further divided into C1 and D1 (energy density: 3.75 J/cm2) and C2 and D2 (energy density: 5.625 J/cm2). Cells in groups A, B, C, and D were irradiated by LLL before irradiation. At 0, 12, 24, 48, and 72 h, the supernatants of the cell cultures were extracted at regular intervals, and the protein expression levels of IL-6, TNF-α, OPG, and RANKL were detected by enzyme-linked immunosorbent assay.@*RESULTS@#1) The levels of IL-6 and TNF-α secreted by HPDLCs increased gradually with time under static pressure stimulation. After 12 h, the levels of IL-6 and TNF-α secreted by HPDLCs in group A were significantly higher than those in groups B, C1, and C2 (P<0.05), which in group B were significantly higher than those in groups D1, and D2 (P<0.01). 2) The OPG protein concentration showed an upward trend before 24 h and a downward trend thereafter. The RANKL protein concentration increased, whereas the OPG/RANKL ratio decreased with time. Significant differen-ces in OPG, RANKL, and OPG/RANKL ratio were found among group A and groups B, C1, C2 as well as group B and groups D1, D2 (P<0.05).@*CONCLUSIONS@#1) In the high glucose+stress stimulation environment, the concentrations of IL-6 and TNF-α secreted by HPDLCs increased with time, the expression of OPG decreased, the expression of RANKL increased, and the ratio of OPG/RANKL decreased. As such, high glucose environment can promote bone resorption. After LLL therapy, the levels of IL-6 and TNF-α decreased, indicating that LLL therapy could antagonize the increase in the levels of inflammatory factors induced by high glucose environment and upregulate the expression of OPG in human HPDLCs, downregulation of RANKL expression in HPDLCs resulted in the upregulation of the ratio of OPG/RANKL and reversed the imbalance of bone metabolism induced by high glucose levels. 2) The decrease in inflammatory factors and the regulation of bone metabolism in HPDLCs were enhanced with increasing laser energy density within 3.75-5.625 J/cm2. Hence, the ability of LLL therapy to modulate bone remodeling increases with increasing dose.

Research progress on ankylosis of primary molars / 口腔疾病防治

@#Ankylosis of primary molars is a kind of eruption abnormality of the teeth, where the periodontal membrane disappears, owing to a bony union between bone and root. Studies have shown that the common proportion of ankylosed primary molars is 1.3%~8.9% with an equal occurrence. In the primary dentition, the mandibular first primary molar is the most commonly affected tooth, while in the middle mixed dentition stage of development, the second primary molar is more affected. Its etiology may be related to genetics, signaling pathways of mineralization metabolism of local alveolar bone or cementum, cytokines secreted by epithelial rest cells of Malassez, and enhanced inflammatory reactions during physiological absorption of roots. Ankylosis of primary molars can be diagnosed by clinical symptoms and imaging and is classified as mild, moderate and severe according to the degree of infraocclusion. As it may cause a series of complications, such as occlusal disturbances, delayed exfoliation and incomplete alveolar process development, multidisciplinary treatment, including in the departments of pediatric dentistry, orthodontics, periodontics and prosthodontics, should be adopted, and long-term treatment is determined based on the patient's age, severity of infraocclusion, and presence of permanent teeth. This review summarizes the etiology, diagnosis, complications and treatment of ankylosed primary molars to provide a reference for the clinical diagnosis and treatment of decidual molar fixation.

Hippo-YAP signaling pathway regulates autophagy of human periodontal ligament cells under cyclic tensile stress / 华西口腔医学杂志

OBJECTIVES@#This work aimed to investigate the molecular mechanism of cyclic tensile stress (CTS) stimulating autophagy in human periodontal ligament cells (hPDLCs).@*METHODS@#hPDLCs were isolated and cultured from normal periodontal tissues. hPDLCs were loaded with tensile stress by force four-point bending extender to simulate the autophagy of hPDLCs induced by orthodontic force du-ring orthodontic tooth movement. XMU-MP-1 was used to inhibit the Hippo signaling pathway to explore the role of the Hippo-YAP signaling pathway in activating hPDLC autophagy by tensile stress. The expression levels of autophagy-related genes (Beclin-1, LC3, and p62) in hPDLCs were detected by real-time quantitative polymerase chain reaction. Western blot was used to detect the expression levels of autophagy-related proteins (Beclin-1, LC3-Ⅱ/LC3-Ⅰ, and p62) and Hippo-YAP pathway proteins (active-YAP and p-YAP) in hPDLCs. Immunofluorescence was used to locate autophagy-related proteins (LC3-Ⅱand p62) and Hippo-YAP pathway proteins (active-YAP) of hPDLCs.@*RESULTS@#CTS-activated autophagy in hPDLCs and expression of autophagy-related proteins initially increased and then decreased; it began to increase at 30 min, peaked at 3 h, and decreased (P<0.05). CTS increased the expression of active-YAP protein and decreased the expression of p-YAP protein (P<0.05). When XMU-MP-1 inhibited the Hippo-YAP signaling pathway (P<0.05), active-YAP protein was promoted to enter the nucleus and autophagy expression was enhanced (P<0.05).@*CONCLUSIONS@#The Hippo-YAP signaling pathway is involved in the regulation of autophagy activation in hPDLCs under CTS.

Astaxanthin inhibits inflammation of human periodontal ligament cells induced by lipopolysaccharide / 中南大学学报(医学版)

OBJECTIVES@#Human periodontal ligament cells (hPDLCs) are important source of periodontal tissue reconstruction. Under chronic inflammation, the multi-directional differentiation potential and chemotaxis in hPDLCs are decreased. Therefore, inhibiting inflammatory microenvironment and improving the functional characteristics of stem cells can better promote periodontal tissue reconstruction. This study was to investigate the effect of astaxanthin (AST) on lipopolysaccharide (LPS)-induced inflammation in hPDLCs and the underlying mechanisms.@*METHODS@#hPDLCs were isolated and cultured in vitro, and vimentin and keratin immunocytochemical staining were used to identify hPDLCs. CCK-8 assay was used to measure the effects of AST (1, 5, 10, 20, 50, 100, and 200 μmol/L) on proliferation of hPDLCs. Quantitative RT-PCR (RT-qPCR) and ELISA were used to measure the mRNA and protein expression of inflammatory factors (IL-6, IL-1β, and TNF-α) in the control (Con) group, the LPS group, and the LPS+AST (5, 10, 20, and 50 μmol/L) group. Western blotting was used to detect the protein expression of IKBα, phosphorylated IKBα (p-IKBα), and p65 in the Con group, the LPS group, the AST (20 μmol/L) group, and the LPS+AST (20 μmol/L) group. After 10 μmol/L PDTC treatment, the mRNA and protein expressions of IL-6, IL-1β, and TNF-α were detected by RT-qPCR and ELISA.@*RESULTS@#Cell morphology and immunocytochemical staining showed that the cells were in line with the characteristics of hPDLCs. Treatment with AST could promote the proliferation of hPDLCs, which reached the peak at 20 μmol/L. The mRNA and protein expressions of IL-6, IL-1β, and TNF-α in the LPS group were higher than those in the Con group (all @*CONCLUSIONS@#AST promotes the proliferation of hPDLCs, which is related to suppression of LPS-induced the secretion of inflammatory factors via inhibiting the activation of NF-κB signaling pathway.

Effect of endoplasmic reticulum stress on the osteogenic differentiation of periodontal ligament cells under continuous static pressure / 口腔疾病防治

Objective@#To study the effect of continuous static pressure on the endoplasmic reticulum of human periodontal ligament cells (hPDLCs) and the mechanism of osteogenic differentiation.@*Methods@#hPDLCs cultured in vitro were subjected to 1 g/cm 2 of continuous compressive pressure (CCP) by custom-made, round, glass panes for 0, 2, 4, and 6 h, respectively. Alkaline phosphatase staining was used to detect osteogenic differentiation, and real-time quantitative PCR was used to detect the expression of protein kinase receptor-like ER kinase (PERK), eukaryotic translation initiation factor 2α (eIF2α), and transcription activation factor 4 (ATF-4). The 0 h loading group was the control group.@*Results@#After CCP treatment, the alkaline phosphatase staining of hPDLCs was blue-violet and significantly stronger than that of cells in the control group. The expression levels of PERK and ATF4 in the hPDLCs after CCP treatment were higher than those of cells in the control group (P < 0.05) and increased over time (P < 0.05). The expression of eIF2α was lower in the experimental groups than in the control group (P < 0.05) and decreased over time (P < 0.05).@*Conclusion @#Mechanical stimulation can activate ERS in hPDLCs, leading to enhanced PERK-eIF2α-ATF4 signaling and inducing osteogenic differentiation.

Influence of 3,3'-diindolylmethane on expression of inflammatory cytokines in periodontal ligament cells induced by lipopolysaccharide / 上海交通大学学报（医学版）

Objective: To investigate the effect of 3,3'-diindolylmethane (DIM) on the expression of inflammatory cytokines in human periodontal ligament cells (hPDLCs) induced by lipopolysaccharide (LPS) and to study the related mechanism. Methods: hPDLCs were isolated and cultured, and CCK-8 method was used to detect the effect of DIM on the proliferation of hPDLCs. hPDLCs were randomly divided into 4 groups: blank group (without LPS and DIM), LPS group (10 μg/mL LPS), 10 μg/mL LPS+6.25 μg/mL DIM, 10 μg/mL LPS+12.50 μg/mL DIM. The cells of all groups were cultured for 12 h. The protein levels of TNF-α, IL-1β and IL-6 in supernatant were detected by enzyme linked immunosorbent assay. The change of mitogenactivated protein kinase (MAPK) and nuclear factor κB (NF-κB) signaling pathways were detected by Western blotting. Results: The cell viability was not affected when the DIM concentration was less than 50 μmol/L (P>0.05). DIM at 6.25 and 12.50 μg/mL reduced the LPS-induced expression of TNF-α, IL-1β and IL-6 at protein levels (P<0.05). DIM inhibited the activation of the NF-κB signaling pathway. Conclusion: DIM can reduce the LPS-induced inflammatory cytokine expression in hPDLCs via restraining the activation of the NF-κB signaling pathway.

Anti-inflammation effects of psoralen and angelicin on human periodontal ligament cells in vitro / 上海交通大学学报（医学版）

Objective: To investigate the effects of psoralen and angelicin on inflammation cytokine expression of human periodontal ligament cells (hPDLCs). Methods: hPDLCs were primarily cultured using tissue explant method. Effects of psoralen and angelicin on the cell viability were tested by CCK-8 assay. hPDLCs were stimulated by Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) after treatment with different concentrations of psoralen and angelicin for 2 h. mRNA expression of IL-1β and IL-8 were determined by real-time PCR. Enzyme-linked immunosorbent assay (ELISA) was used to measure the secretion of IL-1β and IL-8. Results: hPDLCs were cultured successfully by tissue explant method. Psoralen and angelicin (≤ 12.5 μg/mL) did not show significant effects on the cell viability of hPDLCs. Pg-LPS markedly elevated the mRNA expression of IL-1β and IL-8, which could be attenuated by psoralen and angelicin in a dose-dependent manner. Likewise, the up-regulated protein secretion of IL-1β and IL-8 could be significantly blocked by psoralen and angelicin. Conclusion: Psoralen and angelicin could attenuate the inflammatory response of hPDLCs induced by Pg-LPS. Therefore, psoralen and angelicin may act as natural agents to prevent and treat periodontitis.

microRNA-146a reverses the inhibitory effects of Porphyromonas gingivalis lipopolysaccharide on osteogenesis of human periodontal ligament cells / 中华口腔医学杂志

Objective@#To investigate the effects and mechanisms of microRNA-146a (miR-146a) on osteogenic differentiation of human periodontal ligament cells (hPDLC) stimulated by lipopolysaccharide (LPS) of Porphyromonas gingivalis (Pg).@*Methods@#hPDLC were cultured in vitro and induced to the phase of osteogenic differentiation. These cells were divided into five groups: non-osteogenic differentiation cells, osteogenic differentiation cells, osteogenic differentiation cells treated with Pg LPS, osteogenic differentiation cells treated with Pg LPS and miR-146a mimic, osteogenic differentiation cells treated with Pg LPS and miR-146a negative control. Osteogenic markers and mineralization were detected via quantitative real-time PCR (qPCR) and alizarin red staining, respectively. Meanwhile, non-radioactive transcription factor assay was applied to explore the nuclear activity of nuclear factor kappa B (NF-κB) p65 in nuclear extracts of hPDLC.@*Results@#Compared with cells of osteogenic differentiation in non-LPS-stimulated groups, Pg LPS could decrease the markers of osteogenic differentiation of hPDLC such as collagen Ⅰ (Col-Ⅰ), alkaline phosphatase (ALP), Runt-related transcription factor-2 (RUNX2) and osteocalcin (OCN) (P<0.05), inhibit mineralization, and stimulate NF-κB p65 nuclear activity expression (non-LPS stimulated group: 1.023±0.217, LPS stimulated group: 6.252±0.613, P=0.008). However, compared with cells in Pg LPS/miR-146a negative control group, miR-146a increased Col-Ⅰ (P=0.007) and OCN (P=0.049) mRNA expression, rather than ALP (P=0.167) and RUNX2 (P=0.580) at day 3; miR-146a also upregulated mRNA levels of Col-Ⅰ, ALP, RUNX2 and OCN (P<0.05) at day 7 and day 14, and enhance mineralization. Meanwhile, miR-146a mimic could decrease the nuclear activity of NF-κB p65 induced by Pg LPS in hPDLC (miR-146a: 2.427±0.354, negative control: 5.863±0.482, P=0.019).@*Conclusions@#miR-146a could reverse the inhibitory effects of Pg LPS on osteogenic differentiation of hPDLC through enhancing the expression of osteogenic markers and decreasing inflammatory pathway in hPDLC.

Influence of 3,3'-diindolylmethane on expression of inflammatory cytokines in periodontal ligament cells induced by lipopolysaccharide / 上海交通大学学报（医学版）

Objective·To investigate the effect of 3,3'-diindolylmethane (DIM) on the expression of inflammatory cytokines in human periodontal ligament cells (hPDLCs) induced by lipopolysaccharide (LPS) and to study the related mechanism.Methyls· hPDLCs were isolated and cultured,and CCK-8 method was used to detect the effect of DIM on the proliferation of hPDLCs.hPDLCs were randomly divided into 4 groups:blank group (without LPS and DIM),LPS group (10 μg/mL LPS),10 μg/mL LPS+6.25 μg/mL DIM,10 μg/mL LPS+12.50 μg/mL DIM.The cells of all groups were cultured for 12 h.The protein levels of TNF-α,IL-1β and IL-6 in supernatant were detected by enzyme linked immunosorbent assay.The change of mitogenactivated protein kinase (MAPK) and nuclear factor κB (NF-κB) signaling pathways were detected by Western blotting.Results· The cell viability was not affected when the DIM concentration was less than 50 μmol/L (P＞0.05).DIM at 6.25 and 12.50 μg/mL reduced the LPS-induced expression of TNF-α,IL-1β and IL-6 at protein levels (P＜0.05).DIM inhibited the activation of the NF-κB signaling pathway.Conclusion· DIM can reduce the LPS-induced inflammatory cytokine expression in hPDLCs via restraining the activation of the NF-κB signaling pathway.

Anti-inflammation effects of psoralen and angelicin on human periodontal ligament cells in vitro / 上海交通大学学报（医学版）

Objective·To investigate the effects of psoralen and angelicin on inflammation cytokine expression of human periodontal ligament cells (hPDLCs).Methods· hPDLCs were primarily cultured using tissue explant method.Effects ofpsoralen and angelicin on the cell viability were tested by CCK-8 assay,hPDLCs were stimulated by Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) after treatment with different concentrations of psoralen and angelicin for 2 h.mRNA expression of IL-1β and IL-8 were determined by real-time PCR.Enzyme-linked immunosorbent assay (ELISA) was used to measure the secretion of IL-1β and IL-8.Results · hPDLCs were cultured successfully by tissue explant method.Psoralen and angelicin (≤ 12.5 μg/mL) did not show significant effects on the cell viability of hPDLCs.Pg-LPS markedly elevated the mRNA expression of IL-1β and IL-8,which could be attenuated by psoralen and angelicin in a dose-dependent manner.Likewise,the up-regulated protein secretion of IL-1β and IL-8 could be significantly blocked by psoralen and angelicin.Conclusion · Psoralen and angelicin could attenuate the inflammatory response of hPDLCs induced by Pg-LPS,Therefore,psoralen and angelicin may act as natural agents to prevent and treat periodontitis.

A preliminary study on the expression of caspases in stretch-induced apoptosis in human periodontal ligament cells / 医用生物力学

Objective To study the expression of caspases in stretch-induced apoptosis in human periodontal ligament cells (HPDLCs). Methods HPDLCs in vitro were subjected to mechanical stretch with 20% strain for 6 h or 24 h. The apoptotic rates were analyzed by flow cytometry. The protein expression of caspase-3, -5, -7, -8 and -9 was detected by Western blot, and the activity of caspase-3, -5, -8 and -9 was measured using colorimetric assay. Results Mechanical stretches with 20% strain for 6 and 24 h could induce apoptosis in HPDLCs. Compared with non-stretching control group, the protein expression level and activity of caspase-3, as well as the protein expression level of caspase-7 were up-regulated by 24 h-stretch. The protein expression level and activity of caspase-5, -8, -9 were up-regulated after stretches for 6 h and 24 h. Conclusions Mechanical stretch with 20% strain can induce apoptosis in HPDLCs in vitro, with the activation of caspase-3, -5, -7, -8 and -9.

The effects of platelet-rich fibrin extract on human periodontal ligament cells stimulated by TNF-α in vitro / 实用口腔医学杂志

Objective:To investigate the effects of platelet-rich fibrin extract (PRFe) on the osteogenetic differentiation and mineralization of human periodontal ligament cells (hPDLCs) stimulated by tumor necrosis factor-α (TNF-α) in vitro.Methods:hPDLCs were cultured and identified.PRFe was obtained by Choukroun's protocols.The cells were treated as the 4 groups:① the blank control group,②TNF-α(10 ng/ml),③ PRFe and ④PRFe + TNF-α(10 ng/ml) respectively.Alkaline phosphatase activity was detected by the ALP kit.The alizarin red dye was used to observe the mineralization of the cells.Runx2 and Osterix expression was quantified by Western blotting.Results:The ALP activity,mineralization level and the expression of Runx2 and Osterin of the TNF-oα group were lower that those of the control group(P ＜ 0.05).The examined indexs of PRFe group were higher than that of the control group(P ＜0.05).The indexs of the PRFe +TNF-α group were higher than that of TNF-α group(P ＜0.05).The indexs of PRFe group were higher than that of PRFe + TNF-α group(P ＜ 0.05).Conclusion:PRFe may promote the osteogenetic differentiation of hPDLCs stimulated by TNF-α in vitro.

Resveratrol attenuates LPS-induced cell injury and TLR4/NF-κB activation in human periodontal ligament cells / 实用口腔医学杂志

Objective:To investigate the protective effect of resveratrol on lipopolysaccharide (LPS)-induced cell injury in human periodontal ligament cells(hPDLCs).Methods:hPDLCs were cultured and identified.The cultured hPDLCs were divided into 5 groups:control group and LPS(10 μg/ml) + RES(0/30,60 and 90 μmol/L respectively) groups.The cell proliferation was detected by MTI assay.The secretion of TNF-α and IL-6 of hPDLCs was detected by ELISA kit.The expression of TLR4/NF-κB mRNA and protein was determined by PCR and Western blot analyses,respectively.Results:The cultured cells were negative for cytokeratin and positive for vimentin staining.Compared with the control group,cell proliferation was decreased,the secretion of TNF-α/IL-6 levels and the expression of TLR4/NF-κB mRNA and protein were increased after treatment with LPS.Whereas,with 30-90 μmol/L resveratrol pretreatment,the proliferation ability of hPDLCs was enhanced(P ＜ 0.05),the secretion levels of TNF-α and IL-6 and the expression of TLR4/NF-κB mRNA and protein were reduced (P ＜ 0.01) in a dose-dependent manner.Conclusion:Resveratrol may attenuate LPSinduced cell injury by inhibiting TLR4/NF-κB pathway in hPDLCs.

The inhibitory effects of aloe vera extract on lipopolysaccharide-induced inflammatory response in human periodontal ligament cells / 实用口腔医学杂志

Objective:To explore the effects of aloe vera extra (AVE) on lipopolysaccharide (LPS)-induced inflammatory response of human periodontal ligament cells(hPDLCs).Methods:hPDLCs were induced with LPS at 1 μg/ml for the simulation of periodontitis model(model group) and then treated by AVE at 0.05,0.1,0.2 mg/ml respectively(AVE group).Cell viability was examined by MTT assay.The level of interleukin-6(IL-6) from cell culture medium was measured by ELISA.The expression of Toll like receptor 4(TLR4) protein was detected by immunocytochemistry staining and the transfer of nuclear factor-kappa B p65 (NF-κB-p65) was observed by immunofluorescence staining.Results:There was no significant difference of the cell viabilities among the groups.IL-6 in culture medium,the expression of TLR4 protein and the transfer of NF-κB-p65 into the nucleus were increased in model group.AVE at 0.05-0.2 mg/ml inhibited the secretion of IL-6 in the cell culture supernatant down-regulated the TLR4 expression,attenuated the transfer of NF-κB-p65 into the nucleus in a concentration-dependent manner.Conclusion:Aloe vera extract can inhibit the inflammation response of hPDLCs induced by LPS through TLR4/ NF-κB-p65 signaling pathway.

A preliminary study on the expression of caspases in stretch-induced apoptosis in human periodontal ligament cells / 医用生物力学

Objective To study the expression of caspases in stretch-induced apoptosis in human periodontal ligament cells (HPDLCs).Methods HPDLCs in vitro were subjected to mechanical stretch with 20％ strain for 6 h or 24 h.The apoptotic rates were analyzed by flow cytometry.The protein expression of caspase-3,-5,-7,-8 and -9 was detected by Western blotting,and the activity of caspase-3,-5,-8 and-9 was measured using colorimetric assay.Results Mechanical stretches with 20％ strain for 6 h and 24 h could induce apoptosis in HPDLCs.Compared with non-stretching control group,the protein expression level and activity of caspase-3,as well as the protein expression level of caspase-7 were up-regulated by 24 h-stretch.The protein expression level and activity of caspase-5,-8,-9 were up-regulated after stretches for 6 h and 24 h.Conclusions Mechanical stretch with 20％ strain can induce apoptosis in HPDLCs in vitro,with the activation of caspase-3,-5,-7,-8 and-9.

The effects of Nicotine on the expression of lncRNA NEAT1 in human periodontal ligament cells / 医学研究生学报

Objective Several physiological reactions of human periodontal ligament cells (hPDLCs) are effected by a varie-ty of long-chain non-coding RNA , and nicotine as the major risk factor of smoking-related periodontitis , would lead to many cytokines changes in hPDLCs .The aim of the study was to explore the effect of nicotine on the expression of lncRNA NEAT 1 and IL 8 in the nucleus of hPDLCs. Methods We cultured the hPDLCs in vitro and used the 4th generation cells for subsequent experiments .hPDLCs were randomly divided into 4 groups:control group , nicotine group ,α-BTX group, nicotine+α-BTX group.Control group:hPDLCs without any ir-ritation;nicotine group: 10-5 mol/L nicotine stimulated hPDLCs for 4h;α-BTX group:10-8 mol/Lα-BTX stimulated hPDLCs; nicotine+α-BTX group:10-5 mol/L nicotine+10-8 mol/Lα-BTX stimulated hPDLCs .Real-time quantitative PCR was taken to detect the expres-sion level of NEAT1 and downstream IL 8 mRNA.In addition, the protein expression of IL-8 was tested by ELISA. Results After we primary cultured the periodontal ligament tissue for 3 weeks, the cells around the tissue were arranged radially .After a stable passage, microscopic observation showed that hPDLCs were long spindle-shape or spindle-shape with clear contour and adherent growth .Com-pared with nicotine group , the expression of NEAT1-1, NEAT1-2, IL-8 mRNA in hPDLCs in other three groups were all decreased ( P0.05). Conclusion Nicotine can promote the expression of NEAT1 and IL-8 in hPDLCs, which suggets that NEAT 1 may be involved in the development of smoking-related periodontitis by promoting early inflammatory reaction .

A preliminary study on the expression of caspases in stretch-induced apoptosis in human periodontal ligament cells / 医用生物力学

Objective To study the expression of caspases in stretch-induced apoptosis in human periodontal ligament cells (HPDLCs).Methods HPDLCs in vitro were subjected to mechanical stretch with 20％ strain for 6 h or 24 h.The apoptotic rates were analyzed by flow cytometry.The protein expression of caspase-3,-5,-7,-8 and -9 was detected by Western blotting,and the activity of caspase-3,-5,-8 and-9 was measured using colorimetric assay.Results Mechanical stretches with 20％ strain for 6 h and 24 h could induce apoptosis in HPDLCs.Compared with non-stretching control group,the protein expression level and activity of caspase-3,as well as the protein expression level of caspase-7 were up-regulated by 24 h-stretch.The protein expression level and activity of caspase-5,-8,-9 were up-regulated after stretches for 6 h and 24 h.Conclusions Mechanical stretch with 20％ strain can induce apoptosis in HPDLCs in vitro,with the activation of caspase-3,-5,-7,-8 and-9.

A preliminary study on the expression of caspases in stretch-induced apoptosis in human periodontal ligament cells / 医用生物力学

Objective To study the expression of caspases in stretch-induced apoptosis in human periodontal ligament cells (HPDLCs).Methods HPDLCs in vitro were subjected to mechanical stretch with 20％ strain for 6 h or 24 h.The apoptotic rates were analyzed by flow cytometry.The protein expression of caspase-3,-5,-7,-8 and -9 was detected by Western blotting,and the activity of caspase-3,-5,-8 and-9 was measured using colorimetric assay.Results Mechanical stretches with 20％ strain for 6 h and 24 h could induce apoptosis in HPDLCs.Compared with non-stretching control group,the protein expression level and activity of caspase-3,as well as the protein expression level of caspase-7 were up-regulated by 24 h-stretch.The protein expression level and activity of caspase-5,-8,-9 were up-regulated after stretches for 6 h and 24 h.Conclusions Mechanical stretch with 20％ strain can induce apoptosis in HPDLCs in vitro,with the activation of caspase-3,-5,-7,-8 and-9.

A preliminary study on the autophagy level of human periodontal ligament cells regulated by nicotine / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To explore the effect of nicotine on the autophagy level of human periodontal ligament cells (hPDLCs).</p><p><b>METHODS</b>Periodontal tissues collected from premolars for orthodontic treatment reasons were used to culture hPDLCs. Western blot analysis was performed to test the most optimal time and concentration of nicotine on the autophagy level of the hPDLCs. Transmission electron microscope and immunofluorescence observation were carried out to detect the form of autophagosomes and expression of autophagy related protein LC3 in hPDLCs under this optimal condition.</p><p><b>RESULTS</b>Protein expression of LC3Ⅱ was up regulated with the 12 h nicotine stimulating. Besides that, the up regulation of the protein expression of LC3Ⅱ was concentration dependent and nicotine with a concentration of 1×10⁻⁵ mol·L⁻¹ was the most optimal condition. Transmission electron microscope and immunofluorescence observations indicated that nicotine would activate the autophagy level of hPDLCs by increasing the number of autophagosomes and up regulating the expression of autophagy related protein LC3.</p><p><b>CONCLUSIONS</b>Nicotine could increase autophagy level of hPDLCs, thus affecting the occurrence and development of smoking related periodontitis.</p>

Effects of cytosolic bacteria on cyclic GMP-AMP synthase expression in human gingival tissues and periodontal ligament cells / 华西口腔医学杂志

<p><b>OBJECTIVE</b>This work aims to determine the effect of cytosolic bacteria on the expression of cyclic GMP-AMP synthase (cGAS) in human periodontal ligament cells (hPDLCs) and gingival tissues.</p><p><b>METHODS</b>The ability of Porphyromonas gingivalis (P. gingivalis) to invade hPDLCs was detected using laser scanning confocal microscope assay at a multiplicity of infection of 10. P. gingivalis-infected cells were sorted by fluorescence-activated cell sorting (FACS). Then, quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were used to detect cGAS expression in infected cells. Finally, the location and expression of cGAS in inflammatory and normal gingival tissues were investigated by immunohistochemistry.</p><p><b>RESULTS</b>P. gingivalis actively invaded hPDLCs. Moreover, cGAS expression significantly increased in P. gingivalis-infected cells. Although cGAS was expressed in the epithelial and subepithelial cells of both inflamed and normal gingival tissues, cGAS expression significantly increased in inflamed gingival tissues.</p><p><b>CONCLUSIONS</b>Cytosolic bacteria can upregulate cGAS expression in infected cells. These data suggest that cGAS may act as pattern-recognition receptors and participate in recognizing cytosolic nucleic acid pathogen-associated molecular patterns.
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