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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.
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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.
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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.
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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.
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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.
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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.
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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.
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Objective:To observe the effects of lactoferrin(LF)on the proliferation,migration and osteogenic differentiation of human periodontal ligament cells(HPDLCs)in vitro.Methods:HPDLCs were cultured and identified.The proliferation and migration of HP-DLCs cultured with 0,10 and 20 μg/ml lactoferrin respectively were tested by MTT assay,Transwell assay and scratch test.The os-teogenic differentiation of the cells was evaluated using alizarin red staining and real-time PCR.Results:Lactoferrin at 10 and 20μg/ml increased the proliferation(P <0.05),increased the quantities of mineralized nodules and the expression of alkaline phospha-tase(ALP),osteocalcin(OCN)and osteopontin(OPN)(P <0.05).Conclusion:Lactoferrin promotes the proliferation,migration and osteogenic differentiation of HPDLCs.
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Objective To study the effect of mechanical stretch on expression of inflammasome related factors in human periodontal ligament cells (HPDLCs). Methods HPDLCs were subjected to mechanical stretch with a 20% elongation magnitude for 6 h or 24 h, respectively. The mRNA and protein expression levels of IL-1β, Caspase-1 and NLRP3 were detected by real-time quantitative PCR and Western blot. The levels of IL-1β in the cell-culture medium of HPLCs in response to mechanical stretch for 1, 2, 4, 6 h were detected by ELISA, respectively. In control group, HPDLCs were cultured in similar conditions but not subjected with stretch. Results The mRNA and protein expression levels of IL-1β, Caspase-1 and NLRP3 were up-regulated with mechanical stretch for 6 h (P<0.05). The protein expression level of NLRP3 was up-regulated with mechanical stretch for 24 h (P<0.05). Compared with control group, the content of IL-1β in the cell-culture medium of HPLCs was increased significantly in response to mechanical stretch for 4 h and 6 h(P<0.05). Conclusions The expression of NLRP3/Caspase-1/IL-1β related factors in HPDLCs can be induced by 20% mechanical stretch for 6 h.
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Objective:To observe the effects of hydrogen on osteogenic capacity of human periodontal ligament cells(hPDLCs)stim-ulated with P.g-LPS.Methods:hPDLCs were cultured and divided into 4 groups:control(C)group,osteogenic induction(OI) group,OI +1 00 ng/ml LPS(OILPS)group and OIPLS +3%H2 (H2 OIPLS)group,and treated respectively.Alizarin red staining (ARS)was carried out 3 weeks after treatment.ALP and OC mRNA expression of the cells was examined by RT-PCR after 7-d treat-ment.Results:LPS decreased A value of ARS(P <0.01 ),ALP mRNA expression(P <0.001 )and OC mRNA expression(P <0.001 )of the cells.H2 increased the A value(P <0.05),ALP mRNA expression(P <0.01 )and OC mRNA(P <0.01 )of the cells treated by LPS.Conclusion:High concentration of P.g-LPS can inhibit osteogenic capacity of hPDLCs,while hydrogen can impair the P.g-LPS induced suppression of hPDLC's osteogenesis.
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Objective:To survey the expression of MMP-1,MMP-2 of human periodontal ligament cells(HPDLCs)treated by tea polyphenols(TP)and lipopolysaccharide(LPS).Methods:HPDLCs were in vitro cultured in vitro and treated by TP(200 μg/ml) and /or LPS(100 μg/ml)for 24,48 and 72 h respectively,the secretion of MMP-1 and MMP-2 were examined by ELISA,MMP-1 and MMP-2 mRNA expression was examined by real-time PCR.Results:The secression and mRNA expression of MMP-1 and MMP-2 of HPDLCs increased by LPS treatment and significantly inhibited by TP at the different times.Conclusion:TP can inhibit the col-lagen degradation of HPDLCs mediated by LPS.
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Objective:To study the effects of smokeless tobacco extract(ST)on the proliferation and the heat shock protein 70 (HSP70)expression of human periodontal ligament cells(hPDLCs).Methods:hPDLCs were cultured in vitro and identified by im-munohistochemistry(IHC).The cells were stimulated with ST at 0.01 6 -50 g/L respectively for 24 h,the proliferation was examine by MTT assay,HSP70 expression was detected by immunehistochemical staining and Western Blot.Results:ST inhibited the prolifer-ation and increased HSP70 expression in cytoplasm and nucleus at 0.4 -50 g/L dose dependantly.Conclusion:ST may inhibit the proliferation and increase HSP70 expression of hPDLCs in a dose depandant manner.
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Objective To investigate the effects of different magnitudes of mechanical strain on the expression of MMP-13/TIMP-1 and try to determine the signal transduction pathways in response to mechanical strain in human periodontal ligament cells (HPDLCs) in vitro. MethodHPDLCs were subjected to 0%，6%，12% or 18% elongation for 24 h by using cell stress loading system simultaneously. Then the MMP-13/TIMP-1 mRNA and protein expression in cells were tested by reverse transcription polymerase chain reaction (RT-PCR) and western blotting respectively. Furthermore, specific inhibitors were employed to examine the role of different signal transduction pathway on the expression of strain induced MMP-13 /TIMP-1 in HPDLCs. ResultsThe expression of MMP-13 /TIMP-1 in HPDLCs significantly increased in groups of 6%，12%, 18% elongation in a magnitude-dependent manner compared with the control group (0%) after the mechanical strain treatment for 24 h. PD098059 and cycloheximide could inhibit the increase in MMP-13 and TIMP-1 mRNA expression in response to mechanical strain respectively. Conclusions Different magnitudes of mechanical strain can affect the expression of MMP-13/TIMP-1 in HPDLCs in a magnitude dependent manner, and further affect the periodontium remodeling with the characteristics of degradation and synthesis of extracellular matrix in response to mechanical strain. The ERK-MAPK pathway is involved in strain induced MMP-13 expression while the strain-induced TIMP-1 expression depends on de novo protein synthesis.
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Objective:To investigate the effect of active components Chinese medicine for tonifying kidney extracted by alcohol on the proliferation and function of cultured human periodontal ligament cells.Methods:The human periodontal ligament cells were cultured in vitro and the effect of active components of Chinese medicine for tonifying kidney extracted by alcohol on the proliferation ability and alkaline phosphatase(ALP)activity of hPDLCs were detected.Results:The active components extracted by alcohol strengthened the proliferation ability and ALP activity of cultured human periodontal ligament cells.Conclusions:The active components of Chinese medicine for tonifying kidney extracted by alcohol had great promotion on the proliferation ability and function of cultured human periodontal ligament cells.