| Background and ObjectivePeriodontitis is a bacterial infection of the oral cavity,which is mainly associated with the inflammation caused by bacteria,the destruction of local bone tissue caused by immune response and the imbalance of homeostasis caused by reconstruction.During the development of periodontitis,several inflammatory factors,such as tumor necrosis factor ?(TNF-?),interleukin-1(IL-1),and IL-6,are involved in the destruction of periodontal tissues.Among them,TNF-? is considered as an important endogenous proinflammatory factor in the inflammatory microenvironment of periodontal tissues,which plays a critical role in the occurrence and development of periodontitis,and is also an important factor affecting periodontal regenerationThe ultimate goal of periodontal therapy is to reconstruct periodontal tissues which are damaged by inflammatory factors.Periodontal ligament stem cells(PDLSCs)are the important cells in the study of periodontal tissue regeneration.PDLSCs are mesenchymal stem cells isolated from human periodontal ligament tissue.PDLSCs have the ability of multi-differentiation,and can differentiate into fibroblasts,osteoblasts and osteoblasts.These functions make PDLSCs become important seed cells for periodontal tissue regeneration and repair.The proliferation and differentiation of PDLSCs are the key factors to determine the success of periodontal regeneration.In the process of periodontal tissue regeneration,the activity of PDLSCs is affected by the periodontal inflammatory microenvironment,and the inflammatory factor TNF-? could reduce the osteogenesis ability of PDLSCs.TNF-? can promote inflammation by mediating the production of other inflammatory mediators,induce the formation of osteoclasts to promote alveolar bone absorption,inhibit the functional transformation of periodontal membrane cells into osteoblasts to inhibit bone formation,and inhibit the regeneration and repair of periodontal tissues.Therefore,antagonism to TNF-? and its associated pathways is expected to be used as adjuvant therapy for periodontitis.TNF-?-mediated inhibition of osteoblastic differentiation is mainly associated with several signaling pathways,such as nuclear factor-?B(NF-?B)and Eph B4 signaling.TNF-? has two receptors:TNF receptor-1(TNFRI)and TNF receptor-2(TNFR2).TNFR1 is expressed on all cell types and is the primary signaling receptor for TNF-?.TNFR1 promotes inflammatory response and cell apoptosis,and activates NF-?B signaling pathway to inhibit osteogenic differentiation.TNFR2 is mainly expressed in immune cells,neurons and endothelial cells,and is involved in signal transmission,and T cell proliferation,and plays a role in promoting osteogenic differentiation.PGRN is an autocrine multi-functional growth factor,which is widely distributed in various tissues and cells.It plays important roles in the process of inflammation,wound healing and chondral development.It has been found that PGRN can not only directly promote osteoblast differentiation and bone defect healing,but also competitively bind TNFR to block the TNF-?-mediated osteoclast differentiation and the inhibitory effect of osteoblast differentiation.Compared with the traditional anti-TNF-? treatment regimen,PGRN is expected to be a new generation of periodontitis therapeutic biological factor that can not only induce the osteogenic differentiation of PDLSCs,but also have anti-inflammatory effects.In our previous experiments,we have confirmed that PGRN promotes regeneration of inflammatory periodontal defects in rats by anti-inflammatory,inhibiting osteoclasts and promoting osteogenic differentiation,and has anti-inflammatory and protective effects on gingival tissues of mice and patients with periodontitis.In addition,in vitro experiments have also preliminarily confirmed that PGRN directly promotes the osteoblastic differentiation of PDLSCs and has an antagonistic effect on TNF-?-mediated inhibition of the osteoblastic differentiation of PDLSCs.The mechanism,however,is unclear.PGRN shows a high affinity with TNFRs and TNFRs are TNF-? receptors.Therefore,on the basis of the construction of lentivirus transfected PDLSCs target cells with TNFR1 and TNFR2 gene knockout,this study intends to explore the related signaling pathways and mechanisms of PGRN directly promoting osteogenic differentiation and antagonizing TNF-a-mediated inhibition of PDLSCs osteogenic differentiation,including TNFRs and its downstream signaling pathways.This work would provide experimental basis for further research on the role of PGRN in the treatment of periodontal disease.Materials and Methods1.Isolation,culture and identification of PDLSCs in healthy people in vitro1.1 Isolation,culture and cloning of PDLSCs in vitroThe periodontal ligament tissues were selected from healthy premolars or third molars extracted from donors for orthodontic reasons in Stomatological Hospital of Shandong University.The samples were scraped from 1/3 of the root.PDLSCs were isolated and cultured in vitro by the tissue mass culture method,and cloned by the limited dilution method.The morphological characteristics of PDLSCs in the primary culture were observed,and the clone formation rate of PDLSCs was detected.The cells in good growth state were cryopreserved for subsequent experiments.1.2 Multidirectional differentiation ability of PDLSCsP3-P5 cells in good growth state were used to induce differentiation of PDLSCs into lipid-forming,osteogenic and neuron-forming cells to identify the multidirectional differentiation ability of PDLSCs.For adipogenic differentiation detection,P3 PDLSCs with good cell status were selected and induced to differentiate into adipocytes with lipid induction medium.After 21 days of induction,oil red O staining was performed to observe the formation of adipocytes.For osteogenesis differentiation,the cells were cultured with the prepared bone mineralization inducer,and the formation of calcified nodules was observed by alizarin red staining 28 days after induction,meanwhile the color development was observed by BClP/NBT alkaline phosphatase staining kit 7 days after culture.For neurogenic differentiation ability,PDLSCs of P3-P5 generation in good growth state were cultured in the prepared neural induction solution for 2-3 days,and immunofluorescence staining results were observed under confocal fluorescence microscope.1.3 Identification of PDLSCs related phenotypesPDLSCs from P3-P5 generations with good growth were treated with pre-packed and diluted CD 105,CD34,CD45,CD90,CD44 and STR-1 related monoclonal antibodies,respectively,and expression levels of surface related phenotypic molecules of PDLSCS were detected by flow cytometry.2.Mechanism of PGRN promoting the osteogenic differentiation of PDLSCs2.1 Expressions of TNFR1 and TNFR2 in PDLSCsPDLSCs of P3-P5 generation with good growth were selected.The expressions of TNFRI and TNFR2 in PDLSCs were detected by cellular immunofluorescence assay,and the mRNA expressions of TNFR1 and TNFR2 in PDLSCs were detected by qRT-PCR.2.2 TNFR1 and TNFR2 gene knockout in PDLSCsCRISPR/Cas9 technology was used to knock out TNFR1 and TNFR2 genes.We first designed sgRNAs to identify TNFR1 and TNFR2 gene targets respectively.Lentiviral vectors expressing sgRNA and lentiviral vectors expressing Cas9 were constructed and co-transfected into PDLSCs.PDLSCs were infected with lentiviral particles of LV-TNFR1-sgRNA(02682,02683,02684),LV-TNFR2-sgRNA(02673,02674,02675)and LV-sg-NC,and became PDLSCs-sh-TNFR1 with TNFR1 gene knockout,PDLSCs-sh-TNFR2 with TNFR2 gene knockout and the corresponding control cells PDLSCs-NC.Cell morphology was observed using fluorescence microscope.Flow cytometry technology was used to detect the infection efficiency of lentivirus to PDLSCs.The optimal MOI value and transfection conditions were determined.qRT-PCR and Western blotting were used to verify the mRNA and protein expression of TNFR1 and TNFR2 in PDLSCs,and judge the efficiency of gene knockout.The optimal lentivirus particles were selected for subsequent transfection to obtain target cells.2.3 Effect of PGRN on expression of osteoblastic differentiation regulators in PDLSCs with/without TNFR2 gene knockoutIn our previous study,25 ng/mL PGRN had the most obvious effect on the osteogenic differentiation of PDLSCs,so the dose of PGRN in this study was 25 ng/mL.25 ng/mL PGRN was applied to PDLSCs-sh-TNFR2 and PDLSCs-NC groups,and the cells in each group were divided into two groups:blank control group and 25 ng/mL PGRN group.The cells were cultured in osteogenic induction medium for 3 d and 7 d,respectively.mRNA and protein expressions of osteogenic markers Runx2 and ALP were detected by qRT-PCR and Western blotting experiments.2.4 Effects of PGRN on MAPK-related signaling pathways regulating the osteogenic differentiation of PDLSCs(1)PDLSCs were cultured in sera-free medium with PGRN concentration of 25 ng/mL for 0,5,15,30,60 min and 2 h.Then,the expression levels of MAPK pathway related proteins(ERK1/2/p-ERkl/2,JNK/p-JNK,p38/p-p38)were detected by Western blotting.(2)According to the above results,the processing time of PGRN was selected to be 15 min.25 ng/mL PGRN was used to stimulate PDLSCs-sh-TNFRI,PDLSCs-sh-TNFR2 and PDLSCs-NC cells for 15 min.Western blotting was used to detect the expressions of p-ERk1/2 and p-JNK.(3)In order to further clarify whether ERK1/2 and JNK signaling pathways are associated with the osteogenetic differentiation under PGRN stimulation,PDLSCs ware cultured in the osteogenesis induction medium.According to the literature and the preliminary experiments,the concentration of ERK1/2 and JNK signaling pathway inhibitors U0126 and SP600125 were selected respectively.U0126(10 ?m/L)and SP600125(20 ?m/L)were selected to block the corresponding signaling pathways,respectively.Then,25 ng/mL PGRN was added into medium for stimulation 3 d and 7 d.mRNA and protein expressions of osteogenic markers Runx2 and ALP were detected by qRT-PCR and Western blotting,respectively.3.Mechanism of PGRN antagonizing TNF-?-mediated inhibition of osteoblastic differentiation of PDLSCs3.1 Effects of PGRN on the osteogenic differentiation of PDLSCs-NC stimulated by TNF-?PDLSCs-NC were cultured in osteogenic induction medium containing ? 10 ng/mL TNF-?,?25 ng/mL PGRN,?10 ng/mL TNF-?+25 ng/mL PGRN and?blank control for 3 d and 7 d,respectively.qRT-PCR and Western blotting were used to detect mRNA and protein expressions of osteogenic markers Runx2 and ALP.3.2 Effects of PGRN on osteoblastic differentiation of PDLSCs with TNFR gene knockout under TNF-? stimulationPDLSCs-sh-TNFR1 and PDLSCs-sh-TNFR2 were cultured in osteogenic induction medium containing? 10 ng/mL TNF-?,?25 ng/mL PGRN,? 10 ng/mL TNF-?+25 ng/mL PGRN and? blank control for 3 d and 7 d,respectively.qRT-PCR and Western blotting were used to detect mRNA and protein expressions of osteogenic markers Runx2 and ALP.3.3 Effect of PGRN on NF-?B signaling pathway in PDLSCs stimulated by TNF-?PDLSCs were cultured in sera-free medium with 10 ng/mL TNF-? stimulation for 0,5,15,30,60 min and 2 h.Western blotting was used to detect the expression of p-p65,an important member of NF-?B pathway.The treatment time of 5 min was selected because the expression of p-p65 can significantly increase at 5 min,and decrease with time.Then,PDLSCs-sh-TNFR1,PDLSCs-sh-TNFR2,and PDLSCs-NC were divided into 4 groups,which were stimulated using ? 10 ng/mL TNF-?,? 25 ng/mL PGRN,?10 ng/mL TNF-?+25 ng/mL PGRN and?blank control medium for 5 min.The protein expression of p-p65 was detected by Western blotting.Results1.PDLSCs isolated and cultured in vitro have the characteristics of mesenchymal stem cells1.1 Isolation,culture and cloning of PDLSCs in vitroThe primary cells generally crawled out of the adherent wall around the tissue block to grow and proliferate in about a week.After subculture cloning of the primary cells,the cells were mostly round and spindle shaped,but all showed that the nuclei were clustered in the center with obvious nucleoli.The cytoplasm was radiating outward,showing the morphological characteristics of adult stem cells.The clones have obvious growth incubation period,logarithmic growth period and platform growth period,which accords with the growth law of cell biology.Clusters of purple cells can be observed by staining with crystal violet.1.2 Multidirectional differentiation ability of PDLSCsPDLSCs were stained with oil red O after adipogenic induction,and a large number of lipid droplets were observed in the experimental group under optical microscope Alizarin red staining was observed 28 days after osteogenesis mineralization induction.The results showed that obvious mineral ized nodules were observed under light microscope in the experimental group,and the alkaline phosphatase staining test was positive.Immunostaining results showed that Nestin,a surface specific marker of neural stem cells,was highly expressed on the cell surface.This indicates that PDLSCs derived from single-cell clones have multiple differentiation abilities of mesenchymal stem cells1.3 PDLSCs express surface phenotypic molecules of mesenchymal stem cellsCell surface markers detected by flow cytometry showed that PDLSCs showed positive expression of CD44(99.9%),CD90(100%),CD105(100%)and STR-1(14.2%)on the surface markers of mesenchymal stem cells,and negative expression of CD34(0.76%)and CD4(0.25%)on the surface markers of hematopoietic molecules2.Mechanism of PGRN promoting the osteogenesis of PDLSCs2.1 Positive expression of TNFR1 and TNFR2 in PDLSCsImmunofluorescence staining results showed that both TNFR1 and TNFR2 were positively expressed in PDLSCs.qRT-PCT results showed that both TNFR1 and TNFR2 were positively expressed in PDLSCs,while the expression level of TNFR1 was higher than that of TNFR22.2 Decreased expressions of TNFR1 and TNFR2 in PDLSCs after transfection The optimal MOI value of lentivirus transfection was 30,and P3 cells were selected for transfection.LV-TNFR1-sgRNA-02682 and LV-TNFR2-sgRNA-02673 in PDLSCs showed the highest knockout effects of TNFR1 and TNFR2,respectively.After PDLSCs were infected with LV-TNFR1-sgRNA and LV-TNFR2-sgRNAS respectively,the mRNA and protein expression levels of TNFR1 and TNFR2 were significantly decreased compared with that in the control group(P<0.05).LV-TNFR1-sgRNA-02682 and LV-TNFR2-sgRNA-02673 were used for further gene knockout analysis in the transfection experiment.Therefore,PDLSCs transfected with LV-TNFR1-sgRNA-02682 and LV-TNFR2-sgRNA-02673(named PDLSCs-sh-TNFR1 and PDLSCs-sh-TNFR2,respectively)were used for further post-knockout analysis.2.3 PGRN promotes the osteogenic differentiation of PDLSCs through TNFR2(1)PGRN promoted the osteogenic differentiation of PDLSCs.Compared with the control group,qRT-PCR and Western blotting results showed that mRNA and protein expressions of RUNX2 and ALP in PDLSCs treated with PGRN were significantly increased(P<0.05),indicating that PGRN could promote the osteogenic differentiation of PDLSCs.(2)PGRN promotes the osteogenic differentiation of PDLSCs through TNFR2.qRT-PCR and Western blotting results showed that there was no significant difference in mRNA and protein expression of RUNX2 and ALP after PGRN-treated PDLSCs-sh-TNFR2 compared with the control group(P>0.05),and PGRN-mediated osteogenic differentiation in PDLSCs-sh-TNFR2 disappeared,suggesting that PGRN-mediated osteogenic differentiation of PDLSCs may play a role by binding to TNFR2.2.4 The osteogenic differentiation of PDLSCs promoted by PGRN is related to the activation of MAPK signaling pathway2.4.1 PGRN activates ERK1/2 and JNK signaling pathways Western blotting results showed that p-ERK1/2/ERK1/2 and p-JNK/JNK protein level in PDLSCs increased significantly under the stimulation of PGRN(P<0.05).2.4.2 PGRN activating JNK signaling pathways is related to TNFR2 After the PDLSCs-sh-TNFR1,PDLSCs-sh-TNFR2,and PDLSCs-NC cells were stimulated with 25 ng/mL PGRN for 15 min,Western blotting results showed that the protein expressions of p-ERK1/2 and p-JNK in the PDLSCs-NC and PDLSCs-sh-TNFR1 cells were significantly increased compared with the control group(P<0.05).However,there were no significant changes in p-JNK protein expression in PDLSCs-sh-TNFR2 cells compared with the control group(P>0.05),while the protein expression of p-ERK1/2 in PDLSCs-sh-TNFR were significantly increase(P<0.05).It can be seen that TNFR2 in particular mediated PGRN-stimulated activation of JNK signaling pathway.2.4.3 ERK and JNK pathway inhibitors inhibit PGRN-mediated osteogenic differentiationCompared with the control group,the expression levels of ALP and RUNX2 in PDLSCs treated with PGRN were significantly increased in both protein and mRNA levels(P<0.05).However,after ERK1/2 and JNK signaling pathway inhibitors U0126 and SP600125 were added to block the corresponding signaling pathway,the expressions of ALP and RUNX2 in PDLSCs significantly decreased at the protein and mRNA levels(P<0.05).Thus,PGRN enhanced the expression of osteogenic regulatory factors ALP and RUNX2,thus promoting the osteogenic differentiation process associated with the activation of the ERK 1/2 and JNK signaling pathways3.Mechanism of PGRN antagonizing TNF-a-mediated osteoblastic differentiation inhibition of PDLSCs3.1 Effect of PGRN on expression of osteoblastic regulators in TNF-?-induced PDLSCsCompared with the control group,qRT-PCR and Western blotting results showed that TNF-? treatment significantly decreased the mRNA and protein expressions of RUNX2 and ALP in PDLSCs(P<0.05),and PGRN treatment increased the expressions of RUNX2 and ALP(P<0.05).The mRNA and protein expressions of RUNX2 and ALP in the PGRN+TNF-? treated group were increased relative to that in TNF-? treated group(P<0.05).The results suggested that PGRN can antagonize TNF-?-mediated inhibition of osteoblastic differentiation of PDLSCs.3.2 Antagonistic effect of PGRN on TNF-?-induced osteoblastic inhibition ofPDLSCs is mediated by TNFR1There was no significant difference in mRNA and protein expression of RUNX2 and ALP in PDLSCs-sh-TNFR1 treated with TNF-? compared with the control group(P>0.05),and the mRNA and protein expressions of RUNX2 and ALP were increased in the PGRN+TNF-? treatment group relative to that in the control group and TNF-?treatment(P<0.05).In PDLSCs-sh-TNFR2,the mRNA and protein expressions of RUNX2 and ALP in the TNF-? group were significantly lower than those in the control group(P<0.05).The mRNA and protein expressions of RUNX2 and ALP in the TNF-?+PGRN treatment group were significantly lower than those in the control group(P<0.05),while were significantly higher than those(except mRNA expression of RUNX2 at 3 days)in the TNF-? group(P<0.05).Since PGRN is a known TNFR1 blocker,it is speculated that PGRN reversed TNF-?-inhibited expression of osteoblastic regulators in PDLSCs is mediated by TNFR1.3.3 PGRN inhibits TNF-?-mediated NF-?B signaling activationIn PDLSCs,p-p65 protein expression first increased and then decreased with the extension of TNF-? action time,indicating that TNF-? plays an osteoblastic inhibition role by activating the NF-?B signaling pathway.In PDLSCs-sh-TNFR1,p-p65 expression in TNF-? and TNF-?+PGRN stimulation groups showed no significant difference(P>0.05),while PGRN antagonized TNF-?-enhanced p-p65 expression in PDLSCs-NC and PDLSCs-sh-TNFR2(P<0.05).These results,together with our previous results,suggested that PGRN antagonized TNF-?-mediated osteogenesis inhibition by reversing TNF-? activation of the NF-?B signaling pathway by binding TNFR.Conclusions1.PGRN can directly promote osteoblastic differentiation of PDLSCs.This effect is related to the activation of ERK1/2 and JNK signaling pathways,and TNFR2 mediates the activation of PGRN on JNK signaling pathway.and antagonize TNF-?-mediated osteoblastic inhibition o.2.TNF-? can activate NF-?B signaling pathway in PDLSCs through-FNFR1 to inhibit the osteogenic differentiation of PDLSCs.3.PGRN reverses TNF-?-mediated osteogenesis inhibition of PDLSCs by competitively antagonising TNF-? through TNFR1,thereby inhibiting the activation of NF-?B signaling pathway. |
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