| Chronic periodontitis is a common oral disease,mainly manifested as inflammation of the gums and destruction of periodontal tissues,which eventually leads to loose teeth.Current conventional treatments for periodontitis can prevent the aggravation of the lesion,but the problem of repair and regeneration of the alveolar bone that be absorpted cannot be completely solved.Stem cell-based tissue regeneration is a new type of periodontal treatment,that can regenerate and repair the absorbed alveolar bone tissue,thereby fundamentally solving the problem of alveolar bone resorption.Periodontal ligament stem cells（PDLSCs）have the ability to differentiate into osteoblasts and are ideal seed cells for periodontal tissue regeneration.However,inflammatory damage will affect the osteogenic differentiation ability of PDLSCs.Therefore,how to improve the adverse effects of inflammation on PDLSCs has become an urgent problem to be solved.Lipopolysaccharide（LPS）is a highly active pathogenic substance unique to Gram-negative bacteria.It plays an important role in the occurrence and development of periodontitis.Studies have found the content of LPS in gingival crevicular fluid It is significantly related to the degree of inflammation of periodontal tissue.The higher the LPS content,the more severe the inflammation of periodontal tissue.LPS can cause local allergic reactions in the periodontal,such as:activation of monocytes to produce cytokines,activation of complement to release allergic mediators,etc.,resulting in the destruction of periodontal tissues.In addition,studies have shown that LPS can inhibit the osteogenic differentiation of PDLSCs in vitro.Therefore,in this study,PDLSCs cultured in vitro with appropriate concentrations of LPS were selected to simulate the inflammatory microenvironment in vitro.Exendin-4（Ex-4）is an analogue of glucagon-like peptide-1（GLP-1）,which is a new type of hypoglycemic agent that has been used clinically.Ex-4 can stimulate insulin secretion and inhibit glucagon secretion.In addition,it can promote the proliferation and differentiation of isletβcells.In recent years,it has been discovered that Ex-4 also has a bone-promoting and anti-inflammatory effect.However,the role of Ex-4 in regulating the proliferation and osteogenic differentiation of PDLSCs in an inflammatory environment is currently unclear.In this study,lipopolysaccharide（LPS）was used to mimic the inflammatory microenvironment in vitro,to investigate the effect of Ex-4on the proliferation and osteogenic differentiation of human periodontal ligament stem cells in the inflammatory microenvironment,and to explore its mechanism.Divide the experiment into the following four sections:PART 1:PERIODONTAL LIGAMENT STEM CELLS CULTURE AND IDENTIFICATIONObjective:To isolate and culture PDLSCs from periodontal ligament tissues,and to identify their biological characteristics such as surface markers and multi-directional differentiation ability,and lay the foundation for the next study of the effects of Exendin-4 on the proliferation and osteogenic differentiation of PDLSCs in the inflammatory microenvironment basis.Methods:PDLSCs were isolated and cultured by tissue enzyme digestion.The cell clone formation ability was detected by crystal violet staining.The osteogenic differentiation of cells to form mineralized nodules was measured using Alizarin Red staining.The adipogenic differentiation of cells to form lipid droplets was detected by Oil Red O staining.Immunofluorescent staining method was used to identify cell stemness.Results:After 5-8 days of culture,PDLSCs crawled out of the tissue.Crystal violet staining showed that the cells adhered and survived after colonization and formed colonies.After 21 days of osteogenic induction,Alizarin Red staining showed the formation of mineralized nodules.14days after induction of adipogenesis,Oil Red O staining showed the formation of lipid droplets.Mesenchymal stem cell phenotype molecular markers STRO-1 and CD146 were positive by immunofluorescence staining.Conclusion:The tissue enzyme digestion can successfully culture PDLSCs,and the cultured cells have strong clone forming ability,and can differentiate into osteoblasts and adipocytes.The cell surface expresses mesenchymal stem cell phenotype molecular markers STRO-1 and CD146,which can be used in subsequent experiments.PART 2:EFFECTS OF EXENDIN-4 ON THE PROLIFERATION AND OSTEOGENIC DIFFERENTIATION OF PERIODONTAL LIGAMENT STEM CELLS IN THE INFLAMMATORY MICROENVIRONMENTObjective:To investigate the effect of Ex-4 on the proliferation and osteogenic differentiation ability of PDLSCs in the LPS simulated inflammatory microenvironment in vitro.Methods:1.Using different concentrations of LPS to simulate the inflammatory microenvironment and detecting its effect on the proliferation of PDLSCs,grouped as follows:N1（blank control group）,0.1μg/ml LPS,1μg/ml LPS,10μg/ml LPS,100μg/ml LPS.The third generation PDLSCs were grouped as described above,and different concentrations of LPS were added to the common media of each group except the blank control group for 7 days.The CCK-8 method was used to detect the cell proliferation ability on days 1,3,5,and 7.2.Select 10μg/ml LPS to simulate the inflammatory microenvironment,and examine the effect of adding different concentrations of Ex-4 on the proliferation of PDLSCs under the inflammatory microenvironment,grouped as follows:N1（blank control group）,LE1（1 nmol/l）,LE2（10 nmol/l）,LE3（50 nmol/l）,LE4（100nmol/l）.The third-generation PDLSCs were grouped as described above,and 10μg/ml LPS and different concentrations of Ex-4（1 nmol/l,10 nmol/l,50 nmol/l,100 nmol/l）were added to the common media of each group except the blank control group for 7 days.The CCK-8 method was used to detect the cell proliferation ability on days 1,3,5,and 7.3.Select 10 nmol/l Ex-4 to simulate PDLSCs,and examine the effect of Ex-4 on the osteogenic differentiation of PDLSCs in the inflammatory microenvironment,grouped as follows:N2（osteoblast induction group）,Ex-4（10 nmol/l）Group,LPS（10μg/ml）group,LPS（10μg/ml）+Ex-4（10 nmol/l）group.Take the 3-5 generations of PDLSCs according to the above grouping,add 10μg/ml LPS or（and）10 nmol/l Ex-4 to the osteogenic differentiation induction medium of each component except the osteogenic control group for 21 days.The amount of mineralized nodules were measured by Alizarin Red staining.RT-PCR was used to detect the expression of osteogenic genes（including Runx2,ALP,Osx）.Results:1.On the third day of cell culture,the proliferation ability of PDLSCs in the 10μg/ml LPS group and the 100μg/ml LPS group was significantly increased compared with the blank control group（N1）（the difference was statistically significant（P<0.05））,There was no significant difference in cell proliferation ability between the remaining dose groups and the blank control group.On the fifth day of cell culture,the proliferation capacity of the 100μg/ml LPS group increased compared with the blank control group（the difference was statistically significant（P<0.05））,and the cell proliferation capacity of the remaining dose groups had no significant difference compared with the blank control group.On the 7th day of cell culture,the cell proliferation capacity of all dose groups decreased compared with the blank control group,but the 100μg/ml LPS group showed the most significant decrease（the difference was statistically significant（P<0.05））.2.Compared with the blank control group,the combined effect of LPS and Ex-4 has no significant effect on the proliferation ability of PDLSCs（P>0.05）.The LE2（10 nmol/l）group had higher cell proliferation ability than the other groups except the blank control group,but the difference was not statistically significant（P>0.05）.3.The Alizarin Red staining results showed that,compared with the osteogenic induction group,on day 21,the mineralized nodules formed in the Ex-4 group increased significantly,and the mineralized nodules formed in the LPS group decreased significantly.Compared with the LPS group,following Ex-4 treatment,mineralized nodules of the PDLSCs significantly increased in the presence of LPS.RT-PCR results showed that the expression of Runx2,ALP,Osx was significantly increased in the Ex-4treatment groups at day 14 compared to the osteogenic induction group.LPS treatment decreased the expression of the osteogenic genes that were recovered by Ex-4 co-treatment.The differences were statistically significant（P<0.05）.Conclusion:10μg/ml LPS promotes the proliferation ability of PDLSCs in the early stage and inhibits the proliferation ability of PDLSCs in the late stage.10μg/ml LPS can also inhibit the osteogenic differentiation ability of PDLSCs.Ex-4 can restore the proliferation of PDLSCs in the inflammatory microenvironment and promote the osteogenic differentiation of PDLSCs in the inflammatory microenvironment.PART 3:EFFECTS OF EXENDIN-4 ON NF-ΚB SIGNALING OF PERIODONTAL LIGAMENT STEM CELLS IN INFLAMMATORY MICROENVIRONMENTObjective:To investigate whether Ex-4 exerts anti-inflammatory effects on PDLSCs by affecting the NF-κB signaling pathway in the inflammatory microenvironment.Methods:Grouping:N1（blank control group）,Ex-4（10 nmol/l）group,LPS（10μg/ml）group,LPS（10μg/ml）+Ex-4（10 nmol/l）group.The 3-5generations of PDLSCs were cultured in general medium and added with LPS（10μg/ml）or（and）Ex-4（10 nmol/l）for 24h.RT-PCR was used to detect TNF-αand IL-6 gene expression;Western Blot was used to detect TNF-α,IL-6,IκBαand p-IκBαprotein expression;ELISA was used to detect soluble TNF-αand IL-6 protein level in the medium supernatant;Immunofluorescent staining was used to detect the expression of NF-κB/p65 proteins in the nucleus and cytoplasm.Results:Western blot analysis showed that both TNF-αand IL-6expression significantly increased after 24h of LPS（10μg/ml）induction compared to N1.However,the expression of TNF-αand IL-6 significantly decreased following LPS（10μg/ml）and Ex-4（10 nmol/l）treatment compared to LPS alone.RT-PCR and ELISA analysis showed comparable results.IκBαexpression increased after 24 h of LPS induction.In contrast,IκBαaccumulation was significantly lower in the Ex-4 treatment groups.LPS significantly induced IκBαphosphorylation that was inhibited by Ex-4.In addition,immunofluorescent analysis revealed that the NF-κB/p65complex localized to the cytoplasm of untreated PDLSCs,but translocated to the nucleus after 24 h of LPS induction.Ex-4 treatment partially blocked this translocation.Conclusion:Ex-4 exerts anti-inflammatory effects on PDLSCs by inhibiting the NF-κB signaling pathway in the inflammatory microenvironment.PART 4:EFFECTS OF EXENDIN-4 ON WNT SIGNALING PATHWAY OF PERIODONTAL LIGAMENT STEM CELLS IN THE INFLAMMATORY MICROENVIRONMENTObjective:To investigate whether Ex-4 could promote osteogenic differentiation of PDLSCs by affecting the Wnt signaling pathway in the inflammatory microenvironment.Methods:Grouping:N2（osteoblast induction group）,Ex-4（10 nmol/l）group,LPS（10μg/ml）group,LPS（10μg/ml）+Ex-4（10 nmol/l）group.The 3-5 generations of PDLSCs were cultured in general medium and added with LPS（10μg/ml）or（and）Ex-4（10 nmol/l）for 7 days.Western Blot was used to detect Runx2,β-catenin,GSK-3βand p-GSK-3βprotein expression.Results:Compared with the osteogenic induction group,the expression ofβ-catenin protein in the cytoplasm and nucleus increased in the Ex-4 group.In the LPS group,β-catenin levels were significantly higher in the nuclear fraction,with decreased cytoplasmic expression.Interestingly,cotreatment of Ex-4 with LPS significantly decreasedβ-catenin expression in the nuclear fraction and increased its cytoplasmic expression.In addition,the phosphorylation of GSK-3βsignificantly decreased in the Ex-4 treatment group compared to the osteogenic induction group,whilst the expression of total GSK-3βincreased.LPS treatment significantly promoted the phosphorylation of GSK-3β,but reduced total GSK-3βexpression.LPS and Ex-4 co-treatment increased total GSK-3βexpression and reduced p-GSK-3βlevels compared to the LPS group.We also observed increased Runx2 expression in the Ex-4treatment group and reduced Runx2 expression in the LPS treatment group compared to the osteogenic induction group.LPS and Ex-4 cotreatment led to significantly increased Runx2 expression levels compared to LPS treatment alone.The differences were statistically significant（P<0.05）.Conclusion:Ex-4 can promotes osteogenic differentiation of PDLSCs by regulating the Wnt signaling pathway in the inflammatory microenvironment. |
