The Effect And Mechanism Of IL-17 On The Proliferation Of Human Periodontal Ligament Fibroblasts In Vitro

Objective: Periodontitis is a kind of disease with high morbidity in our country,and also a public health problem to be solved worldwide.IL-17 is a proinflammatory cytokines mainly produced by Th17 cell.And the IL-17 level in the serum,saliva and gingival crevicular fluid and gingival tissue of patients with periodontitis was significantly higher than those with healthy periodontium.Human periodontal ligment fibroblasts(h PDLFs)are the most commom cells in the periodontium,playing an important role in the process of periondontal tissue regeneration.In addition,many studies have shown that Notch signaling pathway plays an important role in the proliferation and differentiation phase of different kinds of cells.Therefore,this study aims to explore effects of IL-17 on the proliferation of h PDLFs and the mechanism of Notch signaling pathways involving in the process of IL-17 affectting the h PDLFs' proliferation.Materials and Methods: Part 1,effects of IL-17 on the proliferation and cell cycle of periodontal ligament fibroblasts: We collected the premolars or wisdom teeth from orthodontic patients with their permission.Periodontal ligament fibroblasts were isolated from the periodontal tissue scraped from the middle third of the roots using the enzyme digestion and tissue pieces culture method.The primary cells were identified using keratin and vimentin staining;The study was divided into four groups: the control group,10ng/ml IL-17,50ng/ml IL-17 and 100ng/ml IL-17,respectively.After culturing for 1,3,5 and 7 days,the ability of cell proliferation was tested by CCK-8 assay.And then,we chose a concentration of IL-17 that had the biggest influence on the proliferation of h PDLFs to use in the later study;The influence of IL-17 on the cytoskeleton,clone formation rate,ALP activity and cell cycle of h PDLFs was also detected by the immunofluorescent staining,crystal violet staining,ALP staining and flow cytometry,respectively.Part 2,the effect of Notch signaling pathway in the process of IL-17 affectting the proliferation capacity of h PDLFs: after treated with ?-secretase inhibitor(DAPT),we used western blot to detect the expression of Notch signaling pathway related proteins(NICD and Hes1)and the ability of cell proliferation by CCK-8.Secondly,we detected the effects of IL-17 on the expression of the cell cycle related genes and proteins(cyclin D1 and p21),NICD and Hes1 by q PCR and western blot.Finally,the experiment was divided into three groups: the control group,the IL-17 group and IL-17+Notch signaling pathway activator(Jagged1)group;The expressions of cyclin D1 and p21 were detected by western blot,and the ability of cell proliferation was detected by CCK-8 assay.Results: Part 1,the primary cultured cells appeared as long shuttle and growed as rioactive.Besides,these cells showed a positive staining for vimentin whereas they were negative for cytokeratin by immunofluorescence analysis,demonstrating their mesenchymal origin and proving that they were periodontal ligament fibroblasts.Results of CCK-8 showed that there was no statistical significance(P>0.05)between the four groups in the first day;But there was statistically lower optical density(OD)in the experiment groups(P<0.05)in the third,fifth and seventh day;Furthermore,the OD value of 50ng/ml group was the lowest one.Therefore,it was determined to apply 50ng/ml as the final concentration of IL-17 in subsequent experiments.IL-17 had less effect on the cytoskeleton of h PDLFs.The clone formation rate of the control group and the IL-17 group were 37.66%±4.04% and 27.33%±3.21%,respectively;And there was statistical significance between the two groups(P<0.05).The ALP staining of IL-17 group was significantly lighter than that of the control group,indicating that IL-17 inhibited osteogenic ability of h PDLFs.Changes of cell cycle showed that: when compared with the control group,the percentage of h PDLFs at G1 phase in the IL-17 group increased,while there was no significant difference(P>0.05);And there was no significant difference between them at G2 phase(P>0.05);But the percentage of h PDLFs at S phase in the IL-17 group significantly decreased(P<0.05).Part 2,the ability of h PDLFs' proliferation was inhibited and the expressions of Notch signaling pathway related proteins(NICD and Hes1)decreased after treated with DAPT.In addition,IL-17 can reduce the expressions of m RNA and proteins(cyclin D1 and p21)associated with the cell cycle,and also reduce the expressions of NICD and Hes1.Further studies showed that the effect of IL-17 on the production of cyclin D1 and p21 and the ability of cell proliferation were reversed by the Jagged1,which was a proof that Notch signaling participated in the process of h PDLFs' proliferation and reversed the inhibiting effect of IL-17 on the h PDLFs' proliferation.Conclusion: Notch signaling pathways involves in the process of IL-17 inhibiting the h PDLFs' proliferation.The mechanism is: IL-17 may firstly down-regulate Notch signaling pathway related proteins(NICD and Hes1),then inhibits the expression of cell cycle related genes and proteins(cyclin D1 and p21),resulting in the inhibition of cell cycle from G1 phase to S phase.Finally,IL-17 inhibits the proliferation of h PDLFs.