| Apical periodontitis, one of the most common inflammatory bone resorption dental diseases, is primarily caused by bacterial infection through infected root canals. It is characterized by infiltration of inflammatory cells, production of pro-inflammatory cytokines, elaboration of lytic enzymes and activation of osteoclasts, leading to alveolar bone destruction surrounding the tooth apex. Periodontal ligament fibroblasts are a prominent cell type in the periodontal ligament, which play an important role in the periodontal integrity maintenance, modulating homeostasis and periodontal tissue regeneration.Glycogen synthase kinase 3? (GSK3 ?) is a ubiquitous serine/threonine protein kinase. It was initially described for its function to inhibit glycogen synthesis through phosphorylation of glycogen synthase. GSK3P regulates various cellular responses, such as cell growth, differentiation, apoptosis and inflammation. GSK3? is also involved in cell membrane to nucleus signal transducting, gene transcription and translation,cytoskeletal organization, and the regulation of cell cycle regulation and survival.GSK3? activity is regulated by numerous signalling pathways, including PI3K, protein kinase A and protein kinase C. They can inhibit GSK3P kinase by phosphorylating the Ser 9 residue in the N-terminal domain. GSK3P negatively regulates downstream signaling mechanisms, inactivation of GSK3? in fact stimulates many cellular functions by removing the negative constraint imposed by GSK3?. GSK3?has been found to regulated the activation of proinflammatory nuclear factor ?B (NF-?B) activation and expression of NF-?B-mediated chemoattractants. Recent studies have implicated GSK3P in both human and experimental inflammatory diseases, such as Alzheime's disease, diabetes, acute renal failure, chronic graft kindney nEPhropathy, rheumatoid arthritis (RA) and cancer. GSK.3P can regulate the production of inflammatory cytokines and inflammatory cell migration in inflammatory response. Inhibiting GSK3P greatly reduces the production of pro-inflammatory cytokines (such as IL-1?, IL-6, IL-12, and TNF-?) and increases the production of anti-inflammatory cytokine IL-10. Therefore, reducing the inflammatory reaction and showing certain protective role. Based on the above theory, we hypothesize that GSK3? might participate in apical periodontitis. Besides, inhibition of GSK3P can be used for the treatment of periapical lesions.The current study was divided into two parts:the in vivo experiments aim to investigate the expression of GSK3? and p-GSK3?(Ser 9) in rat and mouse periapical lesions, finding out the possible role of GSK3? in the pathogenesis of apical periodontitis, also assessing the therapeutic effect and mechanisms by GSK3? inhibitor systemic administration in induced mouse periapical lesions model. Moreover, through the inhibitor SB216763 intervention, we detected the production of RANKL and inflammatory cytokines, observed the change of NF-?B p65 activity in normal and LPS-induced inflammatory human periodontal ligament cells, further dissected the role of GSK3? in apical periodontitis.Part 1:Dissecting the role of GSK3? in apical periodontitisExperiment 1:Identifying the expression and mechanisms of GSK3P in experimentally induced rat periapical lesionsObjective:To investigate the kinetics of GSK3? and p-GSK3? (Ser 9) expression in experimentally induced rat periapical lesions and to explore their possible functions in the pathogenesis of periapical lesions.Methods:Periapical lesions were established in Wistar rats by occlusal pulp exposure in mandibular first molar teeth. The animals were killed on days 0,7,14,21 and 28. Micro-computed tomographic, histological and enzyme histochemical analyses were performed to detect the progression of periapical lesions. Immunohistochemistry, double-dye immunofluorescence and Western blot were performed to determine the expression of GSK3p and p-GSK3p (Ser 9) in periapical tissues.Results:From day 0 to day 28, the lesion volume and area gradually expanded, and the GSK3? positive cells gradually ascended. A few p-GSK3? (Ser 9) positive cells and osteoclasts appeared on day 7 and then climaxed on day 14. The numbers then simultaneously decreased from day 21 to day 28.Western blot analysis revealed that p-GSK3? (Ser 9) and GSK3? proteins were expressed at all time-points. The positive cells and protein expression ratio of p-GSK3P (Ser 9) against GSK3P increased from day 0 to day 14 and then decreased from day 14 to day 28. Finally, double-dye immunofluorescence assay revealed that p-GSK3?(Ser 9) positive and RANKL positive cells were co-localized around periapical lesions on days 14 and 28.Conclusions:GSK3? and p-GSK3? (Ser 9) can be observed and may be involved in alveolar bone resorption and inflammatory response in periapical lesions, as well as associated with periapical lesion pathogenesis.Experiment 2:Induction of mouse periapical lesions model and the expression of GSK3P in experimentally induced mouse periapical lesionsObjective:The purpose of the present study was to build up the induced mouse periapical lesions model, observing and investigating the alveolar bone resorption and inflammatory response during the development of periapical lesions, detecting the expression of GSK3?, p-GSK3?(Ser9) and RANKL positive cells in experimentally induced mice periapical lesions.Methods:Forty two adult male C57BL/6 mice were divided into six groups, each group contained seven mice. Periapical lesion was induced in each mouse by pulp exposure on both mandibular first molars. Mice were sacrificed at 0,7,14,21,28 and 35 days after pulp exposure and the jaws contained the first molar were harvested for X-ray imaging, micro-computed tomography scanning, histological analysis, immunohistochemistry, and enzyme histochemistry.Results:Results from X-ray imaging, Micro-CT scanning and histological observation showed that the volume and area of the periapical lesions increased from day 0 to day 28, and then seemed to be stabilized on day 35. The inflammatory cells infiltration started at day 7, peaking on day 21, and then remained comparably stable from day 28 to day 35. A few p-GSK3?(Ser 9) positive cells, RANKL positive cells and osteoclasts appeared on day 7, and then climaxed on day 14. From day 21 to day 35, the expression of p-GSK3?(Ser 9) and RANKL protein decreased, and fewer osteoclasts could be observed. The number of GSK3? positive cells increased from day 0 to day 28, and then seemed to be stabilized on day 35.Conclusions:(1) These findings showd that mouse's molar pulp exposure is an efficient way to establish experimental periapical lesions, which can simulate the whole process of human periapical lesions. Based on the rat periapical disease model, this model was successful and reliable, which layed a good foundation for the establishment of transgenic mouse model of periapical lesions. (2) The results demonstrated that GSK3? and p-GSK3? (Ser 9) might be involved in the acute and chronic inflammatory response, and associated with the development and conversion process of periapical lesions.Experiment 3:Expression of GSK3? in human chronic periapical lesions Objective:The purpose of the present study was to investigate the expression and localization of GSK3p, p-GSK3p (Ser 9) and NF-?B p65 in the human chronic periapical lesions.Methods:Nineteen human periapical lesion samples (nine cysts and ten granulomas) as well as six healthy periapical samples were obtained in the clinic. Ten periapical lesion samples were selected for histologic analysis to observe the histopathological changes of periapical lesions, and immunohistochemical analysis to determine the presence of GSK3?, p-GSK3? (Ser 9) and NF-?B p65. The remaining periapical lesion samples and healthy periapical samples were used for Western blot analysis to examine the protein of GSK3?, p-GSK3?(Ser 9) and NF-?B p65.Results:Results from histological observation showed that the human periapical lesions were composed of fibroblasts, inflammatory cells and new capillaries. It could be observed that a large amount of inflammatory cells infiltrate in the periapical lesions, such as lymphocytes, neutrophils and monocytes. From the immunohistochemical staining, we can see that GSK3?, p-GSK3?(Ser 9) and NF-?B p65 positive cells expressed in periapical lesions, they are mainly expressed in inflammatory cells, endothelial cells and fibroblasts. Western blot analysis revealed that compared with healthy periapical samples, the expressions of GSK3? and NF-?B p65 protein were remarkable elevated in periapical lesions, the expression of p-GSK3? (Ser 9) protein also slightly increased.Conclusions:These findings indicated that GSK3?, p-GSK3? (Ser 9) and NF-?B p65 can be observed in periapical lesions, and they may be involved in human chronic periapical lesions.Part 2:Inhibition of GSK3? attenuates periapical lesions in miceExperiment 4:GSK3? inhibitors attenuate the experimentally induced mice periapical lesionsObjective:To investigate the effects of systemically administered GSK3? inhibitors on inflammatory response and alveolar bone resorption in mice subjected to experimental periapical lesions.Methods:Seventy-five adult male C57BL/6 mice were divided into five groups (fifteen mice in each group), including:28 day experimental group (SB216763 treatment group, TDZD-8 treatment group, LiCL treatment group),28 day experimental control group (DMSO treatment group) and the blank control group (0 day). The pulp chambers of their mandibular first molars were exposed to the oral environment to induce periapical lesions. SB216763 (10mg/kg intraperitoneally), TDZD-8(1mg/kg intraperitoneally), and LiCL(100mg/kg intraperitoneally) or vehicle (10% dimethyl sulfoxide) was administered 1 d prior to the periapical lesion induction and every other day thereafter until sacrifice.28 days after pulp exposure, the mice were killed and then the blood serum and tissue RNA were collected, the mandibles were then dissected out. Alveolar bone loss and inflammatory response were dectected by X-ray scanning, Micro-CT scanning, histologic analysis, immunohistochemistry, real-time PCR, and ELISA analysis. The new alveolar bone formation was observed by alizarin red and calcein staining.Results:Results from X-ray imaging, Micro-CT scanning and histological observation showed that the periapical lesion volume and area in the GSK3P inhibitor groups significantly decreased compared with the experimental control group on day 28. The pro-inflammatory cytokines, including IL-1?,IL-6, IL-12, IL-17 and TNF-a expression were suppressed, while concurrently the anti-inflammatory cytokine IL-10 expression was augmented by pharmacological inhibition of GSK3?.Immunohistochemical analysis revealed that the expression of NF-?B p65 in GSK30 inhibitor treatment groups were mainly concentrated in the nucleus, and the expression was lower than that of the experimental control group; the expression of p-GSK3?(ser 9) was significantly increased in GSK3? inhibitor treatment groups compared with the experimental control group, while the expression of RANKL in the GSK3P inhibitor treatment groups was lower than that of the experimental control group. Besides, the bone formation in the experimental control group was less than that of GSK3? inhibitor treatment groups.Conclusions:Inhibition of GSK3? could selectively regulate the production of pro-inflammatory cytokines and anti-inflammatory cytokines, regulate bone destruction and bone formation in mice periapical lesions, consequently, reduce the inflammatory reaction and bone resorption. These findings showed that the potent GSK3? inhibitors SB216763, TDZD-8, and LiCL could reduce the inflammatory response and alveolar bone resorption in a mouse model of apical periodontitis, suggesting that GSK3? might be a potential target for treatment of periapical lesions.Part 3:Effect of GSK3? inhibitor on the expression of RANKL and inflammatory cytokines in hPDLFs stimulated by LPSExperiment 5:Effect of GSK3P inhibitor on the expression of RANKL and inflammatory cytokines in hPDLFs stimulated by LPSObjective:The aim of the present study was to determine the effect of GSK3? inhibitor SB216763 on the expression of RANKL and inflammatory cytokines in hPDLFs stimulated by LPS,and explore the related mechanism.Methods:The hPDLFs were divided into three groups:control group. LPS-induced inflammatory model group, and inflammatory model with SB216763 intervention group. The mRNA levels of RANKL, IL-1?,IL-6, IL-12, TNF-a, and IL-10 were evaluated via real-time quantitative PCR, whereas the protein was assessed via enzyme-linked immunosorbent assay; Differential expression of inflammation-related protein NF-?B p65 was detected by immunofluorescence staining and Western blotting analysis.Results:GSK3? inhibitor SB216763 was found to significantly down-regulate the expression of RANKL and pro-inflammatory cytokines IL-1?, IL-6, IL-12, IL-17, and TNF-a, but up-regulate the expression of anti-inflammatory cytokine IL-10 in hPDLFs stimulated by LPS. Immunofluorescence staining and Western blotting analysis showed that the expression of NF-kB p65 transfered from cytoplasm to nucleus in LPS-induced hPDLFs, and this nuclear translocation can be blocked by GSK3? inhibitor SB216763.Conclusions:SB216763 could inhibit the activation of NF-?B pathway in hPDLFs by inhibiting GSK3? activity, and then down-regulate the expression of RANKL and proinflammatory cytokines IL-1?, IL-6, IL-12, IL-17, and TNF-a, but up-regulate the expression of antiinflammatory cytokine IL-10,finally reduce the inflammatory reaction of hPDLFs. This process might be one of the mechanisms underlying the ability of GSK3P inhibitor to ameliorate human periapical lesions. |
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