The Study Of Effects Of Metformin On The High Glucose-induced RANKL Expression In Human Periodontal Ligament Cells And The RANKL Expression In The Healing Process Of Tooth Extraction Socket In Diabetic Rats

Periodontal disease is becoming one of common complication associated with diabetes. As previous studies have described, the development of periodontal disease is closely related to abnormality of glucose levels. Receptor activator of nuclear factor-kB (RANK), receptor activator of nuclear factor-kB ligand (RANKL), and osteoprotegerin(OPG) are the cytokines which regulate the differentiation and activation of osteoclast. It is well known that periodontal ligament (PDL) cells play a pivotal role in the healing of wounds and the regeneration of periodontal tissues, and its cellular function may be affected by high glucose. Recent reports have shown that PDL cells can express the receptor activator nuclear factor kappa B ligand (RANKL), suggesting a regulatory role of these cells on bone resorption in periodontal tissue homeostasis and repair processes via RANK-RANKL signaling. In this study, we aim to investigate the mechanisms underlying the effect of glucose levels on RANKL expression in human PDL (hPDL) cells.Metformin, as an anti-hyperglycemic drug, is commonly used in patients with diabetes and metabolic syndrome, and recent report revealed that metformin can exert beneficial effects on alveolar bone loss, possibly by mediating cellular activity and functions in periodontal tissues. Meanwhile, metformin acts as a negative regulator of osteoclastic activity via AMPK activation. It suggested that metformin may be a useful agent for inhibiting alveolar bone loss in periodontal diseases associated with diabetes. In this study, we aim to investigate the mechanisms underlying the effect of glucose levels on RANKL expression in human PDL(hPDL) cells. We also investigate whether metformin reverses the effects of high glucose on RANKL mRNA and protein expression in hPDL cells via AMPK activation.Professor Liu proposed that anti-hyperglycemia drug formulated in controlled-release delivery could be benefit for bone remodeling around dental implant, and he successfully have used this form of drug delivery around the dental implant in diabetics. In present, the effect of metformin on bone remodeling in the healing process of tooth extraction when chitosan-based hydrogel insulin injected is not been discussed. In our study, we utilized the chitosan/β-glycerophosphate hydrogel (chitosan/β-glycerophosphate gels, C/GP) system loaded appropriate physiological concentrations of metformin, injected when the sol to gel in the tooth extraction socket in GK rats. We observed the change of RANKL expression and AMPK phosphorylation, aimed to explore the effect of metformin in the healing process of teeth extraction socket in diabetic rats.PartⅠ:The effect of high glucose on the RANKL expression and AMPK activityObjective:This study investigated the direct effect of high glucose on the expression of RANKL and AMPK activity in human periodontal ligament (PDL)Methods:Isolated, cultured and identified hPDL were treated with physiological glucose concentration and high glucose concentration of culture medium. We examined the expression of OPG and RANKL in hPDL cells cultured at different glucose levels using Real-time PCR and Western blot analysis. AMPK phosphorylation in hPDL cells was studied by immunoprecipitate kinase assay for SAMS and Western Blotting.Results:High glucose levels caused an increase in RANKL mRNA and protein expression in hPDL cells. Moreover, the amount of p-AMPK and AMPK activity was lower in hPDL cells exposed to high glucose levels than in cells exposed to normal glucose levels.Conclusion:High glucose up-regulated the RANKL expression and decreased the AMPK activity in hPDL cell.PartⅡ:The mechanism underlying the effect of metformin on RANKL expression in hPDL cell at high glucoseObjective:To investigate whether high glucose-induced RANKL expression is related to high glucose, and can be mediated by metformin via AMPK activation in hPDL cells.Method:RANKL mRNA and protein expression in hPDL cells at normal glucose with metformin and Compound C respectively, were examined by Real-time PCR and Western blot analysis. AMPK phosphorylation and activity in these cells was studied by immunoprecipitate kinase assay for SAMS and Western Blotting. We also examined the RANKL mRNA and protein expression in hPDL cells at high glucose with metformin, in the presence or absence of compound C, using Real-time PCR and Western blot analysis.Results:Compound C decreased AMPK phosphorylation and activity, whereas metformin increased its phosphorylation and activity in hPDL cells, as seen by Western blotting and a functional AMPK assay. Meanwhile, Real-time PCR and Western blot analysis showed that compound C up-regulated RANKL expression and metformin down-regulated RANKL expression in hPDL cells at both the mRNA and protein levels, compared with the control group. We also found that metformin decreased RANKL mRNA and protein expression, and co-incubation with compound C remarkably reversed the metformin-mediated decrease in RANKL protein in hPDL cells with high glucose.Conclusion:We found that suppression of AMPK by compound C augmented RANKL expression, whereas activation of AMPK by metformin decreased RANKL expression in hPDL cells at the mRNA and protein level. These findings, taken together, imply that high glucose-induced up-regulation of RANKL is associated with decreased AMPK activity. We also confirmed that metformin reverses the effects of high glucose on RANKL mRNA and protein expression in hPDL cells via AMPK activation.PartⅢ:The effect of local administration of metormin gel on RANKL expression and AMPK phosphorylation in the healing process of tooth extraction sockets in GK rats.Objective:To observe the effect of local control-release metformin on RANKL expression and AMPK phosphorylation in the healing process of tooth extraction socket in GK rat, and preliminary analyze the effect of metformin, as a AMPK activator, on the RANKL expression in the bone remodeling of tooth extraction socket.Methods:Extraction of the rat incisor; local administration of the chitosan-based hydrogel metformin in tooth extraction socket. Randomized:Wistar group, only the extraction group (WN); GK rats,only the extraction group (GN), tooth extraction with chitosan/GP group (G1), tooth extraction of chitosan/GP contained metformin group (G2). After tooth extraction,7 days,14days,21 days and 28days, rats were sacrifced to remove the mandible quickly. To observe the change of RANKL expression and AMPK phosphorylation in each group, using Real-time PCR and Western blot analysis. Results:HE staining in G2 group at 28 days there was no C/GP; Real-time PCR and Western blot analysis showed that RANKL mRNA and protein expression in GN group were higher than in WN group during the period of tooth socket from 7d to-28d. The RANKL expression between G1 and GN showed no significant difference. Compared with G1 and GN group, RANKL mRNA and protein expression in G2 group are down-regulated. We also found during the period of tooth socket from 7d to-28d, no change of AMPK phosphorylation in WN group, and higher than in GN group. AMPK phosphorylation between G1 and GN showed no significant difference. Compared with G1 and GN group, AMPK phosphorylation in G2 group are elevated.Conclusion:Overexpression of RANkL and suppression of AMPK were found in the healing process of tooth extraction sockets in GK rats. We also revealed that metformin can decrease the RANKL expression via AMPK activation in GK rats. It implied that application of metformin in tooth extraction sockets could improve the alveolar bone remodeling in diabetic rats.