The Effect Of Macrophage Migration Inhibitory Factor In The Pathogenisis Of Periapical Lesions

Macrophage migration inhibitory factor (MIF) has been defined as a key cytokine in regulation of innate and adaptive immunity, whose rapid release is triggered by stress, inflammation, and infections. In spite of T cells being the main source of MIF production, they are also produced and secreted by other cell types such as macrophages, lymphocytes and vascular endothelial cells. Receptor activator of nuclear factor kappa B ligand (RANKL), which is mainly expressed by osteoblasts or T cells, has an essential role in osteoclastogenesis. Increasing the expression of RANKL in the periapical lesion has been associated with the pathogenesis process of periapical bone destruction, and using drugs to inhibit RANKL production has been suggested to have a protective effect in periapical disease in rats.Because apical periodontitis is a sequel of a root canal infection, it can result in the destruction of the pulp architecture and, ultimately, the development of a periapical lesion with alveolar bone resorption around the root apex. Although MIF has been shown to play a role in mediating inflammatory and bone loss in a number of diseases, the role of MIF in mediating alveolar bone loss in apical disease is unknown. T cells play an important role in the maintenance of a balanced immune defense against oral bacteria and inflammatory bone resorption. MIF and RANKL are produced in large quantities by T cells. Therefore, we hypothesize that MIF might be involved in the pathogenesis of periradicular lesions, particularly the role it might play in both inflammatory response and alveolar bone destruction. The aim of this study is to examine the presence of MIF in the periapical lesions of rat and human and discuss the mechanism of rhMIF on RANKL production in human periodontal ligament fibroblast cells. Moreover, application of metformin to interfere rats periapical lesions.Experiment OneThe expression of macrophage migration inhibitory factor in induced rat periapical lesionsObjective:The purpose of this study was to investigate the immunohistochemical localization of MIF and its relationship with receptor activator of nuclear factor kappa B ligand (RANKL) protein during the development of periapical lesions in rats.Materials and Methods:Apical periodontitis was induced in36Wistar rats by occlusal pulp exposure in mandibular first molar teeth. The animals were randomly killed at0,7,14,21,28, and35days after pulp exposure. The jaws that contained the first molar were obtained and were prepared for histologic analysis, enzyme histochemistry, immunohistochemistry, and double immunofluorescence staining.Result:From day0to day35, the areas of periapical bone loss increased and seemed to be stabilized on day35. A few MIF-positive and RANKL-positive cells and osteoclasts could be observed on day7, and all climaxed on day14. From day21to day35, the expression of MIF and RANKL protein decreased, and fewer osteoclasts could be observed. From day21to day35, the number of MCP-1-positive cells increased.Conclusion:These findings showed that MIF involved the pathogenesis progress of periapical lesions. In the early stage of periapical lesions MIF might be associated with the differentiation of osteoclasts. MIF contributes to the protective effect of the periapical lesions through the induction of MCP-1protein in the late stage.Experiment Two Macrophage migration inhibitory factor enhances osteoclastogenesis through upregulation of RANKL expression from periodontal ligament fibroblastsObjective:The aim of this study was to localize the MIF and RANKL in the human periapical lesions and hypothesised that MIF may target specialised human periodontal ligament fibroblasts (hPDLFs) for production of receptor activator of nuclear factor-κB ligand (RANKL), which is known to play a crucial role in bone destruction in periapical diseases.Materials and Methods:Thirty-two human periapical lesion samples were obtained in the clinic for histological and immunohistochemicals to determine the presence of MIF and RANKL. The cells were stimulated with rhMIF (0.1,1,5, and10ng/ml) for different time periods. The expression levels of RANKL were evaluated via Immunofluorescence analysis, quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. To further understanding the mechanisms by which rhMIF regulates RANKL production in hPDLFs, we evaluated these intracellular signalling pathway.Results:From the Immunohistochemical staining, we can see that both MIF-and RANKL-positive cells expressed in periapical lesion, but MIF is mainly expressed in lymphucytes, meanwhile majority ECs expressed Rankl protein. rhMIF significantly up-regulated RANKL mRNA and protein expression in hPDLFs, meanwhile, rhMIF stimulation resulted in rapid activation of Akt, p38mitogen-activated protein kinase (MAPK), extracellular signalregulated kinase (ERK)1/2, c-Jun-N-terminal kinase (JNK), nuclear factor-kappaB (NF-κB) in hPDLFs. Blocking of p38MAPK, AKT, or NF-κB signalling led to a marked reduction in RANKL expression. Moreover, rhMIF-initiated NF-κB activation was blocked by Akt, p38MAPK inhibition.Conclusion:This study determined that both MIF-and RANKL-positive cells expressed in periapical lesion, and up-regulating expression of RANKL by rhMIF stimulation in hPDLFs. And the expression of RANKL is mediated via pathway involving Akt-, p38MAPK-dependent NF-κB signalling versus the JNK/ERK1/2pathway.Experiment ThreeProtective Effect of Metformin on Periapical Lesions in RatsObjective:The current study aimed to investigate the effects of systemically administered metformin on MIF expression and on the ratio of receptor activator of nuclear factor kappa B ligand/osteoprotegerin (RANKL/OPG) in rats subjected to experimental periapical lesions.Materials and Methods:Forty adult male Wistar rats were divided equally into control and experimental groups, and the pulp chambers of their mandibular first molars were exposed to the oral environment to induce periapical lesions. The experimental group received daily intramuscular injections of metformin at40mg/kg doses, whereas the control group received only the saline vehicle. The injections were initiated1day before the periapical lesion induction and then were continued daily throughout the entire experimental period. Two or4weeks after pulp exposure, the rats were killed. First, the mandibles including the molars were analyzed by X-ray and Micro-CT, at last the mandibles were prepared for histologic analysis, enzyme histochemistry, immunohistochemistry, and immunofluorescence staining.Results:The number of MIF-and RANKL-positive and tartrate-resistant acid phosphatase (TRAP)-positive cells in the metformin-treated groups decreased on day14, whereas the number of OPG positive cells increased on day28. The periapical bone loss area in the metformin-treated group significantly decreased on day28 compared with the control group.Conclusions:Metformin inhibits the periapical lesions possibly by lowering the MIF expression and the RANKL/OPG ratio, subsequently reducing the number of osteoclasts and bone loss.