The Study On The Mechanism Of MMPs In Regulating Glucocorticoid Induced Osteoporosis

Purpose:Glucocorticoid (GC) have a multitude of effects on the immune response at several sites and are both anti-inflammatory and immunosuppressive when administered therapeutically. Although GC are effective for the treatment of a variety of disorders ranging from autoimmune diseases to acute situations such as organ transplantation and tumors, long-term therapy with GC causes osteoporosis, which has become a big clinical problem. Glucocorticoid-induced osteoporosis, the most frequent form of secondary osteoporosis, is characterized by bone loss, bone microarchitechtural damage, decreased bone strength and high risk of bone fracture. The mechanism and prevention of GIO has caused more attention at present. Lone-term administration of GC inhibits the proliferation of osteoblasts, increases the apoptosis of osteoblasts and osteocytes and promotes the activities of osteoclasts, resulting in decreased bone strength and bone loss. Matrix metal loproteinases (MMPs), a growing family of metalloendopeptidases, can cleave the protein components of the extracellular matrix. Meanwhile, the tissue inhibitor of metalloproteinases (TIMP) tightly control MMPs activities by combined with them. Most MMPs expressed at low levels in resting-state adult tissues, which are regulated by cytokines and growth factors. Therefore, the research of MMPs may propose new therapeutic strategies for GIO. In our study, the MMP-2, MMP-9 and MMP-13 expression were examined both in vivo and vitro to explore the mechanism of osteocytic osteolysis and GIO, providing theoretical evidence of treatment and prevention for GIO.Materials and Methods:In the first stage of the experiment, twenty 7-week-old Kunming mice were divided into two groups:GC-treated group and control group. After 1 week acclimation, mice were administered prednisolone (15mg/kg) or PBS as control intragastrically every other day. Two weeks later, the mice were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer by transcardial perfusion and tibiae were harvested for histomorphology analysis. During the second stage of in vitro experiment, various dose dexamethasone (Dex) were added into osteocytic MLO-Y4 cells. And then, cell vitality was assessed by MTT and the expression of matrix metalloproteinase-2 (MMP-2) and MMP-13 were evaluated through qRT-PCR analysis.Results:The first stage:1. Trabecular bone:After prednisolone administration, the mass of trabecular bone, the height of grow plate and the width of cortical bone decreased significantly. Trap positive osteoclasts in the trabecular bone of tibiae increased dramatically, and CK positive osteoclasts increased. The expression of MMP-2, MMP-9 and MMP-13 in trabecular bone of tibiae increased.2. Cortical bone:After prednisolone administration, the number of osteocytes in cortical bone of tibiae decreased obviously. The osteocyte lacunae enlarged at various levels and empty lacunae augmented. Meanwhile, some crevices were presented in the narrowing cortical bone, showing lighter eosin staining. The expression of DMP-1 decreased and the expression of SOST increased after GC treated. The expression of MMP-2 in cortical bone increased remarkably, especial in osteocytes and lacunae. The expression of MMP-13 also increased, primarily in the crevices of cortical bone. There was no significant difference of MMP-9 between the two groups.The second stage:1.MTT indicated that the proliferation of MLO-Y4 cells were inhibited after dexamethasone administration, which were dose and time dependent.2. The results of qRT-PCR showed that dexamethasone increased the expression of MMP-2 and MMP-13 mRNA in MLO-Y4 cells, which were dose and time dependent.Conclusion:After glucocorticoid administration, the expression of MMP-2, MMP-9 and MMP-13 in the trabecular bone of tibiae increased significantly, which may be response for bone loss. Meanwhile, osteocytic osteolysis could be observed in cortical bone. The expression of MMP-2 and MMP-13 augmented both in vivo and in vitro, which were mainly localized in osteocytes and cortical crevices. The proliferation of MLO-Y4 cells were inhibited and the expression of MMP-2 and MMP-13 mRNA increased obviously after glucocorticoid administered in vitro. These results suggested that MMP-2 and MMP-13 may play important roles in the mechanism of GIO, which might be new potential therapeutic targets for prevention and treatment of GIO, especially osteocytic osteolysis.