Clinical Study Of Osteocyte Necroptosis Inducing Postmenopausal Osteoporosis

Part I: Osteocyte necroptosis occurs in patients with postmenopausal osteoporosis Objective:To analyze osteocyte necroptosis rate and apoptosis rate in patients with different stages of postmenopausal osteoporosis.To identify whether osteocyte necroptosis happened in various stages of postmenopausal osteoporosis.To explore if there were various expressions in different stages of necroptosis and apoptosis.And to clarify necroptosis is one of the vital ways of osteocyte death in patients with postmenopausal osteoporosis.Methods:1.According to the dual X-ray absorptiometry(DXA)identified by the international society for clinical bone mineral density(ISCD),we detected the bone mineral density(BMD)of postmenopausal patients who underwent hip replacement surgery in the first affiliated Hospital of Hainan Medical University from January2020 to January 2021.Those patients were divided into the normal bone mass group,osteopenia group,osteoporosis group,and severe osteoporosis group by BMD.2.According to the inclusion and exclusion criteria,60 patients’ data and postoperative bone tissue in the femoral Ward’s triangle were collected(15 patients in each group).Each patient’s bone tissue were performed HE staining,transmission electron microscope,immunofluorescence and immunohistochemistry.3.The trabecular microstructure of bone tissue was observed in each HE staining specimen under microscope.4.The morphology of osteocytes was observed by transmission electron microscope.5.Immunofluorescence experiments were carried out on immunofluorescence specimens of each group in turn to detect the expression of necroptosis related proteins RIP1,RIP3 and MLKL in osteocytes,and the expression of apoptosis marker protein was detected by TUNEL+cleaved caspase-3.6.Immunohistochemical experiments were carried out on Immunohistochemical specimens of each group in turn to detect the expression of necroptosis marker proteins RIP3 and the expression of apoptosis marker protein Cleved caspase-3 in bone tissue.Results:1.The HE staining showed that as the osteoporosis aggravated,trabecula became thinner and even broken,and the trabecular gap was increased in postmenopausal patients.2.Typical necrotic morphological features can be observed in the patients’ bone tissue of the osteoporosis group and severe osteoporosis group,which included swelling of cells and organelles,rupture of the plasma membrane,spilling intracellular contents out,and intracellular vacuoles extensive formed.However,those features were not seen in the normal bone mass group and osteopenia group.3.The results of immunofluorescence and immunohistochemistry show that RIP1,RIP3,MLKL and cleaved Caspase-3 were expressed in cytoplasm of all patients’ osteocytes.Compared with the normal bone mass group and osteopenia group,the positive rate of RIP3 and cleaved Caspase-3,which indicated necroptotic and apoptotic cells,were significantly increased in the osteoporosis group and severe osteoporosis group(P<0.01).And the positive rate of RIP3 was higher than cleaved Caspase-3(P<0.05).Conclusions:1.In the process of postmenopausal osteoporosis,osteocytes may occur Necroptosis in the stage of osteoporosis and severe osteoporosis.2.Necroptosis may play a more important role than apoptosis in the stage of postmenopausal osteoporosis and severe osteoporosis.Part II : RANKL was upregulated in patients’ osteocytes of postmenopausal osteoporosis inducing aggravation of osteoporosis Objectives:To analyze the expression of RANKL and OPG in postmenopausal patients’ osteocytes.To clarify necroptotic osteocyte induce osteoporosis development by upregulated RANKL,which leads to the imbalance of RANKL/RANK/OPG system,during estrogen deficiency.Methods:1.The expression of RANKL and OPG in osteocytes were detected by immunofluorescence.2.The levels of RANKL and OPG in osteocytes were detected by immunohistochemistry.3.The correlation of RANKL,OPG and osteocyte necroptotic rate was analyzed through Pearson correlation test.Result:1.According to immunofluorescence and immunohistochemistry,both RANKL and OPG were expressed in cytoplasm of all group patients’ osteocytes.The level of RANKL was upregulated as osteoporosis aggravated and OPG was reversed.2.The result of Pearson correlation test show that necroptotic rate of osteocyte was positively related to RANKL(r = 0.834)and negatively correlated with OPG(r=-0.566).Conclusions:Osteocyte necroptosis may lead to osteoporosis development by upregulated RANKL,which causes the imbalance of RANKL/RANK/OPG system,during postmenopausal osteoporosis.