Bacterial LPS Induces Osteocyte Apoptosis And Realeasing High Mobility Group Protein1

Background and objectiveOsteocyte is the most numerous and longest-lived cell type in bone. Recent researches show that osteocyte is not just’silence cell’. They extend numerous long cell processes to connect with each other or other cell types on the bone surface like lining cells and osteoblasts. Osteocyte was reported to sense changes in mechanical, hormones, inflammatory factors, et al. They also send these changes to other cell types using canaliculus. Now osteocyte is believed to play an important role in bone remodeling. Apoptosis of osteocyte effect on the formation and activation of osteoclast. Some studies show that activated osteoclasts was seen close to apoptosis ostecytes in bone. It has found that the osteocyte may play an important role in lead the activation and localization of osteoclast. However, the specific mechanism needs more study.MethodsCell culture:the MC3T3-E1murine osteoblast-like cells were culture in a-MEM containing10%FBS. The MLO-Y4murine osteocyte-like cells were culture in a-MEM containing5%FBS and5%CS. After cell proliferation, this two cell styles were seed in6-well plates at an initial density of4×105cell/well in a-MEM and LPS (Oug/ml,0.5ug/ml, lug/ml,5ug/ml,10ug/ml). Approximately6h,18h,24h,48h and72h after seeding, these cells and culture medium were collected for ELISA, RT-PCR and DAPI staining.High mobility group box-1protein (HMGB1) is a non-histone protein. It participates in DNA replication, recombination, transcription and repair. In the other hand, HMGB1also functions as an extracellular signaling molecule during inflammation, immunity, cell differentiation, cell migration and tissue regeneration. As an pro-inflammation factor, HMGB1can stimulate bone resorption. Some research shows that MLO-Y4apopotosis will improve HMGB1release level. In this study, we use LPS stimulates MLO-Y4and MC-3T3apoptosis and discover HMGB1releasing situation. ResultsELISA results shows that on18hrs, MLO-Y4has a significant increase in HMGB1realeasing level and the increase maintaining to48hrs. Compared with MLO-Y4, the level of HMGB1in MC-3T3culture supernatant reached a peak between18hrs and24hrs after seeding, but the level of HMGB1do not has a significant increase in some group.Real-time quantitative PCR assay shows that the level of HMGBl mRNA expression in MLO-Y4has a time-connection increasing. The level in MC-3T3have little increase.DAPI staining shows that MLO-Y4is more sensitive to LPS stimulates than MC-3T3., and MLO-Y4apoptosis has a time-connection increasing, but MC-3T3apoptosis cells only have a peak between18hrs and24hrs.ConclusionHMGB1can promote inflammation and also can stimulate bone resorption. Under the condition of LPS-stimulates and simulated ischemia, mouse osteocyte-like cell are more sensitive to mouse osteoblast-like cell, and realease a higher level of HMGB1condition. It suggests that osteocyte can release HMGB1themselves to promote inflammation and bone resorption.