The Research Of Atuophagy Protect End-plate Chondrocytes From Mechanical Tension (ICMT) Induced Calcification In Vitro

Objective:To investigate the relationship between autophagy and the ICMT-inducedcalcification of endplate chondrocytes.Summary of Backgroud data: Autophagy, anevolutionarily conserved process for the bulk degradation of cytoplasmic components,serves as a cell survival mechanism.Indaequate cellular homeostasis and functionsprimarily contributed to calcification of endplate chondrocyes Previous study showed thatintermittent cyclic mechanical tension contrubite to the calcification of endplatechondrocyes in vitro,but there was no clear ecidence to indicate the relationship ofautophagy and tension-induced calification of endplate chondrocyes in vitro.Methods. Rat end plate chondrocytes were cultured and ICMT (strain at0.5Hzsinusoidal curve at10%elongation) was applied for25days,4hours a day. End platechondrocytes calci fi cation was stained by alizarin red S (AR-S). Endplate chondrocytesviability was examined by LIVE/DEAD viability/cytotoxicity kit (Invitrogen, Carlsbad,CA). Related gene expression was examined by reverse transcription-polymerase chainreaction and Western blot. Immunofluorescence examination and fluorescent microscopymicroscopy were used to visualize autophagy in the cytoplasm.Results. Immunofluorescence examination and fluorescent microscopy were used tovisualize autophagy in end-plate chondrocytes.The reslut of LIVE/DEAD assay betweenthe nonloading (NC) group and the ICMT group verified that end plate chondrocytesremained viability and adherent after ICMT. The Beclin-1and LC3genes expression ofboth mRNA and protein levels increased after short-term of ICMT and inhibited after long-term of ICMT.Alizarin red staining and Alkaline phosphatase staining showed thecalcifcation level of end plate chondrocytes after ICMT and rapamycin.Real-time reversetranscription-polymerase chain reaction showed that mRNA expression of typeII,aggrecan,and Sox-9decreased after ICMT. Compared with NC group, the alkalinephosphatase activities significantly increased in ICMT group,moreover Compared withICMT group,the alkaline phosphatase activities significantly decreased in ICMT andRapamycin group.Conclusion. Our results directly showed that autophagy takes place in end-platechondrocytes,and then the increased autophagy activity induced by rapamycin isaccompanied by a insignificant calcification of end plate chondrocytes, on the contrary,the suppressive autophagy inhibited by the long-term ICMT is accompanied by a moresignificant calcification.