The Mechanism Of Articular Cartilage Damage Induced By Deoxynivalenol

Objective : A large number of epidemiological studies have confirmed that mycotoxin,such as deoxynivalenol(DON),is one of the major pathogenic factors of Kashin-Beck disease(KBD).However,how DON results in articular cartilage damage is unclear.This study aims to investigate the effect of DON on chondrocyte proliferation,death,and matrix metabolism,exploring the regulatory mechanism of DON in articular cartilage,and revealing the relationship between mycotoxins in grain and articular cartilage injury in order to provide new insight for the prevention and treatment of degenerative diseases of articular cartilage related to DON.Methods:1.The normal articular chondrocytes of adult human knees and newly born SD rats(24 hr after birth)were isolated and cultured in vitro,cells were identified by collagen ? antibody with immunohistochemical staining.2.After normal chondrocytes were treated with DON,the IC50 value of DON was measured with CCK8 assay.The proliferation of chondrocytes was determined with CCK8 assay,Real Time Cellular Analysis,and Western Blotting.Meanwhile,chondrocytes were treated with solvent acetonitrile(ACN)at the same concentration for evaluating the toxic effects of ACN.3.After normal chondrocytes were treated with DON,the expression levels of collagen?,AGG,ADAMTS-5,MMP-13,and TIMP-1 proteins were detected by Western Blotting;the m RNA levels of collagen?,AGG,ADAMTS-5,MMP-13,and TIMP-1were measured with RT-PCR;the concentrations of collagen?,AGG,MMP-13,and TIMP-1 in the cell culture supernate were detected by ELISA.4.After normal chondrocytes were treated with DON,the apoptotic bodies or autophagosomes were observed under transmission electron microscopy(TEM);the levels of cleaved-caspase-3,cleaved-PARP,LC3 BII /I,P62,and MMP-13 protein were detected by Western Blotting.5.Normal chondrocytes were treated with specific inhibitor of apoptosis(Z-VAD)or autophagy(3-MA),respectively,prior to DON treatment.The levels of cleaved-caspase-3,cleaved-PARP,LC3BII/I,P62,collagen?,AGG,ADAMTS-5,MMP-13,TIMP-1 protein were then assessed with Western Blotting.Results:1.Culture cells from adult human normal knees and newly born SD rats express higher level of collagen II,representing the character of chondrocyte.2.The results of CCK8 assay and western blotting indicated that DON inhibited chondrocyte proliferation.3.The results of western blotting,RT-PCR,and ELISA showed that DON accelerated the degradation of articular cartilage.4.Under transmission electron microscope,autophagosomes were dominant in chondorcytes treated by lower concentration of DON,while apoptotic bodies appeared as well as the increase of DON concentration.Meanwile,the levels of cleaved-caspase-3?cleaved-PARP?LC3BII/I?P62?MMP-13 increased in a DON concentration-dependent manner.5.The addition of autophagy inhibitor down-regulated Col II and up-regulated MMP-13,while cleaved-caspase-3 and cleaved-PARP increased.The addition of apoptosis inhibitor up-regulated Col II and down-regulated MMP-13,but p62 level increased.Conclusions:DON could inhibit the proliferation of chondrocytes and the matrix synthesis of normal chondrocyte,resulting in articular cartilage degeneration.Both apoptosis and autophagy were involved in the regulation of DON in chondrocytes,which played different role.We suggest that apoptosis could be a pivotal element to result in chondrocyte death through suppressing autophagy,which was accompanied with the degradation of cartilage matrix.Autophagy could protect chondrocyte from destruction induced by low concentration of DON through antagonizing apoptosis,buy this protective effect could be inhibited under higher concentration of DON?...