Protection Of Raffinose On Chondrocytes And The Underlying Mechanism

ObjectiveTo investigate the protection of raffinose against H₂O₂-induced injury in chondrocytes and the related mechanism.Methods1.Rat chondrocytes were isolated and cultured in vitro.CCK-8 assay was used to detect the effect of raffinose on the viability of chondrocytes exposed to H₂O_2.The effect of raffinose on the expression of P16INK4a and Cleaved Caspase-3 in chondrocytes under H₂O₂ was evaluated by Western blot.2.Cells were randomly divided into 5 groups and treated with 100 mM raffinose for different times?0,6,12,24,48 h?.Western blot was used to detect the phosphorylation of AMPK together with the expression of autophagy-related proteins including Atg12-5,LC3 and P62.Immunofluorescence was used to evaluate the effect of raffinose on the accumulation of LC3 puncta.Cells were randomly divided into 4 groups:the control group cultured by routine assay;the model group exposed to 400?M H₂O₂ for 24 h;the raffinose-pretreated group stimulated with 100 mM raffinose for 2 h,followed by H₂O₂ exposure for 24 h;the 3-MA group exposed to 10mM 3-MA for 1 h,followed by the co-treatment of raffinose and H₂O₂.Western blot was used to detect the levels of Atg12-5,LC3 and P62.SA-?-Gal staining was used to detect the rate of senescent cells.Apoptosis incidence was evaluated by Annexin V-FITC/PI staining.The expression of senescence-related proteins including p-P53,P53,P21,P16INK4a along with apoptosis-related Bax,Bcl-2,Cleaved Caspase-3was also detected using Western blot.3.Cells were randomly divided into 4 groups:the control group cultured by routine assay;the model group exposed to 400?M H₂O₂ for 24 h;the raffinose-pretreated group stimulated with 100 mM raffinose for 2 h,followed by H₂O₂exposure for 24 h;the Compound C group exposed to 10?M Compound C for 1 h,followed by the co-treatment of raffinose and H₂O₂.Western blot was used to detect the phosphorylation of AMPK together with the expression of autophagy-related proteins.Results1.Pretreatment of chondrocytes with raffinose significantly relieved the inhibition of H₂O₂ on cell viability.The optimal protection of raffinose was achieved at 100 mM.Western blot showed that raffinose suppressed the expression of P16INK4a and Cleaved Caspase-3 induced by H₂O₂ in a dose dependent manner.2.Raffinose at 100 mM time-dependently induced the phosphorylation of AMPK.Besides,the expression of Atg12-5 and the ratio of LC3-II/I was upregulated by raffinose while the protein level of P62 was downregulated.Immunofluorescence analysis revealed an accumulation of LC3 puncta in cells treated with 100 mM raffinose for 24 h.Besides,raffinose also significantly upregulated Atg12-5expression,LC3-II/I ratio and downregulated the level of P62 in chondrocytes under H₂O₂.While the effects of raffinose on autophagy-related proteins were reversed by3-MA,an autophagy inhibitor.Compared with the model group,both the rate of senescence and the incidence of apoptosis were suppressed by raffinose pretreatment.While the suppression of raffinose on the rates of senescence and apoptosis was significantly relieved by 3-MA.Consistently,the inhibition of raffinose on senescence-related makers and apoptosis-related makers were also reversed by 3-MA.3.Compared with the model group,raffinose notably induced the phosphorylation of AMPK.While the promotion of raffinose on AMPK phosphorylation was suppressed by Compound C.Moreover,the regulation of raffinose on the expression of autophagy-related proteins was also reversed by Compound C.Conclusion1.Raffinose protects chondrocytes against H₂O₂-induced senescence and apoptosis.2.Raffinose induces autophagy in an AMPK-dependent manner,which confers protection against H₂O₂-induced senescence and apoptosis in chondrocytes.