Effect of SREBP-2 silencing on tunicamycin-induced endoplasmic reticu-lum stress in chondrocytes / 中国病理生理杂志

[ ABSTRACT] AIM:To explore the effect of sterol regulatory element-binding protein 2 ( SREBP-2) on tunicamy-cin-induced endoplasmic reticulum stress ( ERS) in chondrocytes.METHODS:After isolation of human normal chondro-cytes and osteoarthritis ( OA) chondrocytes, the normal cells were cultured and treated with tunicamycin and SREBP-2 siR-NA.After 24 h treatment, fluorescent quantitative RT-PCR ( RT-qPCR) was applied to quantify microRNA-185 ( miR-185) levels.The cell apoptotic rate was determined by flow cytometry.The expression of SREBP-2 and ERS-related pro-teins, C/EBP homologous protein (CHOP), phosphorylated eukaryotic initiation factor-2α(p-eIF2α) and activating tran-scription factor 4 (ATF4), and the expression of apoptosis-related proteins, Bcl-2, Bax and caspase-3, were determined by Western blot.The caspase-3 activity kit was used to determine the caspase-3 activity.RESULTS: Compared with hu-man normal chondrocytes, both SREBP-2 up-regulation and miR-185 down-regulation were observed in OA chondrocytes (P<0.05).SREBP-2 siRNA transfection enhanced tunicamycin-inhibited miR-185 level (P<0.05).miR-185 overex-pression reduced tunicamycin-induced SREBP-2 expression ( P <0.05 ) .OA control group and tunicamycin treatment group consistently resulted in ERS and cell apoptosis with concomitant enhancement of CHOP, p-eIF2αand ATF4 proteins, increases in Bax and caspase-3 proteins, and reduction of Bcl-2 (P<0.05).However, SREBP-2 silencing significantly re-versed these effects ( P<0.05) .The apoptotic rates were consistent with the expression tendency of apoptosis-related pro-teins (P<0.05).SREBP-2 siRNA transfection markedly down-regulated tunicamycin-induced caspase-3 activity, which was notably blocked by miR-185 inhibition (P<0.05).CONCLUSION:SREBP-2 silencing may inhibit tunicamycin-in-duced ERS and cell apoptosis via up-regulating miR-185 expression.

Effects of BAPTA-AM on acid-induced autophagy of rat articular chondrocytes and its possible mechanisms / 中国药理学通报

Aim To observe the effect of BAPTA-AM on extracellular acid-induced autophagy in rat articular chondrocytes and its possible mechanisms.Methods Rat articular chondrocytes were isolated from Sprague-Dawley rats and incubated with different pH medium. The states of autophagy were examined by acridine or-ange (AO ) staining .Moreover,the expressions of LC3 ,Beclin-1 ,ULK1 ,CaMKKβ,AMPK and mTOR were detected using Western blot or quantitative real-time PCR (qRT-PCR ). Intracellular calcium ([Ca2+]i )was analyzed by a Ca2+-imaging method. Results Compared with pH 6.0 group,BAPTA-AM could significantly decrease the activation of autophagyinduced by acid exposure,and the expressions of autophagy markers including LC3 Ⅱ,Beclin1 and ULK1were also decreased,accompanied with reduced acidinduced [Ca2 +]i influx,decreased proteins expressionof CaMKKβand phosphorylatedAMPK,and increasedphosphorylation of mTOR.Conclusion BAPTAAMcan significantly restrain acidinduced autophagy in ratarticular chondrocytes,the mechanism of which may beassociated with decreased Ca2 + influx.