Hydroxytyrosol Promotes The Autophagy Against Advanced Oxidation Protein Products-Induced Damage In Chondrocytes

Background and objectiveImpaired chondrocyte repair mechanisms,due to inflammation,oxidative stress and autophagy,play essential roles in the pathogenesis of rheumatoid arthritis(RA).Our previous study has illustrated that advanced oxidation protein products(AOPPs),as a marker of oxidative stress and a mediator of inflammation,triggered NADPH oxidase(NOX)-dependent ROS in the pathophysiology of RA.Autophagy acts as an adaptive response to protect chondrocytes from various environmental changes.Silent information regulator 1(SIRT1)is a nicotinamide adenine dinucleotide(NAD+)-dependent histone deacetylase,which plays a critical role in regulating cellular physiological processes,such as cell degeneration,cell growth,and cell survival.It is also a main signal pathway to regulate autophagy.Hydroxytyrosol(HT),a phenolic compound in olive oil,which possesses anti-inflammatory,antioxidant and autophagy-enhancing activities.Later experiments showed that HT could induce autophagy via SIRT1 signaling pathway in primary OA chondrocytes and human C-28/I2 chondrocytes,using its anti-oxidant and anti-inflammation when challenged with H2O2.However,the protective role of HT regulating Sirtl signaling against AOPPs in the pathophysiology of RA is still mostly unknown.Our study aims to investigate whether HT influences AOPPs and the possible underlying mechanisms.In-vitro studies,rat chondrocytes were treated with HT before AOPPs exposure.These results offer a novel molecular mechanism for the protective effects of HT and potential clinical application of HT to RA therapy.Methods1.The effect of HT on the rat chondrocytes under AOPPsOur first aim was to evaluate the fatal effects of AOPPs,chondrocytes cultures were subjected to increasing concentrations of AOPPs(0,50,100,200and 400ug/mL)for 24 h.Cell viability was determined by CCK-8 assay.The DPCs were stimulated with AOPPs 200ug/ml for 2 h,the intracellular reactive oxygen species(ROS)levels was detected by fluorescent DCFH-DA.These data determine that AOPPs treatment causes damage to chondrocytes.To further observe the protective effect of HT on chondrocytes under AOPPs,chondrocytes were pretreated with HT 75umol/ml for 30 minutes and then added AOPPs for 2h.The effects of HT on the biological activity and characteristics of chondrocytes after AOPPs treatment were detected by PCR,Western Blot,ELISA and DCFH-DA.2.HT regulates autophagy against AOPPs damage in rat chondrocytesAutophagy-associated protein ATG5,ATG7,P62 and LC3 were detected by western blot:the chondrocytes were treated with 200ug/ml for different time;200ug/ml AOPPs and different dose HT cotreatment for 2 hours.To further clarify the role of autophagy in regulating the action of HT on chondrocytes under AOPPs,the autophagy inhibitor chloroquine(CQ)was used to detect the relevant biological activities and characteristics.3.HT promotes autophagy in chondrocytes through SIRT1 stimulated with AOPPsThe chondrocytes were pretreated with HT 75?M for 30min,and then stimulated with AOPPs for 2h.SIRT1 levels were also determined by western blotting.The chondrocytes transfected with SIRT1 siRNA were used,and then detect the autophagy and relevant biological activities and characteristics.Results1.The effect of HT on the rat chondrocytes under AOPPsThe CCK-8 results showed that the activity of AOPPs decreased the cell viability of chondrocytes in a dose-dependent manner compared with the blank control group.After AOPPs treatment,ROS levels and inflammatory factors level and NADPH oxidase were significantly increased.Thus,AOPPs is cytotoxic to chondrocytes.In contrast,the pretreatment of HT significantly inhibits AOPPs-induced oxidative stress damage to chondrocytes.2.HT regulates autophagy against AOPPs damage in rat chondrocytesThe AOPPs treatment significantly decreased the expression of ATG5,ATG7,P62 and LC3,suggesting that AOPPs inhibits the expression of autophagy.In contrast,the pretreatment of HT in chondrocytes reversed this trend and significantly enhanced autophagy expression.To further verify the protection in the HT-mediated autophagy against AOPPs,autophagy inhibitor chloroquine(CQ)was used:the protection of HT in chondrocytes was significantly reversed,further demonstrating that regulating autophagy is an important mechanism for HT to protect chondrocytes from AOPPs damage.3.HT promotes autophagy in chondrocytes through SIRT1 stimulated with AOPPsHT significantly up-regulated the expression of SIRT1 protein compared with the AOPPs group in chondrocytes.The results showed that knockdown of SIRT1 reduced the effect of HT on the autophagy expression and increased NADPH oxidase level(NOX2 and NOX4).ConclusionHT-induced autophagy decreases intracellular ROS generation and inflammation in chondrocytes stimulated with AOPPs by SIRT1 pathway.