Study On The Protective Effect Of Proanthocyanidins Against Hydrogen Peroxide-induced Oxidative Damage In Sheep Temporomandibular Joint Disc Cells

Objective:Oxidative stress is one of the main causes of temporomandibular joint（TMJ）degeneration,and H₂O₂-mediated oxidative stress may play an important role in TMJ degeneration.Proanthocyanidins are natural polyphenolic compounds with antioxidant,apoptosis-inhibiting and anti-inflammatory effects,but their role in TMJ degeneration is not yet clear.In this experiment,a model of oxidative stress in sheep TMJ disc cells was established using hydrogen peroxide（H₂O₂）to investigate the effect of H₂O₂on oxidative stress damage in TMJ disc cells.The protective effect of proanthocyanidins（PC）on H₂O₂-induced oxidative damage in TMJ disc cells was further investigated.Methods:1.Fresh 3-month-old sheep heads were selected,TMJ disc cells were isolated and cultured in vitro,and passed to the second generation.TMJ disc cells were treated with concentrations of 0,50,100,200,400 and 800μM H₂O₂for 12,24 and 48 h.Cell viability was detected by CCK-8,reactive oxygen levels by DCFH-DA fluorescence probe,and SOD activity and MDA content by colorimetric assay to screen the H₂O₂-induced oxidative stress injury model in TMJ disc cells.The optimal concentration and treatment time of H₂O₂-induced oxidative stress damage model were screened1.Different concentrations of PC（0,5,10,20,40μM）were applied to treat TMJ disc cells for 24 h.The effect of PC on TMJ disc cell viability was detected by CCK-8.Then the experiments were grouped into control group,H₂O₂group,low concentration PC group（cells were pretreated with 5μM PC for 24 h and then incubated with 400μM H₂O₂for 24 h）,medium concentration PC group（cells were pretreated with 10μM PC for 24 h and then incubated with 400μM H₂O₂for 24 h）,and high concentration PC treatment group（cells were pretreated with 20μM PC 24h and then incubated with 400μM H₂O₂for 24h）,and cell viability was detected using CCK-8 method,reactive oxygen species（ROS）level was detected by DCFH-DA fluorescent probe,apoptosis rate was detected by flow cytometry,and colorimetric assay(Superoxide dismutase（SOD）activity,MDA（malondialdehyde,MDA）content,q RT-PCR to detect inflammatory factors including interleukin-1β（IL-1β）,tumor necrosis factor-α(tumornecrosis factor-α（TNF-α）,interleukin-6（IL-6）,extracellular matrix anabolic factors including typeⅠcollagen（COLⅠ）,Aggrecan（Agg）,extracellular matrix catabolic factors including matrix metalloproteinase（MMP）-1,3,9 m RNA expression levels.Results:1.The inhibitory effect of cell viability was not significant when the H₂O₂concentration was 100μM,and cell viability decreased with the increase of H₂O₂concentration when the H₂O₂concentration exceeded 100μM,which could inhibit the growth of TMJ disc cells（p<0.05）.The cell survival rate of TMJ disc cells treated with 400μM H₂O₂for 24 h was 60.57%,which was significantly lower compared with the control group（p<0.05）.And the ROS level and MDA content in TMJ disc cells in the H₂O₂group were significantly higher than those in the control group,while the SOD activity was significantly lower compared with the control group（p<0.05）.2.Compared with the control group,TMJ disc cell viability was significantly reduced,ROS level,MDA content was significantly increased and SOD activity was decreased in the H₂O₂group.Compared with H₂O₂group,5,10 and 20μM PC treatment groups could increase TMJ disc cell viability under H₂O₂induction（p<0.05）,decrease ROS level,MDA content,and increase intracellular SOD activity in a dose-dependent manner.The flow results showed that the apoptosis rate was higher in the H₂O₂group compared with the control group,while the apoptosis rate was significantly lower after pretreatment with PC than in the H₂O₂group（p<0.05）.the expression of intracellular inflammatory factors（IL-1β,TNF-α,IL-6）was significantly higher in the H₂O₂group,while the expression of IL-1β,TNF-α,IL-6was decreased after PC pretreatment（p<0.05）.In addition,PC increased（p<0.05）the expression of COLⅠand Aggrecan under H₂O₂induction,and also inhibited（p<0.05）the expression of MMP-1,3,and 9 and suppressed extracellular matrix degradation.Conclusions:1.The optimal action condition for establishing TMJ disc cell oxidative stress model was 400μM H₂O₂for 24h.2.Based on a model of H₂O₂-induced oxidative stress injury in TMJ disc cells,PC exerts protective effects against oxidative damage in TMJ disc cells by antioxidant,anti-inflammatory,inhibiting apoptosis and slowing down extracellular matrix degradation.PC could be a potential drug in the clinical treatment of TMJ degeneration.