Study On The Protective Effect Of Emodin On Oxidative Stress Damage In Rat Chondrocyte

Osteoarthritis（OA）is a chronic degenerative joint disease that seriously affects human and livestock health.The pathogenesis of OA is related to many factors,of which oxidative stress is one of the key factors.The antioxidant system regulated by Nrf2 is an important way to resist oxidation.Activation of Nrf2/ARE signaling pathway can inhibit cartilage matrix degradation.Emodin has antioxidant pharmacological effects,and its effect on oxidative damage of chondrocytes has not been studied.In this experiment,H ₂O₂ was used to induce chondrocytes to undergo oxidative stress,and the changes of ROS level,Nrf2/ARE signal pathway activity and matrix degrading enzyme protein content in chondrocytes after emodin treatment were observed.A rat OA model was established to observe the effect of emodin on the content of oxidative stress markers in rats.We evaluate its function value on OA and provide new ideas for prevention and treatment of OA.In vitro tests were divided into control group,H ₂O₂ group,low-dose Emodin group（5μM）,medium-dose emodin group（10μM）and high-dose emodin group（20μM）.Chondrocytes from the2nd generation were treated with 400μM H₂O₂ alone in H₂O₂ group,and Emodin group was treated with different concentrations of emodin for 6h,followed by H ₂O₂ and Emodin combined treatment for 12h.ROS levels in each group were detected by reactive oxygen species detection kit,and the expression of Nrf2 in nucleus,Nrf2 in cytoplasm,total protein HO-1,NQO1,MMP-3 and MMP-13 were detected by western blot.In vivo experiment,50 SD rats were randomly divided into control group,model group,low-dose Emodin group（20 mg/kg）,medium-dose Emodin group（50 mg/kg）and high-dose Emodin group（80 mg/kg）.The model group and Emodin group used ACLT surgery to establish rat OA models.At the 4th week after surgery,rats in Emodin group were injected intraperitoneally with Emodin,while rats in control group and model group were injected intraperitoneally with DMSO and saline（once every 2 days）.At the 6th week after t he operation,the rats in each group were decapitated and executed,and the femoral condyle and tibial plateau of the right hind limb were taken for general observation.Then,pathological sections were made and stained with HE and safranine O to observe the pathological changes of femoral condyle and tibial plateau in OA rats.Blood was taken from eyeball of rats.Serum was reserved,and the content and concentration changes of SOD,GSH,MDA,CTX-I and BGP were detected.In vitro test results showed that chondrocytes treated with H₂O₂ alone had a strong ROS level,and the expression levels of Nrf2,total proteins MMP-3 and MMP-13 in cartilage cytoplasm were significantly increased,while the expression levels of total proteins HO-1 and NQO1 were significantly decreased.After Emodin combined treatment,the intracellular ROS level gradually decreased.And the expression levels of Nrf2 in cytoplasm,MMP-3 and MMP-13 significantly decreased,and the expression levels of Nrf2 in nucleus,total proteins HO-1 and NQO1significantly increased.In vivo test results showed that HE staining of femoral condyle and tibial plateau in model group showed that articular cartilage surface structure was seriously missing,faults were obvious,and chondrocytes were atrophic and deformed.In Emodin group,the surface structure of cartilage gradually recovered and chondrocytes increased.Safranine O solid green staining showed that cartilage erosion was serious,red staining disappeared and chondrocytes decreased in model group.In Emodin group,cartilage layer structure gradually recovered,matrix red staining increased and chondrocytes increased.Serum tests showed that SOD and GSH contents in the model group were significantly lower than those in the control group,and the seru m contents in emodin group were significantly higher.The contents of MDA,CTX-Iand BGP in the serum of rats in the model group were significantly higher than those in the control group,while those in emodin group were significantly lower.Conclusion:（1）Emodin can activate Nrf2/ARE signaling pathway,improve the antioxidant capacity of chondrocytes and slow down the degradation of chondrocytes extracellular matrix induced by H₂O₂.（2）Emodin can alleviate the pathological changes of knee joint in OA rats,delay the progression of OA and protect articular cartilage.（3）Emodin can improve the antioxidant level in the body of rats and alleviate the oxidative damage and cartilage matrix degradation.