| Osteoarthritis(OA)is a common joint disease characterised by cartilage damage,the pathogenesis of which is still unclear.Under physiological conditions,a dynamic balance is maintained between the differentiation and death of chondrocytes and the synthesis and breakdown of cartilage matrix.In OA,this balance is disturbed and a large number of factors such as inflammation and oxidative stress in the joint tissue area can trigger excessive chondrocyte death,leading to degradation of the cartilage matrix and ultimately synergistically exacerbating articular cartilage degeneration in OA.Excessive chondrocyte death leads to cartilage degeneration,which in turn leads to loss of joint function,so research into the mode of chondrocyte death and its regulatory mechanisms will be useful in the prevention and treatment of OA.Ferroptosis is a form of death caused by iron-dependent oxidative damage and plays an important role in the process of cartilage injury.It is mainly caused by irondependent lipid peroxidation and reactive oxygen species(ROS)-induced damage resulting in changes such as cell membrane disruption and mitochondrial shrinkage,and is characterised by an overload of iron and ROS aggregates and involves a range of abnormal gene expression and signalling.In previous studies,our research team found that curcumin has a protective effect on OA cartilage damage,and there are literature reports that curcumin can inhibit ferroptosis,but its role in chondrocyte ferroprosis is still unclear,so it is important to find the key molecules regulating chondrocyte ferroptosis.Peroxiredoxin6(Prdx6)is a member of the peroxiredoxin family of enzymes,which are structurally unique and are protein molecules with multiple activities and involved in different cellular signalling pathways,and are key regulators of antioxidant defence and maintenance of cellular phospholipid homeostasis,scavenging ROS and related peroxides and protecting cells from damage caused by oxidative stress.It can scavenge ROS and related peroxides to protect cells from oxidative stress.Prdx6 has been shown to play an important role in a number of diseases such as tumours,inflammatory diseases and ischaemic stroke,and the expression and function of Prdx6 in other tissues has also received a lot of attention from researchers.In addition,it has been reported that curcumin can up regulate the expression of Prdx6 and protect cells from oxidative stress.However,whether curcumin regulates Prdx6 plays a role in the ferroptosis of articular chondrocytes remains to be determined.The occurrence of ferroptosis depends on the accumulation of lipid ROS and its mediated multiple signal cascades.Some studies have found that the activation of ASK1-p38 pathway induces ferroptosis,while Prdx6 negatively regulates ROS and then negatively regulates ferroptosis.So whether the negatively regulated ROS of Prdx6 regulates ASK1,and then affects the inhibitory effect of curcumin on ferroptosis of chondrocytes?Therefore,we investigated the role and molecular mechanism of Prdx6 in the inhibition of ferroptosis in articular chondrocytes by curcumin through in vivo and in vitro experiments.The following relevant experiments were carried out in this project.1.Effect of curcumin on ferroptosis and Prdx6 expression in articular chondrocytesHistopathological staining and histoprotein assays were performed on cartilage tissues from OA patients and OA rat models,divided into Normal and OA groups,to investigate the damage to cartilage tissues in OA patients and OA rats.The results showed that ferroptosis was present during osteoarthritis,and a significant decrease in Prdx6 protein expression may be associated with ferroptosis.The results showed that curcumin inhibited ferroptosis in chondrocytes in a concentration-dependent manner,increased cell viability and decreased cytotoxicity by MTT assay and LDH assay.The effects of curcumin on the mitochondrial morphology,mitochondrial membrane potential,lipid peroxidation level and ferroptosis marker proteins of chondrocytes induced by Erastin were examined by transmission electron microscopy,Calcein/PI staining,Rh123 staining,flow cytometry,GSH kit,Western Blot method and immunofluorescence method.The results showed that curcumin could up-regulate Prdx6 protein expression,reverse Erastin-induced mitochondrial damage and lipid ROS accumulation in chondrocytes,and protect chondrocytes from ferroptosis.These results suggest that curcumin may inhibit chondrocyte ferroptosis by up-regulating Prdx6 protein expression.2.Role and molecular mechanism of Prdx6 in curcumin inhibiting ferroptosis of articular chondrocytesThe effects of Prdx6 protein overexpression on chondrocyte ferroptosis were examined by LDH,Western Blot and flow cytometry.The results showed that overexpression of Prdx6 inhibited Erastin-induced chondrocyte ferroptosis,reduced cytotoxicity and lipid peroxidation levels,downregulated the expression of ACSL4 and ASK1,and exerted anti-oxidative stress effects,whereas the Prdx6-i PLA2 inhibitor MJ33 exacerbated Erastin-induced ferroptosis in chondrocytes and significantly increased lipid peroxidation levels.The binding of curcumin molecules to Prdx6 protein was examined by molecular docking assay and the results showed a strong interaction between the two.The effects of ASK1 inhibitor TC ASK10 on Erastin-induced ferroptosis cell viability,cytotoxicity,mitochondrial membrane potential,lipid peroxidation levels and ferroptosis marker proteins in chondrocytes were examined by MTT assay,LDH assay,Calcein/PI staining,Rh123 staining,flow cytometry,GSH kit and Western Blot assay.The results showed that TC ASK10 was able to reverse Erastininduced mitochondrial damage and lipid ROS accumulation in chondrocytes and protect chondrocytes from ferroptosis.These results suggest that overexpression of Prdx6 protein can scavenge lipid ROS,exert anti-oxidative stress effects,inhibit the activation of ROS-ASK1 signalling axis and ultimately inhibit ferroptosis in OA articular chondrocytes.3.Curcumin regulates the effect of Prdx6 on ferroptosis of articular chondrocytes in OA rats and the protective effect of arthritisThe rat osteoarthritis model was established by ACL severance with medial meniscus removal.The rats were divided into six groups,namely Normal,OA,MJ33,Cur,MJ33 + Cur and HA groups.The effects of curcumin and MJ33 on the cartilage tissues of OA rats were examined by OARSI cartilage damage score,H&E staining,and staining with red solid green.The results showed that the OA group had a defective joint surface and curcumin had a protective effect on OA cartilage,while the MJ33 group had severe cartilage damage and could inhibit the protective effect of curcumin.The expression of Prdx6 and ACSL4,a marker of ferroptosis,was detected by tissue immunofluorescence.The results showed that the expression of Prdx6 protein was significantly reduced and that of ACSL4 protein was significantly increased in the OA group,and that MJ33 exacerbated the process of OA and affected the protective effect of curcumin on articular cartilage.The above results suggest that the Prdx6-i PLA2 inhibitor MJ33 can inhibit the arthritic protective effect of curcumin,further demonstrating that Prdx6 plays a key role in curcumin’s inhibition of ferroptosis in OA articular chondrocytes.In summary,our results suggest that Prdx6 negatively regulates the activation of the ROS-ASK1 axis,which in turn inhibits OA chondrocyte ferroptosis,and that curcumin inhibits chondrocyte ferroptosis by upregulating Prdx6 expression,exerting its protective effect against OA.In conclusion,Prdx6 regulation of the ROS-ASK1 signaling axis plays an important role in the inhibition of chondrocyte ferroptosis by curcumin,which provides a new target and strategy for the prevention and treatment of OA. |
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