The Role And Mechanism Of Chondrocyte Ferroptosis In The Progression Of Osteoarthritis

Objective1.To investigate whether inflammatory environment and iron overload induce chondrocyte ferroptosis.2.To investigate the effect of ferroptosis on the phenotype of chondrocytes and cartilage.3.To investigate the activation level of Nrf-2 antioxidant system in inflammatory environment and iron overload,and its relation with chondrocyte ferroptosis.4.To investigate the effect of ferrostatin-1,a classic ferroptosis inhibitor,on the pathological progress of OA.Methods1.Mouse chondrocytes were treated with IL-1β to simulate the inflammatory environment in vitro,and treated by ferric ammonium citrate(FAC)to simulate the iron overload environment in vitro.The morphological changes of mitochondria were observed by transmission electron microscope;the expression level and intracellular distribution of GPX4,as well as the expression levels of three common protein markers of ferroptosis(ACSl4,p53 and SLC7A11),were detected;The levels of Reactive oxygen species(ROS),Lipid ROS and Malondialdehyde(MDA)were measured.2.Mouse chondrocytes were treated with IL-1β or FAC with or without Ferrostatin-1,and the intracellular ROS,Lipid ROS and MDA were detected.CCK-8 assay were used to detect the protective effect of ferrostatin-1 on the cytotoxicity of IL-1β and FAC.The effects of ferrostatin-1 on the expression of four protein markers of ferroptosis,includes GPX4,ACSL4,P53 and SCL7A11,were detected by Western blot;the effects of Ferrostatin-1 on the expression of GPX4 were visualized by immunofluorescence staining.3.Mouse chondrocytes were treated with Erastin,the most classic inducer of ferroptosis,and the expression level of GPX4 were detected by Western blot to verify the efficiency of erastin.The expression levels of MMP13 and collagen Ⅱ were detected by Western blot.Further,Erastin were injected into the articular cavity of mice,and the expression of MMP13 and collagen Ⅱ in articular cartilage were detected by immunohistochemical staining to verify the results of in vitro experiment.4.Mouse chondrocytes were treated with Ferrostatin-1,a classic inhibitor of ferroptosis,in inflammatory environment(IL-1β treatment)or iron overload(FAC treatment).The expression levels of MMP13 and collagen Ⅱ were detected by Western blot.5.Nrf-2 expression of mouse chondrocytes were knocked-down by siRNA,and chondrocytes were then treated with IL-1β or FAC.The expression level of GPX4 were detected by western blot to verify the inhibitory effect of Nrf-2 on ferroptosis.Furthermore,Mouse chondrocytes were treated with IL-1 β or FAC with or without ferrostatin-1,then,the expression levels of Nrf-2 and its downstream antioxidant molecules HO-1,NQO-1 and Trx were detected by western blot.6.In order to establish a OA model,mouse were surgically constructed destabilization of medial meniscus(DMM).The treatment group received intraarticular injection of ferrostatin-1(0.1mg/kg in low concentration group,1mg/kg in high concentration group,twice a week for 8 weeks)while sham surgery group and OA group received equal volume of vehicle.The degeneration of articular cartilage was evaluated by safranin O-fast green staining,and the progression of arthritis was evaluated by OARSI score.The expression levels of GPX4 and collagen Ⅱ in articular cartilage were detected by immunohistochemistry staining.Results1.We found mitochondria with characteristic morphological changes of ferroptosis in IL-1β and FAC treated chondrocytes.Also,IL-1β and FAC decreased the protein expression levels of GPX4 and SLC7A11 while increased the protein expression levels of ACSL4 and P53.Immunofluorescence staining also showed that the expression level of GPX4 protein was significantly decreased.The granular fluorescence of GPX4 in cytoplasm also decreased or disappeared.Further,both IL-1β and FAC can lead to excessive accumulation of ROS,Lipid ROS and MDA in mouse chondrocytes.2.Ferroptosis inhibitor ferrostatin-1 inhibited the cytotoxicity of IL-1β and FAC.It also significantly reduced the production of intracellular ROS,Lipid ROS and MDA,increased the protein expression levels of GPX4 and SLC7A11,and decreased the protein expression levels of ACSL4 and P53 in inflammatory environment(IL-1β treatment)and iron overload(FAC treatment).3.Ferroptosis inducer Erastin inhibited the protein expression of collagen Ⅱ while up-regulated MMP13 expression both in vitro and in vivo.4.Ferrostatin-1 rescued the collagen Ⅱ expression that inhibited by IL-1β and FAC while it did not inhibit the expression of MMP13 induced by IL-1β and FAC.5.Knock-down of Nrf-2,when chondrocytes were treated with IL-1β or FAC,inhibited the expression of GPX4 protein.IL-1β treatment increased the protein expression levels of Nrf-2 and its downstream antioxidant molecules(HO-1,NQO-1 and Trx)while Ferrostatin-1 further increased the expression of these proteins.FAC treatment had no significant effect on the expression of Nrf-2,HO-1,NQO-1 and Trx while Ferrostatin-1increased the expression of these proteins.6.The expression of GPX4 protein in OA group was lower than control group.Intraarticular injection of 1 mg/kg ferrostatin-1 mitigated the degeneration of articular cartilage and reduced the OARSI score.Meanwhile,intraarticular injection of 1 mg/kg ferrostatin-1 increased the protein expression of GPX4 and collagen Ⅱ in articular cartilage.Conclusions1.Both inflammatory environment(IL-1β treatment)and iron overload(FAC treatment)induced chondrocyte ferroptosis.2.Erastin,the classic inducer of ferroptosis,suppressed the expression of collagen Ⅱ while enhanced the the expression of MMP13 in vivo and in vitro.Ferroptosis inhibitor ferrostatin-1 reversed the inhibition of collagen Ⅱ expression induced by IL-1β and FAC.These results indicate that ferroptosis led to phenotype changes that similar to OA chondrocytes.3.Inactivation of Nrf-2 antioxidant system facilitated the chondrocytes ferroptosis in inflammatory environment and iron overload environment.Although Nrf-2 antioxidant system was activated when treated with IL-1β,its activation level was low.Ferrostatin-1could significantly activated Nrf-2 antioxidant system in inflammatory environment.Although iron overload led to oxidative stress,Nrf-2 antioxidant system was not activated in iron overload environment.Ferrostatin-1 activated Nrf-2 antioxidant system in iron overload environment.Therefore,the low activation level or inactivation of Nrf-2antioxidant system might be an important cause of ferroptosis under oxidative stress.4.The GPX4 protein level in OA cartilage was significantly decreased.Intraarticular injection of 1 mg/kg ferrostatin-1 attenuated the degeneration of articular cartilage,increased the expression of GPX4 and collagen Ⅱ and delayed the progression of OA.5.Our study indicates that chondrocyte ferroptosis was involved in the pathological progress of OA,and inhibition of ferroptosis could be a potential ideal target for novel therapies of OA.