The Role And Mechanism Of Iron Ion Homeostasis In The Progress Of Osteoarthritis

Objective 1.To investigate the changes of iron homeostasis in inflammatory environment of osteoarthritis and the effect of iron overload on osteoarthritis 2.To explore the role and mechanism of iron-dependent oxidative stress injury in osteoarthritis cartilage induced by iron overload 3.To study the protective effect and mechanism of icariin on iron overload induced bone loss and to explore the application of traditional Chinese medicine in the treatment of iron overload induced diseasesMethods 1.Primary mouse chondrocytes were cultured and pro-inflammatory factor IL-1? or TNF-? were used to detect the changes of iron ion influx and efflux,then the changes of iron regulatory proteins were further detected.10-week-old C57/BL6 male mouse was used to establish a mouse model of DMM osteoarthritis by inducing medial meniscus instability in the knee joint.The left leg was given a sham operation as control.The expression of TFR1 and FPN was detected.Finally,the effect of iron overload on chondrocytes was examined by an iron overload cell model.2.Chondrocytes were treated with different concentrations of ferric ammonium citrate (FAC),the ROS level and mitochondrial membrane potential in chondrocytes were examined.Then antioxidant N-acetyl-L-cysteine(NAC)was used to detect the role of iron overload-induced oxidative stress and mitochondrial function disruption in the progression of osteoarthritis caused by iron overload.Western Blot was then used to detect the expression of the mitochondrial division-associated protein MFF/DRP1.3.To establish iron overloaded mice model,mice were treated intraperitoneally once a week for 2 months with iron dextran(500mg/kg).Icariin was intragastrically administered 100mg/kg per day.After 2 months,Micro-CT was used to quantitatively assess the bone status.ELISA was used to detect serum levels of bone turnover markers TRAP,CTX-1,ALP and OCN.The MC3T3-E1 osteoblast cell line was used to detect the protective effect of icariin on osteogenic inhibition caused by iron overload,and the mechanism of icariin was further investigated by detecting reactive oxygen species levels and mitochondrial function.Results 1.We found that IL-1? or TNF-? treatment could promote the expression of TFR1,DMT1 and inhibit the expression of FPN.Thereby promoting the influx of iron ions,inhibiting the outflow of iron ions.Animal experiments also confirmed that osteoarthritis articular cartilage increased TFR1 expression and decreased FPN expression.Chondrocytes were then treated with ferric ammonium citrate to mimic chondrocytes iron overload and found that FAC promoted the expression of MMPs, while iron chelator intervention blocked iron ion flow and decreased MMPs expression.2.FAC treatment could promote the production of reactive oxygen species and decrease the mitochondrial membrane potential in chondrocytes.Western Blot results suggested that mitochondrial fission protein expression was elevated,while antioxidant NAC could not only reverse FAC induced damage to mitochondrial function,but also inhibit FAC induced chondrocyte apoptosis and increase the expression of MMPs.3.Iron overload could cause osteoporosis in mice,and bone mineral density was significantly increased after icariin administration.Iron overload could lead to an increase in reactive oxygen species and a decrease in mitochondrial membrane potential in MC3T3-E1 osteoblasts.Icariin intervention significantly reversed this trend.Icariin administration could also significantly reduce the deposition of iron in the bone marrow.Conclusion 1.Our results demonstrate that pro-inflammatory cytokines in osteoarthritis could promote iron ion influx by up-regulating the expression of TFR1 and DMT1,and down-regulating the expression of FPN to inhibit iron ion efflux,resulting in iron overload of chondrocytes.Iron overload could promote the expression of MMPs in chondrocytes.2.Our results demonstrate that iron overload may disrupt mitochondrial function by promoting the production of reactive oxygen species,leading to increased chondrocyte apoptosis and expression of matrix metalloproteinase MMPs.3.Our results also indicate that icariin can be used to treat iron overload-related diseases...