AAPH-induced Ferroptosis Influence Bone Development Of Chick Embryo

Oxidative stress is a condition that an imbalance occurs in oxidants and antioxidants system and the oxidants are prevailing.Oxidative stress linked with the pathophysiology of osteoporosis,hyperostosis,cervical spondylosis induced by diabetes and joint inflammatory diseases,on account of oxidative stress related to chondrocyte hypertrophy,osteoclastogenesis,bone resorption and bone mineral density.However,there is a lack of well-accepted and effective animal models to mimic oxidative stress-induced skeletal dysplasias,and the underlying mechanism remains inconclusive.To investigate oxidative stress-induced bone abnormal development of the newborns,we established a new 2,2’-azobis[2-methylpropionamidine] dihydrochloride(AAPH)chronic oxidative stress model in the chick embryos.First of all,we used chicken embryo to develop an in vivo AAPH induced oxidative stress model.The chick embryos were treated with different concentrations of AAPH at 0.25,0.50,1.00,2.00,4.00,8.00,10.00 μmol per egg,chick simple saline(control,0.72% sodium chloride)were injected into the albumen near the embryos topically on EDD 1.5,3.5 and 5.5 or every two days from EDD 1.5 to EDD 9.5.Embryos were futher incubated to reach EDD 7 or EDD 17,death rate and body weight were recorded.The internal characterization of embryos’ tibia was observed by paraffin section and HE staining.Through observing the model,we found that AAPH caused increasing death and growth retardation.The length of tibia was affected.The paraffin section showed that the proliferative zone was significantly shortened after AAPH-treated.To determine these abnormalities were caused by AAPH,oxidative stress damage of the tibia cartilages on EDD17 was confirmed by measuring the Malondialdehyde(MDA)content,Superoxide dis-mutases(SOD)activities and Glutathione(GSH)contents.Results showed that the cartilage of chick embryos suffered from severe oxidative stress.The result of iTRAQ indicated that ferroptosis was connected with AAPH-induced short limbs.The death rate,embryos weight,bone development,proliferative zone,gene and protein were observed after treating with AAPH(0.50 μmol/egg)and ferroptosis’ inhibitor Ferrostatin-1(Fer-1,50 nmol/egg).Fer-1 recovered the shorten length of limbs induced by AAPH,protected the structure of proliferative zone and suppressed the activated of ferroptosis pathways.ATDC5 cell line was origin from AT805,which can simulate chondrogenesis in the early stage of endochondral ossification.The cell death model was established by using ATDC5 cell with AAPH and Erastin.The inhibition rate,cartilage formation processes,lipid peroxidation,gene and protein were examined after treated with AAPH and Fer-1.There are various reports about the death caused by AAPH,however,we first discovered that AAPH-induced cell death included ferroptosis in ATDC5 cell.AAPH influenced the cartilage differentiation,elevated lipid peroxidation condition and activated the pathways of ferroptosis.Meainwhile,Fer-1 could significantly recover the damage caused by AAPH.In conclusion,this study has successfully developed an AAPH-induced oxidative stress model in chicken embryos.The mechanism of oxidative stress on bone abnormal development was further studied.It was confirmed that ferroptosis involved in the development of bone by affecting the proliferative zone of the growth plate,which provided a new therapeutic target for treatment.