Effect Of Hypoxia/Reoxygenation On The Biological Of IGF System And The Inflammatory Mediators In Synoviocytes

Objective Hypoxia/reoxygenation(H/R)plays an important role in the pathogenesis of osteoarthritis.Fibroblast-like synoviocytes(FLS),which are highly sensitive to H/R,are thought to be associated with cartilage degradation during osteoarthritis development.In this study,we investigate the effect of hypoxia/reoxygenation(H/R)on the expression of insulinlike growth factor(IGF)system and the inflammatory mediators in cultured fibroblast-like synoviocytes(FLS).Method Isolation and culture of normal primary synovial cells and identification by immunofluorescence.Establishment of H/R model: Cell flasks were placed in turn in hypoxia conditions for 2 h and normoxic conditions for 2 h,repeating for 3 cycles.Cell culture supernatant was collected and blood oxygen analyzer was used to monitor the oxygen partial pressure under culture condition.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of chemokines CCL-5,interleukin-1 ?(IL-1?)and interleukin-6(IL-6)in cell culture supernatant.The m RNA levels of insulin-like growth factor-1(IGF-1)and insulin-like growth factor binding protein-3(IGFBP-3)in FLS were detected by PCR.The protein levels of IGF-1,IGFBP-3,COX-2,ADAMTS-5,MMP-13,INOS,P-P65,P65,P-I?B and I?B in FLS were detected by western blot.Reactive oxygen species(ROS),mitochondrial membrane potential(MMP)and mitochondrial permeability transition pore(MPTP)were measured by flow cytometry.The morphological changes of mitochondria were detected by transmission electron microscopy.The nuclear translocation of P65 in FLS was detected by immunofluorescence.Results H/R increased levels of TNF-?-induced C-C chemokine ligand 5(CCL5),interleukin-1?(IL-1?)and interleukin-6(IL-6)in cell-free culture supernatants.H/R also increased the expression of TNF-?-induced insulin-like growth factor binding protein 3(IGFBP-3),downregulated the expression of insulin-like growth factor 1(IGF-1),promoted the loss of mitochondrial membrane potential(MMP),the openness of mitochondrial permeability transition pore(MPTP),the release of intracellular reactive oxygen species(ROS),and mitochondrial matrix swelling,outer membrane rupture and decrease in cristae.Furthermore,H/R induced the expression of catabolic factors and activated the NF-?B signaling pathway in FLS.Conclusion H/R may play a role in inducing inflammation and increase TNF-?-induced inflammatory effect in FLS,contributing to osteoarthritis pathogenesis.