The Research Of The Mechanism Of NOX/NADPH Oxidase Mediated ROS Generaton In Steroid Induced Osteoblast Apoptosis

Objective:Osteoblast,which plays an important role in formation and repair of bone tissue,is closely related to steroid-induced osteonecrosis of femoral head(SANFH).Accumulating evidences have demonstrated excessive glucocorticoid(GC)usage can induce oxidative stress in bone tissue,which could eventually lead to osteoblast aopotosis.In addition,reactive oxygen species(ROS)is an important molecule in mediating oxidative stress.However,the exact source and pathogenesis of ROS in SANFH remained uncertain.In this study,to clarify the role of nonphagocytic nicotinamide adenine dinucleotide phosphate-reduced oxidase(NADPH oxidase,NOX)in ROS generation and osteoblast apoptosis,dexamethasone was used to establish a high-dose GC environment in vitro.Methods:According to culture conditions,murine osteoblastic MC3T3-E1 cells at passage 3 were divided into control group;Dexamethasone group(DEX group);DEX+ROS scavenger n-acetyl-l-cysteine(NAC)group(DEX+NAC group);NAC group;DEX+NOX inhibitor diphenyleneiodonium(DPI)group(DEX+DPI group);DPI group.At 24 hours after culture,the morphology of osteoblast was observed by inverted phase contrast microscope.Cell viability was determined by MTT assay.The generation of ROS in osteoblast was measured using a fluorescent probe DCFH-DA.The apoptosis of each group was detected with Annexin V-FITC/PI,and then cell apoptotic rate was quantitated by flow cytometry.The mRNA level and protein expression of NOX1,NOX2 and NOX4 were detected with real-time quantitative PCR and Western-Blot.In addition,after targeted silence of NOX1,NOX2 and NOX4 with small interfering RNA(siRNA),osteoblast was divided into control group;negative control group;DEX group;siRNA group;DEX+siRNA group,the ROS generation was detected by a fluorescent probe DCFH-DA and cell apoptosis was observed with TUNEL assay.Results:1.After treatment with 1000 nM dexamethasone for 24 hours,compare to control group,the result of inverted phase contrast microscope and MTT showed that osteoblast in DEX group exhibited obvious signs of shrinkage and deformation with decreased cell viability.After intervene with NAC and DPI,morphology of osteoblast was good with increased viability of osteoblast.The difference has statistical significance(P<0.05).2.Compared to control group,the production of ROS and apoptotic rate in DEX group were significantly increased(P<0.05).In addition,compare to the result of DEX+NAC group and DEX+DPI group,it presented that NAC or DPI significantly decreased the formation of ROS and apoptotic rate of osteoblast(P<0.05).3.Compare to control group,the mRNA level and protein expression of NOX1 and NOX4 of osteoblast were significantly increased in DEX group(P<0.05).In addition,the expression of NOX2 has no significant difference(P>0.05).4.NOX1,NOX2,and NOX4 siRNAs were used to confirm the role of specific NOX in ROS generation of osteoblast.After siRNA transfection,osteoblasts were cultured for 24 h with 1000 nM DEX.Then,the production of ROS was detected by immunofluorescence.The result showed that NOX1 or NOX4 knockdown significantly decreased the fluorescence intensity(P<0.05).However,such effects were not observed following NOX2 knockdown(P>0.05).5.After siRNA significant transfection,osteoblasts were cultured for 24 h with 1000 nM DEX.Then,the result of TUNEL assay showed that NOX1 or NOX4 knockdown significantly decreased the osteoblast apoptosis(P<0.05).However,such effects were not observed following NOX2 knockdown(P>0.05).Conclusion:High-dose DEX induces a marked increase in NOX1-derived and NOX4-derived pathological ROS generation in osteoblast,which could lead to osteoblast apoptosis.However,there was no significant different in mRNA and protein expression of NOX2.Therefore,NOX1 and NOX4 are probably important targets in contributing to bone repair in early steroid-induced avascular necrosis of the femoral head.