铁蓄积通过还原型烟酰胺嘌呤二核苷酸磷酸氧化酶4激活活性氧自由基抑制间充质干细胞与体内成骨功能
Iron accumulation induces reactive oxide species to inhibits mesenchymal stem cells and osteogenesis via nicotinamide adenine dinucleotide phosphate reduced oxidase 4 activation
目的 研究铁蓄积对间充质干细胞(MSCs)影响,探索铁蓄积在MSCs层面对成骨功能抑制的可能机制.方法 体内实验,将8周龄C57雄性小鼠,分为对照组(Ctrl)和实验组(FAC),实验组腹腔注射枸橼酸铁铵(FAC)每周0.1 g/kg,8周后两组均检测:血清铁蛋白(FER)、成骨标志物(P1NP)、股骨远端骨小梁三维形态重建和空间结构参数、MSCs占骨髓细胞比例.体外实验,取C57雄性小鼠骨髓分离培养MSCs细胞,分为细胞对照组和细胞实验组,细胞实验组加FAC干预24h,两组均行活性氧自由基(ROS)水平、还原型烟酰胺嘌呤二核苷酸磷酸氧化酶4(NOX4)表达水平检测.结果 FAC组血清铁蛋白[(14.72±0.97)μg/L]高于对照组[(4.92±0.82) μg/L],P=0.002;FAC组骨髓MSCs占骨髓比例[(2.08±0.98)%]低于对照组[(3.98±0.82)%],P=0.004;FAC组细胞ROS的Mean值(56.26 ±8.36)高于对照组(257.65±12.69),P=0.000;FAC组细胞NOX4蛋白表达上升;FAC组P1NP[(4.81±0.51)ng/ml]低于对照组[(10.06±0.96) ng/ml],P=0.001.micro-CT检测的FAC组骨密度[(53.12±9.86) mg/mm3]低于对照组[(103.76±7.92) mg/mm3],P =0.001;FAC组骨小梁空间结构参数显示:FAC组骨体积分数(BV/TV):[(11.23±1.86)%]低于对照组[(18.63±2.02)%],P=0.002;FAC组骨小梁厚度(Tb.Th):[(0.057±0.018) mm]低于对照组[(0.083±0.006) mm],P=0.003;FAC组骨小梁数目(Tb.N):[(0.92±0.22) N/mm]低于对照组[(1.25 ±0.18) N/mm],P=0.004.结论 铁蓄积后,成骨指标降低可能与MSCs受抑制有关,MSCs受抑制可能与铁蓄积诱导NOX4激活ROS相关.
更多Objective To study iron accumulation affect on mesenchymal stem cells (MSCs),and to explore iron accumulation in MSCs level pathogenesis.Methods In vivo,6-8 weeks C57 male mice were devived into control group and experimental group,the experimental group was intraperitoneally injected with ferric ammonium citrate (FAC) of 0.1 g/kg/week intervention in eight weeks,the two groups were tested:serum ferritin (FER),osteogenesis markers procollagen type 1 amino-terminal propeptide (P1NP),distal femur trabecular bone reconstruction of three dimensional form and spatial structure parameter,MSCs accounted for the proportion of bone marrow cells.In vitro,6-8 weeks male C57 mice bone marrow tissue were separation,MSCs were cultivated and devided after reaching a certain number in the control group and experimental group.After 24 h FAC intervention in experimental group,two groups were tested:reactive oxygen species (ROS),nicotinamide adenine dinucleotide phosphate reduced oxidase 4 (NOX4) expression level.Statistical analyses were performed.Results FAC group serum FER [(14.72 ±0.97) μg/L] was elevated compared to control group [(4.92 ± 0.82) μg/L],P =0.002.FAC group bone marrow MSCs [(2.08 ± 0.98) %] was reduced compared to control group [(3.98 ± 0.82%)%],P =0.004.FAC group MSCs ROS mean value (56.26 ±8.36) was exceed to control group (257.65 ± 12.69),P =0.000;MSCs NOX4 cell protein was increased compared with the control group;Osteogenesis index P1NP in FAC group [(4.81 ±0.51) ng/ml] was decreased compared to control group [(10.06 ±0.96) ng/ml],P=0.001.The micro-CT detection of bone mineral density in FAC group [(53.12 ± 9.86) mg/mm3] was decreased compared to control group [(103.76 ± 7.92) mg/mm3].FAC group spatial structure parameter showed bone volume/ tissue volume (BV/TV):[(11.23 ± 1.86)%] was decreased compared to control group [(18.63 ±2.02)%],P =0.002;FAC group trabecular thickness (Tb.Th):[(0.057± 0.018)mm] was decreased to control group [(0.083± 0.006) mm],P=0.003;FAC group trabecular number (Tb.N):[(0.92±0.22) N/mm] was decreased compared to control group [(1.25 ± 0.18) N/mm],P =0.004.Conclusion The osteogenetic activity is restrained after iron accumulation may be associated with increased MSCs ROS,MSCs ROS increased may be related to iron accumulation induced NOX4 activation.
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