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0.1g／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[（4.924±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／mm^3]低于对照组[（103.76±7.92）mg／mm^3]，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。

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/mm^3] was decreased compared to control group [（103.76±7.92） mg/mm^3]. 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） % J, P = 0.002 ; FAC group trabecular thickness （Tb. Th）： [（0.0572 0.018） mini 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.