活性氧介导铁过载诱导成骨细胞坏死性凋亡的调控机制  

Regulatory mechanism of reactive oxygen species-mediated iron overload to induce necroptotic apoptosis in osteoblasts

作　　者：张松[1] 陈松峰 田青 唐恒涛[1] Zhang Song;Chen Songfeng;Tian Qing;Tang Hengtao(Department of Orthopedics,the First Affiliated Hospital,Zhengzhou University,Zhengzhou 450052,China)

机构地区：[1]郑州大学第一附属医院骨科,450052

出　　处：《中华实验外科杂志》2022年第2期312-315,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金(81902253)。

摘　　要：目的探讨铁过载是否能够诱导成骨细胞发生坏死性凋亡, 并探讨其分子机制。方法采用50、100、200 μmol/L枸橼酸亚铁(FAC)对小鼠胚胎成骨细胞系(MC3T3-E1)进行干预, 以模拟铁过载状态;同样将加入等量生理盐水, 设为对照组。细胞计数试剂盒8(CCK-8)检测铁过载对成骨细胞活性的影响, 流式细胞仪检测铁过载诱导成骨细胞的坏死率, 活性氧荧光探针(DCFH-DA)染色检测铁过载诱导成骨细胞内活性氧的水平, 蛋白印记法(Western blot)检测铁过载干预后成骨细胞内坏死性凋亡标志性分子受体相互作用蛋白激酶1(RIPK1)、受体相互作用蛋白激酶3(RIPK3)以及混合谱系激酶域样蛋白(MLKL)表达水平的变化。为了明确活性氧在铁过载诱导的成骨细胞坏死性凋亡中的作用, 我们应用抗氧化剂N-乙酰胺半胱氨酸(NAC)干预铁过载组的成骨细胞, 观察抑制活性氧后, 坏死性凋亡相关分子RIPK1、RIPK3及MLKL表达的变化。多组间比较应用单因素方差分析, 两组间比较采用t检验。结果 CCK-8结果显示, 铁过载干预120 h后能够明显抑制成骨细胞的活性, 和空白对照组(1.77±0.04)比较, FAC 50 μmol/L组(1.38±0.04)、FAC 100 μmol/L组(1.01±0.08)和FAC 200 μmol/L组(0.81±0.08)成骨细胞活性均受到抑制, 且具有浓度依赖性, 差异有统计学意义(t=19.22、12.22、17.07, P<0.05)。流式细胞仪检测结果显示, 铁过载干预120 h后导致成骨细胞坏死率明显增加, 和空白对照组[(3.42±0.31)%]比较, FAC 50 μmol/L组[(10.25±4.62)%]、FAC 100 μmol/L组[(15.20±6.66)%]和FAC 200 μmol/L组[(41.53±3.97)%], 差异有统计学意义(t=4.116、3.061、16.560, P<0.05)。DCFH-DA染色后, 流式细胞仪检测结果显示, 铁过载干预120 h后导致成骨细胞内活性氧水平明显增加, 和空白对照组(1.00±0.00)比较, FAC 50 μmol/L组[(4.00±1.19)%]、FAC 100 μmol/L组[(8.10±3.63)%]和FAC 200 μmol/L组[(10.63±2.46)%]成骨细胞内活性氧水�Objective To study whether necroptosis contributes to iron overload-induced osteoblastic cell death and related underlying mechanisms.Methods MC3T3-E1 cells were treated with 50,100,200μmol/L ferric ammonium citrate(FAC)to simulate iron overload state.The control group was treated with normal saline.Cell counting kit-8(CCK-8)assay was used to determine the effect of iron overload on osteoblast activity.Flow cytometry was used to determine the necrosis rate of osteoblasts induced by iron overload.Dichloro dihydro fluorescein diacetate(DCFH-DA)staining was used to detect the level of reactive oxygen species(ROS).Western blotting was used to detect the expression of receptor-interacting protein kinase 1(RIPK1),receptor-interacting protein kinase 3(RIPK3)and mixed lineage kinase domain-like(MLKL).In order to investigate the role of ROS in iron overload-induced necrotic apoptosis of osteoblasts,N-acetylcysteine(NAC)was used to interfere with the expression of necrotic apoptoses-related molecules RIPK1,RIPK3 and MLKL.One-way ANOVA was used for comparison between multiple groups,and t test was used for comparison between two groups Results CCK-8 assay results showed that iron overload significantly inhibited the activity of osteoblasts at 120 h after intervention as compared with the blank control group(1.77±0.04).The activity of osteoblasts in FAC 50μmol/L group(1.38±0.04),FAC 100μmol/L group(1.01±0.08)and FAC 200μmol/L group(0.81±0.08)was all inhibited in a concentration-dependent manner.The difference was statistically significant(t=19.22,12.22,17.07,P<0.05).The results of flow cytometry showed that the osteoblast necrosis rate increased significantly after iron overload intervention[for FAC 50μmol/L group,(10.25±4.62)%;for FAC 100 mol/L group,(15.20±6.66)%;and for FAC 200μmol/L group,(41.53±3.97)%]for 120 h as compared with the blank control group[(3.42±0.31)%].(t=4.116,3.061,16.560,P<0.05).After DCFH-DA staining,flow cytometry results showed that iron overload intervention resulted in a significant in

关 键 词：铁过载 成骨细胞 坏死性凋亡 活性氧 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]