鸢尾素对脂多糖诱导小鼠成骨细胞凋亡的保护作用  

Irisin attenuates lipopolysaccharide-induced apoptosis in mouse embryonic osteoblastic precursor cells

作　　者：叶文斌 林达生[1] 王江泽[1] 丁真奇[1] Ye Wenbin;Lin Dasheng;Wang Jiangze;Ding Zhenqi(Orthopaedics Center of PLA,the 909th Hospital of PLA,the Affiliated Southeast Hospital of Xiamen University,Zhangzhou 363000,China)

机构地区：[1]联勤部第九〇九医院(厦门大学附属东南医院)全军骨科中心,漳州363000

出　　处：《中华实验外科杂志》2020年第6期1059-1061,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金(81600696);联勤部第909医院苗圃基金(16Y010)。

摘　　要：目的探讨鸢尾素(Irisin)对成骨细胞炎性反应的保护作用及其机制。方法2018年8月至2019年4月,往小鼠成骨细胞(MC3T3-E1,购自北京北纳生物公司)加入鸢尾素预处理后,再经脂多糖(LPS)刺激。实验分为6组:对照组(Control组)、LPS(5 mg/L)、LPS+Irisin(25μg/L)、LPS+Irisin(50μg/L)、LPS+Irisin(100μg/L)和LPS+Irisin(200μg/L)组。用噻唑蓝(MTT)法检测细胞增殖,2’,7’-二氯荧光黄双乙酸盐(DCFH-DA)检测细胞内活性氧自由基(ROS)含量,流式细胞术检测线粒体活性与细胞凋亡率,蛋白质印迹法(Western blot)检测单磷酸腺苷酸活化蛋白激酶-α(AMPK-α)、p-AMPK-α与半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)蛋白表达。两组比较采用t检验,多组比较采用单因素方差分析(One-way ANOVA),两两比较采用LSD检验。结果(1)与Control组比较(0.864±0.021),LPS组细胞增殖明显下降(0.719±0.018),而100μg/L(0.760±0.033)与200μg/L(0.809±0.025)Irisin预处理组细胞增殖的明显增加(F=23.625,P<0.05),差异有统计学意义;(2)与Control组比较(286.600±33.448),LPS组ROS含量显著增加(402.600±42.694),而Irisin预处理组(25~200μg/L)(323.400±27.437、322.600±28.325、309.000±42.936、307.800±24.045)明显抑制ROS形成(F=6.986,P<0.01),差异有统计学意义;(3)与Control组比较,LPS刺激后明显降低线粒体活力[(31.800±3.242)%、(15.667±1.804)%,t=7.532,P<0.01],细胞凋亡率显著增加[(7.610±0.229)%、(14.057±1.631)%,t=6.777,P<0.01],差异有统计学意义。p-AMPK-α表达显著降低[(0.546±0.178)、(0.186±0.093),t=6.777,P<0.01],而Caspase-3水平明显升高[(0.212±0.031)、(0.324±0.026),t=4.811,P<0.01],差异有统计学意义;(4)与LPS处理组比较,Irisin+LPS组中MC3T3-E1线粒体活性明显升高[(10.157±0.774)%、(27.500±1.572)%,t=17.145,P<0.01],细胞凋亡率显著减少[(11.927±0.385)%、(7.017±1.080)%,t=7.417,P<0.01],p-AMPK-α表达显著升高[(0.356±0.059)、(0.551±0.044),t=4.567,P<0.05],凋亡蛋白Caspase-3显著下降[(0.384Objective To investigate the underlying mechanisms of irisin attenuating lipopolysaccharide(LPS)-induced inflammatory injury in osteoblasts.Methods This study was conducted from August 2018 to April 2019.Mouse embryonic osteoblastic precursor(MC3T3-E1)cells were treated with 5 mg/L of LPS,with or without irisin pretreatment.Methyl thiazolyl tetrazolium(MTT)assay was employed to evaluate the ability of cell proliferation.Intracellular reactive oxygen species(ROS)was measured using 2’,7’-dichlorodihy-drofluorescein diacetate(H2DCF-DA).Mitochondrial activity was measured by flow cytometry using MitoTracker™Deep Redprobe.Apoptosis was detected by flow cytometry using Annexin V-fluorescein isothiocyanate(FITC)/propodium iodide(PI)kit.The expression of phosphorylated adenosine monophosphate activated protein kinase-α(p-AMPK-α),and Caspase-3 was detected by Western blotting.Results(1)Compared with control group(0.864±0.021),5 mg/L of LPS significantly inhibited proliferation of MC3T3-E1 cells(0.719±0.018),and 100μg/L and 200μg/L of irisin pre-treatment could significantly attenuate the inhibitory effect of LPS(F=23.625,P<0.05).(2)Compared with control group(286.600±33.448),5 mg/L of LPS significantly increased intracellular ROS level(402.600±42.694),and irisin pre-treatment(25-200μg/L)(323.400±27.437,322.600±28.325,309.000±42.936,307.800±24.045)could significantly attenuate the inhibitory effect of LPS(F=6.986,P<0.01).(3)LPS inhibited mitochondria activity[(31.800±3.242)%,(15.667±1.804)%,t=7.532,P<0.01],induced apoptosis[(7.610±0.229)%,(14.057±1.631)%,t=6.777,P<0.01],when compared to control group.Meanwhile,the expression of p-AMPK-αwas decreased by LPS exposure[(0.546±0.178),(0.186±0.093),t=6.777,P<0.01],while Caspase-3 expression was up-regulated[(0.212±0.031),(0.324±0.026),t=4.811,P<0.01].(4)Irisin pre-treatment markedly increased mitochondria activity[(10.157±0.774)%,(27.500±1.572)%,t=17.145,P<0.01]and reduced apoptosis rate[(11.927±0.385)%,(7.017±1.080)%,t=7.417,P<0.01].Additionally,

关 键 词：鸢尾素 成骨细胞 脂多糖 细胞凋亡 单磷酸腺苷酸活化蛋白激酶 

分 类 号：R965[医药卫生—药理学]