基于FoxO1通路探讨仙茅苷对H2O2诱导成骨细胞氧化损伤的保护作用及机制

Investigating Protective Effects of Curculigoside on H2O2 Damaged Osteoblastic MC3T3-E1 Cells and Its Mechanism via FoxO1 Pathway

目的探讨仙茅苷(Curculigoside)对H_2O_2诱导成骨细胞氧化损伤的保护作用及机制。方法采用H_2O_2(0.3 mmol·L^(-1))处理MC3T3-E1成骨细胞建立细胞氧化损伤模型,并以阳性对照药氮乙酰半胱氨酸(NAC,2.5 mmol·L^(-1))和不同浓度仙茅苷(10^(-6)、10^(-7)、10^(-8)mol·L^(-1))进行干预。采用MTT法检测成骨细胞的增殖情况;磷酸苯二钠法(p-PNPP-Na)法测定成骨细胞碱性磷酸酶(ALP)活性;茜素红染色法观察成骨细胞的骨矿化结节形成情况;ELISA法检测细胞超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活力;流式细胞仪检测细胞活性氧(ROS)和线粒体膜电位;Western Blot法检测细胞叉形头转录因子1(FoxO1)和SOD-2的蛋白表达水平;免疫荧光组织化学法检测FoxO1、活化转录因子4(ATF4)、β-连环蛋白(β-catenin)在细胞核中的表达。结果与空白对照组比较,模型组成骨细胞在H_2O_2处理36~72 h后,MC3T3-E1细胞的增殖受到显著抑制(P<0.001);成骨细胞活力及ALP活性均明显降低(P<0.05),细胞形成的骨矿化结节的数量和面积明显减少;SOD活性明显降低(P<0.05),CAT活性明显增加(P<0.05);ROS水平增加,线粒体膜电位降低,总FoxO1a、SOD-2蛋白表达下调,FoxO1a在细胞核内的表达下调,在细胞质中的表达上调,在细胞核与细胞质表达的比例降低,但差异没有统计学意义(P>0.05);细胞核中FoxO1a、β-catenin、ATF4表达量升高。与模型组比较,阳性药组和不同浓度仙茅苷组的成骨细胞活力及ALP活性均明显上调(P<0.05),且细胞骨矿化结节的数量和面积明显增加;阳性药组及仙茅苷10^(-6)mol·L^(-1)组的细胞SOD活性明显增加(P<0.05),仙茅苷10^(-6)mol·L^(-1)组细胞的CAT活性明显增加(P<0.05);10^(-7)mol·L^(-1)仙茅苷组细胞的ROS水平明显降低(P<0.05);仙茅苷10^(-8)mol·L^(-1)组的线粒体膜电位明显升高(P<0.05);阳性对照组和10-6mol·L^(-1)仙茅苷组的细胞总FoxO1a蛋白表达明显上调(P<0.05);各给药组细胞的SOD-2蛋白表达均明显上调(P<0.05);10^(-6)mol·L^(-1)仙茅苷组细胞FoxO1a在细胞核与细胞质表达的比值明显升高(P<0.05);10^(-6)mol·L^(-1)仙茅苷组细胞的FoxO1a、ATF4在细胞核的表达上调,β-catenin在细胞核的表达没有明显变化。结论仙茅苷能够保护H_2O_2对MC3T3-E1成骨细胞的氧化损伤,可能与其增加FoxO1在细胞核内的表达,增强FoxO1a与ATF4相互作用,以及增强SOD及CAT活性有关。

Objective To investigate the protective effects and mechanism of curculigoside on osteoblast damaged by H2 O2.Methods MC3T3-E1 osteoblasts were treated with H2O2(0.3 mmol·L-1) to induce oxidative damage model,further intervened with positive control drug N-Acetyl-cysteine(NAC,2.5 mmol·L-1) and different concentrations of curculigoside(10-6,10-7,10-8 mol·L-1).The proliferation of cells was determined by MTT assay;alkaline phosphatase(ALP) activity was detected by p-PNPP-Na method;bone mineralized nodules were stained with alizarin red;the activities of superoxide dismutase(SOD) and catalase(CAT) were detected by ELISA kits;reactive oxygen species(ROS) and mitochondrial membrane potential were evaluated by flow cytometry;the protein levels of Forkhead box O1(FoxO1) and SOD-2 were analyzed by Western Blot.The expression of nucleus FoxO1,ATF4 and β-catenin were detected by fluorescence immunohistochemical staining.Results Compared with the blank control group,the proliferation of MC3 T3-E1 cells in the model group was significantly inhibited after H2O2 treatment for 36-72 h(P <0.001);cell viability and ALP activity were significantly reduced(P <0.05),and the number and area of bone mineralization nodules were significantly reduced;SOD activity was significantly reduced(P <0.05),CAT activity was significantly increased(P <0.05);ROS levels increased,mitochondrial membrane potential decreased,and total FoxOl a and SOD-2 protein expressions were down-regulated;the expression of FoxO1 a in the nucleus was down-regulated,and its expression in the cytoplasm was up-regulated.The ratio of expression in the nucleus to the cytoplasm decreased,but the difference was not statistically significant(P> 0.05).The expression of FoxOla,β-catenin,and ATF4 in the nucleus increased.Compared with the model group,the osteoblast viability and ALP activity of the positive drug group and the different concentrations of curculigoside groups were significantly up-regulated(P <0.05),and the number and area of cellular bone mineralization nodules increased significantly;the SOD activities of cells in the positive drug group and the curculigoside 10-6 mol·L-1 group were significantly increased(P <0.05),and the CAT activity of the cells in the curculin 10-6 mol·L-1 group was significantly increased(P <0.05);the ROS level of cells in the 10-7 mol·L-1 curculigoside group was significantly reduced(P <0.05);the mitochondrial membrane potential of the 10-8 mol·L-1 group was significantly increased(P <0.05);the expression of total FoxO1 a protein in the cells of the positive control group and the 10-6 mol·L-1 curculigoside group was significantly up-regulated(P <0.05);the expression of SOD-2 in the cells of each administration group was significantly up-regulated(P <0.05);the expression ratio of FoxO1 a in the nucleus to the cytoplasm of cells in the 10-6 mol·L-1 curculigoside group was significantly increased(P <0.05);the expression of FoxOla and ATF4 in the nucleus of the cells in the 10-6 mol·L-1 curculigoside group up-regulated,the expression of β-catenin in the nucleus showed no change.Conclusion Curculigoside can protect the oxidative damage of MC3 T3-E1 osteoblasts caused by H2O2,which may be related to increasing the expression of FoxO1 in the nucleus,increasing the interaction between FoxO1 a and ATF4,and enhancing the activities of SOD and CAT.