梓醇对H_(2)O_(2)诱导成骨细胞损伤的影响及机制

Effects of catalpol on H2O2-induced osteoblast injury and its mechanism

目的探究梓醇对H_(2)O_(2)诱导成骨细胞损伤的影响及机制。方法将小鼠成骨细胞MC3T3-E1分为对照组、模型组、空载组(转染空载质粒)、梓醇组(100μmol/L)、梓醇+叉头框蛋白O3(FoxO3)过表达组(100μmol/L梓醇+转染FoxO3过表达质粒),经梓醇和转染处理后,除对照组细胞不作处理外,其余各组细胞以H_(2)O_(2)诱导建立成骨细胞氧化应激模型。检测各组细胞活力、凋亡率、碱性磷酸酶(ALP)活性、钙结节光密度(OD)值、活性氧(ROS)的平均荧光强度(MFI)、抗氧化酶[超氧化物歧化酶(SOD)、过氧化氢酶(CAT)]活性、炎症因子[白细胞介素6(IL-6)、IL-1β]水平、FoxO3/Wnt/β-连环蛋白(β-catenin)信号通路相关蛋白表达水平。结果与对照组比较,模型组细胞活力、ALP活性、钙结节OD值、抗氧化酶活性和Wnt、β-catenin蛋白表达水平均显著降低,凋亡率、ROS的MFI、炎症因子水平和FoxO3蛋白表达水平均显著升高(P<0.05);与模型组比较,空载组细胞上述指标均无明显变化(P>0.05),梓醇组细胞上述指标均显著逆转(P<0.05);与梓醇组比较,梓醇+FoxO3过表达组细胞上述指标的逆转效果均被显著削弱(P<0.05)。结论梓醇可通过下调FoxO3激活Wnt/β-catenin信号通路,抑制H_(2)O_(2)诱导的MC3T3-E1细胞氧化应激和炎症反应,提高细胞活力与成骨分化能力,抑制细胞凋亡。

OBJECTIVE To investigate the effects of catalpol on H_(2)O_(2)-induced osteoblast injury and its mechanism.METHODS The osteoblasts MC3T3-E1 were separated into control group,model group,empty group(transfected with empty plasmid),catalpol group(100μmol/L),catalpol+forkhead box O3(FoxO3)overexpression group(100μmol/L catalpol+transfected with FoxO3 overexpression plasmid).After catalpol treatment and transfection,except for control group,other groups were induced with H_(2)O_(2) to establish osteoblast oxidative stress model.The cell viability,apoptotic rate,alkaline phosphatase(ALP)activity,optical density(OD)value of calcium nodule,mean fluorescence intensity(MFI)of reactive oxygen species(ROS),antioxidant enzyme activity[superoxide dismutase(SOD),catalase(CAT)],the levels of inflammatory factors[interleukin-6(IL-6),IL-1β],and the expressions of FoxO3/Wnt/β-catenin signaling pathway-related proteins were detected in each group.RESULTS Compared with the control group,the cell viability,ALP activity,OD value of calcium nodule,activities of antioxidant enzyme,and the protein expressions of Wnt andβ-catenin were decreased significantly in the model group,while apoptotic rate,MFI levels of ROS,inflammatory factor levels and the protein expression of FoxO3 were all increased significantly(P<0.05).Compared with the model group,above indicators of the empty group had no significant change(P>0.05),while those of catalpol group were reversed significantly(P<0.05).Compared with the catalpol group,the reversal effect of the changes in the above indicators was significantly weakened in the catalpol+FoxO3 overexpression group cells(P<0.05).CONCLUSIONS Catalpol can activate Wnt/β-catenin signaling pathway by down-regulating FoxO3,thereby inhibiting H_(2)O_(2)-induced MC3T3-E1 oxidative stress and inflammation reaction,enhancing cell viability and osteogenic differentiation activity,and alleviating apoptosis injury.