雌马酚对高糖高脂诱导的成骨细胞线粒体损伤的保护作用  

Protectiveeffect of equol on mitochondrial damage induced by high glucose and fat in osteoblasts

作　　者：李正宙 倪向敏 白倩 梁馨予 朱文艺 王建 LI Zhengzhou;NI Xiangmin;BAI Qian;LIANG Xinyu;ZHU Wenyi;WANG Jian(Department of Nutrition,Second Affiliated Hospital,Army Medical University(Third Military Medical University),Chongqing,400037,China)

机构地区：[1]陆军军医大学(第三军医大学)第二附属医院营养科,重庆400037

出　　处：《陆军军医大学学报》2023年第11期1152-1160,共9页Journal of Army Medical University

基　　金：国家自然基金面上项目(81973040)。

摘　　要：目的 观察雌马酚(equol, Eq)干预对高糖高脂诱导的成骨细胞的影响,并探讨其作用机制。方法 体外培养ROS1728成骨细胞,以不同浓度的Eq、棕榈酸钠(sodium palmitate, PA)和葡萄糖(glucose, GLU)处理48 h,通过HE染色观察细胞核凋亡表现及CCK-8法检测细胞存活率确定GLU、PA及Eq干预的适宜浓度。将细胞分为对照(Control)组(GLU,5.5 mmol/L)、模型(Model)组(GLU,30 mmol/L+PA,250μmol/L)、LEq组(Model+1×10^(-8)mol/L Eq)、MEq组(Model+1×10^(-7)mol/L Eq)、HEq组(Model+1×10^(-6)mol/L Eq)。CCK8检测细胞存活率,流式细胞术检测细胞凋亡率,Hoechst33342染色和HE染色观察细胞核凋亡表现,透射电镜观察线粒体结构,Mito-Tracker Red CMXRos(线粒体红色荧光探针)染色检测细胞线粒体膜电位(mitochondrial membrane potential, MMP)变化情况,Western blot检测Sirt1、PGC-1α、Bcl-2、Bax和Cleaved caspase-3蛋白表达。结果 与对照组相比,当GLU和PA浓度分别为30 mmol/L和250μmol/L时细胞存活率显著降低(P<0.05),凋亡率升高,确定为诱导成骨细胞高糖高脂环境的适宜浓度;与模型组相比,Eq干预后细胞存活率显著升高、凋亡率显著降低(P<0.05),线粒体结构受损明显改善、MMP显著升高(P<0.05),同时ROS显著减少(P<0.05);细胞Sirt1、PGC-1α、Bcl-2表达水平显著升高,Bax、Cleaved caspase-3表达水平显著降低(P<0.05)。结论 Eq干预可有效减少高糖高脂诱导的成骨细胞线粒体损伤、氧化应激和细胞凋亡,其部分机制可能与Eq促进Sirt1、PGC-1α表达有关。Objective To observe the effect of equol(Eq)intervention on osteoblasts induced by high glucose and fat,and to explore its mechanism of action.Methods After ROS1728 osteoblasts were treated with different concentrations of Eq,sodium palmitate(PA)and glucose(GLU)for 48 h,nuclear apoptosis was observed by HE staining and cell survival rate was detected by CCK-8 assay in order to determine the optimal concentrations of GLU,PA and Eq interventions to induce cellular model of diabetic osteoporosis.ROS1728 cells were then divided into control group(5.5 mmol/L GLU),model group(30 mmol/L GLU+250μmol/L PA),and low,medium and high dose Eq treatment groups(model+1×10^(-8),1×10^(-7) and 1×10^(-6) mol/L Eq).CCK8 assay was used to detect cell viability,and flow cytometry for cell apoptosis.Hoechst33342 staining and HE staining were employed to observe apoptotic nuclei.Mitochondrial structure,mitochondrial membrane potential(MMP)changes,and protein levels of Sirt1,PGC-1α,Bcl-2,Bax and Cleaved caspase-3 were observed by transmission electron microscopy,Mito-Tracker Red CMXRos(mitochondrial red fluorescent probe)staining,and Western blotting,respectively.Results Compared with the control group,treatment of 30 mmol/L GLU and 250μmol/L PA significantly reduced the cell viability rate(P<0.05)and increased the apoptotic rate(P<0.05),and the optimal concentrations were determined to induce high glucose and fat environment of osteoblasts.While Eq intervention significantly increased the survival rate and decreased the apoptotic rate(P<0.05),attenuated the damage of mitochondrial structure,elevated MMP(P<0.05)and decreased ROS level(P<0.05),as well as up-regulated the expression levels of Sirt1,PGC-1αand Bcl-2 and down-regulated those of Bax and Cleaved caspase-3(P<0.05).Conclusion Eq intervention could effectively reduce mitochondrial damage,oxidative stress and apoptosis of osteoblasts induced by high glucose and fat,which might be partially related to its activation of Sirt1 and PGC-1α.

关 键 词：雌马酚 ROS1728细胞 高糖高脂 线粒体 氧化应激 凋亡 

分 类 号：R285.5[医药卫生—中药学] R322.71[医药卫生—中医学] R362