脂多糖刺激小鼠MLO-Y4骨细胞白细胞介素6及核因子κB受体活化因子配体的表达  

Lipopolysaccharide stimulates the expression of interleukin-6 and nuclear factor kappa B receptor activator ligand in mouse MLO-Y4 osteoblasts

作　　者：刘奇奇 刘敏 杨健 余科 Liu Qiqi;Liu Min;Yang Jian;Yu Ke(Department of Oral Implantology,2Department of Prosthodontics,School/Hospital of Stomatology,Southwest Medical University,Luzhou 646000,Sichuan Province,China;Department of Oral Implantology,School/Hospital of Stomatology,Southwest Medical University,Luzhou 646000,Sichuan Province,China)

机构地区：[1]西南医科大学附属口腔医院种植科,四川省泸州市646000 [2]西南医科大学附属口腔医院修复科,四川省泸州市646000

出　　处：《中国组织工程研究》2022年第20期3121-3126,共6页Chinese Journal of Tissue Engineering Research

基　　金：四川省教育厅2017年科研计划(17ZB0481),项目负责人:余科;中国口腔医学会西部口腔医学临床研究基金(CSA-W2018-03),项目负责人:余科;泸州市人民政府-西南医科大学战略合作项目(2020LZXNYDJ31),项目负责人:余科。

摘　　要：背景:前期研究证实,脂多糖刺激小鼠MLO-Y4骨细胞上调炎症相关细胞因子白细胞介素6的表达,白细胞介素6上调破骨细胞生成相关因子核因子κB受体活化因子配体(receptor activator of nuclear factor-κB ligand,RANKL)的表达,而PI3K/AKT信号通路是骨代谢中的重要调节通路,验证其是否参与脂多糖诱导骨细胞炎症因子的产生,对研究细菌炎症性骨吸收的机制具有重要意义。目的:验证PI3K/AKT信号通路是否参与脂多糖刺激下小鼠MLO-Y4骨细胞白细胞介素6及RANKL的表达。方法:①分别以0,100μg/L的脂多糖刺激MLO-Y4骨细胞,1,2,4,6,8 h后,采用RT-qPCR检测白细胞介素6和RANKL的mRNA表达,Western blot法检测RANKL和p-AKT蛋白表达;②取对数生长期的MLO-Y4骨细胞,分7组处理:不做任何处理(对照组)、100μg/L脂多糖、1μmol/L PI3K抑制剂+100μg/L脂多糖、2.5μmol/L PI3K抑制剂+100μg/L脂多糖、5μmol/L PI3K抑制剂+100μg/L脂多糖、10μmol/L PI3K抑制剂+100μg/L脂多糖、20μmol/L PI3K抑制剂+100μg/L脂多糖,采用RT-qPCR检测白细胞介素6和RANKL的mRNA表达;③将MLO-Y4骨细胞分4组处理:不做任何处理(对照组)、100μg/L脂多糖、二甲基亚砜+100μg/L脂多糖、10μmol/L PI3K抑制剂+100μg/L脂多糖,采用RT-qPCR检测RANKL的mRNA表达,Western blot法检测RANKL与p-AKT蛋白表达。结果与结论:①脂多糖刺激后,骨细胞内白细胞介素6与RANKL的mRNA表达呈先升高后降低的趋势,各时间点的两基因表达量均高于无脂多糖刺激组(P<0.05);骨细胞内RANKL和p-AKT蛋白表达呈先升高后降低的趋势,各时间点的两蛋白表达量均高于无脂多糖刺激组(P<0.05);②1-10μmol/L的PI3K抑制剂呈浓度依赖性降低脂多糖诱导的骨细胞白细胞介素6和RANKL的mRNA表达量升高,浓度越高降低效果越明显,当浓度达到20μmol/L时降低效果与10μmol/L无明显差异;③二甲基亚砜与PI3K抑制剂均可降低脂多糖刺激诱导的骨细胞RANKL蛋白与mBACKGROUND:Previous studies have confirmed that lipopolysaccharide could up-regulated the expression of interleukin-6,an inflammation-related cytokine,in mouse MLO-Y4 osteocytes,and interleukin-6 could further up-regulate the expression of receptor activator of nuclear factorκB ligand(RANKL),a factor related to osteoclastogenesis,in osteoblasts.PI3K/AKT signaling pathway is an important regulatory pathway in bone metabolism.Verifying whether this signaling pathway is involved in the production of inflammatory factors induced by lipopolysaccharides is greatly beneficial to studying the mechanism of bone resorption that is caused by bacterial inflammation.OBJECTIVE:To verify whether PI3K/AKT signaling pathway is involved in the expression of interleukin-6 and RANKL in mouse MLO-Y4 osteocytes stimulated by lipopolysaccharides.METHODS:MLO-Y4 osteoblasts were stimulated with 0 or 100μg/L lipopolysaccharides for 1,2,4,6,and 8 hours.Then the mRNA expressions of interleukin-6 and RANKL were detected by real-time quantitative polymerase chain reaction,and the protein expression of RANKL and p-AKT were detected by western blot.MLO-Y4 osteocytes in logarithmic growth was divided into seven groups:no treatment group(control group),100μg/L lipopolysaccharide group,1μmol/L PI3K inhibitor+100μg/L lipopolysaccharide group,2.5μmol/L PI3K inhibitor+100μg/L lipopolysaccharide group,5μmol/L PI3K inhibitor+100μg/L lipopolysaccharide group,10μmol/L PI3K inhibitor+100μg/L lipopolysaccharide group,20μmol/L PI3K inhibitor+100μg/L lipopolysaccharide group.Real-time quantitative polymerase chain reaction was used to detect the mRNA expression of interleukin 6 and RANKL.MLO-Y4 osteocytes were divided into four groups:no treatment group(control group),100μg/L lipopolysaccharide group,dimethyl sulfoxide+100μg/L lipopolysaccharide group,and 10μmol/L PI3K inhibitor+100μg/L lipopolysaccharide group.Real-time quantitative polymerase chain reaction was used to detect the mRNA expression of RANKL,and western blot was used to detect

关 键 词：骨细胞 白细胞介素6 脂多糖 PI3K/AKT 核因子ΚB受体活化因子配体 骨吸收 磷酸化 PI3K抑制剂 

分 类 号：R459.9[医药卫生—治疗学] R364.5[医药卫生—临床医学]