高糖状态下脂多糖介导βtc6细胞自噬的机制  

Lipopolysaccharides mediate autophagy of mouse insulinomaβtc6 cells in high glucose state

作　　者：蔡智国 都沙沙 杨琨 赵娜 刘琪 Cai Zhiguo;Du Shasha;Yang Kun;Zhao Na;Liu Qi(Department of Periodontology,Affiliated Stomatological Hospital of Zunyi Medical University,Zunyi 563000,Guizhou Province,China)

机构地区：[1]遵义医科大学附属口腔医院牙周科,贵州省遵义市563000

出　　处：《中国组织工程研究》2022年第20期3127-3132,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金项目(81860196),项目负责人:刘琪,课题名称:2型糖尿病伴牙周炎牙周膜干细胞凋亡转归的线粒体损伤、自噬失调机制研究。

摘　　要：背景:作者团队前期证实糖尿病促进牙周炎的发生发展,而牙周炎是否促进糖尿病发展?目的:探索牙周炎促进糖尿病发展的分子机制。方法:体外培养小鼠胰岛素瘤βtc6细胞,分为对照组、葡萄糖组、脂多糖组、葡萄糖+脂多糖组,采用不同浓度葡萄糖(0,25,50,100 mmol/L)和脂多糖(0,10,20,40 mg/L)单独或联合刺激βtc6细胞12 h,检查每组胰岛素分泌量。另外分别加入自噬抑制剂3-甲基腺嘌呤(5μmol/L)和自噬激活剂雷帕霉素(10 mmol/L)对磷脂酰肌醇3-激酶/蛋白激酶B/雷帕霉素靶蛋白(phoshatidylinositol-3-kinase/rotein kinase B/the mammalian target of rapamycin,PI3K/AKT/mTOR)信号通路进行干预,分为雷帕霉素+脂多糖组、雷帕霉素+葡萄糖组、雷帕霉素+葡萄糖+脂多糖组及3-甲基腺嘌呤+脂多糖组、3-甲基腺嘌呤+葡萄糖组、3-甲基腺嘌呤+葡萄糖+脂多糖组。CCK-8法检测各组βtc6细胞增殖情况,活性氧荧光探针检测各组βtc6细胞中活性氧聚集量,透射电镜观察各组βtc6细胞中自噬小体生成量,Western Blot法检测自噬蛋白及相关通路蛋白表达。结果与结论:①葡萄糖浓度超过50 mmol/L时,胰岛素分泌量不受葡萄糖调节(P>0.05);脂多糖质量浓度大于20 mg/L时,胰岛素分泌量受到抑制(P<0.05);②加入3-甲基腺嘌呤后,3-甲基腺嘌呤+葡萄糖组、3-甲基腺嘌呤+脂多糖组、3-甲基腺嘌呤+脂多糖+葡萄糖组中Becline1、p-PI3K、p-AKT/AKT、p-mTOR/mTOR表达较对照组显著降低(P<0.05);p62蛋白表达则呈相反趋势(P<0.05);③加入雷帕霉素后,Becline1、p-PI3K、p-mTOR/mTOR表达较对照组显著增加(P<0.05);④提示脂多糖能诱导胰岛β细胞过度自噬下调胰岛素分泌,该机制可能与沉默PI3K/AKT/mTOR信号通路有关。BACKGROUND:Our previous studies have confirmed that diabetes mellitus promotes the occurrence and development of periodontitis.However,it is unclear whether periodontitis can promote the development of diabetes.OBJECTIVE:To explore the molecular mechanism by which periodontitis promotes the development of diabetes mellitus.METHODS:Mouseβtc6 insulinoma cells cultured in vitro were divided into control group,glucose group,lipopolysaccharide group,and glucose+lipopolysaccharide group.The cells in the latter three groups were treated with different concentrations of glucose(0,25,50,100 mmol/L)and lipopolysaccharide(0,10,20,40 mg/L)alone or in combination for 12 hours.Insulin secretion was measured in each group.In addition,3-methyladenine(an autophagy inhibitor,5μmol/L)and rapamycin(an autophagy activator,10 mmol/L)were used to intervene with the phosphatidylinositol 3-kinase/rotein kinase B/the mammalian target of rapamycin(PI3K/AKT/mTOR)signaling pathway.The cells were then further divided into rapamycin+lipopolysaccharide group,rapamycin+glucose group,rapamycin+glucose+lipopolysaccharide group,3-methyladenine+lipopolysaccharide group,3-methyladenine+glucose group,and 3-methyladenine+glucose+lipopolysaccharide group.The cell counting kit-8 method was used to detect the proliferation ofβtc6 cells.Fluorescent probes were used to detect the amount of reactive oxygen species inβtc6 cells.Production of autophagosomes inβtc6 cells was observed by transmission electron microscopy.Western blot method was used to detect the expression of autophagy proteins and PI3K/AKT/mTOR signaling pathway-related proteins.RESULTS AND CONCLUSION:The insulin secretion was out of control as the glucose concentration exceeded 50 mmol/L(P>0.05),whereas the insulin secretion was inhibited as the lipopolysaccharide concentration exceeded 20 mg/L(P<0.05).After addition of 3-methyladenine,the expression levels of Becline1,p-PI3K,p-AKT/AKT,p-AKT/AKT,p-AKT/AKT,and p-PI3K were significantly decreased in the 3-methyladenine+glucose group,3-methy

关 键 词：牙周炎 糖尿病 脂多糖 自噬 3-甲基腺嘌呤 PI3K/AKT/mTOR信号通路 

分 类 号：R459.9[医药卫生—治疗学] R587.1[医药卫生—临床医学]