吉林大学学报(医学版) ›› 2018, Vol. 44 ›› Issue (04): 709-717.doi: 10.13481/j.1671-587x.20180404

• 基础研究 • 上一篇    下一篇

铁皮石斛对小鼠和胰岛瘤细胞胰岛素抵抗的改善作用

陈瑛波1,2,3, 宋丹1,2,3, 吴晋1,2, 景作磊1,2, 刘利平1,2,3, 姜如娇1,2, 孙捷4, 吴英杰1,2,3,5   

  1. 1. 大连医科大学 重大疾病基因工程模式动物研究所, 辽宁 大连 116044;
    2. 大连医科大学 基因工程模式 动物国际联合研究中心, 辽宁 大连 116044;
    3. 大连医科大学中西医结合研究院, 辽宁 大连 116044;
    4. 吉林大学第一医院干部病房, 吉林 长春 130021;
    5. 西奈山医学院内分泌、糖尿病和骨疾病系, 纽约 100029
  • 收稿日期:2018-04-24 出版日期:2018-07-28 发布日期:2018-07-27
  • 通讯作者: 吴英杰,教授,博士研究生导师(Tel:0411-86110232,E-mail:yingjiewu@dmu.edu.cn);孙捷,教授,主任医师,硕士研究生导师(Tel:0431-88782272,E-mail:sunjiesusan@126.com) E-mail:yingjiewu@dmu.edu.cn;sunjiesusan@126.com
  • 作者简介:陈瑛波(1989-),女,山东省济南市人,医学硕士,主要从事糖尿病和糖脂代谢方面的研究。
  • 基金资助:
    国家自然科学基金面上项目资助课题(81471000);吉林省科技厅科研项目资助课题(20150414009GH)

Improvement effects of Dendrobium candidum on insulin resistance in MKR mice and MIN6 cells

CHEN Yingbo1,2,3, SONG Dan1,2,3, WU Jin1,2, JING Zuolei1,2, LIU Liping1,2,3, JIANG Rujiao1,2, SUN Jie4, WU Yingjie1,2,3,5   

  1. 1. Institute of Genome Engineered Animal Models for Human Disease, Dalian Medical Univerty, Dalian 116044, China;
    2. International Research Center of Gene Engineering Animal Models, Dalian 116044, China;
    3. Institute of Integrated Chinese andWestern Medicine, Dalian Medical University, Dalian 116044, China;
    4. Cadre Ward, First Hospital, Jilin University, Changchun 130021, China;
    5. Division of Endocrinology, Diabetes and Bone Disease, Icahn Mount Sinai School of Medicine, New York 100029, USA
  • Received:2018-04-24 Online:2018-07-28 Published:2018-07-27

摘要: 目的:探讨铁皮石斛(DC)对MKR小鼠和MIN6细胞中胰岛素抵抗的作用,并阐明其作用机制。方法:9周龄MKR小鼠随机分为4组,分别灌胃给予10、20、40 g·kg-1·d-1DC和生理盐水(空白对照组),连续给药6周,每2周检测小鼠的血糖,以确定DC降糖作用最佳浓度。8周龄MKR雄鼠随机分为对照组、DC组、二甲双胍(Met)组和联合用药(Met+DC)组,灌胃给予生理盐水和相应药物8周,同时检测小鼠血糖,给药结束后对小鼠进行葡萄糖耐量实验(GTT)和胰岛素耐量实验(ITT),采用qPCR法检测小鼠肝脏和皮下脂肪组织中糖脂代谢相关基因表达水平。体外培养MIN6细胞,分为对照组、高浓度棕榈酸(H-PA)组、高浓度葡萄糖(H-Glu)组、H-PA+H-Glu组、H-PA+DC组、H-Glu+DC组和H-Glu+H-PA+DC组,进行葡萄糖刺激下胰岛素分泌能力实验(GSIS),采用MTT法和ELISA法检测各组细胞增殖活性和胰岛素分泌水平,流式细胞术检测各组细胞凋亡率,Western blotting法检测各组细胞中PI3K/AKT通路蛋白表达水平。结果:在小鼠体内,DC降糖作用最佳浓度为20 g·kg-1·d-1。与对照组比较,DC组、Met组和联合用药组小鼠血糖水平降低(P<0.05);与DC组和Met组比较,联合用药组小鼠血糖水平进一步降低(P<0.05)。GTT和ITT实验,与DC组和Met组比较,联合用药组小鼠糖耐量和胰岛素敏感性明显提高,胰岛素抵抗情况明显改善。与对照组比较,联合用药组小鼠肝组织中磷酸烯醇式丙酮酸羧激酶(Pepck) mRNA表达水平明显降低(P<0.05),葡萄糖激酶(Gck) mRNA表达水平明显升高(P<0.05),Met组和联合用药组小鼠皮下脂肪组织中氧化物酶增殖物激活受体γ(Ppar-γ) mRNA表达水平明显升高(P<0.05)。在MIN6细胞中,经筛选,选择160 μmol·L-1 H-PA和30 mmol·L-1 H-Glu诱导MIN6细胞胰岛素抵抗,以1.44g·L-1 DC处理各组细胞。GSIS,分别与H-PA组、H-Glu组和H-PA+H-Glu组比较,加入DC后,H-PA+DC组、H-Glu+DC组和H-PA+H-Glu+DC组MIN6细胞胰岛素分泌水平明显升高(P<0.05),细胞凋亡率降低,磷酸化胰岛素受体底物1(p-IRS-1)、磷酸化磷酸肌醇3激酶(p-PI3K)和磷酸化蛋白激酶B (p-AKT)蛋白表达水平明显升高(P<0.05); H-PA+H-Glu+DC组MIN6细胞磷酸化糖原合成酶激酶3β(p-GSK3β)蛋白表达水平明显降低(P<0.05)。结论:DC可以改善MKR小鼠的胰岛素抵抗,与Met联合用药后效果更加明显;在体外,DC可以通过PI3K/AKT信号通路改善H-PA和H-Glu诱导的MIN6细胞的胰岛素抵抗,并可增加胰岛素分泌水平,降低细胞凋亡率。

关键词: MIN6细胞, 铁皮石斛, 糖代谢, 小鼠, 胰岛素抵抗, MKR

Abstract: Objective:To investigate the effects of Dendrobium candidum (DC)on the insulin resistance in the MKR mice and the MIN6 cells, and to illuminate the possible mechanisms. Methods:The MKR mice aged 9 weeks were randomly divided into 4 groups,and the mice in various groups were treated with 10,20,40 g·kg-1 DC and normal saline(blank control group) by gavage,respectively,for 6 weeks.The blood glucose levels of the mice in various groups were detected bi-weekly,and the optimum concentration of DC in reducing the glucose level.The male MKR mice aged 8 weeks were randomly divided into control group,DC group,Met group and Met+DC group.The mice were treated with normal saline and the corresponding drugs for 8 weeks,and the blood glucose levels of the mice were determined.Glucose tolerance test(GTT) and insulin tolerance test(ITT) were performed after administration.The qPCR method was used to detect the expression levels of genes related to glucose and lipid metabolism in liver and subcutaneous fat tissues of the mice.The MIN6 cells cultured in vitro were divided into control group,high concentration of palmitic acid(H-PA) group,high concentration of glucose (H-Glu) group,H-PA+H-Glu group,H-PA+DC group,H-Glu+DC group,and H-Glu+H-PA+DC group.MTT method and ELISA method were used to detect the proliferation activities and insulin secretion levels of cells in various groups.Flow cytometry was used to measure the apoptotic rates in various groups.Western blotting method was used to detect the protein expression levels of PI3K/AKT/signaling pathway in the cells in various groups. Results:In vivo,the optimum concentration of DC in reducing the blood glucose level was 20 g·kg-1·d-1.Compared with control group,the blood glucose level of the mice in DC,Met and Met+DC groups were decreased significantly (P<0.05);compared with DC group and Met group,the blood glucose level of the mice in Met+DC group was decreased (P<0.05). Compared with DC group and Met group,the glucose tolerance and insulin sensitivity were increased,and the insulin resisitance was improved.Compared with control group,the Pepck mRNA expression level in liver tissue of the mice in Met+DC group was decreased significantly(P<0.05),and the GCK mRNA expression level was increased significantly (P<0.05);the Ppar-γ mRNA expression level in subcutaneons fat tissue of the mice in Met group and Met+DC group were increased significantly(P<0.05).In the MIN6 cells,160 μmol·L-1and 30 μmol·L-1 were confirmed to use as the end concentrations of H-PA and H-Glu to induce the insulin resistance,and the cells in various groups were treated with 1.44 g·L-1 DC.Compared with H-PA group,H-Glu group and H-PA+H-Glu group,the insulin secretion levels of MIN6 cells in H-PA-DC group,H-Glu+DC group and H-PA+H-Glu+DC group were increased significantly(P<0.05),the apoptotic rates were decreased,and the expression levels of p-IRSp-1, p-PI3K and p-AKT proteins was increased significantly(P<0.05);the expression level of p-GSK3β protein in H-PA+H-Glu+DC group was significantly decreased(P<0.05). Conclusion: DC can ameliorate the insulin resistance in the MKR mice. The effect of hypoglycemic therapy is more obvious with metformin in combination. In vitro, DC can improve the palmitic acid and glucose-induced insulin resistance through the PI3K/AKT signaling pathway and increase the insulin secretion level and reduce the apoptotic rates.

Key words: glucose metabolism, Dendrobium candidum, mice, MKR, MIN6 cells, insulin resistence

中图分类号: 

  • R285.5