睡茄交酯类成分通过Akt/mTOR和Sirt3信号通路改善棕榈酸诱导的骨骼肌L6细胞胰岛素抵抗

Improvement of palmitic acid-induced insulin resistance in the L6 myoblast cells by withanolides through Akt/mTOR and Sirt 3 signal pathways

关键词:  酸浆属药用植物  睡茄交酯类成分  胰岛素抵抗  活性氧  自噬  Akt/mTOR信号通路  Sirt3信号通路

Key words:  Physalis genus  withanolides  insulin resistance  ROS  autophagy  Akt/mTOR signal pathway  Sirt3 signal pathway

摘要:

[目的] 探讨酸浆属药用植物来源的睡茄交酯类成分改善大鼠骨骼肌胰岛素抵抗的作用机制。[方法] 利用棕榈酸（PA）作用于L6大鼠成肌细胞9 h建立胰岛素抵抗细胞模型；采用葡萄糖氧化酶法试剂盒和噻唑蓝法（MTT）法检测3种酸浆属药用植物来源的15种睡茄交酯类成分对骨骼肌细胞葡萄糖消耗和生存率的影响；采用流式细胞术分别结合2’，7’-二氯荧光素二乙酸酯（DCFH-DA）染色和丹酰尸胺（MDC）染色检测细胞中活性氧（ROS）含量和自噬阳性细胞比例；采用蛋白免疫印迹法（Western blot）检测磷脂酰肌醇3激酶（PI3K）/蛋白激酶B（Akt）/哺乳动物雷帕霉素靶蛋白（mTOR）、沉默信息调节蛋白2同源的去乙酰化酶3（Sirt3）信号通路及自噬相关蛋白的表达水平。[结果] 与模型组比较，6种睡茄交酯类成分在10 μmol/L剂量下能够明显缓解PA诱导的L6细胞葡萄糖摄取减少，且对细胞的毒性较小；与空白对照组相比，模型组的ROS含量和自噬阳性染色明显增加，6种睡茄交酯类成分明显减少ROS含量和自噬阳性细胞数量；同时模型组的微管关联蛋白-轻链3 Ⅱ（LC3 Ⅱ）和肌球蛋白样BCL2结合蛋白（Beclin1）蛋白表达量明显增加，p-Akt/Akt蛋白表达比例和Sirt3的蛋白表达量明显减少；其中，ZM6、ZM11、XGY21、XGY24和XGY29能够抑制LC3Ⅱ的表达，ZM3、ZM6、XGY21和XGY29能够抑制Beclin1蛋白的表达；XGY21、XGY24和XGY29能够逆转p-Akt/Akt和p-mTOR/mTOR的下调；ZM3、XGY21和XGY24亦能够促进Sirt3的表达。[结论] 酸浆属药用植物来源的睡茄交酯类成分能够通过降低ROS和自噬缓解PA引起的L6细胞胰岛素抵抗，其调节机制可能与Akt/mTOR与Sirt3信号通路相关。

Abstract:

[Objective] To investigate the mechanism of the effects of withanolides from the medicinal plants of Physalis genus on improving insulin resistance. [Methods] L6 rat myoblasts were incubated with palmitic acid（PA） for 9 h to build the insulin resistance cell model（IR-L6）. The glucose oxidase assay and the Methylthiazolyldiphenyl-tetrazolium bromide（MTT） assay were used to measure the glucose consumption and cell viability of IR-L6 cells treated with 15 withanolides. Flow cytometry after stained with 2’，7’-Dichlorodihydrofluorescein diacetate（DCFH-DA） or Monodansylcadaverine（MDC） were used to test the production of Reactive oxygen species（ROS） and the proportion of autophagy cells，respectively. Western blot was used to measure the expression of the proteins related with phosphatidylinositol-3 kinase（PI3K）/protein kinase B（AKT）/the mammalian target of rapamycin（mTOR），Sirtuin 3（Sirt3） signal pathways and autophagy-related proteins. [Results] The 6 active components at the concentration of 10 μmol/L significantly promoted glucose uptake in the IR-L6 cells with low toxic. Compared with the control group，the levels of ROS and the proportion of autophagic cells were higher，but 6 withanolides could reversed these changes. Meanwhile，the protein expression of microtubule-associated protein-light chain 3 Ⅱ（LC3Ⅱ）and coiled-coil，myosin-like BCL2-interacting protein（Beclin 1） were up-regulated in the model group，but the relative expression of p-Akt/Akt and Sirt3 were down-regulated. In addition，ZM6，ZM11，XGY21，XGY24 and XGY29 down-regulated the expression of LC3Ⅱ and Beclin1，while the ratio of the expression of p-p-Akt/Akt and p-mTOR/mTOR were reduced by XGY21，XGY24 and XGY29. Meanwhile，ZM3，XGY21 and XGY24 promoted the expression of Sirt3. [Conclusion] The withanolides from Physalis alleviated insulin resistance by reducing ROS and autophagy，which may be related to Akt/mTOR and Sirt3 signal pathway.