降糖消渴颗粒含药血清对INS-1细胞磷酸肌醇-3激酶/AKT/FOXO1通路的影响
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摘要
目的:通过研究降糖消渴颗粒含药血清对INS-1细胞PI3K/AKT/FOXO1通路中相关蛋白与基因表达的影响,探讨降糖消渴颗粒含药血清保护胰岛B细胞的分子机制。方法:选取FOXO1高表达INS-1稳定细胞株,分别以1%、5%、10%、15%、20%不同浓度降糖消渴颗粒含药血清干预,用MTT法计算细胞成活率以观察药物毒性,分别检测各组细胞中总FOXO1蛋白水平以确定最佳药物浓度开展后续实验。细胞用或不用PI3K抑制剂LY294002干预后,以Western blotting检测细胞中总FOXO1、p-FOXO1、Akt、p-Akt和细胞核内外的FOXO1、p-FOXO1的蛋白表达,qRT-PCR检测FOXO1mRNA和AktmRNA含量。结果:不同浓度含药血清对细胞均无毒性,其中10%为最佳药物干预浓度。10%含药血清使磷酸化FOXO1和Akt的表达升高;在胞质和胞核中,10%含药血清使FOXO1的表达降低,磷酸化表达升高,抑制剂LY294002使FOXO1和p-FOXO1表达均降低。实时荧光定量PCR(qRT-PCR)结果显示,Akt mRNA表达升高,FOXO1mRNA表达降低,抑制剂使FOXO1mRNA表达降低,结果与Western blotting结果一致。结论:降糖消渴颗粒含药血清可通过PI3K/AKT/FOXO1通路促进FOXO1磷酸化出核以抑制FOXO核转录实现保护胰岛B细胞的作用。
Objective: To study the effects of Jiangtang Xiaoke Granules containing serum on expressions of protein and genes of phosphoinositide-3 kinase( PI3 K)/AKT/FOXO1 signaling pathway in INS-1 cells to explore the molecular mechanism of serum contained Jiangtang Xiaoke Granules on protecting β-cells. Methods: The FOXO1 high expression INS-1 stable cell line was selected and treated with 1%,5%,10%,15% and 20% different concentrations of Jiangtang Xiaoke Granules containing serum.The cell survival rate was calculated by MTT method to observe the toxicity of the drug. The total FOXO1 protein level in each group was detected to determine the optimal drug concentration for subsequent experiments. The cells were transfected with or without the PI3 K inhibitor LY294002. Western blotting was used to detect the expression of FOXO1,p-FOXO1,Akt,p-Akt and FOXO1 and p-FOXO1 in the nucleus. The expression of FOXO1 mRNA and Akt mRNA was detected by qRT-PCR. Results: Different concentrations of drug-containing serum were not toxic to cells,and 10% of them were the optimal drug intervention concentration. The expression of phosphorylated FOXO1 and Akt was increased with 10% drug-containing serum. In the cytoplasm and nucleus,the expression of FOXO1 was decreased and the expression of phosphorylation was increased with 10% drug-containing serum,and the expression of FOXO1 and p-FOXO1 were decreased by the inhibitor LY294002. The results of real-time quantitative PCR( qRT-PCR) showed that the expression of Akt mRNA was increased,the expression of FOXO1 mRNA was decreased,and the expression of FOXO1 mRNA was decreased by inhibitor. The results were consistent with the results of Western blotting.Conclusion: The drug-containing serum of Jiangtang Xiaoke Granules can promote the phosphorylation of FOXO1 through the PI3 K/AKT/FOXO1 pathway to inhibit the transcription of FOXO nuclear to protect islet B cells.
Objective: To study the effects of Jiangtang Xiaoke Granules containing serum on expressions of protein and genes of phosphoinositide-3 kinase( PI3 K)/AKT/FOXO1 signaling pathway in INS-1 cells to explore the molecular mechanism of serum contained Jiangtang Xiaoke Granules on protecting β-cells. Methods: The FOXO1 high expression INS-1 stable cell line was selected and treated with 1%,5%,10%,15% and 20% different concentrations of Jiangtang Xiaoke Granules containing serum.The cell survival rate was calculated by MTT method to observe the toxicity of the drug. The total FOXO1 protein level in each group was detected to determine the optimal drug concentration for subsequent experiments. The cells were transfected with or without the PI3 K inhibitor LY294002. Western blotting was used to detect the expression of FOXO1,p-FOXO1,Akt,p-Akt and FOXO1 and p-FOXO1 in the nucleus. The expression of FOXO1 mRNA and Akt mRNA was detected by qRT-PCR. Results: Different concentrations of drug-containing serum were not toxic to cells,and 10% of them were the optimal drug intervention concentration. The expression of phosphorylated FOXO1 and Akt was increased with 10% drug-containing serum. In the cytoplasm and nucleus,the expression of FOXO1 was decreased and the expression of phosphorylation was increased with 10% drug-containing serum,and the expression of FOXO1 and p-FOXO1 were decreased by the inhibitor LY294002. The results of real-time quantitative PCR( qRT-PCR) showed that the expression of Akt mRNA was increased,the expression of FOXO1 mRNA was decreased,and the expression of FOXO1 mRNA was decreased by inhibitor. The results were consistent with the results of Western blotting.Conclusion: The drug-containing serum of Jiangtang Xiaoke Granules can promote the phosphorylation of FOXO1 through the PI3 K/AKT/FOXO1 pathway to inhibit the transcription of FOXO nuclear to protect islet B cells.
引文
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[13]Liu P,Kao TP,Huang H.CDK1 promotes cell proliferation and survival via phosphorylation and inhibition of FOXO1 transcription factor[J].Oncogene,2008,27(34):4733-4744.
[14]Anuradha R,Saraswati M,Kumar KG,et al.Apoptosis of beta cells in diabetes mellitus[J].DNA Cell Biol,2014,33(11):743-748.
[15]Martinez SC,Cras-Méneur C,Bernal-Mizrachi E,et al.Glucose regulates Foxo1 through insulin receptor signaling in the pancreatic islet beta-cell[J].Diabetes,2006,55(6):1581-1591.
[2]GBD 2015 Risk Factors Collaborators.Global,regional,and national comparative risk assessment of 79 behavioural,environmental and occupational,and metabolic risks or clusters of risks,1990-2015:a systematic analysis for the Global Burden of Disease Study 2015[J].Lancet,2016,388(10053):1659-1724.
[3]Cinti F,Bouchi R,Kim-Muller JY,et al.Evidence ofβ-Cell Dedifferentiation in Human Type 2 Diabetes[J].J Clin Endocrinol Metab,2016,101(3):1044-1054.
[4]Biggelaar LJ,Eussen SJ,Sep SJ,et al.Associations of Dietary Glucose,Fructose,and Sucrose withβ-Cell Function,Insulin Sensitivity,and Type 2 Diabetes in the Maastricht Study[J].Nutrients,2017,9(4).pii:E380.
[5]高思华,龚燕冰,倪青,等.肝脾肾同治法辨证治疗2型糖尿病的临床研究[J].中华中医药杂志,2009,24(8):1007-1010.
[6]莫芳芳,刘海霞,华静,等.降糖消渴颗粒含药血清对INS-1细胞Fox O1表达的影响[J].世界中医药,2017,12(12):3046-3049.
[7]赵丹丹,穆倩倩,方心,等.降糖消渴颗粒含药血清对C2C12细胞胰岛素抵抗的影响[J].中华中医药杂志,2014,29(5):1577-1579.
[8]Cao B,Zhang Z,Zhang Y,et al.Effect of Smilax china L.-containing serum on the expression of POLD1 mRNA in human hepatocarcinoma SMMC-7721 cells[J].Exp Ther Med,2013,6(4):1070-1076.
[9]Potente M,Urbich C,Sasaki K,et al.Involvement of Foxo transcription factors in angiogenesis and postnatal neovascularization[J].J Clin Invest,2005,115(9):2382-2392.
[10]Baracho GV,Cato MH,Zhu Z,et al.PDK1 regulates B cell differentiation and homeostasis[J].Proc Natl Acad Sci U S A,2014,111(26):9573-9578.
[11]Folli F,Okada T,Perego C,et al.Altered insulin receptor signalling andβ-cell cycle dynamics in type 2 diabetes mellitus[J].PLo S One,2011,6(11):e28050.
[12]Kim-Muller JY,Zhao S,Srivastava S,et al.Metabolic inflexibility impairs insulin secretion and results in MODY-like diabetes in triple Fox O-deficient mice[J].Cell Metab,2014,20(4):593-602.
[13]Liu P,Kao TP,Huang H.CDK1 promotes cell proliferation and survival via phosphorylation and inhibition of FOXO1 transcription factor[J].Oncogene,2008,27(34):4733-4744.
[14]Anuradha R,Saraswati M,Kumar KG,et al.Apoptosis of beta cells in diabetes mellitus[J].DNA Cell Biol,2014,33(11):743-748.
[15]Martinez SC,Cras-Méneur C,Bernal-Mizrachi E,et al.Glucose regulates Foxo1 through insulin receptor signaling in the pancreatic islet beta-cell[J].Diabetes,2006,55(6):1581-1591.