高浓度葡萄糖条件下罗格列酮对NIT-1细胞FOXO1、TSC2基因表达及细胞分泌功能的影响

目的：研究罗格列酮在不同浓度葡萄糖条件下，对胰岛β细胞增殖凋亡与胰岛素分泌以及叉头转录因子-1(FOXO1)和结节性硬化症-2(TSC2)表达的影响。方法： 将 NIT-1细胞按每孔5×10个放置于24孔细胞培养板，培养48 h后随机分为各处理组：5.6、7.8、11.1、16.7、22.2 和27.6 mmol/L葡萄糖组，继续培养24 h后再分别施加1×10^-5mol/L罗格列酮，分别于干预24和48 h后取细胞培养上清液，采用放射免疫法检测胰岛素水平和免疫荧光法检测细胞增殖情况，RT-PCR半定量法检测FOXO1和TSC2 mRNA表达水平。结果：①1×10^-6～1×10^-5 mol/L罗格列酮可以分别在不同浓度葡萄糖培养条件下使胰岛NIT-1细胞增殖（P<0.05）,且这种变化趋势随剂量的增加而增加（即1×10^-5 mol/L罗格列酮组＞1×10^-6 mol/L罗格列酮组＞1×10^-7 mol/L罗格列酮组）。1×10^-5 mol/L的罗格列酮干预后，可见细胞凋亡百分率增加趋势随着葡萄糖浓度不断升高；②在同一浓度罗格列酮作用下，当葡萄糖浓度为11.1 mmol/L时，胰岛素分泌水平最高，高于其他各组（均P<0.05），随着葡萄糖浓度增加，胰岛素分泌量逐渐下降（11.1 mmol/L葡萄糖组>16.7 mmol/L葡萄糖组>22.5 mmol/L葡萄糖组>27.6 mmol/L葡萄糖组），而葡萄糖为5.6 mmol/L时，胰岛素分泌量最低；③ 在1×10^-5 mol/L罗格列酮干预后，FOXO1和TSC-2 mRNA的表达水平均较未干预组明显下降，且呈现出5.6 mmol/L组＜11.1 mmol/L组＜16.7 mmol/L组＜22.5 mmol/L组＜27.6 mmol/L组的变化趋势，而且葡萄糖浓度＞16.7 mmol/L的各组均较前面小剂量葡萄糖组（≤11.1 mmol/L各组）表达明显。结论：罗格列酮可以通过直接影响胰岛β细胞内FOXO1和TSC2表达促进胰岛β细胞的增殖及影响细胞胰岛素分泌功能，提示通过调控FOXO1和TSC2表达，可以直接影响胰岛β细胞的生物学功能，如分泌功能、细胞的增殖与凋亡以及改善胰岛素抵抗状况。

Effects of rosiglitazone on expressions of FOXO1 and TSC2 gene and cell secretory function of NIT-1 cells after treated with high concentration glucose

Objective To study the effects of rosiglitazone on FOXO1 and TSC2 gene expressions,insulin secretory function,cell proliferation and apoptosis of pancreatic βcells under high concentration glucose condition.Mothods The NIT-1 cells were put into plates (5×10~4 cells/well) and cultivated for 48 h,then they were randomly divided into treatment groups containing different concentrations of glucose as follows:5.6,7.8,11.1,16.7,22.2,and 27.6 mmol·L~(-1) groups.After cultivated for 24 h,they were intervented by 10~(-5) mmol·L~(-1) rosiglitazone for next 24 and 48 h,then the supernatant was collected.The insulin level was evaluated by radio-immunity technique,the cell proliferation and apoptosis were detected by immunofluorescence staining and MTT assay respectivly.The expressions of FOXO1 and TSC2 mRNA were detected by semi-quantitative RT-PCR assay.Results ① Under different concentrations of glucose,after treated with 10~(-6)-10~(-5) mol·L~(-1) rosiglitazone the proliferation of pancreatic β cells (NIT-1 cell line) was found (P<0.05) and the apoptotic rate of cells was increased in a dose-dependent manner(1×10~(-5) mol·L~(-1) rosiglitazone group>1×10~(-6) mol·L~(-1) rosiglitazone group>1× 10~(-7) mol·L~(-1) rosiglitazone group).② When under same dose of glucose,the insulin secretion level in 11.1 mmol·L~(-1) group was much higher than those in other groups (P<0.05),but the insulin secretion level was reduced gradually following the decrease of glucose concentration(11.1 mmol·L~(-1) group>16.7 mmol·L~(-1) group >22.5 mmol·L~(-1) group>27.6 mmol·L~(-1) group).The insulin secretion level in 5.6 mmol·L~(-1) group was the lowest.③ After intervention of 10~(-5) mol·L~(-1) rosiglitazone,the expression levels of both FOXO1 and TSC2 mRNA were significantly lower than those in control group(5.6 mmol·L~(-1) group<11.1 mmol·L~(-1) group<16.7 mmol·L~(-1) group<22.5 mmol·L~(-1) group<27.6 mmol·L~(-1) group).When the glucose concentration was over 16.7 mmol·L~(-1) the expressions of FOXO1 and TSC2 mRNA were obviously higher than those in the groups with glucose concentration≤11.1 mmol·L~(-1) after the intervention of 10~(-5) mol·L~(-1) rosiglitazone.Conclusion Rosiglitazone can improve the secretion function of pancreatic β cells and cell proliferation and alleviate insulin resistance by directly regulating FOXO1 and TSC2 expressions.