摘要目的 探讨生长分化因子11(GDF-11)对高糖诱导的小鼠胰岛素瘤细胞系MIN6细胞凋亡的影响及可能机制.方法 在体外不同葡萄糖浓度下培养MIN6细胞,分别加入重组GDF-11蛋白(rGDF-11)、转化生长因子β(TGF-β)受体Ⅰ抑制剂SB431542及磷脂酰肌醇?3?激酶(PI3K)抑制剂LY294002等干预因素.根据实验目的及干预因素不同将细胞随机分组为:正常对照组、正常对照组+GDF-11组、高糖组、高糖+GDF-11组、高糖+GDF-11+LY492002组、高糖+GDF-11+SB431542组.采用膜联蛋白V?异硫氰酸荧光素/碘化丙啶双染法检测细胞凋亡率,Western blotting法检测细胞内B淋巴细胞瘤2(Bcl-2)、Bcl-2相关X蛋白(Bax)、活性半胱天冬酶3(Cleaved-caspase3)等凋亡相关蛋白以及蛋白激酶B(Akt)、磷酸化Akt(p-Akt)、叉头转录因子1(FoxO1)、磷酸化FoxO1(p-FoxO1)、核糖体S6蛋白激酶1(S6k1)、磷酸化S6k1(p-S6k1)水平.免疫荧光染色法检测MIN6细胞磷酸化Smad2(p-Smad2)、磷酸化Smad3(p-Smad3)表达水平.多组间比较选用单因素方差分析,组间多重比较用最小显著差异t检验.结果 高糖可明显降低Bcl-2/Bax比例,升高凋亡关键酶Cleaved-caspase3表达,细胞凋亡率增加(分别为0.39±0.03比3.82±0.26、0.83±0.04比0.17±0.02、26.1%±3.0%比7.9%±0.8%,t=23.12、-27.60、-10.11,均P<0.01).而重组GDF-11蛋白干预可上调Bcl-2/Bax比例,减少Cleaved-caspase3表达,抑制细胞凋亡(t=-44.110、19.530、6.535,P<0.01).SB431542或LY294002与MIN6细胞共培养后rGDF-11的抗凋亡作用均明显减弱(均P<0.05).高糖显著降低p-Smad2水平(荧光强度表示),升高p-Smad3水平(t=4.830、5.760,均P<0.01).rGDF-11预处理可恢复细胞内p-Smad2水平,而SB431542明显抑制rGDF-11诱导的MIN6细胞p-Smad2蛋白表达的增加(t=5.55、3.04,均P<0.05).高糖可明显减少p-Akt、p-S6k1、p-FoxO1水平,而rGDF-11预处理后p-Akt、p-FoxO1水平明显高于HG组(均P<0.01).加入LY492002后,显著抑制rGDF-11诱导的细胞内p-Akt、p-FoxO1蛋白表达的增加(均P<0.05).结论 GDF-11可能通过激活TGF-β/Smad2及PI3K-Akt-FoxO1信号通路减少高糖诱导的β细胞凋亡.

abstractsObjective To investigate the effects of growth and differentiation factor 11 (GDF-11) on injury of MIN6 cells induced by high glucose and its possible mechanisms. Methods MIN6 cells were cultured in vitro and divided into the following groups:normal glucose (NG) group, NG+GDF-11 group, high glucose (HG) group, HG+GDF-11 group, HG+GDF-11+phosphatidylinositol?3?kinase (PI3K) inhibitor LY492002 group, and HG+GDF-11+transforming growth factorβ(TGF-β) receptorⅠinhibitor SB431542 group. Apoptosis of MIN6 cells were stained with Annexin V-FITC and propidium iodide (PI), and assessed by flow cytometry. The expression levels of B cell lymphoma 2 (Bcl-2), Bcl-2 associated X protein (Bax), cleaved-caspase3 and protein kinase B (Akt), phosphorylated Akt (p-Akt), forkhead box o1 (FoxO1), phosphorylated FoxO1 (p-FoxO1), ribosomal protein S6 kinase 1 (S6k1) and phosphorylated S6k1 (p-S6k1) were measured by western blotting. The phosphorylation of Smad were assessed by immunofluorescence. Data among multiple groups were compared by using one-way ANOVA and the least significant difference t test. Results Compared with the vehicle control groups, Bcl-2/Bax ratio decreased, the protein levels of cleaved-caspase 3 increased and the percentage of apoptotic MIN6 cells elevated in those groups cultured with HG (0.39±0.03 vs 3.82±0.26, 0.83±0.04 vs 0.17±0.02, 26.1%±3.0%vs 7.9%±0.8%, t=23.12,-27.60,-10.11, P<0.01, respectively). However, the effects were blocked by pretreatment with recombinant GDF-11 (rGDF-11) (t=-44.110, 19.530, 6.535,P<0.01, respectively). Treatment of MIN6 cells with TGF-βreceptorⅠinhibitor SB431542 or PI3K inhibitor LY294002 partially abolished the anti-apoptotic effects of rGDF-11 (all P<0.05). Immunofluorescence analyses showed that Smad 2 phosphorylation (p-Smad 2) decreased in high glucose culturing, whereas Smad 3 phosphorylation increased (t=4.830, 5.760, P<0.01, respectively). Pre-incubation with rGDF-11 restored p-Smad 2 expression, however, rGDF-11-mediated Smad 2 activation was partially blocked by SB431542 (t=5.550, 3.040, P<0.05, respectively). High glucose decreased Akt, FoxO1 and S6k1 phosphorylation in MIN6 cells, but pretreatment with rGDF-11 partly rescued these defects (P<0.01, respectively). The PI3K inhibitor LY294002 exerted a suppressive effect on rGDF-11-mediated Akt and FoxO1 phosphorylation (all P<0.05). Conclusion GDF-11 may prevent apoptosis in high glucose-induced MIN6 cells through activation of TGF-β/Smad 2 and PI3K-Akt-FoxO1 signaling pathway.