肥胖抑制素对高脂诱导的大鼠胰岛β细胞株INS-1凋亡及信号通路的影响

Effects of obestatin on lipotoxicity-induced apoptosis in INS-1 cells

目的 探讨肥胖抑制素对高脂诱导的大鼠胰岛β细胞株INS-1凋亡和相关信号通路的影响.方法 将大鼠胰岛β细胞株INS-1分为5%牛血清白蛋白（BSA）组、5%BSA＋1 nmol/L肥胖抑制素（OB）组、5%BSA＋10 nmol/L OB组、5%BSA＋100 nmol/LOB组、5%BSA＋500 nmol/L OB组、0.5 mmol/L棕榈酸（PA）组、0.5 mmol/L PA＋1 nmol/L OB组、0.5 mmol/LPA＋10 nmol/L OB组、0.5 mmol/L PA＋100 nmol/L OB组、0.5 mmol/L PA＋500 nmol/L OB组,应用细胞计数试剂盒检测各组细胞存活率.另将INS-1细胞分为5%BSA组、100 nmol/L OB组、50 μmol/L LY294002组、0.5mmol/L PA组、0.5 mmol/L PA＋50 μmol/L LY294002组、0.5 mmol/L PA＋100 nmol/L OB组、0.5mmol/L PA＋100 nmol/L OB＋50 μmol/L LY294002组,应用膜连蛋白V/碘化丙啶双染方法检测各组细胞凋亡率.最后,将INS-1细胞分为5%BSA组、0.5 mmol/L PA＋100 nmol/L OB 0 min组、0.5mmol/L PA＋100 nmol/L OB 15 min组、0.5 mmol/L PA＋100 nmol/LOB 30 min组、0.5 mmol/L PA＋100 nmol/L OB 60 min组,应用Westen blot法检测各组细胞蛋白激酶B（Ser473）磷酸化水平.应用单因素方差分析进行组间比较.结果 无PA干预时,OB剂量依赖性地促进细胞存活;与5%BSA组相比,5%BSA＋100 nmol/L OB组细胞存活率增加36%.PA干预后,0.5 mmol/L PA组细胞存活率较5%BSA组下降35%.OB剂量依赖性地阻上PA诱导的脂毒性,与0.5 mmol/L PA组相比,0.5 mmol/LPA＋10 nmol/L OB组细胞存活率增加23%,0.5 mmol/L PA＋100 nmol/LOB组增加35%.0.5mmol/L PA组细胞凋亡率为26%,明显高于5%BSA组.加用100 nmol/L OB后,细胞凋亡率下降15%.0.5 mmol/L PA＋100 nmol/L OB 30 min组、0.5 mmol/L PA＋100 nmol/L OB组磷酸化蛋白激酶B表达水平较高.加用LY294002后磷酸化蛋白激酶B表达明显受抑制,细胞凋亡率增加至20%.结论 OB抑制高脂诱导的胰岛β细胞凋亡,可能与磷脂酰肌醇3激酶/蛋白激酶B信号通路有关.

Objective To investigate the effect of obestatin （OB） on lipotoxicity-induced apoptosis in pancreatic β-cell line INS-1. Methods The INS-1 cells were divided into the following groups： 5%bovine serum albumin （BSA） group,5% BSA ＋ 1 nmol/L OB group,5% BSA ＋ 10 nmol/L OB group,5%BSA ＋ 100 nmol/L OB group,5% BSA ＋500 nmol/L OB group,0. 5 mmol/L palmitic acid （PA） group,0. 5 mmol/L PA ＋ 1 nmol/L OB group,0. 5 mmol/L PA ＋ 10 nmol/L OB group,0. 5 mmol/L PA ＋ 100nmol/L OB group,0. 5 mmol/L PA ＋ 500 nmol/L OB group. Cell survival rate was evaluated by using CCK-8. To investigate cell apoptosis,the INS-1 cells were then divided into 7 groups,including 5% BSA group,100 nmol/L OB group,50 μmol/L LY294002 group,0. 5 mmol/L PA group,0. 5 mmol/L PA ＋50μmol/L LY294002 group,0. 5 mmol/L PA ＋ 100 nmol/L OB group,and 0. 5 mmol/L PA ＋ 100 nmol/LOB ＋50 μmol/L LY294002 group. The INS-1 cells were finally divided into 5% BSA group,0. 5 mmol/LPA ＋ 100 nmol/L OB 0 min group,0. 5 mmol/L PA ＋ 100 nmol/L OB 15 min group,0. 5 mmol/L PA ＋100 nmol/L OB 30 min group,and 0. 5 mmol/L PA ＋ 100 nmol/L OB 60 min group to assess total PKB and p-PKB （Ser 473 ） levels. Data were analyzed by using One-way ANOVA. Results OB alone dosedependently promoted cell growth,and 100 nmol/L OB produced best result when compared with 5% BSA alone. Cell viability was decreased by 35% after PA treatment. Treatment with OB dose-dependently prevented PA-induced toxicity,most effectively by 35% at 100 nmol/L,with 10 nmol/L being the lowest active concentration by 23% when compared with PA alone. The percentage of apoptosis reached to 26%after PA treatment for 24 h,which was significantly increased when compared with BSA alone. Treatment with OB at 100 nmol/L reversed apoptosis rate by 15%. Obestatin at 100 nmol/L induced rapid activation of PKB and reached the maximal effect at 30 min. Obestatin-induced activation of PKB phosphorylation was markedly blocked by LY294002. The percentage of apoptosis increased to 23% after LY294002 treatment. Conclusions Obestatin inhibits lipotoxicity-induced apoptosis in pancreatic β-cell line INS-1 through PI3K/PKB signal pathway and the peptide may have potential in treating diabetes.