化合物CX09040抑制PTP1B和保护胰岛β细胞的作用

The effects of compound CX09040 on the inhibition of PTP1B and protection of pancreatic β cells

本研究旨在探讨2-(4-甲氧羰基-2-十四烷氧基苯基)氨甲酰基苯甲酸(CX09040)对胰岛β细胞的保护作用。经尾静脉注射四氧嘧啶(alloxan)破坏ICR小鼠的胰岛β细胞,建立胰岛β细胞受损小鼠模型,给予小分子化合物CX09040进行治疗。阳性对照药为利拉鲁肽(liraglutide)。以病理切片中胰腺的胰岛数量和总面积评价胰岛细胞量;以葡萄糖刺激胰岛素释放(GSIS)试验评价β细胞分泌功能;采用口服葡萄糖耐量试验(OGTT)评价动物的糖代谢状态;Western blotting法检测胰腺组织的蛋白表达。分别用体外酶活性、细胞内酶活性及动物胰腺蛋白表达等评价对分子靶点蛋白酪氨酸磷酸酶1B(PTP1B)的作用。结果显示,化合物CX09040对β细胞受损小鼠具有明显胰岛β细胞保护作用,增加胰岛数量(P<0.05)和总面积(P<0.05)、升高葡萄糖刺激的胰岛素分泌量(P<0.01)和早期相胰岛素分泌(AIR)指数(P<0.01);具有明显改善葡萄糖耐量低减的作用,降低葡萄糖负荷后AUC值(P<0.01)。经CX09040治疗后,模型小鼠胰腺β细胞增殖相关的p-Akt/Akt(P<0.01)、p-Fox O1/Fox O1(P<0.001)水平升高,PDX-1表达上调(P<0.01)。化合物CX09040抑制重组h PTP1B酶活性的IC50为2.78×10-7 mol·L-1;对PTP1B高表达293T细胞内PTP1B活性的抑制率为72.8%(P<0.001);使β细胞受损小鼠胰腺PTP1B表达降低(P<0.001)、其靶蛋白p-IRβ/IRβ水平升高(P<0.01)。提示化合物CX09040可通过抑制分子靶点PTP1B,从而调控β细胞增殖,增加胰岛细胞量及葡萄糖刺激的胰岛素分泌,发挥保护胰岛β细胞的作用。

To investigate the effects of 2-（4-methoxycarbonyl-2-tetradecyloxyphenyl）carbamoylbenzoic acid （CX09040） on protecting pancreatic fl cells, the β cell dysfunction model mice were induced by injection of alloxan into the caudal vein of ICR mice, and were treated with compound CX09040. Liraglutide was used as the positive control drug. The amount and the size of islets observed in pathological sections were calculated to evaluate the β cell mass; the glucose stimulated insulin secretion （GSIS） test was applied to estimate the β cell secretary function; the oral glucose tolerance test （OGTT） was taken to observe the glucose metabolism in mice; the expressions of protein in pancreas were detected by Western blotting. The effects on the target protein tyrosine phosphatase β （PTP1B） were assessed by the PTP1B activities of both recombinant protein and the intracellular enzyme, and by the PTP1B expression in the pancreas of mice, separately. As the results, with the treatment of CX09040 in alloxan-induced β cell dysfunction mice, the islet amount （P〈0.05） and size （P〈0.05） increased significantly, the changes of serum insulin in GSIS （P〈0.01） and the values of acute insulin response （AIR, P〈0.01） were enhanced, compared to those in model group; the impaired glucose tolerance was also ameliorated by CX09040 with the decrease of the values of area under curve （AUC, P〈0.01）. The activation of the signaling pathways related to β cell proliferation was enhanced by increasing the levels of p-Akt/Akt （P〈0.01）, p-FoxO1/FoxO1 （P〈0.001） and PDX-1 （P〈0.01）. The effects of CX09040 on PTP1B were observed by inhibiting the recombinant hPTP 1B activity with IC 50 value of 2.78 × 10-7 mol.L-1, reducing the intracellular PTP1B activity of 72.8% （P〈0.001）, suppressing the PTP1B expression （P〈0.001） and up-regulating p-IRβ/IRβ （P〈0.01） in pancreas of the β cell dysfunction mice, separately. In conclusion, compound CX09040 showed significant protection effects against the dysfunction of β cell of mice by enlarging the pancreatic β cell mass and increasing the glucose-induced insulin secretion; its major mechanism may be the inhibition on target PTP1B and the suceedent up-regulation of β cell proliferation.