MAPK通路在G蛋白偶联受体40介导的小鼠胰岛NIT-1细胞脂性凋亡中的作用

Role of MAPK pathway in GPR40 mediated NIT-1 cells lipoapoptosis

目的:探讨丝裂原活化蛋白激酶(MAPK)信号通路在G蛋白偶联受体40(GPR40)介导的小鼠胰岛NIT-1细胞脂性凋亡中的作用.方法:利用小RNA干扰(siRNA)技术抑制GPR40在NIT-1细胞表达,观察对细胞脂性凋亡(棕榈酸、油酸干预)的影响.采用Hoechst33342染色、TUNNEL及流式细胞仪检测细胞凋亡.Western Blot检测棕榈酸、油酸孵育NIT-1细胞各时间点及GPR40 siRNA转染NIT-1细胞丝裂原活化蛋白激酶(MAPK)通路激酶磷酸化水平.并予相应激酶阻断剂预处理后观察细胞脂性凋亡变化.结果:棕榈酸孵育后空转组,对照siRNA转染和GPR40 siRNA转染细胞凋亡率差异无统计学意义.予棕榈酸和油酸共孵育,GPR40 siRNA转染细胞凋亡率明显高于空转组细胞;油酸孵育使ERK1/2呈时间依赖性地激活(磷酸化),ERK的特异性抑制剂预处理NIT-1细胞后,继予棕榈酸和油酸共孵育,NIT-1细胞凋亡率较对照组明显升高.油酸刺激GPR40 siRNA转染细胞的ERK磷酸化水平较空转组明显下降.结论:棕榈酸诱导NIT-1细胞脂性凋亡不依赖GPR40,而不饱和脂肪酸油酸对NIT-1细胞脂性凋亡的保护作用至少部分通过GPR40介导.其可能的假设机制为不饱和脂肪酸通过受体GPR40,激活ERK-MAPK通路,导致抗凋亡效应产生.

AIM： To evaluate the role of MAPK pathway in GPR40 mediated NIT-1 cells lipoapoptosis. METHODS： The effect of silencing GPR40 gene in NIT-1 cells on lipoapoptosis of NIT-1 cells was evaluated and cell apoptosis was detected by Hoechst 33 342, TUNNEL and flow cytometry analysis （ Annexin V/PI）. The effects of palmitate, oleate or GPR40 siRNA on MAPK signaling pathway and the effects of PD98059 on oleate-inhibited lipoapoptosis were also observed. JNK, ERK and p38 kinase phosphorylation were detected by Western blot. RESULTS： Palmitate induced 13 cell apoptosis, which was not mediated through GPR40. The anti-lipoapoptotic activity induced by oleate was suppressed by transfection with GPR40 siRNA. ERK was markedly activated by treatment with oleate. Pretreatment with PD098059 significantly suppressed the anti-lipoapoptotic effect of oleate. Knock-down of GPR40 inhibited the level of ERK1/2 phosphorylation stimulated by oleate. CONCLUSION： Palmitate induces 13 cell apoptosis by no mediation through GPR40. Oleate protects NIT-1 cells from palmitate-induced lipoapoptosis, partly by mediating through GPR40. The possible mechanism is that oleate promotes the activation of ERK MAPK pathway via GPR40, leading to the anti-lipoapoptotic effect on NIT-1 cells.