FGF21对人胰腺癌细胞炎症环境中星状细胞活化的抑制及机制

Inhibition and mechanism of FGF21 on activation of stellate cells in inflammatory environment of pancreatic cancer cells

摘要:

目的： 探讨成纤维细胞生长因子21（fibroblast growth factors 21，FGF21）对胰腺癌细胞炎症环境中星状细胞增殖和活化的影响及机制。方法： RT-qPCR检测胆囊收缩素（cholecystokinin，CCK）作用于人胰腺癌细胞（pancreatic cancer cells，PCC）后白细胞介素（interleukin，IL）-6及IL-8 mRNA的表达水平变化，确立适宜的时间建立炎症微环境。将PCC（Capan-1和MIA PaCa-2）CCK上清液和PCC（Capan-1和MIA PaCa-2）CCK+FGF21上清液作用于人胰腺星状细胞（pancreatic stellate cells，PSC）。用CCK-8和Western blot分别检测各组上清液对胰腺星状细胞活性及其活化标志物α-平滑肌蛋白（α-smooth muscle protein，α-SMA）表达水平的影响。RT-qPCR和Western blot检测FGF21对CCK处理后的PCC EGR1和PDGFB mRNA及蛋白表达的影响。Western blot进一步检测AMPK抑制剂（compound C）加入PCC CCK组和PCC CCK+FGF21组后对AMPK-κB信号通路中p-AMPK和EGR1蛋白表达的影响。结果： CCK处理Capan-1 6 h与处理MIA PaCa-2 24 h的IL-8 mRNA表达量明显高于其他组（P<0.05）。PCC CCK+FGF21上清液组与PCC CCK上清液组相比，其PSC细胞增殖明显受抑制（P<0.05），α-SMA蛋白表达明显降低（P<0.001）。FGF21干预PCC CCK后EGR1和PDGFB mRNA和蛋白表达均明显低于CCK组（P<0.05）。Compound C加入PCC CCK+FGF21组后，p-AMPK蛋白表达水平均降低（P<0.01），EGR1蛋白表达水平均增高（P<0.05）。结论： FGF21可能通过抑制胰腺癌细胞炎症环境中星状细胞的增殖和活化来实现其对肿瘤的抑制作用，其机制可能与其激活炎症环境下胰腺癌细胞AMPK通路并下调EGR1的表达及减少PDGFB的表达有关。

Abstract:

Objective: To investigate the effect and mechanism of fibroblast growth factor 21 (FGF21) on the proliferation and activation of stellate cells in the inflammatory environment of pancreatic cancer cells. Methods: The appropriate time to establish an inflammatory microenvironment was determined by RT-qPCR to detect interleukin (IL) -6 and IL-8 mRNA expression level of cholecystokinin (CCK) on human pancreatic cancer cells (PCC) such as Capan-1 and MIA PaCa-2. PCC (Capan-1, MIA PaCa-2) CCK supernatant and PCC (Capan-1, MIA PaCa-2) CCK+FGF21 supernatant were acted on human pancreatic stellate cells (PSC). CCK-8 and Western blot were used to detect the effect of each group’s supernatant on the activity of pancreatic stellate cells and the expression of α-smooth muscle protein (α-SMA), a marker of activation. RT-qPCR and Western blot were used to detect the effect of FGF21 on the expression of PCC EGR1 and PDGFB mRNA and protein after CCK treatment. Western blot further detected the effect of AMPK inhibitor (compound C) added to PCC CCK group and PCC CCK+FGF21 group on the expression of p-AMPK and EGR1 protein in AMPK-κB signaling pathway. Results: The relative expressions of IL-8 mRNA in Capan-1 6 h group and in MIAPaCa-2 24 h group treated with CCK were significantly higher than those in other groups (P<0.05). Compared with the PCC CCK supernatant group, the PCC CCK+FGF21 supernatant group had significantly inhibited the proliferation of PSC cells (P<0.05), the expression of α-SMA protein was significantly reduced (P<0.001). Intervention with FGF21, EGR1, PDGFB mRNA and protein expressions were significantly lower than those in CCK group (P<0.05). After compound C was added to the PCC CCK+FGF21 group, the expression level of p-AMPK protein decreased (P<0.01), and the expression level of EGR1 protein expression increased (P<0.05). Conclusion: FGF21 may prevent tumor by inhibiting the proliferation and activation of stellate cells in the inflammatory environment of pancreatic cancer cells and the mechanism may be related to its activation of the AMPK pathway of pancreatic cancer cells in the inflammatory environment, down-regulation of EGR1 expression and reduction of PDGFB expression.