非律平菌素干扰甲基硝基亚硝基胍对FL细胞鞘脂代谢的影响

Filipin Interferes with the Effects of N-Methyl-N′-nitro-N-nitrosoguanidine on Sphingolipids Metabolism in Human Amnion FL Cells

甲基硝基亚硝基胍(MNNG)可通过脂筏诱导细胞表面受体聚簇并激活NF-κB信号通路.本研究拟探讨脂筏干扰剂非律平菌素(filipin)对MNNG作用的影响.利用脂类组学方法分别研究了MNNG、filipin单独处理及先用filipin再用MNNG处理情况下对人羊膜FL细胞鞘脂代谢的影响,用MALDI-TOF质谱法分析细胞鞘脂组成的变化,酶联免疫吸附法检测NF-κB通路的活化,RT-PCR法检测鞘脂代谢通路中关键酶的表达.结果表明,MNNG和filipin都可影响FL细胞鞘脂类代谢,但MNNG作用更显著.Filipin预处理可部分抑制MNNG对细胞鞘脂类代谢的影响,且能够抑制MNNG对NF-κB的活化;但filipin、MNNG单独或联合处理都不影响鞘脂代谢关键酶丝氨酸棕榈酰转移酶、酸性鞘磷脂酶和鞘磷脂合成酶在mRNA水平的表达.以上结果说明,filipin预处理会导致甲基硝基亚硝基胍引起FL细胞鞘脂代谢以及NF-κB活性的改变.而可能的机制在于,filipin破坏脂筏结构从而引起一系列信号途径的改变,而非通过改变脂类代谢关键酶的表达.

N-methyl- N＇-nitro- N-nitrosoguanidine （MNNG） induces cell surface receptor clustering through lipid rafts and the activation of NF-κB. In the present study, we investigated whether the lipid raft-interfering agent filipin influences the effects of MNNG on sphingolipids metabolism. The effects of MNNG, filipin, and pretreatment with filipin before MNNG exposure on human amnion FL cell sphingolipids metabolism were analyzed by lipidomic approaches; sphingolipids profiles were analyzed by MALDI-TOF Mass spectrometry, NF- κB activation was measured by ELISA; and the expression levels of key enzymes in sphingolipids metabolism were examined by RT-PCR. It was found that both MNNG and filipin changed the sphingolipids metabolism profiles, while the effect of MNNG was more profound. Filipin preincubation partially inhibited the effects of MNNG on sphingolipids, as well as the activation of NF-κB. However, there were no significant changes of gene expressions of key sphingolipid metabolism enzymes （serine palmitoyhransferase, acid sphingomyelinase, and sphingomyelin synthase） compared with the expression levels of control groups. Together, these data indicated that filipin interfered with the effects of MNNG on sphingolipids metabolism and the activity of NF-VB in FLcells through the disruption of lipid raft structure rather than increase the expression of the key enzymes in the sphingolipid metabolism pathway.