脂筏介导的内源性大麻素对HepG2细胞的作用及其机制

Membrane cholesterol mediates the endocannabinoids-anandamide effection on HepG2 cells

目的研究内源性大麻素（AEA）以脂质为基础的信号途径对肝癌细胞株HepG2的作用机制，探讨AEA在肝癌发生和发展中的作用。方法免疫荧光检测脂肪酸水解酶、大麻素受体（CB）1和CB2在胎肝细胞株L02和肝癌细胞株HepG2中的定位。以不同浓度的AEA及膜胆固醇耗竭剂甲基-β-环糊精（MCD）处理，分为AEA10μmol／L组、AEA20μmol／L组、AEA40μmol／L组、MCD10mmol／L＋AEA10μmol／L组、MCD10mmol／L＋AEA20μmol／L组和MCD10mmol／L＋AEA40μmol／L组，分别孵育L02细胞和HepG2细胞，流式细胞术碘化丙啶单染法检测细胞的坏死率。WesternBlot检测L02和HepG2细胞中脂肪酸水解酶、CBl和CB2的蛋白表达及其下游信号通路磷酸化P38促分裂原活化蛋白激酶（P—P38MAPK）和磷酸化CJun氨基端激酶（P—JNK）的表达变化。组间均数的比较采用独立样本t检验或单因素方差分析。结果AEA可有效地导致肝癌细胞坏死，以浓度为AEA40μmol／L组达到最大效应，F=108．594，P〈0．05，差异有统计学意义。MCD10mmol／L预孵育后的HepG2细胞坏死率在AEA10μmol／L组、AEA20μmol／L组和AEA40μmol／L组分别为7盘3％±2．13％，16．30％±0．94％，43．09％±5．10％，MCD处理前AEA10μmol／L组、AEA20μmol／L组、AEA40μmol／L组分别为13．64％±1．69％、20．28％±0．91％、52．71％±4．29％，处理前后比较，t值分别为3．702，5．274和3．503，P值均〈0．05，差异均有统计学意义。同时，AEA能激活HepG2细胞中P38MAPK和JNK，以AEA40μmol／L组作用明显，F值分别为11．908和26．054，P值均〈0．05，差异均有统计学意义，MCD作用前P—P38MAPK和P—JNK／D-肌动蛋白灰度值分别为1．63±0．06，1．60±0．31，MCD作用后PP38MAPK／β-肌动蛋白、P-JNK灰度值分别为1．14±0．01、1．17±0．29，作用前后相比，t值分别为2．801和12．829，雅均〈0．05，差异均有统计学意义。结论AEA可以有效地导致HepG2细胞坏死而对L02细胞无影响，AEA激活了HepG2细胞pP38MAPK和PJNK相关信号传导途径，且此过程与脂筏有关。

Objective To study the effect of anandamide （AEA） on necrosis in HepG2 cells and to explore the role of AEA in progression of liver cancer. Methods Localization of the fatty acid hydrolytic enzyme （FAAH）, cannabinoid receptors 1（CB1） and cannabinoid receptors2 （CB2） proteins was detected in L02 and HepG2 cells using immunofluorescence. L02 and HepG2 cells were treated with different concentrations of AEA and methyl-β -cyclodextrin, and the rates of cells necrosis were examined by PI stain. Meanwhile, the expression levels of FAAH, CB 1 and CB2 receptor proteins, as well as P38 mitogen-activated protein kinase （p-P38 MAPK） and c-Jun-NH2-terminal kinase （p-JNK） proteins, were analyzed by Western blot. Results The FAAH, CB 1 and CB2 receptor proteins were observed both in cytoplasm and on membrane in L02 and HepG2 cells. The expression level of FAAH protein was higher in HepG2 than in L02 cells. The expression level of CB 1 receptor protein was very low in both L02 and HepG2 cells. The expression level of CB2 receptor protein was high in both L02 and HepG2 cells. AEA treatment induced necrosis in HepG2 cells but not in L02 cells. Methyl-β -cyclodextrin treatment prevented necrosis in HepG2 cells （t = 3.702; 5.274; 3.503, P 〈 0.05）. The expression patterns of FAAH, CB 1 and CB2 receptor protein in L02 and HepG2 cells were confirmed by western blot, which were consistent with the immunofluorescence results. AEA treatment increased the levels of p-P38MAPK and p-JNK proteins in a dose-dependant manner in HepG2 cells （F = 11.908; 26.054, P 〈 0.05） and the increase can be partially by prevented by MCD （t = 2.801; t = 12.829, P 〈 0.05）. Conclusion AEA treatment induces necrosis in HepG2 cells via CB1 and CB2 receptors and lipid rafts.