葛根素对RAW264.7源性泡沫细胞胆固醇摄取及外排功能的影响

Effect of puerarin on cholesterol influx and efflux in RAW264.7-treated foam cells

目的:探讨葛根素(puerarin,Pur)对RAW264.7源性泡沫细胞胆固醇摄取及外排功能的影响。方法:以氧化低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL)诱导RAW264.7源性泡沫细胞。细胞经处理后,利用荧光探针Di I-ox-LDL检测泡沫细胞摄取ox-LDL的能力,NBD标记胆固醇外排实验检测泡沫细胞的胆固醇外排功能,Western blot法检测LC3II、P62、CD36、ATP结合盒转运蛋白A1(ATP-binding cassette transporter A1,ABCA1)、溶酶体酸性脂肪酶(lysosomal acid lipase,LAL)以及p-AMPK的蛋白水平。结果:RAW264.7源性泡沫细胞经Pur处理后胆固醇摄取减少、胞内胆固醇外排增加并且自噬相关蛋白LC3II表达增加,P62表达减少,脂质吸收相关蛋白CD36表达减少,胆固醇外排相关蛋白ABCA1和LAL表达增加,其变化与自噬激动剂雷帕霉素处理后改变类似。Pur与自噬抑制剂3-甲基腺嘌呤共处理后,胆固醇摄取增加、胆固醇外排功能减弱,并且ABCA1、LAL和p-AMPK的蛋白水平减少,但CD36表达未有明显改变。结论:Pur可使得泡沫细胞LAL和ABCA1介导的胆固醇外排能力有所增强,其机制可能是通过AMPK通路增强自噬对胆固醇外排的调控;又可通过对CD36负向调节而使得泡沫细胞对脂质的摄取减少。

AIM： To study the protective effect of puerarin on the atherosclerosis of RAW264. 7-derived foam cells. METHODS： The model of foam cells was established by incubating the RAW264. 7 cells with ox-LDL. The cholesterol uptake was evaluated by a Di I-ox-LDL binding assay. The ability of cholesterol efflux of the RAW264. 7-derived foam cells was detected by cholesterol efflux assay. The protein levels of LC3 II,P62,CD36,ABCA1,LAL and p-AMPK were determined by Western blot. RESULTS： Puerarin treatment reduced the cholesterol uptake capacity and enhanced the cholesterol efflux rate. The protein levels of LC3 II,ABCA1 and LAL in puerarin group were higher than that in ox-LDL group,while the protein levels of P62 and CD36 were obviously decreased,and those in rapamycin treatment group had the same change as puerarin group. The protein levels of LC3 II,ABCA1 and LAL were obviously decreased and the protein level of p-AMPK was increased after co-treated with 3-MA. CONCLUSION： Puerarin promotes LAL and ABCA1-mediated cholesterol efflux in ox-LDL-treated RAW264. 7 macrophages,which might enhance autophagy through AMPK-dependent pathway for cholesterol efflux regulation,and reduce the uptake of lipids by CD36 negative regulation.