儿茶素对Caco-2细胞胆固醇摄取的影响及机制研究

Effect of catechin on cholesterol uptake in Caco-2 cells and its mechanism

目的:研究儿茶素(Catechin,Cat)对Caco-2细胞胆固醇摄取的影响,以及可能的作用机制。方法:利用胆固醇:甲基β环糊精复合物(Chol:MβCD)建立Caco-2细胞脂质蓄积模型。不同浓度的儿茶素(20、40、60μmol·L-1)处理细胞24 h,结合油红O染色法观察细胞内的脂质蓄积,酶法测定细胞内胆固醇含量及分布,qRT-PCR及Westernblot检测胆固醇代谢相关基因NPC1L1和SREBP-2的表达。结果:与空白组相比,Chol:MβCD处理组细胞内红色脂滴颗粒以及胆固醇含量明显增加。20~60μmol·L-1儿茶素不仅可以不同程度地减少细胞内红染脂滴的形成,而且可以显著降低细胞内总胆固醇和游离胆固醇的含量,以及胆固醇酯在总胆固醇中的比例。此外,儿茶素可以剂量依赖性地降低胆固醇代谢相关基因NPC1L1及SREBP-2的mRNA和蛋白表达。其中以60μmol·L-1 Cat处理组的作用最为显著(P<0.01)。结论:儿茶素可能通过下调胆固醇代谢相关基因NPC1L1和SREBP-2的表达,进而减少Caco-2细胞摄取胆固醇及蓄积。

OBJECTIVE To study the effect of catechin(Cat)on cholesterol uptake in Caco-2 cells and its possible mechanism.METHODS The Caco-2 cells lipid accumulation model was established with Cholesterol:Methyl-β-cyclodextrins Complex(Chol:MβCD).Cells were treated with different concentrations of catechin(20,40,60μmol·L-1)for 24 hours.Meanwhile,the intracellular lipid accumulation was observed by oil red O staining.Enzymatic method was used to determine the content of intracellular free cholesterol(FC)and total cholesterol(TC).Real-time polymerase chain reaction(qPCR)and Western blot were conducted to analyzed the influences of Cat on the expressions of the cholesterol metabolism related genes,such as NPC1 L1 and SREBP-2.RESULTS Compared with the blank group,the number of red lipid droplets in cells of Chol:MβCD group was significantly increased.Cat(20-60μmol·L-1)could not only alleviate the formation of intracellular lipid droplets in different degrees,but also significantly reduce the levels of FC and TC,and the ratio of cholesteryl ester to TC.In addition,the mRNA levels and protein expression of NPC1 L1 and SREBP-2 in Caco-2 cells were reduced in Cat treated groups with a concentration-dependent manner when compared with Chol:MβCD group.The effect was most obvious in 60μmol·L-1 Cat treated group(P<0.01).CONCLUSION Catechin could inhibit Caco-2 cell uptake cholesterol and the mechanism might be associated with down-regulation the expression of the cholesterol metabolism related genes NPC1 L1 and SREBP-2.