姜黄素下调IDOL水平促进肝细胞摄取血浆LDLC

Curcumin promotes the hepatocytes-uptake of plasma LDLC by down-regulating IDOL level

目的明确姜黄素可通过下调低密度脂蛋白受体诱导降解蛋白(IDOL)来升高低密度脂蛋白受体(LDLR)的表达水平,从而促进肝细胞摄取低密度脂蛋白胆固醇(LDLC)。方法利用过表达IDOL和干扰IDOL慢病毒(OE/RNAi-IDOL)感染HepG2和LO2两种体外培养的肝细胞,倒置荧光显微镜观察病毒感染情况;Western blot检测IDOL及LDLR蛋白表达;油红O染色观察细胞中脂质情况;酶法检测细胞内胆固醇含量;DiI-LDL摄取实验检测肝细胞对LDLC的摄取能力;流式细胞术检测肝细胞膜LDLR分布丰度。结果与明场比较,暗场下可见胞内呈绿色荧光;与对照组比较,三种不同干扰IDOL慢病毒感染的HepG2和LO2细胞中,仅RNAi-IDOL-2的IDOL蛋白表达显著降低,且相应组的LDLR蛋白表达显著增强(P<0.01),选择为后续RNAi-IDOL组所用;相反,OE-IDOL慢病毒感染的HepG2和LO2细胞中,IDOL表达显著增高,相应组内LDLR表达减弱;以上结果表明已获得RNAi-IDOL-2及OE-IDOL慢病毒感染的HepG2和LO2细胞模型。与无药物处理的Control组相比,RNAi-IDOL-2及OEIDOL慢病毒感染的肝细胞内经25μmol/L姜黄素处理24 h后,油红O染色及胆固醇含量测定结果显示:细胞内脂滴含量和相对胆固醇含量均显著增加(P<0.01);且DiI-LDL摄取及免疫流式细胞结果显示:肝细胞摄取LDLC的能力增强(P<0.01);肝细胞膜表面LDLR分布丰度增多(P<0.01);以上结果与阳性药物对照组的结果趋势一致。结论姜黄素可以下调肝细胞内IDOL水平,提高细胞膜LDLR分布丰度,从而促进肝细胞摄取LDLC。

Aim To explore the effect of Curcumin on that inducible degrader of the low density lipoprotein receptor(IDOL)regulating the uptake of low density lipoprotein cholesterol(LDLC)of hepatocytes.Methods HepG2 and LO2 cells(two kinds of hepatocytes)were infected with the constructed overexpression or RNA-interference IDOL(OE/RNAi-IDOL)lentivirus.The efficiency of the lentiviral infection experiment was evaluated by fluorescence microscopy.The expression of IDOL and low density lipoprotein receptor(LDLR)proteins was detected by Western blot.After HepG2 and LO2 cells were treated with Curcumin for 24 hours,intracellular lipid droplets were determined by red oil O staining;cholesterol content was detected by using cholesterol testing kits;the uptake of LDLC by hepatocytes was detected by DiI-LDL uptake experiment;LDLR abundances of hepatocytes surfaces were determined by immune flow cytometry.Results Compared with white light view,the phenomenon of the green fluorescence was observed both in HepG2 and LO2 cells infected by OE-IDOL and RNAi-IDOL lentivirus;Western blot results showed that both in HepG2 and LO2 cells infected by RNAi-IDOL-2 lentivirus,IDOL protein expression were decreased,while LDLR expression was increased(P<0.01);on the contrary,in HepG2 and LO2 cells infected by OE-IDOL lentivirus,IDOL protein expression was increased,while LDLR expression was decreased,the above results indicated that both HepG2 and LO2 cells infected by OE/RNAi-I-DOL lentivirus had been acquired.Compared with the control group of HepG2 and LO2 cells without any treatment,after 25μmol/L Curcumin treatment for 24 hours in the OE/RNAi-IDOL lentivirus infected cells,both the intracellular lipid droplet content and relative cholesterol content were increased in the treatment group(P<0.01),meanwhile the uptake LDLC ability and cell surface LDLR abundances of hepatocytes were also enhanced(P<0.01),the same trend was also observed in the results of Rosuvastatin treatment group.Conclusion The levels of IDOL protein in liver cells were down-regulated by Curcumin,which further promotes the uptake of LDLC of liver cells.