The Mechanism Of PCSK9 By Curcumin Regulating Uptake Of Plasma LDL-C By Hepatocytes

Aim:To confirm the role of curcumin in promoting uptake LDL of hepatocytes via regulating PCSK9.METHODS:HepG2 and normal hepatocyte LO2 cells were used as the study subjects,which were divided into 4 groups,such as blank group（basal culture）,control group(25 mg·L^-1 LDL),curcumin group(25 mg·L^-1 LDL+25μmol·L^-1 Curcumin),and positive control group(25 mg·L^-1 LDL+10μmol·L^-1 Rosuvastatin).HepG2 and LO2 cells were infected with lentivirus（overexpressing PCSK9 and RNA interfering PCSK9 which have been existing in our Lab.）with puromycin(5μg·mL^-1)to screen for 3days.The ratio of lentivirus infection was detected by inverted fluorescence microscopy.The effect of overexpression and RNA interference PCSK9 was detected by Western Blotting;lipid depositional effect of Curcumin on cells was detected by oil red O staining analysis,and cholesterol levels in cells were analyzed by enzymatic detection.RESULTS:1.Cells were evenly distributed with green fluorescence under an inverted fluorescence microscope.2.Compared with the control group,PCSK9 protein expression was significantly decreased by lentivirus interference PCSK9 in HepG2 cells line（RNAi-PCSK9-1）（P<0.01）,and RNAi-PCSK9-1 LDLR protein expression was correspondingly increased（P<0.01）,RNAi-PCSK9-3PCSK9 protein expression was significantly decreased in LO2 cells group（P<0.01）with increased LDLR protein expression（P<0.01）,whereas when overexpressed PCSK9（OE-PCSK9）,PCSK9 protein expression was significantly increased in HepG2/LO2 group（P<0.01）with decreased LDLR protein expression（P<0.01）.3.After adding LDL(25 mg·L^-1 LDL)lipid,the oil red O staining results showed that the Curcumin-treated for 24 h RNAi-PCSK9-1 HepG2group（P<0.05）were compared with the drug-free group,the lipid content increased in the group.The lipid content in the RNAi-PCSK9-1 HepG2group（P<0.001）increased after Rosuvastatin treatment for 24h,and the LO2 cell results were the same as above,after the Curcumin treatment RNAi-PCSK9-3（P<0.05）increased lipid content;Rosuvastatin-treated RNAi-PCSK9-1 group（P<0.001）and OE-PCSK9 group（P<0.001）increased lipid content.4.The results of cholesterol content showed that total cholesterol and free cholesterol were increased in the RNAi-PCSK9 HepG2 group were treated with Curcumin（P<0.001）.Curcumin-treated in the RNAi-PCSK9LO2 group after treatment TC and FC content increased（P<0.001）.Rosuvastatin-treated RNAi-PCSK9 HepG2 group increased TC and FC content（P<0.001）,the TC in the RNAi-PCSK9 LO2 group increased,the FC content increased（P<0.001）.5.DiI-LDL results showed that compared with Control,orange-red fluorescence was significantly increased in the over-expressed PCSK9 and interfering PCSK9 groups（P<0.001）after Curcumin,orange-red fluorescence was significantly higher in the Rosuvastatin-treated group（P<0.001）than in the Control group,suggesting that Curcumin promotes DiI-LDL uptake by hepatocytes.6.Flow cytometry results showed that compared with the control group,curcumin increased HepG2 cells overexpressing PCSK9（P<0.001）group and LO2 cells overexpressing PCSK9（P<0.001）.After rosuvastatin was added,the LDLR abundance of the HepG2 overexpressed PCSK9（P<0.01）group increased with the control group.Conclusion:Curcumin increases the abundance of LDLR on the cell membrane surface by inhibiting PCSK9 and promotes the uptake of LDL-C by liver cells.