| 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+25?mol·L-1 Curcumin),and positive control group(25 mg·L-1 LDL+10?mol·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(5?g·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. |
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