An in vitro investigation of the effects of Urolithins A and B on low-density lipoprotein uptake and its regulatory genes

Abstract

Abstract Background Elevated plasma levels of low-density lipoprotein cholesterol (LDL-C) is a causal risk factor for atherosclerosis. A major pathway of LDL-C clearance from bloodstream is its uptake by hepatic LDL receptors (LDLRs), which is mainly restricted by proprotein convertase subtilisin/kexin type-9 (PCSK9). Purpose This study aimed to evaluate the possible role of Urolithin A (UA) and Urolithin B (UB) on the induction of LDL uptake and the expression of its regulatory genes. Methods Curcumin (20 uM), berberine (50 uM), UA (80 uM) and UB (80 uM) were used as the treatments in the experimental tests. LDL uptake in HepG2 cells was detected by LDL uptake cell-based assay kit and the degrees of LDL uptake as well as the distribution of cell surface LDLR were assessed using fluorescent microscopy. Moreover, the mRNA expression levels of LDLR and PCSK9 genes in HepG2 cells were assessed using qPCR. Results The LDL uptake as well as cell-surface LDLR were higher in cells treated with UA in comparison with cells treated with UB, and even in relation to the cells treated with curcumin and berberine as positive controls. In addition, cells treated with UB showed approximately 2 times greater LDLR expression level compared with curcumin (FC: 2.144, P=0.013) and berberine (FC: 2.761, P=0.006). However, UA treatment resulted in a significantly lower expression levels of LDLR compared with curcumin (FC: 0.274, P<0.001) and berberine (FC: 0.352, P=0.009). UB demonstrated approximately 8 times greater LDLR expression levels when compared with UA (FC:7.835, P=0.001) (Figure 1A). Compared with UB, as well as curcumin and berberine as positive controls, UA was more efficient in reducing PCSK9 expression levels. Although UB did not show any significant differences compared with curcumin and berberine, it illustrated higher levels of PCSK9 expression when compared with UA group (FC: 3.694, P<0.001) (Figure 1B). Conclusion The present results support that UA was more effective than UB in promoting LDL uptake as well as cell surface LDLR in HepG2 cells. This effect seems to be mostly mediated through the suppression of PCSK9 expression but not induction of LDLR expression. Figure 1. HepG2 (A) LDLR and (B) PCSK9 fold change expression in treatment groups. All data were normalized to control group (PBS).