Dyslipidemia is a type of metabolic disease recognized as a significant public health concern worldwide(27). Chinese herbals can be a primary source of active compounds for anti-dyslipidemia. Recently, component-based Chinese medicine has been characterized by safety, high efficiency, controllable quality, and low toxicity and side effects. In addition, component-based Chinese medicine acts on multiple targets and participates in complex pathological processes of disease(28). Component compatibility not only maintains the core advantages of traditional Chinese medicine, but also integrates modern drug design technology to lay the foundation for the discovery of new traditional Chinese medicine preparations. Drug homologous foods such as Astragalus and Nelumbinis folium have favorable effects on dyslipidemia and related metabolic diseases(23, 29). Among the bioactive compounds from medicinal foods, saponins and alkaloids have demonstrated promising lipid-regulating effects(30, 31). ATS and NFTA are the main components of Astragalus and Nelumbinis folium. Although previous studies have reported that ATS and NFTA can regulate lipid metabolism, it is not explicit that the potential mechanisms that regulation of RCT can improve dyslipidemia. Here, we found that AS-NFA can improve lipid metabolic disorders and then reduce hepatic pathological damage and lipid deposition in HepG2 cells. The underlying mechanisms can activate SR-B1/CYP7A1/FXR signaling pathway and promote RCT.
The liver is the main lipid production organ, thus controlling lipid aggregation, secretion, and catabolism (32, 33). When lipid metabolism or transport in the body is imbalanced, aberrantly raised TC and TG in the serum may be deposited in liver tissue, thus resulting in severe lipid lesions. In particular, abnormally high blood cholesterol levels are a major cause of lipid deposition(34). The levels of TBA, ALT, and AST in blood can evaluate the degree of liver injury in clinical practice. Here, treatment with AS-NFA not only markedly reduced serum TC, TG, LDL-C, TBA, AST, and ALT levels—and remarkably increased serum HDL-C levels—but also ameliorated hepatic and feces TC and TBA levels. Moreover, AS-NFA improved the morphological changes of the liver tissue in HFD rats. The Oil red O staining suggests that AS-NFA decreased the lipid contents. Accordingly, our experiments demonstrated that the AS-NFA effectively regulated blood lipids, improved hepatocyte steatosis, protected liver function, reduced lipid accumulation, and exerted a better effect on preventing and controlling dyslipidemia.
RCT is the process by which HDL transports excess FC from peripheral tissue or cells to the liver, where it is metabolized to produce bile acid (BA) for excretion(35). RCT is an endogenous defense mechanism to protect cells from excessive cholesterol accumulation in disorders of lipid metabolism(9, 36). HDL acts both as a receptor and a carrier for cholesterol in the RCT, thus playing an important role in the RCT process(37, 38). In particular, the uptake of lipids by HDL bound to SR-B1 in hepatocytes is the initiating factor of RCT(39, 40).
Previously, quercetin-pretreated HepG2 cells exhibited a potent increase in selective lipid uptake and Dil-HDL binding mediated by SR-B1—this trend is consistent with an increase in SR-B1 at the transcriptional and translational levels(41). Here, Dil-HDL was co-incubated with HepG2 cells. Uptake of HDL by HepG2 cells in the model group was significantly reduced after cellular uptake. However, the administration of AS-NFA increased HDL uptake and attenuated the ability of BLT-1 to inhibit HDL uptake by the HepG2 cells, thus further confirming that lipid uptake by HepG2 cells was mediated by SR-B1.
The RCT process relies on SR-B1-mediated selective HDL-C uptake(41, 42). Therefore, hepatic SR-B1 is essential for HDL-C clearance. SR-B1, located on the cell surface, is a receptor for HDL and selectively delivers CE to hepatocytes by binding to HDL particles that lack cholesterol. They then release HDL particles back into circulation(43, 44). Knockdown of SR-B1 caused excessive accumulation of CE in HDL, thus reducing hepatic uptake of cholesterol and inhibiting HDL-mediated RCT(45).
CES1 is highly expressed in the liver and exhibits cholesteryl ester hydrolase activity; it hydrolyzes SR-B1-delivered CE to free cholesterol (FC)(15). Prior work found that CES1−/− mice developed hepatic steatosis, increased obesity, and hyperlipidemia(46). In contrast, specific overexpression of CES1 in liver reduced hepatic FC level and attenuated diet-induced dyslipidemia(47). The upregulated expression of CES1 is directly associated with SR-B1-delivered HDL-CE hydrolysis—this was accompanied by increased SR-B1 expression(19). Recombinant cytochrome P450 7A1 (CYP7A1) is only expressed in the liver to maintain the balance between cholesterol and BA by catalyzing the metabolism of FC generated to BA(16, 48). Previous studies demonstrated that increased CYP7A1 promotes FC-catalyzed metabolism to produce BA and activate the receptor FXR, thus enhancing RCT and improving hypercholesterolemia(49).
FXR is a nuclear receptor of BA involved in BA and lipid metabolism(50). An increase in BA concentration can effectively activate FXR(51, 52). SHP is a transcriptional repressor directly regulated by FXR(53, 54). LRH-1 is an important target of CYP7A1 activation that promotes CYP7A1 expression(55). FXR is activated and upregulates SHP, which binds to LRH-1 to form a heterodimer to inactivate LRH-1 and inhibit CYP7A1 transcription(56, 57). This study showed that the FXR-SHP signal could regulate the expression of CYP7A1 and thus promote BA metabolism(58). Moreover, the activation of FXR increased SRB1 expression via FXR-JNK-HNF4α -SRB1 pathway to reinitiate RCT(51).
We found here that the mRNA and protein expression levels of SR-B1, CES1, CYP7A1, FXR, and SHP were decreased in liver tissue of HFD rats and HepG2 steatosis cells, but these conditions were reversed by AS-NFA. AS-NFA administration alone significantly decreased the mRNA and protein expression levels of SR-B1, CES1, CYP7A1, FXR, and SHP. Moreover, if SR-B1 was blocked by BLT-1 in HepG2 cells, then the mRNA and protein expression levels of SR-B1, CES1, CYP7A1, FXR, and SHP all decreased. In fact, unaltered LRH-1 was observed in each group of rats, which might be a sign of impaired binding of SHP and LRH-1. This in turn explains the increased CYP7A1. The effect of AS-NFA was blocked by BLT-1 after the intervention with BLT-1 and AS-NFA. Therefore, our study indicates that AS-NFA could activate SR-B1/CYP7A1/FXR signaling pathway to promote RCT. This then effectively improves lipid accumulation, thus indicating that this might be one of the underlying mechanisms of AS-NFA protection against dyslipidemia.