Establishment of RS UPLC fingerprint
Compared to the retention time of each reference substance, the following eight compounds were identified from the extraction of Rubus suavissimus: gallic acid, caffeic acid, rutin, ellagic acid, hyperoside, isoquercetin, rubusoside, and kaempferol (Fig. 1).
RS alleviates lipid metabolism disorder in high-fat diet golden hamsters
Next, we verified the preventive effect of RS on the development of lipid metabolism disorder in Syrian golden hamsters fed a high-fat diet. First, we examined the effect of RS on food intake, body weight, and energy intake. At the beginning of the experiment, there were no significant differences in daily food intake, energy intake, and body weight between the groups; however, throughout the later stages of the experiment, the food- and energy intake as well as body weight of golden hamsters in HFD + RSL, HFD + RSH, and HFD + XZK groups were significantly lower compared to the HFD group (Fig. 2a-c). These observations indicated that RS can maintain the body weight of golden hamsters on a high-fat diet and limit their energy intake.
Then, we analyzed whether RS affects the lipid metabolism on hamsters. For that, we analyzed the liver index and measured liver and serum levels of total cholesterol (TC) and triglyceride (TG) as well as serum levels of low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C).
The liver of golden hamsters on a high-fat diet was white and cloudy with a hard texture whereas the livers of animals belonging to the other groups were more ruddy and shiny with a soft texture. Based on oil Red O staining of liver tissue sections, animals in HFD had more liver lipid droplets compared to animals in ND. However, animals on a high-fat diet supplemented with XZK –a preparation of red yeast rice and a traditional Chinese medicine currently used to lower cholesterol– or RS had less hepatic lipid droplets compared to animals in HFD (Fig. 3).
Compared with ND (liver index 2.91%, liver TC and TG were 367.58 and 705.35 µmol/L, serum TC, TG, and LDL-C were 3.35, 1.52, and 0.66 mmol/L, respectively), animals in the HFD group showed an enhanced liver index (4.63%), increased liver TC (1015.62 µmol/L) and TG (838.07 µmol/L) levels, as well as enhanced serum TC (11.71 mmol/L), TG (8.37 mmol/L), and LDL-C (4.43 mmol/L) levels (Fig. 2d-j). Compared to HFD, a high-fat diet supplemented with RS resulted in a dose-dependent reduction of liver TC (779.17 and 757.57 µmol/L for RSL and RSH) and TG (715.67 and 589.57 for RSL and RSH), serum TC (9.68 and 7.71 mmol/L for RSL and RSH), TG (3.51 and 2.18 mmol/L for RSL and RSH) and LDL-C (3.28 or 2.14 mmol/L for RSL and RSH). Of note, supplementation of a high-fat diet with a low-dose RS increased the level of HDL-C (4.47 and 4.01 mmol/L) in serum (Fig. 2g). Whereas animals in HFD had significantly higher serum levels of alanine aminotransferase (ALT, 289.17 U/L) and aspartate aminotransferase (AST, 248.33 U/L) compared to ND (ALT: 183.83, AST: 94.83 U/L), this was prevented by supplementation of the high-fat diet with RS (ALT: 171.57 and 164.63 U/L; AST: 88.83 and 50.55 U/L in RSL and RSH, respectively, Fig. 2k-l). These results indicated that RS can regulate abnormal blood lipids and liver lipids in high-fat diet golden hamsters and attenuate liver injury.
RS regulates lipid metabolism disorders through an up-regulation of the PPAR signaling pathway
In order to verify whether the PPAR signaling pathway is involve in the RS-mediated attenuation of the lipid metabolism disorder process, we examined the expression of PPARα, PPARγ as well as their downstream targets aP2, C/EBPα, Glut4, and LPL in the liver of golden hamsters at both the mRNA and protein level. Upon activation, PPARs regulate the expression of their downstream targets such as aP2, Glut4, LPL, and CCAAT binding protein enhancer α (C/EBPα). Interestingly, while being regulated by PPARs, C/EBPα can also bind its recognition site within the PPARγ promoter region and co-regulate the expression of aP2 [4]. Furthermore, activation of PPARα promotes lipid oxidative metabolism through enhancing the expression levels of fatty acid transporters, lipoprotein lipase (LPL), apolipoprotein, and the fatty acid oxidase system in the liver. The above process stimulates the uptake of fatty acids and thus reduces the concentration of blood lipids [22].
Compared to ND, animals in the HFD group had significantly lower mRNA levels of PPARα and PPARγ as well as aP2, C/EBPα, Glut4 and LPL (Fig. 4a-f). Relative to HFD, animals in HFD + XZK had similar levels of PPARα and PPARγ, but significantly higher levels of aP2. Compared to HFD, animals in HFD + RSL had significantly higher levels of PPARα and the downstream targets aP2 and Glut4, whereas animals in HFD + RSH had significantly higher mRNA levels of PPARα and PPARγ as well as aP2, Glut4, and C/EBPα. These observations suggested that supplementation of RS prevents the high-fat diet-induced inhibition of PPARα and PPARγ mRNA in the liver of golden hamster and preserved the expression of aP2, C/EBPα, and Glut4.
At the protein level, animals in HFD had significantly lower PPARγ levels compared to ND as well as significantly lower levels of the downstream targets aP2, Glut4, C/EBPα, and LPL. Animals in HFD + XZK had significantly higher levels of PPARα as well as Glut4, C/EBPα, and LPL compared to HFD. Animals in HFD + RSL had significantly higher levels of PPARα and the downstream targets aP2, Glut4, C/EBPα, and LPL whereas animals in HFD + RSH had significantly higher levels of PPARα as well as Glut4, C/EBPα, and LPL (Fig. 4k).
RS inhibits lipid synthesis through down-regulation of the SREBP signaling pathway
Sterol regulatory element binding proteins (SREBPs) are cholesterol-sensitive transcription factors located on the endoplasmic reticulum and nuclear membrane [23] which regulate the metabolism of fatty acids, glucose, and TG by regulating the expression of, among others, fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC1), and stearoyl-CoA dehydrogenase 1 (SCD1) [5–8, 24, 25]. Under normal circumstances, continuous activation of SREBP promotes the expression of key mediators of lipid synthesis leading to lipid accumulation. Upon high cellular cholesterol levels, SREBP shear activation protein (SCAP) and insulin-induced gene (INSIG) form a complex with SREBP and lock the latter on the endoplasmic reticulum membrane thereby reducing the expression of cholesterol and fatty acid synthase [26]. Upon activation, SREBP1 promotes lipid synthesis through upregulation of downstream target genes, such as ACC1, FAS, and SCD-1 [6, 7].
In order to verify whether RS attenuates the lipid metabolism disorder through the SREBP1 signaling pathway, we examined the expression of SREBP1 and its downstream targets in the liver of golden hamsters at both the mRNA and protein level. Compared to ND, animals in HFD had significantly higher mRNA levels of SREBP1 and of its targets FAS and SCD-1 in the liver. Compared to HFD, animals in HFD + XZK, HFD + RSL, and HFD + RSH had significantly lower mRNA levels of SREBP1 as well as ACC1, FAS, and SCD-1 (Fig. 4g-j). At the protein level, animals in HFD had significantly higher levels of ACC1 compared to ND. Compared to HFD, animals in HFD + XZK had significantly lower levels of SREBP1, ACC1, FAS, and SCD-1. Compared to HFD, animals in HFD + RSL had significantly lower levels of ACC1, FAS, and SCD-1 whereas animals in HFD + RSH had significantly lower levels of SREBP1, ACC1, FAS, and SCD-1 (Fig. 4k). These observations suggested that RS can prevent the high-fat diet-induced activation of the SREBP1 pathway.