To elucidate the effect of Ruanjian Qingmai granules on arteriosclerosis obliterans from the perspective of cholesterol efflux

Abstract

Aim of the study: To investigate the effect of Ruanjian Qingmai granules (RJQM) on arteriosclerotic obliterans (ASO) and identify its potential bioactive components.

Materials and methods: Separate zebrafish atherosclerosis models and cellular lipid metabolism disorder models were established, and RJQM was administered at different concentrations for intervention. The lipid deposition was examined by using Nile Red staining. The expression levels of cholesterol metabolism-related genes were determined by using quantitative real-time PCR (qRT-PCR). The CYP7A1 inhibitor was utilized to elucidate the target of RJQM. Through network pharmacology and serum pharmacochemistry approaches, potential bioactive components were systematically identified and subsequently validated through experimental assays.

Results: Ruanjian Qingmai granules significantly decreased lipid deposition and significantly increased the expression of CYP7A1 mRNA in both zebrafish and HepaRG cells. And this effect was attenuated by CYP7A1 inhibitors. Serum pharmacochemistry and network pharmacological analysis indicated that kaempferol and isorhamnetin were potential bioactive components in RJQM for the treatment of ASO. Both components could significantly reduce lipid deposition in zebrafish and HepaRG cells, and this effect was also diminished by CYP7A1 inhibitors. Molecular docking also confirmed that CYP7A1 might be the target of kaempferol and isorhamnetin, and qRT-PCR results also verified that both components could significantly up-regulate the mRNA expression level of CYP7A1.

Conclusion: Ruanjian Qingmai granules exerts a therapeutic effect on ASO by up-regulating the expression level of CYP7A1 mRNA, thereby reprogramming lipid metabolism. Kaempferol and isorhamnetin are likely the main active components of RJQM in lipid metabolic reprogramming.

Keywords: CYP7A1; Ruanjian Qingmai granules; arteriosclerosis obliterans; lipid reprogramming; potential bioactive components.

FIGURE 1

Ruanjian Qingmai granules (RJQM) significantly reduces lipid deposition in zebrafish and cellular models. (A,B) Representative Nile Red staining images (A) and fluorescence quantification (B) in wild-type AB zebrafish after 5 days treatments. (C,D) Representative Nile Red staining images (C) and fluorescence quantification (D) in wild-type AB zebrafish after 15 days treatments (n = 5). (E,F) Nile Red staining (E) and fluorescence quantification (F) in Fli1a-EGFP transgenic zebrafish after 5 days treatment. (G,H) Nile Red staining (G) and fluorescence quantization (H) in Fli1a-EGFP transgenic zebrafish after 15 days treatment (n = 3). Control, normal diet; Model, 8% high-cholesterol diet; RJQML, RJQMM, and RJQMH, co-treated with 8% high cholesterol diet + 0.025, 0.05, or 0.1 mg/ml/d RJQM. (I) Representative Nile Red staining images in HepaRG cells after 24 h treatment. (J) Quantitative analysis of Nile Red fluorescence intensity in HepaRG cells. Control, 10% control serum; Model, 10% control serum + 0.25 mM oleic acid (OA); RJQML, RJQMM, RJQMH, co-treated with 0.25 mM OA + 5%, 10%, or 20% RJQM drug-containing serum, respectively. ***p < 0.001 compared with the control group; ^##p < 0.01, ^###p < 0.001 compared with the model group.

FIGURE 2

Ruanjian Qingmai granules (RJQM) upregulates CYP7A1 mRNA expression in zebrafish and cellular models. (A–L) Zebrafish atherosclerosis model construction and, quantitative real-time PCR (qRT-PCR) analysis of cholesterol metabolism-related genes after 5 days of RJQM intervention (8∼10 embryos per group, n = 3) Control, normal diet; Model, 8% high-cholesterol diet; RJQML, RJQMM, RJQMH, co-treated with 8% high cholesterol diet + 0.025, 0.05, or 0.1 mg/ml/d RJQM, respectively (M,N) qRT-PCR detection of CYP7A1 and BSEP mRNA expression in HepaRG cells after 24 h treatment n = 3. Control, 10% control serum; Model, 10% control serum + 0.25 mM OA; RJQMM, co-treated with 0.25 mM OA + 10% RJQM drug-containing serum. *p < 0.05 < 0.01, **p < 0.01, ***p < 0.001 compared with the control group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 compared with the model group.

FIGURE 3

Ruanjian Qingmai granules (RJQM)-mediated lipid metabolism reprogramming is attenuated by CYP7A1 inhibition. (A,B) Nile Red staining (A) and fluorescence quantification (B) in zebrafish atherosclerosis model after 5 days of RJQM treatment (n = 5). Control, normal diet; Model, 8% high-cholesterol diet; RJQMM, co-treated with 8% high cholesterol diet + 0.05 mg/ml/d RJQM; OCA, co-treated with 8% high cholesterol diet + 4 μM of obecholic acid (OCA). OCA + RJQMM, co-treated with 8% high cholesterol diet + 4 μM OCA + 0.05 mg/ml/d RJQM. (C,D) Nile Red staining (C) and fluorescence quantification (D) in HepaRG cells after 24 h of RJQM treatment (n = 3). Control, 10% control serum; Model, 10% control serum + 0.25 mM OA; RJQMM, co-treated with 0.25 mM OA + 10% RJQM drug-containing serum; OCA, co-treated with 0.25 mM OA + 30 μM OCA. OCA + RJQMM, co-treated with 0.25 mM OA + 30 μM OCA + 10% RJQM drug-containing serum **p < 0.01, ***p < 0.001 compared with the control group; ^##p < 0.01, ^###p < 0.001 compared with the model group.

FIGURE 4

Screening of potential bioactive components of Ruanjian Qingmai granules (RJQM). (A,B) LC-MS chromatograms of RJQM in positive ion mode (A) and negative ion mode (B). (C,D) KEGG pathway enrichment analysis of kaempferol (C) and isorhamnetin (D). (E) Docking sites and docking fractions of kaempferol, isorhamnetin and CYP7A1 molecules.

FIGURE 5

Kaempferol and isorhamnetin reprogrammed lipid metabolism in the zebrafish model. Zebrafish atherosclerosis model was utilized. (A,B) Representative Nile Red staining images (A) and quantification results (B) after 5 days treatment with different doses of kaempferol. (C,D) Representative Nile Red staining (C) and fluorescence quantification results (D) after 5 days treatment with different doses of isorhamnetin. n = 5. Control, normal diet; Model, 8% high-cholesterol diet; KPFL, KPFM and KPFH, co-treated with 8% high-cholesterol diet +1, 5, or 10 μg/ml/d kaempferol, respectively; ISOL, ISOM and ISOH, co-treated with 8% high-cholesterol diet +1, 5, or 10 μg/ml/d isorhamnetin, respectively. ***p < 0.001 vs. control group; ^###p < 0.001 vs. model group.

FIGURE 6

Kaempferol and isorhamnetin reprogrammed lipid metabolism in the cellular model. (A,B) Nile Red staining images (A) and quantizative fluorescence analysis (B) of HepaRG cells treated with different doses of kaempferol for 24 h. (C,D) Nile Red staining imgaes (C) and fluorescence quantification (D) following 24 h treatment with different doses of isorhamnetin. (E,F) qRT-PCR analysis of CYP7A1 mRNA expression levels after 24 h treatment with kaempferol (E) or isorhamnetin (F), n = 3. Model, HepaRG treated with 0.25 mM OA for 24 h; KPFL, KPFM and KPFH, co-treatment with 0.25 mM OA and kaempferol (5, 10, or 20 μM) for 24 h; ISOL, ISOM and ISOH, co-treatment with 0.25 mM OA and isorhamnetin (5, 10, or 20 μM) for 24 h. Control, give equal volume of solvent. **p < 0.01 vs. control group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 compared with the model group.

FIGURE 7

In Zebrafish and cellular models, the reprogramming of lipid metabolism by kaempferol and isorhamnetin was attenuated by CYP7A1 inhibitors. (A,B) and (C,D) The Nile Red staining images and fluorescence quantification results at 5 days after kaempferol (A,B) or isorhamnetin (C,D) intervention, with or without CYP7A1 inhibitors, in zebrafish atherosclerosis model. Model, 8% high-cholesterol diet; KPFM, co-treated with 8% high-cholesterol diet + 5 μg/ml/d kaempferol; ISOM, co-treated with 8% high-cholesterol diet + 5 μg/ml/d isorhamnetin; OCA, co-treated with 8% high-cholesterol diet +4 μM of OCA. OCA + KPFM and OCA + ISOM, co-treated with 8% high-cholesterol diet + 4 μM OCA + 5 μg/ml/d KPFM or ISOM; Control, was given equal volume of egg water. (E,F) Typical Nile Red staining images (E) and quantitative results (F) of kaempferol or isorhamnetin intervention for 24 h, with or without CYP7A1 inhibitors, in HepaRG cells. Model, 0.25 mM OA. KPFM and ISOM: co-treated with 0.25 mM OA + 10 μM kaempferol or isorhamnetin, respectively. OCA, co-treated with 0.25 mM OA + 30 μM OCA. OCA + KPFM and OCA + ISOM: co-treated with 0.25 mM OA + 30 μM OCA + 10 μM kaempferol or isorhamnetin. Control, was given equal volume of solvent. **p < 0.01, ***p < 0.001 compared with the control group; ^##p < 0.01, ^###p < 0.001 compared with the model group.