Combination of Plasma Pharmacochemistry, RNA-Seq, and Molecular Docking Strategies to Reveal the Mechanism of the Alkaloid Fraction of Nelumbinis folium for the Treatment of Hyperlipidemia

Abstract

Nelumbinis folium (N. folium) exhibits hypolipidemic effects and shows great potential for application in lipid-lowering drugs and healthcare products. This study aimed to investigate the mechanism underlying the hypolipidemic effects of the alkaloid fraction of N. folium (AFN). Animal experiments demonstrated that AFN significantly reduced blood lipid levels and ameliorated liver damage in hyperlipidemic mice. RNA-seq analysis identified 26 reverse-regulated differentially expressed genes (DEGs), which were primarily involved in the PPAR signaling pathway, fat digestion and absorption, and fatty acid degradation. Using UPLC-MSⁿ, 30 plasma-absorbed components were identified, including 13 prototype alkaloids. Among these, three key active components-nuciferine, N-nornuciferine, and N-methylisococlaurine-were screened via network topology analysis. Molecular docking revealed strong binding affinities between these compounds and key targets. The results showed that N-methylisococlaurine bound to SLC27A4 and CPT1A with strong affinity, while nuciferine and N-nornuciferine bound to ACADVL and PPARA. RT-qPCR results confirmed that AFN modulates the expression of FABP1, SLC27A4, PPARA, CPT1A, ACAA2, APOC3, and APOA4. These findings suggest that AFN exerts its hypolipidemic effects through multi-component, multi-target, and multi-pathway mechanisms.

Keywords: alkaloid fraction of Nelumbinis folium; hyperlipidemia; molecular docking technology; plasma pharmacochemistry; transcriptome-sequencing technology.

Figure 1

Determination of four alkaloid components in AFN. (A) Plot of four alkaloid components as controls under UV 285 nm conditions. (B) Determination results of four alkaloid components in AFN prepared from the eighth batch of N. folium medicinal materials under ultraviolet light at 285 nm. Peak 1 is armepavine, peak 2 is 2-hydroxy-1-methoxyaporphine, peak 3 is N-nornuciferine, and peak 4 is nuciferine.

Figure 2

AFN reduced blood lipids and ameliorated liver damage in mice. (A) Weight change within 8 weeks and final body weight. (B) Effects of AFN on serum biochemical indices TC, TG, LDL-C, HDL-C, ALT, and AST in hyperlipidemic mice. (C) AST and ALT levels in liver tissues. (D) Weight of livers. All data are shown as the Mean ± SD (n = 6). * p < 0.05, ** p < 0.01 vs. the Model group. ^# p < 0.05, ^## p < 0.01 vs. the Simvastatin group.

Figure 3

(A). Transcriptomic analysis of mouse liver tissues induced by AFN on high-fat diets. (A1) DEGs statistical plot (A2) Venn diagram of DEGs between groups. The threshold value was FC > 2 or FC < 0.5, and the p-adjust value was <0.05. (B). Transcriptomic analysis of mouse liver tissues induced by AFN on high-fat diets. Volcano plot of model vs. control and AFN 341.25 mg/kg group vs. Model.

Figure 3

(A). Transcriptomic analysis of mouse liver tissues induced by AFN on high-fat diets. (A1) DEGs statistical plot (A2) Venn diagram of DEGs between groups. The threshold value was FC > 2 or FC < 0.5, and the p-adjust value was <0.05. (B). Transcriptomic analysis of mouse liver tissues induced by AFN on high-fat diets. Volcano plot of model vs. control and AFN 341.25 mg/kg group vs. Model.

Figure 4

(A). GO functional enrichment analysis plot of control vs. model, AFN 341.25 mg/kg group vs. model. (B). KEGG pathway enrichment analysis plot of control vs. model, AFN 341.25 mg/kg group vs. model.

Figure 4

(A). GO functional enrichment analysis plot of control vs. model, AFN 341.25 mg/kg group vs. model. (B). KEGG pathway enrichment analysis plot of control vs. model, AFN 341.25 mg/kg group vs. model.

Figure 5

The total ion chromatographs (TIC) of UPLC-LTQ-Orbitrap-MS. AFN extraction in the positive ion mode (A) and negative ion mode (B).

Figure 6

Two-dimensional molecular docking diagram of key active ingredients and proteins. (A) Molecular docking diagram of N-Methylisococlaurine with SLC27A4. (B) Molecular docking diagram of N-Methylisococlaurine with CPT1A. (C) Molecular docking diagram of Nuciferine with ACADVL. (D) Molecular docking diagram of Nuciferine with PPARA. (E) Molecular docking diagram of N-nornuciferine with ACADVL. (F) Molecular docking diagram of N-nornuciferine with PPARA.

Figure 7

The results of key gene expression detected by RT-qPCR. All data are shown as the Mean ± SD (n = 6). * p < 0.05, ** p < 0.01 vs. model group.