High-throughput metabolomics for evaluating the efficacy and discovering the metabolic mechanism of Luozhen capsules from the excessive liver-fire syndrome of hypertension

Abstract

Essential hypertension (EH) is a chronic disease characterized by a variety of causes of elevated systemic arterial pressure, which often causes functional or organic damage to important organs such as the heart, brain, and kidney. Hypertension of excessive liver-fire syndrome is a type of classification for young people with essential hypertension. The disease is slower in its onset and its symptoms are more ambiguous, and thus its pathogenesis is complicated and still unclear. In this study, aconite, dried ginger and cinnamon extracts were combined with l-NAME to establish a model of excessive liver-fire hypertension. Blood pressure (systolic blood pressure), ANGII, NE and 5-HT were used as evaluation indicators to establish the model. Urinary metabolomics based on ultra-high performance liquid chromatography coupled with quadruple time-of-flight mass spectrometry was used to characterize the metabolic changes and potential biomarkers in modeled rats. Compared to the treatment group, 32 potential biomarkers were initially identified in the model using multivariate statistical analysis involving 11 metabolic pathways. After oral administration of Luozhen capsules, eight biomarkers that can be adjusted in high, medium and low doses of Luozhen capsules in urine were preliminarily determined, mainly involving two metabolic pathways of amino acid metabolism and lipid metabolism. In conclusion, this study explored the metabolomic changes in rats with hypertension of liver-fire hyperactivity syndrome and the post-dose metabolomics, determined the relevant biomarker groups, and clarified the metabonomic connotation of Luozhen capsules in the treatment of liver-fire excessive type hypertension.

Fig. 1. Blood pressure levels of the rats in each group (*P < 0.05, **P < 0.01, compared with the control group). K: control group and M: model group.

Fig. 2. Biochemical indicators of the rats in each group (*P < 0.05, **P < 0.01, compared with K group). K: control group, M: model group, A: ANGII, B: NE, and C: 5-HT.

Fig. 3. Score plot of rat urine during PCH establishment of the hypertensive model (PCA analysis). A: positive ion mode and B: negative ion mode.

Fig. 4. Liver-fire type hypertensive model established on the 28th day, rat urine metabolism score plot (PCA analysis). A: positive ion mode and B: negative ion mode.

Fig. 5. Liver-fire type hypertensive model established on the 28th day of rat urine metabolism score plot (OPLS-DA analysis). A: positive ion mode and B: negative ion mode.

Fig. 6. S-plot map of urine metabolism profile of the rats in the positive and negative ion mode on the 28th day of the liver-fire type hypertensive rat model (OPLS-DA analysis). A: positive ion mode and B: negative ion mode.

Fig. 7. Urine metabolism profile of the rats in the positive and negative ion mode on the 28th day of the liver-fire type hypertensive rat model (OPLS-DA analysis). A: positive ion mode and B: negative ion mode.

Fig. 8. Liver BPI of the rats in positive ion mode on the 28th day established by the model of high blood pressure. A: model group and B: control group.

Fig. 9. Liver BPI of the rats in the negative ion mode established on the 28th day of the liver-fire hypertensive model. A: model group and B: control group.

Fig. 10. Changes in the urine content of potential biomarkers for liver-fire type hypertension (compared with the control group, *P < 0.05, **P < 0.01).

Fig. 11. Main metabolic pathways of potential biomarkers in liver-fire type hypertension. (1) Ubiquinone and other steroidal biosynthesis; (2) vitamin B6 metabolism; (3) phenylalanine, tyrosine and tryptophan biosynthesis; (4) tyrosine metabolism; (5) pentose and glucuronic acid conversion; (6) phenylalanine metabolism; (7) starch and sucrose metabolism; (8) histidine metabolism; (9) lysine degradation; (10) tryptophan metabolism; and (11) aminoacyl-tRNA biosynthesis.

Fig. 12. Network diagram of the potential biomarkers associated with the liver-fire hypertensive model rats based on the KEGG network.

Fig. 13. Effect of Luozhen capsules on the blood pressure of rats with liver-fire type hypertensive model (*P < 0.05, **P < 0.01, compared with group K; ^#P < 0.05, ^##P < 0.01, compared with the Model); K: control group; M: model group; Y: positive group; L: LZC low-dose group; Z: LZC medium-dose group; and H: LZC high-dose group.

Fig. 14. Effect of Luozhen capsules on the clinical biochemical parameters of rats with liver-fire type hypertensive model (*P < 0.05, **P < 0.01 compared with the control; ^#P < 0.05, ^##P < 0.01, compared with the Model). K: control group; M: model group; Y: positive control group; L: LZC low dose group; Z: LZC medium dose group; and H: LZC high dose group. A: ANGII; B: NE; and C: 5-HT.

Fig. 15. In positive ion mode, after oral administration of Luozhen capsules, urine BPI map of the 56th day with hypertensive model rat of excessive liver-fire syndrome. A: control group; B: model group; C: positive control group; D: low dose group; E: medium dose group; and F: high dose group.

Fig. 16. In negative ion mode, after oral administration of Luozhen capsules, urine BPI map of the 56th day of liver-fire hypertensive rats. A: control group; B: model group; C: positive control group; D: low dose group; E: medium dose group; and F: high dose group.

Fig. 17. Score plot of urine metabolism profile on day 56 of hypertensive rats after oral administration of Luozhen capsules (PCA analysis). A: positive ion mode and B: negative ion mode.

Fig. 18. Changes in the relative urine content of biomarkers in liver-burning hypertensive patients after oral administration of Luozhen capsules (*P < 0.05, **P < 0.01 compared with blanks; ^#P < 0.05, ^##P < 0.01, compared with the model) K: control group; M: model group; Y: positive group; L: LZC low dose group; Z: LZC medium dose group; and H: LZC high dose group.