Tibetan Medical Formula Shi-Wei-Gan-Ning-Pill Protects Against Carbon Tetrachloride-Induced Liver Fibrosis - An NMR-Based Metabolic Profiling

Abstract

Liver fibrosis is a severe health problem, threatening the life quality and causing death, raising great concerns worldwide. Shi-Wei-Gan-Ning-Pill (SWGNP) is a traditional Tibetan recipe used to treat hepatic injuries; however, its hepatoprotective mechanism has not yet fully clarified. In this study, histological staining, biochemical assays, and elements determination were applied to evaluate the anti-fibrotic efficacy of SWGNP on a carbon tetrachloride (CCl₄) induced hepato-fibrosis rat model. NMR-based metabolomics combined with orthogonal partial least squares-discriminant analysis (OPLS-DA), canonical regression analysis, and correlation networks analysis was used to characterize the potential biomarkers as well as metabolic pathways associated with the hepatoprotective activity of SWGNP. The results showed that SWGNP could significantly attenuate the pathological changes and decrease the levels of fibrosis markers (ColIV, HA, LN, and PCIII), and regulate the disordered elements distribution. Multivariate analysis and correlation network analysis revealed that SWGNP could protect rats against CCl₄-induced liver fibrosis through anti-oxidation, repairing the impaired energy metabolisms and reversing the disturbed amino acids and nucleic acids metabolisms. In conclusion, this integrated metabolomics approach provided new insights into the mechanism of the hepatoprotective effect of SWGNP in liver fibrosis disease.

Keywords: NMR; Shi-Wei-Gan-Ning-Pill; Tibetan medical formula; carbon tetrachloride; liver fibrosis; metabolic profiling.

FIGURE 1

HE staining (A–E), Masson staining (F–J), and semi-quantitative analysis (K–M) of liver tissues. With HE staining (200x), the nuclear was shown blue and cytoplasm was red. With Masson staining (200x), the collagen fiber was shown blue and hepatic cells were red. Liver of control rats exhibiting no obvious pathological changes. Livers of CCl₄ dosed rats showing severe fat vacuoles, significant hepatic cell necrosis, and excessive collagen deposition. SWGNP could decrease the pathological scores and liver indexes in a dose dependent manner. Livers after low dose of SWGNP administration showing severe fat vacuoles and collagen deposition. Livers after medium dose of SWGNP administration showing slight hepatic cell necrosis and less collagen deposition and fat vacuoles. Livers after high dose of SWGNP administration displaying with no obvious collagen deposition without any sign of cell degeneration or necrosis. Values were expressed as mean ± SD (N = 6). ###p < 0.001 vs. control rats; ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 vs. model rats.

FIGURE 2

Boxplots of serum markers of oxidation stress (GPX, SOD, CAT, and MDA), liver function (GOT, GPT, ALB, and TBil), and hepatic fibrosis (LN, PCIII, ColIV, and HA) for rats form control, model, and low, medium, and high dose of SWGNP treated groups. The boxes cover 25% quartile and 75% quartile of the data. The line in the box represents the median value. The extended whiskers show the extent of the rest of the data, and outliers are shown as open circle.

FIGURE 3

Boxplots of elements levels in liver tissues of rats form control, model, and low, medium, and high dose of SWGNP treated groups. The boxes cover 25% quartile and 75% quartile of the data. The line in the box represents the median value. The extended whiskers show the extent of the rest of the data, and outliers are shown as open circle.

FIGURE 4

Boxplots of elements levels in serums of rats form control, model, and low, medium, and high dose of SWGNP treated groups. The boxes cover 25% quartile and 75% quartile of the data. The line in the box represents the median value. The extended whiskers show the extent of the rest of the data, and outliers are shown as open circle.

FIGURE 5

(A) Typical 500 MHz¹H NMR spectra of the serums from rats of control, model, and low, medium, and high dose of SWGNP treated groups: 1. isoleucine, 2. leucine, 3. valine, 4. 3-hydroxyisobutyrate, 5. 3-hydroxybutyrate, 6. ethanol, 7. lactate, 8. alanine, 9. lycine, 10. acetate, 11. glutamine, 12. acetone, 13. acetoacetate, 14. GABA, 15. pyruvate, 16. succinate, 17. citrate, 18. creatine, 19. methanol, 20. glucose, 21. glycine, 22. inosine, 23. tyrosine, 24. histamine, 25. phenylalanine, 26. tryptophan, 27. uridine, and 28. formate. (B) Typical 500 MHz ¹H NMR spectra of the liver extracts from control, model, and low, medium, and high dose of SWGNP-treated rats.1. Isoleucine, 2. leucine, 3. valine, 4. ethanol, 5. 3-hydroxybutyrate, 6. lactate, 7. alanine, 8. lysine, 9. acetate, 10. glutamate, 11. glutamine, 12. succinate, 13. glutathione, 14. 5,6-dihydrouracil, 15. aspartate, 16. dimethylamine, 17. sarcosine, 18. creatine, 19. choline, 20. phosphocholine, 21. sn-glycero-3-phosphocholine, 22. betaine, 23. glucose, 24. glycine, 25. phosphoethanolamine, 26. uracil, 27. uridine, 28. inosine, 29. UDP-glucose, 30. UDP-galactose, 31. NAD⁺, 32. fumarate, 33. tyrosine, 34. histidine, 35. phenylalanine, 36. niacinamide, 37. xanthine, 38. hypoxanthine, and 39. NADPH.

FIGURE 6

Multivariate analysis of ¹H NMR data of liver (A–D) and serum (E–H) extracts for control, model, and low, medium, and high dose of SWGNP treated rats. Score plots (A,E) and corresponding S-/loadings plot (B–D,F–H) for OPLS-DA, color-coded with the absolute value of correlation coefficients.

FIGURE 7

Relationships between metabolites, elements, and biochemical parameters as revealed by canonical regression analysis using metabolites concentrations as x variables and biochemical parameters and elements contents as y variables, with three circles from inner most to outer most denoting radii of 0.50, 0.75, and 1.00, respectively.

FIGURE 8

Correlation network analysis of liver tissues. (A) Control: Normal control group; (B) Model: CCl₄-induced liver fibrosis model group; (C) SWGNP-L: Low dose of SWGNP treatment group; (D) SWGNP-M: Medium dose of SWGNP treatment group; (E) SWGNP-H: High dose of SWGNP treatment group. NMR correlation was connected by dotted lines, colored according to Pearson correlation coefficient and auxiliary biochemical reaction by gray solid lines. Metabolites in red and blue represented significant increase or decrease. Circles were filled by the colors according to corresponding fold changes.

FIGURE 9

Correlation network analysis of serum from control, model and three SWGNP treated groups. (A) Control: Normal control group; (B) Model: CCl₄-induced liver fibrosis model group; (C) SWGNP-L: Low dose of SWGNP treatment group; (D) SWGNP-M: Medium dose of SWGNP treatment group; (E) SWGNP-H: High dose of SWGNP treatment group. NMR correlation was connected by dotted lines, colored according to Pearson correlation coefficient and auxiliary biochemical reaction by gray solid lines. Metabolites in red and blue represented significant increase or decrease. Circles were filled by the colors according to corresponding fold changes.

FIGURE 10

The main metabolic pathways in response to CCl₄ induced liver fibrosis and the treatment effects of SWGNP, showing the interrelationship of the identified metabolic pathways. Metabolites in red and blue color represent significantly increase or decrease.