Targeted Metabolomics of Serum Acylcarnitines Evaluates Hepatoprotective Effect of Wuzhi Tablet (Schisandra sphenanthera Extract) against Acute Acetaminophen Toxicity

Abstract

Possible prevention and therapeutic intervention strategies to counteract acetaminophen (APAP) hepatotoxicity would be of great value. Wuzhi tablet (WZ, extract of Schisandrae sphenanthera) possesses hepatoprotective effects against hepatitis and the hepatic dysfunction induced by various chemical hepatotoxins. In this study, the protective effect of WZ on APAP-induced hepatic injury was evaluated and targeted metabolomics by LC-MS-based metabolomics was used to examine whether WZ influences hepatic metabolism. The results demonstrated significant hepatoprotection of WZ against APAP-induced liver injury; pretreatment with WZ prior to APAP administration blocks the increase in serum palmitoylcarnitine and oleoylcarnitine and thus restores the APAP-impaired fatty acid β-oxidation to normal levels. These studies further revealed a significant and prolonged upregulation of the PPARα target genes Cpt1 and Acot1 by WZ mainly contributing to the maintenance of normal fatty acid metabolism and thus potentially contributing to the hepatic protection of WZ against APAP-induced hepatic toxicity. Taken together, the current study provides new insights into understanding the hepatoprotective effect of WZ against APAP-induced liver toxicity.

Figure 1

WZ protects against APAP-induced liver toxicity. WZ was administered to mice by gavage for 3 days prior to treatment with APAP. H&E staining of livers from control, WZ-treated, WZ/APAP-treated, and APAP-treated mice. Bars represent 100 μm.

Figure 2

WZ protects against APAP-induced liver toxicity. Serum AST and ALT enzyme levels of 2 h (a) or 24 h (b) serum samples from control, WZ-treated, WZ/APAP-treated, and APAP-treated mice (mean ± SD, n = 5 in each group). *P < 0.05, **P < 0.005, ***P < 0.0001, NS: not significant.

Figure 3

Total liver glutathione levels in control, WZ-treated, WZ/APAP-treated, and APAP-treated mice. Hepatic glutathione levels of 2 h (a) or 24 h (b) serum samples from control, WZ-treated, WZ/APAP-treated, and APAP-treated mice were measured by colorimetric methods (n = 5 in each group). *P < 0.05, **P < 0.005, ***P < 0.0001, NS: not significant.

Figure 4

Identification of acylcarnitines as serum biomarkers of WZ protective effect on APAP-induced hepatic toxicity through metabolomic analysis. (a) Scores scatter plot of a PCA model on the serum metabolome of control (∗), WZ-treated (▲), WZ/APAP-treated (•), and APAP-treated (◆) mice. (b) Loadings scatter plot from the PCA analysis of serum from control, WZ-treated, WZ/APAP-treated, and APAP-treated mice. Data points representing palmitoylcarnitine (P1), oleoylcarnitine (P2), myristoylcarnitine (P3), and palmitoleoylcarnitine (P4) were labeled in the plot.

Figure 5

(a) Ion chromatograms of serum palmitoylcarnitine and oleoylcarnitine by extracting ions of 400.34⁺ and 426.35⁺. (b) LC-MS/MS spectra of ions of 400.34⁺ and 426.35⁺ in serum sample.

Figure 6

Quantitation of serum palmitoylcarnitine and oleoylcarnitine level in 2 h (a) and 24 h (b) serum samples from control, WZ-treated, WZ/APAP-treated, and APAP-treated mice (n = 5 in each group). *P < 0.05, **P < 0.005, ***P < 0.0001, NS: not significant.

Figure 7

Relative response of serum myristoylcarnitine (a) and palmitoleoylcarnitine (b) levels in 2 h and 24 h serum samples from control, WZ-treated, WZ/APAP-treated, and APAP-treated mice (n = 5 in each group). *P < 0.05, **P < 0.005, ***P < 0.0001, NS: not significant.

Figure 8

Influence of WZ and/or APAP on lipid metabolism. Triglyceride and free fatty acid levels in 24 h serum samples from control, WZ-treated, WZ/APAP-treated, and APAP-treated mice were measured by colorimetric methods. (a) Serum triglyceride level, (b) serum free fatty acid level (mean ± SD, n = 5 in each group). Triglyceride and free fatty acid levels in control mice were arbitrarily set as 100%. *P < 0.05, **P < 0.005, ***P < 0.0001, NS: not significant.

Figure 9

PPAR-targeted gene expression in livers from control, WZ-treated, WZ/APAP-treated, and APAP-treated mice. Liver samples were collected at 2 h and 24 h after APAP treatment. The gene expression levels were measured by real-time PCR and normalized by β-actin. Gene expression level in control mice was arbitrarily set as 1 (n = 5 in each group). *P < 0.05, compared to the control.