Hepatoprotective Effect of Oplopanax elatus Nakai Adventitious Roots Extract by Regulating CYP450 and PPAR Signaling Pathway

Abstract

O. elatus Nakai is a traditional medicine that has been confirmed to exert effective antioxidant and anti-inflammatory functions, and is used for the treatment of different disorders. However, its potential beneficial effects on drug induced hepatotoxicity and relevant molecular mechanisms remain unclear. This study investigated the protective effect and further elucidated the mechanisms of action of O. elatus on liver protection. O. elatus chlorogenic acids-enriched fraction (OEB), which included chlorogenic acid and isochlorogenic acid A, were identified by HPLC-MS/MS. OEB was administrated orally daily for seven consecutive days, followed by a single intraperitoneal injection of an overdose of APAP after the final OEB administration. The effects of OEB on immune cells in mice liver were analyzed using flow cytometry. APAP metabolite content in serum was detected using HPLC-MS/MS in order to investigate whether OEB affects CYP450 activities. The intestinal content samples were processed for 16 s microbiota sequencing. Results demonstrated that OEB decreased alanine aminotransferase, aspartate aminotransferase contents, affected the metabolism of APAP, and decreased the concentrates of APAP, APAP-CYS and APAP-NAC by inhibiting CYP2E1 and CYP3A11 activity. Furthermore, OEB pretreatment regulated lipid metabolism by affecting the peroxisome proliferator-activated receptors (PPAR) signaling pathway in mice and also increased the abundance of Akkermansia and Parabacteroides. This study indicated that OEB is a potential drug candidate for treating hepatotoxicity because of its ability to affect drug metabolism and regulate lipid metabolism.

Keywords: CYP450; Oplopanax elatus Nakai; PPAR; adventitious roots; gut microbiota; neutrophil.

FIGURE 1

Protective effect of OEB in APAP-induced hepatotoxicity in vivo. (A) Mice were pretreated with 50 mg/kg OEB by intragastric administration for 7 days and administered a single dose of 300 mg/kg APAP by intraperitoneal injection and euthanized after 12 h. Hepatotoxicity was analyzed by measuring serum (B) ALT and (C) AST levels (n = 5). (D) Hematoxylin and eosin-stained liver sections. ***p < 0.001 versus APAP-treated group, ^### p < 0.001 versus NT group.

FIGURE 2

OEB reduced APAP-induced hepatotoxicity through regulating APAP-metabolism. (A) APAP content, (B) APAP-CYS content and (C) APAP-NAC content in mice serum, were measured by HPLC-MS/MS after administering APAP 6 h and 12 h (n = 3). Intensities of (D) APAP, (E) APAP-CYS and (F) APAP-NAC were analyzed by HPLC-MS/MS. *p < 0.05 versus APAP-treated group.

FIGURE 3

OEB reduced APAP-induced hepatotoxicity through regulating cytochrome P450 activity. Drug metabolizing enzymes (A) CYP2E1 and (B) CYP3A11 activities were measured in hepatic microsomes by HPLC-MS/MS (n = 3). Intensities of (C) CYP2E1 and (D) CYP3A11 were analyzed by HPLC-MS/MS. ^### p < 0.001 versus NT group.

FIGURE 4

OEB reduced APAP-induced hepatotoxicity via regulating oxidative enzyme activities. (A) Glutathione (GSH), (B) superoxide dismutase (SOD), (C) catalase (CAT) and (D) malondialdehyde (MDA) levels in the liver tissue were measured. (n = 3). Data are representative of three independent experiments with similar results. *p < 0.05, **p < 0.01, ***p < 0.001 versus APAP-treated group. ^## p < 0.01, ^### p < 0.001 versus NT group.

FIGURE 5

OEB reduced APAP-induced hepatotoxicity by affecting immune cells in the liver. (A) Merged t-SNE plots of flow cytometry data from liver samples of APAP (n = 3) and OEB + APAP (n = 3) groups. (B) Representative flow cytometry data for hepatic CD11b⁺ Gr-1 ^high neutrophil infiltration in the liver of APAP and OEB + APAP groups. The total neutrophil (CD11b⁺ Gr-1 ^high cells) (C) percentages and (D) numbers in the liver of APAP and OEB + APAP groups (n = 5). Data are representative of three independent experiments with similar results. ***p < 0.001 versus APAP-treated group. ^### p < 0.001 versus NT group.

FIGURE 6

RNA-Seq analyses revealed significant difference in PPAR lipid metabolism between the presence or absence OEB pretreated in the APAP treated groups. Hepatotoxicity was analyzed by measuring serum (A) CHO and (B) TG levels (n = 5). (C) KEGG pathway enrichment analyses of the OEB + APAP (n = 3) and APAP (n = 3) groups. (D) GSEA pathway enrichment analysis of pathways related to PPAR signaling pathway. (E) Heatmap of PPAR-related gene expression profiles based on the GSEA results. *p < 0.05, **p < 0.01 versus APAP-treated group. ^## p < 0.01, ^### p < 0.001 versus NT group.

FIGURE 7

16s-Seq analyses revealed significant difference in gut microbiota between the presence and absence of OEB pretreatment in the APAP treated groups. (A) Venn diagram showing the number of specific commensal gut microbiomes and the overlap between APAP (n = 4) and OEB + APAP (n = 4) groups. (B) Wilcoxon rank-sum test bar plot revealed differentially enriched microbiota between APAP (n = 4) and OEB + APAP (n = 4) groups on genus level. (C) COG function classification analysis revealed differentially enriched bacterial functions associated between APAP (n = 4) and OEB + APAP (n = 4) groups. *p < 0.05 versus APAP-treated group.