Hippophae rhamnoides reverses decreased CYP2D6 expression in rats with BCG-induced liver injury

Abstract

In this study, we investigated the effect of Hippophae rhamnoides L. (HRP) on the activity of CYP2D6 via the CAMP/PKA/NF-κB pathway in rats with Bacille Calmette-Guerin (BCG)-induced immunological liver injury. BCG (125 mg/kg) was injected to establish the rat model of liver injury. HRP was administered intragastrically for one week as the intervention drug. Proteomics techniques were used to analyze protein expression levels, obtaining a comprehensive understanding of the liver injury process. ELISA or western blotting was used to detect specific protein levels. Dextromethorphan was detected using high-performance liquid chromatography to reflect the metabolic activity of CYP2D6. BCG downregulated the expression of CYP2D6, cAMP, PKA, IκB, and P-CREB and upregulated that of NF-κB, IL-1β, TNF-α, and CREB in the liver; HRP administration reversed these effects. Therefore, HRP may restore the metabolic function of the liver by reversing the downregulation of CYP2D6 through inhibition of NF-κB signal transduction and regulation of the cAMP/PKA/CREB/CYP2D6 pathway. These findings highlight the role of HRP as an alternative clinical drug for treating hepatitis B and other immune-related liver diseases.

Figure 1

Experimental design and grouping.

Figure 2

Effects of HRP on liver pathology of rats with BCG-induced immune-mediated liver injury. (A–F) Representative light microscope images of rat livers of the (A) control group (H&E staining; original magnification, × 200); (B) HRP group; (C) BCG group (arrows point to a granuloma made up of inflammatory cells); (D) BCG + HRPL group (50 mg⋅kg⁻¹); (E) BCG + HRPM group (100 mg⋅kg⁻¹); and (F) BCG + HRPH group (200 mg⋅kg⁻¹). (G) Representative light microscope images of areas with inflammatory cell infiltration (S) in the liver in each group. Data are expressed as the mean ± standard deviation (SD) (n = 6 rats). BCG Bacille Calmette-Guerin; HRP Hippophae rhamnoides; HRPL HRP low-dose group; HRPM HRP medium-dose group; HRPH HRP high-dose group; H&E Haematoxylin and eosin.

Figure 3

Effects of HRP on the liver weight or biochemical changes of rats with BCG-induced immune-mediated liver injury. (A) Rat liver weight; (B) Rat serum ALT; (C) Rat serum AST. Data are expressed as the mean ± standard deviation (SD) (n = 6 rats). BCG Bacille Calmette-Guerin; HRP Hippophae rhamnoides; ALT Alanine aminotransferase; AST Aspartate aminotransferase.

Figure 4

Effects of HRP on rat liver proteomics. (A) Statistical map of omics identification results. (B) Histogram of the total number of different proteins in the control group and the BCG group (C) GO (Gene Ontology, GO) functional enrichment bubble map (BP) in the biological process in the control versus BCG groups. GO functional enrichment bubble diagram; the closer the bubble to the color red, the stronger the significance, and the larger the bubble, the higher the number of proteins. (D) Histogram of protein quantitative difference results. BCG Bacille Calmette-Guerin; HRP Hippophae rhamnoides. cytochrome P450, family 2 (Cyp2d6, Cyp2d3, Cyp2d4), inhibitor of nuclear factor kap (Ikbkb); protein kinase AMP-activated (Prkag1); protein kinase AMP-activated (Prkab1); phosphorylase kinase catalytic (Phkg2); nuclear factor kappa B subunit (Nfkb1); nuclear factor kappa B subunit (Nfkb2); TNF alpha induced protein (TNF-α); dedicator of cytokine (cytokine D); interleukin 1 receptor antagonist (Il-1rn); interleukin 1 receptor access (Il-1ra).

Figure 5

Effects of HRP on the metabolic activity of CYP2D6 in rats with BCG-induced immune-mediated liver injury. (A) Blood samples were collected 0.25, 0.5, 1, 2, 4, 6, 8, and 12 h after DEM administration. Plasma dextromethorphan was measured using HPLC as described. Data are expressed as the mean ± standard deviation (SD), n = 6. *P < 0.01 compared with the control group. # P < 0.01, compared with the BCG group. Significance was determined by one-way analysis of variance (ANOVA), followed by Tukey’s test. (B) Parameters (AUC, CL, C_max, MRT, Vd, and T1/2) were calculated from the plasma dextromethorphan concentration curve as described. Data are expressed as the mean ± SD (n = 6 rats). (C) Metabolic rate was expressed by the amount of dextromethorphan metabolized after incubation with microsomes in vitro and was normalized according to the control group. Data are expressed as the mean ± SD (n = 6 rats). AUC Area under the curve; BCG Bacille Calmette-Guerin; CL Clearance rate; C_max Maximum blood concentration; DEX Dextromethorphan; HPLC High-performance liquid chromatography; HRP Hippophae rhamnoides; MRT Resident time in vivo; T1/2 Half-life; Vd Apparent volume of distribution.

Figure 6

Effect of HRP on the expression of CYP2D6, PKA, CREB, PCREB, IκB, and NF-κB in rats with BCG-induced immune-mediated liver injury. Rats were administered BCG (single intravenous dose of 125 mg/kg BCG) or BCG + HRP (50, 100, or 200 mg⋅kg⁻¹⋅d⁻¹ orally for 7 d). Liver proteins were extracted to determine the expression levels of CYP2D6, PKA, CREB, PCREB, IκB, and NF-κB. SDS-PAGE was performed using equal amounts (30 μg) of protein, and western blotting was performed using antibodies against CYP2D6, PKA, CREB, PCREB, i-κB, and NF-κB. The results were normalized to tubulin, GAPDH, or β-actin. The protein expression levels of CYP2D6 (a), PKA (b), IκB and NF-κB (c), CREB, and P-CREB (d) in the rat liver were measured by western blotting. The expression levels of CYP2D6, PKA, IκB, NF-κB, CREB, and P-CREB were quantified using the ImageJ software (NIH, Maryland, USA). The data represent the mean ± standard deviation (SD) of three independent experiments. BCG Bacille Calmette-Guerin; HRP Hippophae rhamnoides.

Figure 7

Effect of HRP on cAMP, TNF-α, and IL-1β levels in rats with immune-mediated liver injury. Rats were administered BCG (single intravenous 125 mg/kg dose of BCG) or BCG + HRP (50, 100, or 200 mg⋅kg⁻¹⋅d⁻¹ orally for one week). Each bar represents mean ± standard deviation (SD) of three independent experiments (n = 10 per group). BCG Bacille Calmette-Guerin; HRP Hippophae rhamnoides.