Potential induction of the relative mRNA expression levels of CYP450 by Zhicaowu-Hezi (Aconiti kusnezoffii radix preparata and Terminalia chebula Retz.)

Abstract

Background: Processed Aconiti Kusnezoffii Radix (Aconiti kusnezoffii Radix Preparata, Zhicaowu) and Terminalia chebula Retz. (Hezi) are a classic herb pair in Mongolian medicine, where Hezi mitigates Zhicaowu's hepatotoxicity. Despite extensive studies on their detoxification effects, the role of cytochrome P450 (CYP450) modulation remains unclear.

Aim: This study aimed to systematically evaluate the regulatory effects of Zhicaowu and Hezi combination on both enzymatic activity and mRNA expression of CYP450 isoforms (CYP1a2, CYP2b1, CYP2c11, CYP2c13, CYP2d2, CYP2e1, and CYP3a1), and to explore their correlation with hepatoprotective effect.

Methods: The effects of Zhicaowu-Hezi formulation on CYP450 enzymes were systematically evaluated through integrated in vivo and in vitro approaches. Rats received 14-day oral administrations of either Zhicaowu, Hezi, or their combinations (1:1, 1:3, 3:1 ratios), followed by comprehensive assessment using: (1) cocktail probe drug assays monitoring seven CYP450 isoforms (CYP1a2, 2b1, 2c11, 2c13, 2d2, 2e1, 3a1) with HPLC quantification methods for substrate detection, (2) RT-qPCR analysis of hepatic CYP450 mRNA expression, and (3) parallel in vitro studies employing rat liver microsomes to verify enzyme activity changes. These pharmacological evaluations were correlated with histopathological and biochemical indices to establish mechanistic relationships between CYP450 modulation and hepatotoxicity attenuation.

Results: Pathological and biochemical analyses confirmed Hezi's hepatoprotective effects against Zhicaowu-induced toxicity, with the 1:3 Zhicaowu-Hezi combination showing optimal efficacy. In vivo pharmacokinetic studies revealed that Zhicaowu significantly inhibited CYP1a2, CYP2d2, CYP3a1, and CYP2c11 activities, as demonstrated by marked increases in the AUC_(0-t), AUC_(0-∞)), and C_max values of their respective probe substrates (theophylline, metoprolol, testosterone, and diclofenac), along with significantly prolonged t_1/2 z and reduced CLz/F. It is worth noting that the combined use of Hezi effectively reversed these changes by inducing CYP450, causing significant alterations in the pharmacokinetic parameters of these four substrates. Complementary in vitro studies using liver microsomes consistently showed that Hezi treatment significantly enhanced the metabolic clearance of these four substrates. At the molecular level, RT-qPCR analysis demonstrated that Zhicaowu significantly suppressed hepatic CYP1a2, CYP2d2, CYP3a1, and CYP2c11 mRNA expression, while Hezi co-treatment restored their expression to normal or elevated levels.

Conclusion: Hezi dose-dependently induced CYP450 enzyme activity, reversing Zhicaowu's inhibition of CYP1a2/2d2/3a1/2c11 and markedly improving liver function and histopathology. These results elucidate the scientific basis for toxicity reduction in Zhicaowu-Hezi herb pair through metabolic enzyme regulation, supporting its traditional use. Future studies will focus on toxic alkaloid (e.g., aconitine) pharmacokinetics and their transcriptional regulatory pathways.

Keywords: Aconiti kusnezoffii Radix Preparata; CYP450; Terminalia chebula Retz.; cocktail probe drug method; pharmacokinetics.

FIGURE 1

HPLC chromatograms of seven probe drugs in plasma. (A). Plasma samples (with IS) obtained from rats after oral administration of mixed probe drugs. (B). Blank plasma spiked with a mixture of probe drug and IS. (C). Blank plasma. (D). Mixed probe drugs (with IS). (1. theophylline, 2. IS, 3. metoprolol, 4. omeprazole, 5. bupropion, 6. chlorzoxazone, 7. testosterone, 8. diclofenac).

FIGURE 2

Standard curves of seven probe drugs in rat plasma (Including the regression equation and the correlation coefficient) (n = 3) (y = peak area ratio of probe drugs vs. IS; x = concentration of probe drugs).

FIGURE 3

Blood concentration-time curves for theophylline, metoprolol, omeprazole, bupropion, chlorzoxazone, testosterone and diclofenac.

FIGURE 4

High-performance liquid chromatography (HPLC) chromatogram of seven probe drugs in rat liver microsomes (RLM): (A) Mixed probe drug standard (including internal standard [IS]) (B). RLM samples (including IS) after incubation (C). Blank RLM mixed with the probe drug and IS (D). Blank RLM. (1. Theophylline; 2. IS; 3. metoprolol; 4. bupropion; 5. mephenytoin; 6. chlorzoxazone; 7. testosterone; 8. diclofenac).

FIGURE 5

Standard curves of probe drugs in rat liver microsomes (RLM) (n = 3).

FIGURE 6

Metabolic clearance of seven probe drugs (theophylline, metoprolol, bupropion, mephenytoin, chlorzoxazone, testosterone, and diclofenac) in different groups. Data are expressed as means ± SD (n = 3). Comparing medication groups with the group C, *P < 0.05, **P < 0.01,***P < 0.001.

FIGURE 7

Histopathological examination of rat livers from different groups (Bar = 100 µm) (A) C, (B) Z, (C) H, (D) Z-H1, (E) Z-H2, (F) Z-H3, (G) P. Focal punctate necrosis (red circle).

FIGURE 8

Changes in the plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alanine phosphatase (ALP) in different rat groups. Data are expressed as means ± SD. (n = 5). Comparing medication groups with the group C, *P < 0.05, **P < 0.01,***P < 0.001. Comparing medication groups with the group Z, #P < 0.05, ##P < 0.01.

FIGURE 9

The mRNA expression levels of CYP1a2, CYP2d2, CYP2b1, CYP2c13, CYP2e1, CYP3a1, and CYP2c11 in different groups. Data are expressed as means ± SD. (n = 3). Comparing medication groups with the group C, *P < 0.05, **P < 0.01,***P < 0.001.