Cisplatin and Panax ginseng were obtained from Mayne Pharmaceuticals-UK and Pharco Pharmaceuticals-Egypt, respectively. The treated doses of cisplatin (4 mg/kg BW/week) and Panax ginseng (100 mg/kg BW/day) were chosen according to Jangra et al. (2016) and Voces et al. (2004), respectively. All other chemicals applied in this study were of analytical grade. Additionally, all reagents were employed at concentrations recommended by the manufacturer’s instructions.

Forty male Sprague-Dawley rats weighing from 160 to 180 g were obtained from the Faculty of Medicine, Alexandria University, Egypt. The research was achieved according to the Guide for the Care and Use of Laboratory Animals (International Council for Laboratory Animal Science, ICLAS) and was approved by the local ethical guidelines of the Institutional Animal Care & Use Committee (IACUC), Alexandria University, Egypt (AU14–200922–3-3) and all the methods were performed according to the guidelines and regulations of the same Committee. Also, the study was implemented according to the ARRIVE guidelines ( Percie du Sert, et al., 2020). Additionally, the study was conducted following the Basic & Clinical Pharmacology & Toxicology policy for experimental and clinical studies ( Tveden-Nyborg, et al., 2023).

Animals were housed and kept in a 12-hour light/dark cycle at room temperature. Food and water were available ad libitum. Randomly, all the experimental animals were divided into 4 groups (10 animals in each group). Group 1 served as control; group 2 was treated orally with Panax ginseng (100 mg/kg BW/day). Group 3 was injected (intraperitoneally) with cisplatin at a dose of 4 mg/ kg BW/week. Group 4 (combined group) received an oral dose of Panax ginseng (100 mg/kg BW/day) in addition to an intraperitoneal dose (cisplatin 4 mg/kg BW/week). The animals were treated once a week with cisplatin and daily with Panax ginseng for three months.

Animals were euthanized following anesthesia induced by isoflurane inhalation (approximately 2% delivered in a desiccator chamber). Adequate depth of anesthesia was confirmed by the absence of the pedal withdrawal reflex and loss of response to external stimuli. Euthanasia was completed by maintaining isoflurane exposure until respiratory arrest occurred, followed by cardiac puncture for blood collection. All procedures were conducted in accordance with the recommendations of the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals. Following anesthesia, blood was drawn from the heart and centrifuged for 10 minutes at 860 xg. Serum samples were obtained and stored at −80 °C for laboratory tests. Liver samples were obtained and divided into 2 parts. The first part was rinsed in cold saline solution and homogenized (10%, w/v) in 0.1 M phosphate-buffered (pH 7.4). The supernatant was obtained by centrifugation at 5000 rpm for 15 min. and stored at −80 C for biochemical assay. The second part was used for histopathological examination.

The total hepatic contents of the cytochrome P ₄₅₀, cytochrome b ₅, NADPH-cytochrome C-reductase, amidopyrine N-demethylase, and aniline 4-hydroxylase were estimated by the methods of Omura and Sato (1964), Williams and Kamin (1962), Nash (1953), and Kato and Gillette (1965), respectively.

According to the manufacturer’s instructions, total RNA was isolated from liver tissues using an RNeasy Plus kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The purity and integrity of RNA were determined spectrophotometrically at 260/280 nm. Reverse transcription was performed to convert RNA to cDNA using a reverse transcriptase kit (Maxime RT PreMix kit, iNtRON, Korea). The amplification of CYP1A1, CYP2E1, CYP2D6, and CYP3A4 genes was performed by quantitative PCR assay using Rotor-Gene SYBR® Green with low rox qPCR Kit (Enzynomics, Korea). Specific primers (Life Technologies, Prague, Czech Republic) were used to determine the mRNAs of CYP1A1, CYP2E1, CYP3A4 (CYP3A1 in rats) and CYP2D6. The sequences of the primers used are presented in Table 1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene. The resulting data were analyzed by the ΔΔ ^Ct method.

Malondialdehyde (MDA) was assayed as a lipid peroxidation index in liver homogenate. In which thiobarbituric acid-reactive substances (TBARS) were measured at 532 nm using 2-thiobarbituric acid (2,6-dihydroxypyrimidine-2-thiol; TBA; Sigma-Aldrich, Cat. No. T5500, St. Louis, MO, USA). An extinction coefficient of 156,000 M ⁻¹ Cm ⁻¹ was used for calculation ( Esterbauer and Cheeseman, 1990). Nitric oxide (NO) was measured using the Griess reaction ( Sosroseno et al., 2008). In which about 150 μl of liver extract was mixed with 150 μl Griess reagent (Sigma-Aldrich, Cat. No. G4410), and absorbance was recorded at 540 nm. Superoxide dismutase (SOD; EC 1.15.1.1) activity was measured according to Marklund and Marklund (1974). In which 20 μl tissue supernatant and 10 μl pyrogallol (Sigma-Aldrich, Cat. No. P3130) were added to one ml buffer reagent. The inhibition in pyrogallol autooxidation by SOD was evaluated at 480 nm. The activity of Glutathione peroxidase (GPX, EC 1.11.1.9) was assessed using reduced glutathione (GSH; Cat. No. G4251) with DTNB (Sigma-Aldrich, Cat. No. D8130) as a substrate, and the activity was recorded at 480 nm ( Paglia and Valentine 1967). Glutathione S-transferase (GST; EC 2.5.1.18) activity was measured at 310 nm using P-nitrobenzylchloride (Sigma-Aldrich, Cat. No. N7880) as substrate according to the method of Habig et al. (1974). GST induces the conjugation reaction with glutathione in the mercapturic acid synthesis. The catalase enzyme (CAT; EC 1.11.1.6) catalyzes H ₂O ₂ substrate (Sigma-Aldrich, Cat. No. H1009) into H ₂0. Hydrogen peroxide (Sigma-Aldrich, Cat. No. H1009) was used as the substrate. CAT activity in liver extract was evaluated spectrophotometrically at 240 nm by measuring the rate of H ₂O ₂ degradation by catalase. ( Xu et al., 1997). Reduced glutathione (GSH) content was determined, and the method utilized metaphosphoric acid (Sigma-Aldrich, Cat. No. 102433) for protein precipitation and 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB; Sigma-Aldrich, Cat. No. D8130) for color development and its density was measured at 412 nm. GSH was determined according to the method of Jollow et al. (1974). Glutathione reductase (GR; EC 1.8.1.7) activity was measured according to Goldberg and Spooner (1983). In which glutathione reductase catalyzes the reduction of oxidized glutathione by NADPH. The activity was recorded at 340 nm, monitoring NADPH Sigma-Aldrich, Cat. No. N7505) oxidation and oxidized glutathione reduction in EDTA phosphate buffer. The activity of xanthine oxidase (XO) was measured using the commercial EnzyChromTM Xanthine Oxidase Assay Kit (Bioassay System; catalogue no. EXOX-100; Barcelona, Spain) according to the manufacturer’s protocol. Total antioxidant capacity (TAC) was assayed according to the manual instructions of the Biodiagnostic Kit (catalogue no. TA 25 13; Biodiagnostic Co., Giza, Egypt).

The levels of inflammatory molecules, including Tumor Necrosis Factor-alpha (TNF-α; catalogue number: ER1393), and Interleukin 6 (IL-6) in the liver extract were estimated according to instructions described in the commercial ELISA kit (Ray Biotech, Canada; ELR-IL6–1-RB). Transforming growth factor (TGF-β), and necrosis factor kappa-B (NF-κB) were assayed by using an ELISA kit (Active Motif co. Carlsbad, CA 92008 USA). While Cyclooxygenase-2 (COX-2; Cat. No.: CSB-E13399) was measured according to the kit instructions purchased from Cusabio Biotech Co, China. All the sample volumes and concentrations were applied as recommended by the manufacturer’s instructions.

Tumor suppressor gene p53 (p53), was assayed by using an ELISA kit (Active Motif co., 1914, Palomar Oaks Way, Suite 150, Carlsbad, CA 92008 USA). Caspase-3 was assayed by using the method according to Gen Script USA Inc (Cat. No. L00289).

The activities of serum and liver aspartate transaminase (AST) were determined using the commercial kit (Biosystems S.A., Barcelona, Spain, Cat. No.12531). Alanine transaminase (ALT) activities were evaluated using the commercial kit (Biosystems S.A., Barcelona, Spain, Cat. No.12533), and alkaline phosphatase (ALP) activities were measured using a commercial kit (Biosystems S.A., Barcelona, Spain, Cat. No.11592). Acid phosphatase (ACP) activities were assessed using a commercial kit (Biosystems S.A., Barcelona, Spain, Cat. No.11548), and lactate dehydrogenase (LDH) activities were analyzed by kit (Biosystems S.A., Barcelona, Spain, Cat. No.11580). The activities of the gamma-glutamyl transaminase (GGT) enzyme were evaluated with a commercial kit (Biosystems S.A., Barcelona, Spain, Cat. No.11584). Stored serum samples were analyzed for total protein (TP; Cat. No.11500) and albumin (Cat. No.12547) using commercial kits from Biosystems S.A., Barcelona, Spain, according to the manufacturer’s instructions. All sample volumes and concentrations were applied as recommended by the manufacturer’s instructions. All assays were performed according to the manufacturer’s instructions, using recommended sample volumes and concentrations.

Liver specimens were fixed in 10% formalin solution for 48 hours, then treated with 70% alcohol and xylene. The samples were divided into slices with a thickness of 3–6 μm using a rotary microtome after being immersed in paraffin. For the histological analysis, the liver slices were stained with Hematoxylin and Eosin (H&E) and examined under a microscope ( Scheuer & Chalk, 1986).

Immunohistochemical staining was performed to reveal the expression of p53, Bax, and Bcl2 proteins in the liver. Sections using avidin-biotin complex (ABC) (Elite–ABC, Vector Laboratories, CA, USA; Cat. No. PK-6101). In apoptotic p53 proteins, sections were incubated with an anti-rabbit p53 monoclonal antibody (dilution 1:80; DAKO Japan Co., Ltd., Tokyo, Japan, Cat. No. M3629). In pro-apoptotic Bax expression, liver sections were incubated with Bax polyclonal rabbit antibody (dilution 1:100; Code A3533, Dako Denmark). In anti-apoptotic Bcl2 protein expression, sections were incubated with anti-rabbit Bcl-2 monoclonal antibody (dilution 1:2000; DAKO Japan Co, Ltd, Tokyo, Japan). The slides were counterstained with hematoxylin and cover-slipped using a permanent mounting media, and the prepared sections were examined using a research microscope ( Kos and Matkovich, 2014; Tousson, et al., 2014; He, et al., 2016).

Experimental data were statistically analyzed as mean ± SD by one-way ANOVA analysis (Version 5) using the Biostatistics Software Program with Tukey’s multiple comparison tests (post hoc). The significant results among the groups were evaluated at a p-value of <0.05.

Liver drug metabolizing enzymes (cytochrome P450, cytochrome b5, amidopyrine N-demethylase, aniline 4-hydroxylase, and NADPH cytochrome C-reductase) of male rats treated with ginseng extract, cisplatin, and their combination groups are exhibited in Figure 1. Administration of cisplatin showed a significant (p < 0.05) increase in the activities of liver cytochrome P450, NADPH cytochrome C-reductase, amidopyrine N-demethylase, and aniline 4-hydroxylase, while significantly (p < 0.05) decreased the activity of cytochrome b5 compared to the control values. Ginseng therapy significantly decreased the induction of cytochrome b5 induced by cisplatin alone while significantly improving the activities of liver cytochrome P450, NADPH cytochrome C-reductase, amidopyrine N-demethylase, and aniline 4-hydroxylase compared with cisplatin ( Figure 1).

Figure 2 manifested the changes in the gene expression of hepatic phase I enzymes (cytochrome P1A1, cytochrome P2E1, cytochrome P2D6, and cytochrome P3A4) of male rats treated with cisplatin, ginseng, and their combination. Rats treated with cisplatin alone caused a significant (p < 0.05) induction in cytochrome P1A1, cytochrome P2E1, and cytochrome P3A4, while cytochrome P2D6 significantly (p < 0.05) decreased compared to the control group. In comparison to rats treated with cisplatin alone, animals given ginseng with cisplatin in the combination group displayed a significant (p < 0.05) decrease in cytochromes P1A1, cytochrome P2E1, and cytochrome P3A4, while cytochrome P2D6 considerably (p < 0.05) increased compared to the cisplatin group.

In the liver tissue, cisplatin triggered the oxidative stress markers. Cisplatin considerably (p < 0.05) induced the prooxidant indicators via the increase in the levels of TBARS, Nitric oxide, and xanthine oxidase, and decreased the activity of antioxidant parameters (GST, CPX, CAT, SOD, GR, GSH, and TAC) compared with the control group. Ginseng alone showed improvement in all antioxidant parameters and decreased the oxidative stress markers. The group treated with ginseng and cisplatin showed an increase in the reduction in all antioxidants and a decrease in the induction of the oxidative stress parameters (TBARS, NO, and XO) compared with cisplatin ( Table 2).

Cisplatin treatment triggered the production of inflammatory molecules. Cisplatin significantly (p < 0.05) enhanced the synthesis of TNF-alpha, IL-6, COX-2, TGF-β, and NF-κB levels. However, ginseng alone showed an insignificant decrease in all inflammatory markers, indicating that it had an anti-inflammatory impact. The presence of ginseng with cisplatin reduced the induction in each of the inflammatory parameters ( Figure 3).

As shown in Figure 4, exposure to cisplatin remarkably (p < 0.05) induced apoptosis and cell damage via activating the apoptotic factors caspase-3 and the p53 protein. Co-administration of ginseng reduced the levels of liver p53 and caspase-3 compared to the cisplatin group, restoring these aberrant parameter values.

Treatment with cisplatin resulted in significant (p < 0.05) elevations in the liver function tests (AST, ALT, LDH, ACP, ALP, and GGT) in serum, and suppressed their activities in liver homogenates, and reduced the levels of serum total protein and albumin. The presence of ginseng with cisplatin significantly improved these enzymes and alleviated the levels of total protein and albumin, reversing the liver toxicity induced by cisplatin ( Tables 3, 4).

Figure 5 (A – D) shows photomicrographs of liver sections stained with HE in different groups. Liver sections of the control group (A) and ginseng group (B) showed the normal structure of hepatocytes and central vein. The liver section of rats treated with cisplatin (C) revealed mild to moderate vacuolated hepatocytes, marked cellular infiltration, and congestion in central veins. The liver section of rats treated with cisplatin with ginseng (D) revealed little tissue injury, with only mild cellular infiltration and no congestion in the central veins.

Detection and distribution of P53 immunoreactivity in the liver of the different groups were presented in Figure 6. Liver sections of the control and ginseng groups showed faint positive reactions for P53 ( Figures 6A and B, respectively). In contrast, moderate to strong positive reactions for P53 were detected in the liver of treated rats with cisplatin ( Figure 6C). On the other hand, rats treated with cisplatin and ginseng showed mild to moderate positive reactions for P53 in their liver sections. ( Figure 6D).

The detection and distribution of Bax immunoreactivity in the liver of the different groups were revealed in Figure 7 (A-D). Liver sections in the control and ginseng groups showed faint positive reactions for Bax ( Figures 7A and 8B, respectively). Oppositely, moderate positive reactions for Bax were detected in the liver of treated rats with cisplatin ( Figure 7C). Mild positive reactions for Bax were detected in liver sections in post-treated rats with cisplatin and ginseng ( Figure 7D).

Anti-apoptotic Bcl2 protein immunohistochemistry in the liver: The Bcl2 immunoreactivity in the different groups was revealed in Figure 8 (A-D). Liver sections in the control and ginseng groups showed strong positive reactions for Bcl2 ( Figures 8A and 8B, respectively). While faint positive reactions for Bcl2 were detected in the liver of treated rats with cisplatin ( Figure 8C). On the other hand, moderate positive reactions for Bcl2 were detected in liver sections in post-treated rats with cisplatin and ginseng ( Figure 8D).