Magnesium isoglycyrrhizinate (MgiG) was purchased from Chia Tai Tianqing Pharmaceutical Group Co., Ltd. (Jiangsu, China). Oxaliplatin was obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). ELISA kits for IL-6 (KGEMC138) and von Willebrand factor (vWF) (KGEMC146) were obtained from Nanjing KeyGEN Biotech. Co., Ltd. (Nanjing, China). The alanine aminotransferase (ALT), aspartate aminotransferase (AST), glutathione (GSH) and malondialdehyde (MDA) detection kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Unless indicated otherwise, chemicals used in experiments were purchased from Sigma-Aldrich (Merck KGaA).

Forty male, 10-week-old C57Bl/6 mice, weighing 18-22 g, were obtained from the Laboratory Animal Center of the School of Medicine, Shandong University (Shandong, China). Animals were kept under standard conditions of 55% humidity, 20–25°C, and a controlled 12-h light/dark cycle, and allowed free access to standard food and water. All C57Bl/6 mice were randomly divided into 4 groups. Group I mice were intraperitoneally (i.p.) injected with vehicle (normal saline) as control (n=10). Group II mice received 10 mg/kg intraperitoneal oxaliplatin on a weekly basis for 5 weeks (n=10). Group III mice received an low dose of MgiG [15 mg/kg/day, intraperitoneal (i.p.)] together with intraperitoneal oxaliplatin (n=10). Group IV mice received high-dose MgiG (45 mg/kg/day, i.p.) together with intraperitoneal oxaliplatin (n=10). The drug dosing schedule was based on previously published studies and our preliminary experiments (18,20–22). Mice were sacrificed one week following the final dose of chemotherapy under isoflurane anesthesia by cardiac puncture. The present study was conducted in accordance with Chinese laws and National Institute of Health publications on the use and care of laboratory animals and using a protocol approved by the Institutional Animal Care and Use Committee of Shandong University (Shandong, China).

The human hepatic cell line LO2 and the human colon cancer cell line HT-29 were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were maintained in RPMI1640 medium for LO2 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and DMEM for HT-29 (Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin, and 100 µg/ml streptomycin (Amresco, Inc.; VWR International, Radnor, PA, USA) at 37°C in a 5% CO₂ humidified atmosphere.

AST, ALT, GSH and MDA commercial kits were used to measure the indicators, according to the manufacturers' instructions. In brief, liver homogenates, serum or cell supernatant substrate solutions were added to the reacting system, and the final products were spectrophotometrically measured with various wavelengths. The concentrations of the indicator were calculated according to standard curves. The concentrations of IL-6 and vWF in serum and liver homogenates were measured with ELISA kits, according to the manufacturer's instructions.

Cell viability was determined with a CCK-8 assay. Briefly, cells were plated in 96-well plates at a density of 3,000 cells per well. After 10 h, the medium was replaced with fresh medium containing 300 µM oxaliplatin, with or without other reagents. The doses of oxaliplatin and other reagents were determined based on previous studies and our preliminary experiments (18,23). Experimental groups were treated with various concentrations of MgiG. Untreated cells were used as control. Following incubation for 24 h, the cells were exposed to CCK-8 (100 µl/ml; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) followed by incubation at 37°C for another 2 h. Subsequently, the absorbance of the resulting color in each well was measured using a microplate spectrophotometer at a wavelength of 450 nm.

Formalin-fixed and paraffin-embedded liver tissues were cut into 5 µm-thick slices. Hematoxylin and eosin (H&E) and Sirius red stains were used. The pathological changes of both stained slices were evaluated by a pathologist blinded to the experimental group. The histological changes were scored according to the following criteria: 0, absent; 1, mild; 2, moderate; and 3, severe.

Total RNA of liver homogenates and cells was extracted and purified using TRIzol reagent (Thermo Fisher Scientific, Inc.). The reverse transcription of 1 µg RNA to cDNA was established using the PrimeScript RT reagent kit (cat. no. RR036A; Takara Bio, Inc., Otsu, Japan). qPCR was run on a Bio-Rad iQ5 optical module (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Cycling conditions were: 95°C for 2 min as initial denaturation, 40 cycles of denaturation at 95°C for 15 sec, and annealing/extension at 60°C for 40 sec. Melt curve analysis was set between 65 and 95°C with 0.5°C increments at 5 sec per step. Quantitative values were obtained by the threshold cycle (Cq) value. Relative mean fold change in expression ratios was calculated by the 2^−ΔΔCq method (24). GAPDH was used as internal control. The primer sequences used in the present study were as follows: GAPDH, forward 5′-GCACAGTCAAGGCCGAGAAT-3′ and reverse 5′-GCCTTCTCCATGGTGGTGAA-3′; metallothionein 1 (Mt1), forward 5′-GCTGCTGCTCCTGCTGTCCC-3′ and reverse 5′-CAGCACGTGCACTTGTCCGC-3′; peroxiredoxin 1 (PRDX1), forward 5′-TGTTTCCCCAGCATGTGTACC-3′ and reverse 5′-TGCTCTTATAGAAGACCCAGGTTC-3′; superoxide dismutase 2 (SOD2), forward 5′-CGTGACTTTGGGTCTTTTGAG-3′ and reverse 5′-TAGAGCAGGCAGCAATCTGTAA-3′; IL-6, forward 5′-GAGGATACCACTCCCAACAGACC-3′ and reverse 5′-AAGTGCATCATCGTTGTTCATACA-3′; signal transducer and activator of transcription 3 (STAT3), forward 5′-CCTTCTTGTTCTACGGCTTGC-3′ and reverse 5′-TCGCCTATCTTCTCAACCAGG-3′; vWF, forward 5′-CGGGAAGAGTGTGATGGTTGAC-3′ and reverse 5′-AGCATCTCCCACAGCATTCACC-3′; plasminogen activator inhibitor-1 (PAI-1), forward 5′-GATGCTATGGGATTCAAAGTCA-3′ and reverse 5′-TCCACCTGTTTCACCATAGTCT-3′.

Mice liver samples or cells were lysed in radioimmunoprecipitation assay lysis solution (Beyotime Institute of Biotechnology, Haimen, China) containing phosphatase inhibitors and PMSF. A BCA Protein Assay kit (Beyotime Institute of Biotechnology) was used to determine the protein concentrations. A total of 30 µg protein extracts were resolved by 8 and 15% SDS-PAGE. The proteins were transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA), blocked with 5% nonfat dry milk at room temperature for 1 h and incubated with primary antibodies at 4°C overnight. The following antibodies were used: Metallothionein 1 (cat. no. ab12228; 1:2,000; Abcam, Cambridge, MA, USA), peroxiredoxin 1 (cat. no. 8499; 1:5,000; Cell Signaling Technology, Inc., Danvers, MA, USA), superoxide dismutase 2 (cat. no. 13141; 1:500; Cell Signaling Technology, Inc.), STAT3 (cat. no. 9132; 1:1,000; Cell Signaling Technology, Inc.); vWF (cat. no. 65707; 1:500; Cell Signaling Technology, Inc.), PAI-1 (cat. no. 11907; 1:2,000; Cell Signaling Technology, Inc.). and GAPDH (cat. no. ab22555; 1:2,500; Abcam). The blots were then incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1.5 h (cat. nos. BA1054 and BA1050; 1:50,000; Wuhan Boster Biological Technology, Ltd., Wuhan, China), and visualized by enhanced chemiluminescence detection. GAPDH was used as the loading control. The optical density was analyzed with ImageJ 10.0 software (National Institutes of Health, Bethesda, MD, USA).

Three independent repeats were performed for each experiment. Experimental data were expressed as the mean ± standard deviation. Analyses were performed using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA). Comparisons among datasets were made using one-way analysis of variance followed by Tukey's post hoc test or unpaired Student's t-test. P<05 was considered to indicate a statistically significant difference.

Aminotransferase levels are diagnostic markers of liver damage. LO2 cells exposed to oxaliplatin were cultured in medium supplemented with or without MgiG, and mice receiving oxaliplatin were treated with or without MgiG. AST and ALT activity in serum and cell supernatants was then measured. Following oxaliplatin challenge, both indicators significantly increased in cell supernatants (Fig. 1A). In the MgiG-treated groups, the increase in indicators was significantly attenuated (Fig. 1A). AST and ALT levels continued decreasing when the MgiG concentration was below 100 µM. AST and ALT activity in mouse serum also displayed a significant increase following oxaliplatin administration (Fig. 1B). MgiG effectively attenuated the oxaliplatin-induced changes and this effect was dose-dependent with a more significant reduction observed in the high-dose group (Fig. 1B).

To assess the histological changes in liver parenchyma, H&E-stained liver sections were blindly reviewed by a pathologist and graded according to the degree of histological damage. A large area of hepatocyte necrosis, extensive vacuolization, and inflammatory cell infiltration were observed in the oxaliplatin-treated group (Fig. 1C). MgiG treatment significantly attenuated the pathological damage in the liver of the experimental animals (Fig. 1C). High-dose MgiG had a more significant effect in protecting the liver against oxaliplatin-induced damage (Fig. 1C). Sirius red staining was also performed on the tissue sections to estimate fibrosis in the liver. Collagen deposition was observed within the injured hepatic sinusoids in the liver of oxaliplatin-treated mice (Fig. 1D). This pathological progress was significantly attenuated in the liver of MgiG-treated mice (Fig. 1D).

A cell viability assay was performed on LO2 cells to estimate their response to oxaliplatin challenge and treatment of MgiG at various concentrations. Cell viability decreased by 57% in the oxaliplatin-treated group compared with the control group (Fig. 2A). There was an increase in cell viability in MgiG-treated groups (Fig. 2A). When the concentration of MgiG was >100 µM, the rise in viability occurred in a dose-dependent manner (Fig. 2A). The same experiment was repeated using the colon cancer cell line HT-29. Cell viability also decreased dramatically following oxaliplatin exposure, but MgiG treatment had no effect in the oxaliplatin-mediated loss of viability in the colon cancer cells (Fig. 2B).

It has been well-demonstrated in previous studies that increased oxidative stress is one of the major mechanisms inducing sinusoidal obstruction syndrome (SOS) in patients and animals receiving oxaliplatin treatment (11,21,25,26). To estimate oxidative stress, contents of MDA and GSH in liver homogenates and cell supernatants were measured (27). In LO2 cell supernatants, the content of MDA significantly increased following oxaliplatin exposure, and MgiG treatment significantly attenuated MDA levels (Fig. 2C). In liver homogenates from experimental mice, MDA increased 5.04-fold following oxaliplatin administration, and MgiG treatment remarkably attenuated this effect (Fig. 2D). In addition, a remarkable reduction in the concentration of GSH in LO2 cell supernatants was observed with oxaliplatin exposure, and MgiG significantly restored this (Fig. 2E). In vivo, the GSH levels in the liver homogenates of oxaliplatin-injected mice decreased by 59% compared with control, and MgiG treatment significantly increased the GSH levels by 74 and 113% at low and high-dose, respectively (Fig. 2F).

To explore the role of oxidative stress in the pathogenic process of oxaliplatin-induced liver injury and the mechanism of the protective function of MgiG, a cell viability assay was performed on LO2 cells in the presence of supplementary 1 mM H₂O₂ or 100 µM GSH in the medium. The dose of H₂O₂ for creating oxidative stress in vitro was based on previous studies and our preliminary experiments (28-30). When H₂O₂ was added in the medium, cell viability decreased by 34% in the oxaliplatin group, while high-dose MgiG treatment improved viability by 109, 38% higher than the control group (Fig. 2G). In the viability assay with supplementary GSH, oxaliplatin challenge attenuated cell viability by 30%. The cell viability in MgiG-treated groups also increased (Fig. 2H). High-dose MgiG treatment improved cell viability by 20%, and there was no significant difference between the viabilities of the oxaliplatin-treated group and the low-dose MgiG group (Fig. 2H).

Next, the expression of genes classically associated with anti- and pro-oxidative functions was investigated (27). The expression levels of vital genes implicated in the response to oxidative stress, Mt1, PRDX1 and SOD2, were measured in the liver of oxaliplatin-treated mice by RT-qPCR and western blotting. The results demonstrated that both mRNA and protein expression levels of these genes were increased in the livers of oxaliplatin-treated mice compared with control mice (Fig. 3). MgiG treatment, however, effectively attenuated the increased expression of these genes (Fig. 3). In addition, all three indicators were more improved with high-dose MgiG than with low-dose MgiG (Fig. 3).

Previous studied have reported that the IL-6 pathway is a core signaling pathway activated during oxaliplatin-related liver damage (11,21). IL-6 levels in liver homogenates were measured by ELISA and RT-qPCR. The secreted levels and mRNA expression of IL-6 in liver homogenates were increased following oxaliplatin administration and this effect was significantly attenuated by MgiG treatment (Fig. 4A and B). The levels of IL-6 in the high-dose group were lower compared with the low-dose group. STAT3 is one of the most important transcription factors downstream of IL-6. Its expression in liver homogenates was also tested by RT-qPCR and Western blot. The RT-qPCR results demonstrated that STAT3 expression was increased by 3.4-fold in the oxaliplatin-administered group, and that this increase was reversed by MgiG treatment (Fig. 4C). The western blot analysis demonstrated similar results (Fig. 4D and E).

In most previous studies of oxaliplatin-induced liver injury, activation of the coagulation system was one of the most-discussed mechanisms (11,12,21,26,31). In the present study, ELISA results revealed that levels of vWF, a key component in platelet adhesion, were significantly increased in the livers of animals administered with oxaliplatin (Fig. 4F). MgiG treatment significantly attenuated this increase (Fig. 4F). This effect was also evident at the mRNA and protein level, as measured by RT-qPCR and western blotting (Fig. 4G–I). Expression of PAI-1 (also known as SERPINE1) in the livers of experimental animals was also increased as a result of oxaliplatin exposure, as measured by RT-qPCR (Fig. 4J). MgiG treatment notably inhibited this increase (Fig. 4J). Western blott analysis revealed similar results at the protein level (Fig. 4K and L). In both experiments, high-dose MgiG had an improved protective effect compared with low-dose MgiG.