Male Sprague-Dawley rats (150–180 g; specific pathogen-free grade) were obtained from the Experimental Animal Center of Anhui Medical University (Hefei, China) and housed in rooms with a maintained temperature of 22–25°C and humidity of 50±5% under a 12-h light-dark cycle, and provided food and water ad libitum. Rats were acclimatized for 1 week prior to the initiation of experiments. The animal experiments were approved by the Animal Care and Use Committee of Anhui Medical University. A total of 24 rats were randomly assigned to 3 groups (n=8/group) and treated for 28 consecutive days as follows: Rats in the INH + RIF and PDTC groups were intragastrically administered INH (50 mg/kg/day) and RIF (50 mg/kg/day; Yan'an Pharmaceutical Co., Ltd.) following 3 h of abrosia (13,14). Control rats were intragastrically treated with an equivalent volume of 0.9% sodium chloride. Additionally, the PDTC group received 50 mg/kg/day PDTC (Merck KGaA) via an intraperitoneal injection 2 h following the co-administration of INH and RIF (10,15,16). The rats in the control and model groups were intraperitoneally injected with the equivalent volume of 0.9% sodium chloride (14). RIF and INH were solubilized in 1% sodium carboxymethylcellulose and 0.9% sodium chloride, respectively. PDTC initially was dissolved in DMSO at 10 mg/ml and then diluted by 0.9% sodium chloride to 50 mg/ml. Rats were anesthetized by an intraperitoneal injection of sodium pentobarbital (50 mg/kg) the morning following the final drug treatment. Blood was collected into microtubes from the abdominal aorta. Rat livers were rapidly excised, leaving the left lobe of the liver in 10% neutral formalin-fixed buffer for histological analysis. The remaining livers were snap-frozen and stored in a liquid nitrogen container until required (17). Then, the rats were sacrificed by decapitation. Serum samples were obtained from blood following centrifugation at 4°C, 1,728 × g for 20 min and stored at −20°C for detection.

The serum levels of total bile acid (TBA), total bilirubin (TBIL), direct bilirubin (DBIL), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were measured using common commercial assay kits (TBA, ALT, AST and ALP kits were obtained from Shanghai Kehua Bio-engineering Co., Ltd.; TBIL and DBIL kits were obtained from Roche Diagnostics) using an automatic biochemical analyzer (Roche Diagnostics) in the clinical laboratory of the First Affiliated Hospital of Anhui Medical University. Hepatic superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were detected in the supernatants from 10% liver homogenates, which were prepared according to the manufacturer's protocols. SOD and MDA kits were obtained from Nanjing Jiancheng Bioengineering Institute.

Liver tissues were embedded in paraffin following fixation in 10% formalin at room temperature for 24 h. Tissues were subsequently cut into 4-µm thick sections 24 h later, deparaffinized in xylene and hydrated in a graded series of ethanol, and then routinely stained with hematoxylin for 5 min and eosin for 10 sec at room temperature. From each sample, 5 adjacent, non-overlapping fields were selected at random and observed using routine light microscopy at ×400 magnification.

EMSA was performed according to the instructions of the Light Shift Chemiluminescent EMSA kit (cat. no. 89880; Thermo Fisher Scientific, Inc.) to detect the DNA binding activity of NF-κB. Nuclear extracts from liver tissues were prepared using a nuclear extraction kit (Viagene Biotech, Inc.) and quantified using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Inc.). A biotin-labeled NF-κB probe (Santa Cruz Biotechnology, Inc.) with a 5′-AGTTGAGGGGACTTTCCCAGGC-3′ sequence and nuclear proteins (15 µg) were incubated in a total volume of 15 µl in binding buffer at room temperature for 20 min. The extracts were also incubated with specific NF-κB p65 antibody (2 µg/µl; cat. no. SC-372X; Santa Cruz Biotechnology, Inc.) at room temperature for 20 min for a supershift assay. The specificity of binding was detected by competition with unlabeled 100-fold excess of a cold and a mutant probe with a 5′-TGGGGAACCTGTGCTGAGTCACTGGAG-3′ sequence (Thermo Fisher Scientific, Inc.). Following incubation, the DNA-nuclear protein mixtures (15 µl) were subjected to gel electrophoresis on a 6.5% nondenaturing polyacrylamide gel in 0.25X Tris-borate-ethylenediaminetetraacetic buffer at 120 V for 60 min, and then transferred to a presoaked nylon membrane. When the transfer was complete, DNA was cross-linked to the membrane within commercial UA-light for 10 min. The biotinylated DNA-protein bands were detected with streptavidin-horseradish peroxidase (HRP; 1:750) using a Chemiluminescent Assay (Thermo Fisher Scientific, Inc.).

Total RNA was extracted from liver tissues using TRIzol reagent (Sangon Biotech Co., Ltd.) according to the manufacturer's instructions. RNA concentration was measured by NanoDrop™ 2000 (NanoDrop Technologies; Thermo Fisher Scientific, Inc.). Then, cDNA was synthesized using a First Strand cDNA Synthesis kit (Sangon Biotech Co., Ltd.), according to the manufacturer's protocol. The reverse transcription system was conducted at a final volume of 20 µl, including total RNA (5 µl), primer mix (1 µl), dNTP Mix (2 µl), RNase inhibitor (1 µl), AMV Reverse Transcriptase (2 µl), RNase-free ddH₂O (5 µl), 5X RT buffer (4 µl). The PCR system (25 µl) comprised cDNA (0.5 µl), forward primer (0.5 µl), reverse primer (0.5 µl), Taq polymerase (0.2 µl; Sangon Biotech Co., Ltd.), dNTP (0.5 µl), 10X Taq Buffer (2.5 µl), MgCl₂ (2 µl), RNase-free ddH₂O (18.3 µl). Amplification conditions were as follows: Pre-denaturation at 95°C for 3 min, followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at 56°C for 30 sec, extension at 72°C for 90 sec, final extension at 72°C for 8 min. Primers were synthesized by Sangon Biotech Co., Ltd. Primer sequences were as follows: Tumor necrosis factor (TNF)-α, forward 5′-GTCGTAGCAAACCACCAAGC-3′, reverse 5′-TCACAGAGCAATGACTCCAAAG-3′; bile salt export pump (BSEP), forward 5′-GCACAGTTGCTGGGATTGG-3′, reverse 5′-TTGGCATAGCTCGGAGTATAAGA-3′; β-actin, forward 5′-GCACAGTTGCTGGGATTGG-3′ and reverse 5′-TTGGCATAGCTCGGAGTATAAGA-3′.

All PCR products were examined by gel electrophoresis and visualized by ethidium bromide staining. The analysis of PCR products was performed using a gel documentation system (Shanghai Furi Technology Co., Ltd.).

Liver tissues was homogenized and centrifuged at a speed of 14,917 × g for 15 min at 4°C. Total proteins of liver tissues were extracted using RIPA lysate with PMSF (both Beyotime Institute of Biotechnology). After the protein concentration in the supernatants was measured using a BCA protein assay kit (Beyotime Institute of Biotechnology), the protein samples were mixed with 5X SDS loading buffer at a ratio of 1:4, then denatured at 95°C for 10 min. Equal amounts of protein (30 µg) were separated via 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes (EMD Millipore). Following incubation with blocking buffer (5% nonfat milk) for 2 h at room temperature, membranes were incubated overnight at 4°C with rabbit anti-BSEP (1:500; cat. no. BS71760; Bioworld Technology, Inc.) and anti-β-actin (1:1,000; cat. no. TA-09; OriGene Technologies, Inc.) primary antibodies, then washed with TBST and incubated with an HRP-coupled secondary antibody (1:10,000; cat. no. ZB-2301; Beijing Zhongshan Golden Bridge Biotech Co., Ltd; OriGene Technologies, Inc.) for 1 h at room temperature. The immunoblots were rinsed extensively with TBS-Tween 20 (0.1%) and visualized using an ECL western blot detection kit (Thermo Fisher Scientific, Inc.). Signals were normalized to β-actin levels, which was used as the internal standard. Semi-quantitative evaluation was performed by densitometry using ImageJ software (version 1.51; National Institutes of Health).

SPSS software (version 22.0; IBM Corp.) was used for the statistical analysis. All data are presented as the mean ± SD. Statistical analysis of the differences between groups was performed using one-way analysis of variance (ANOVA) followed by Student Newman-Keuls test as post hoc. P<0.05 was considered to indicate a statistically significant difference.

There was no mortality in any of the groups. Rats in the INH + RIF group showed loss of appetite, listlessness, rough hair coat and slow response to external stimuli compared with the control group. In addition, the skin and mucous membrane of the nose, ears, paws and tail were dyed orange. Feces and urine were also clearly orange. The general behavior of the rats in the PDTC group was almost the same as that of control rats.

Intraperitoneal administration of INH and RIF at a dose of 50 mg/kg/day over a period of 28 days produced hepatotoxicity. As presented in Table I, the co-administration of INH and RIF significantly increased serum TBA, TBIL, DBIL and ALP activity compared with the control (P<0.01). There were no significant alterations in the levels of ALT and AST (P>0.05), whereas PDTC treatment significantly attenuated the elevation of serum TBA, TBIL, DBIL and ALP compared with the INH + RIF group (P<0.01), which was suggestive of reduced injury in liver tissues (Table I). Next, as presented in Fig. 1, all animals in the control group showed normal morphology (Fig. 1A). The INH + RIF group exhibited moderate to severe lobular inflammation and piecemeal necrosis (Fig. 1B). The liver histology of the PDTC group exhibited an almost normal morphology, with only a low level of liver steatosis and occasional infiltration of inflammatory cells (Fig. 1C). Liver histopathology was consistent with serum biochemistry in the present study.

To evaluate the protective effects of PDTC against INH/RIF-induced oxidative stress, SOD activity and MDA content in liver tissue were detected. The SOD activity in liver homogenates was significantly decreased in the INH + RIF group compared with the control (P<0.01). However, the administration of PDTC significantly attenuated the INH/RIF-induced reduction in SOD activity (P<0.01). Similarly, the MDA content was significantly elevated following co-administration of INH and RIF as compared with the control group (P<0.01), whereas PDTC treatment effectively decreased the INH/RIF-induced increase in MDA content (P<0.01; Fig. 2).

EMSA was used to further explore the potential role of NF-κB in INH/RIF-induced liver injury. Following the co-administration of INH and RIF, the NF-κB DNA-binding activity of liver homogenates was upregulated significantly (P<0.01); PDTC treatment markedly inhibited INH/RIF-induced NF-κB activity (P<0.01; Fig. 3A and B). Furthermore, supershift analysis using p65 antibody against NF-κB demonstrated that the band was in accordance with the p65 NF-κB subunit (Fig. 3A). These results suggested that NF-κB activation was involved in the pathogenesis of INH and RIF-induced liver injury.

To further study the possible mechanisms via which PDTC alleviates INH and RIF-induced liver injury, the levels of TNF-α and BSEP mRNA gene expression were detected in liver tissues via RT-PCR, and the protein expression of BSEP was detected via western blot analysis. Compared with the control group, the mRNA expression of TNF-α and BSEP was significantly up- and downregulated, respectively, in the INH + RIF group (both P<0.01; Fig. 4A and B), and the protein expression of BSEP was also significantly decreased in the INH + RIF group (P<0.01; Fig. 5A and B). Conversely, compared with the INH + RIF group, the mRNA expression of TNF-α and BSEP was significantly decreased and increased, respectively, in the PDTC group (P<0.01 and P<0.05, respectively; Fig. 4A and B), and the protein expression of BSEP was also significantly elevated in the PDTC group (P<0.01; Fig. 5A and B). These results revealed that PDTC could prevent INH/RIF-induced increases in TNF-α and decreases in BSEP.