PTS (purity >99%) was purchased from Meilunbio (Dalian, Liaoning Province, China). DOX was purchased from Sigma-Aldrich (St. Louis, MO, United States). Alanine transaminase (ALT) and aspartate transaminase (AST) were from Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). Malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH) kits were purchased from Solarbio (Beijing, China). Hematoxylin–eosin (H & E) and Masson staining kits were from Beyotime Biotechnology (Shanghai, China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was provided by Roche Diagnostics (Basel, Switzerland). TUNEL staining kits (Green) were from Beyotime Biotechnology. The bicinchoninic acid (BCA) protein assay kit was from Thermo Scientific, lysis buffer and phenylmethanesulfonylfluoride (PMSF) were obtained from Beyotime Biotechnology. Human recombinant Trx-1 was from Med Chem Express (HY-P73431).

We used 8-week-old wild type (WT) C57/BL6J mice as experimental animals and divided the mice into four groups (with n = 8 for each group): a control group, a PTS treatment group, a DOX treatment group, and a DOX + PTS treatment group. The animals of the DOX group were injected a dose of 10 mg/kg intraperitoneally. This was conducted on day 1 and day 4 for a total of 2 times (20 mg/kg cumulative dose of DOX). The mice of the DOX + PTS group were injected intraperitoneally with PTS (10 mg/kg/day) every day for a total of 7 times, until one day before DOX treatment. As in the DOX treatment group, afterwards DOX was injected intraperitoneally with a dose of 10 mg/kg. This was conducted on day 1 and day 4 for a total of 2 times (20 mg/kg cumulative dose of DOX). All mice were euthanized 6 days after the initial injection of DOX (Liu D. et al., 2019). All the animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Dalian Medical University (SCXK 2015-2003).

All the mice were sacrificed under anesthesia after our study period. The liver tissue was fixed with 4% paraformaldehyde (PFA) for more than 24 h, followed by paraffin embedding. All sections (4 μm) were subjected to a H & E and Masson staining. The liver tissues were subjected to immunohistochemical staining. For this, the sections were incubated with the primary antibody anti-NLRP3 (Wanleibio, WL02635, 1:200) at 4°C overnight and afterwards with the corresponding secondary antibody. The blots were developed using DAB. Digital images were taken at 200 × magnification and were analyzed with ImageJ software.

The levels of MDA, SOD, and GSH in DOX-treated livers were evaluated by MDA, SOD, and GSH kits (Solarbio), respectively, according to the manufacturer’s instructions.

HepG2 cell were purchased from the American Type Culture Collection. Cells were cultured in DMEM supplemented with 10% fetal bovine serum and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin, Sigma) and were grown in a humidified atmosphere containing 5% CO₂ at 37°C. Hep G2 cells were treated with recombinant Trx-1 at a dose of 1 μg/ml to elucidate the presumed PTS effects (El Hadri, K., et al., 2012).

HepG2 cells were seeded in 96-well plates for 24 h. After 24 h, the medium was removed, 100 μL of sample solution with different concentrations of DOX (0, 1, 2, 5, 8, and 10 mM) was added for different treatment times and a period of 24 h (Song, et al., 2019b). MTT solution (5 mg/ml) was added to each well to a final concentration of 0.5 mg/ml for 4 h. After exposure, DMSO (100 μL/well) was added to dissolve the formed formazan crystals. The absorbance at 490 nm was measured with a microplate reader (Thermo, United States). Based upon these data, a suitable DOX concentration for the induction of cell injury was identified.

HepG2 cells were seeded in 96-well plates for 24 h and then pretreated with different concentrations of PTS (0, 5, 10, and 20 μM) for 16 h before the treatment with DOX (5 μM) for 24 h. DOX group cells were cultured without PTS pretreatment. An MTT assay was used to assess cell viability (Song S. et al., 2019).

HepG2 cells were plated in 6-well culture plates for 24 h and afterwards treated with PTS at a concentration of 10 μM for 4 h before treatment with DOX (5 μM) for 24 h (Song S. et al., 2019). DOX group cells were cultured without PTS pretreatment and the control group was cultured in serum-free DMEM under normal conditions during the entire experiment. Cells were loaded with 10 μM DCFH-DA. After that, the cells were washed 3 times with serum-free DMEM and the images were captured by fluorescence microscopy (Olympus, Japan) with a 200 × overall magnification.

HepG2 cell apoptosis detection was performed by using TUNEL staining (Green, Beyotime Biotechnology) and the assay was performed according to the manufacturer’s instructions. For this, HepG2 cells were plated in 6-well culture plates for 24 h and afterwards treated with PTS at a concentration of 10 μM for 4 h before the treatment with DOX (5 μM) for 24 h. After that, the cells were washed 3 times with PBS and fixed with 4% PFA for 30 min after which the cells were washed 3 times with PBS. Afterwards, the cells were treated with 0.3% Triton X-100 containing PBS solution for 5 min, washed with PBS 3 times and finally the cells were incubated with the TUNEL reaction mixture for 60 min at 37°C in the dark. The cells were evaluated under a fluorescence microscope.

All mice were sacrificed under anesthesia after our study period. The serum of the animals was collected, and AST and ALT were measured by employing commercially available biochemical kits that were used according to the manufacturer’s instructions.

Total proteins were extracted from snap-frozen liver tissues or cells. We use a protein extraction kit (Keygenbio, KGP250) and centrifuge tube (Guangzhou Jet Bio-Filtration Co., Ltd.) for proteins extracting. The protein lysates (30 μg) were separated by electrophoresis in an 8–15% SDS–PAGE gel and transferred to polyvinylidene difluoride (PVDF) membranes. The blots were incubated with appropriate antibodies at 4°C overnight and then incubated with a goat anti-rabbit or mouse conjugated secondary antibody (Sino Biological Inc., 1:3000). All blots were developed using an ECL Plus chemiluminescence system. The following antibodies were used: anti-Trx-1 (CST, #2429, 1:800), anti-NLRP3 (CST, #15101, 1:800), Caspase-1 p20 (Affinity, AF5418, 1:500), IL-1β (Wanleibio, WL0227, 1:500), IL-18 (Wanleibio, WL01127, 1:1000), ASC (Wanleibio, WL02462, 1:500), BAX (CST, #14796S, 1:500), BCL-2 (CST, #3498S, 1:500) and Cleaved Caspase-3 (CST, #9664, 1:500). ImageJ software was used for densitometry analysis and GAPDH was used as an internal control.

According to the manufacturer’s instructions, we used TRIzol reagent (Invitrogen, New York) to purify the total RNA from the fresh livers and cells. The first-strand cDNA (1–2 μg) was synthesized using a Superscript II kit (TAKARA, Japan). All the primers were synthesized by Sangon Biotech Company (Shanghai, China). The primer sequences were as follows: NLRP3: forward 5′-AGC CAA GAA TCC ACA GTG TAA CC-3′ and reverse 5′-AGT GTT GCC TCG CAG GTA AG-3′; IL-1β: forward 5′-TGC CAC CTT TTG ACA GTG ATG-3′ and reverse 5′-TTC TTG TGA CCC TGA GCG AC-3′; IL-18: forward 5′- GCA AAG CTT ATG ACC ATG AGA CAC AAC TG-3′ and reverse 5′-GCG AAT TCG TCG ACT TTA ACC CTG CTG TGG ACT-3′; NOX-1: forward 5′-GCT ACG CCT TCA ACA CCA AG-3′ and reverse 5′-AGT TCG TCC CCT TCT CCT GT-3′; NOX-4: forward 5′-GCA CGC TGT TGA TTT TTA TGG-3′ and reverse 5′-GCG AGG CAG GAG AGT CAG TA-3′; GAPDH: forward 5′-CAT CAA GAA GGT GGT GAA-3′ and reverse 5′-TGT TGA AGT CAG AGG AGA-3′. We used GAPDH as the internal control and normalized the resulting transcript levels to those of GAPDH gene. The results were analyzed using the ΔΔCt technique.

All data are expressed as the mean ± SD. The statistical analyses were performed with GraphPad Prism 9 software. One-way ANOVA followed by Tukey’s comparison test was used to analyze significant differences among multiple groups. Values of p < 0.05 were considered as being statistically significantly different.

To explore the effects of PTS on DOX-induced hepatotoxicity, we pre-treated the mice with PTS (10 mg/kg) before DOX administration (Figure 1A). H & E staining (Figure 1B) revealed that the liver of control group mice displayed a normal architecture whereas apparent injuries were found in the DOX-treated group that could be restored by PTS. In addition, Masson staining revealed that administration of DOX in mice markedly increased the collagen deposition compared to the control group and that PTS remarkably reduced the DOX-induced fibrosis (Figure 1C). As shown in Figure 1D, after treatment with DOX, compared with the control group, the levels of ALT and AST were increased, respectively. The pre-treatment of PTS significantly reduced the ALT and AST levels in mice compared to the DOX-treated group. We next detected the expression of Trx-1 protein. Compared to the control group, the expression of Trx-1 was obviously decreased after DOX treatment but could be rescued by PTS (Figure 1E). We next evaluated the SOD, GSH, and MDA levels in DOX-treated mice. As shown in Figure 1F, the SOD and GSH levels were both markedly decreased after treatment with DOX compared to the control group and the MDA level in the DOX-treated group was higher than that of the control group. After PTS treatment, the level of MDA was decreased and the levels of SOD and GSH were both increased compared to the DOX-treated group. Therefore, PTS was able to prevent the increase in ROS and the decrease of Trx-1 in DOX-treated mice.

Oxidative stress frequently results in inflammatory reactions (Ventura et al., 2017). To further elucidate the effects of PTS, we performed immunohistochemical staining to detect the expression of NLRP3 in DOX-treated mice. As shown in Figure 2A, the expression of NLRP3 was upregulated in the DOX-treated group and PTS was able to alleviate the increase in NLRP3 expression. We then evaluated the levels of NLRP3, IL-1β, and IL-18 mRNA. As shown in Figure 2B, PTS significantly reduced the levels of NLRP3, IL-1β and IL-18 mRNA compared to those in the DOX-treated group. In addition, we evaluated the expressions of NLRP3 and its downstream proteins. Compared to the DOX-treated group, the expression of NLRP3, ASC, Caspase-1 p20, IL-1β, and IL-18 was significantly decreased after pretreatment with PTS (Figure 2C). In addition, we detected the expressions of Cleaved Caspase-3, BAX, and BCL-2 proteins. As shown in Figure 2D, the expression of Cleaved Caspase-3 and BAX were increased, and the expression of BCL-2 was significantly decreased after treatment with DOX. Compared to the DOX-treated mice, the pretreatment of PTS reduced the expression of BAX and upregulated the expression of BCL-2.

HepG2 cells were treated with different DOX doses (0, 1, 2, 5, 8, 10 μM) for 24 h. As shown in Figure 3A, the viability of HepG2 cells treated with 5 μM DOX for 24 h was decreased to nearly 75%, which is why we treated the cells in the following experiments with 5 μM DOX. We used PTS at concentrations of 0, 5, 10, and 20 μM to check whether PTS could protect cells against DOX-induced injury in a dose dependent manner. HepG2 cells were pretreated with different PTS concentrations for 4 h and afterwards treated with DOX (5 μM) for 24 h. Compared to the DOX-treated group, PTS at a 10 and 20 μM concentration significantly increased the viability of HepG2 cells (Figure 3B) so that we treated the cells in the following experiments with a PTS concentration of 10 μM.

To clarify whether the DOX-induced upregulation of NLRP3 was mediated by the reduction in Trx-1 levels, we pretreated the HepG2 cells with recombinant Trx-1 (1 μg/ml) for 4 h and afterwards with DOX (5 μM) for 24 h. Upon administration of recombinant Trx-1, the levels of both NOX-1 and NOX-4 were decreased compared to those of the DOX-treated group (Figure 4A). As shown in Figure 4B, this was also true for the NLRP3 and IL-1β mRNA levels. Similarly, the expressions of NLRP3, IL-1β, and IL-18 protein were decreased (Figure 4C). The results confirmed that the upregulation of Trx-1 was able to decrease the NLRP3 signal and inflammasome stimulation.

PTS inhibits DOX-induced oxidative stress in cells through increasing Trx-1 expression.

We used a DCFH-DA staining to detect the effect of PTS on DOX-treated cells. HepG2 cells were pretreated with PTS (10 μM) for 4 h and afterwards treated with DOX (5 μM) for 24 h. As shown in Figure 5A, treatment with DOX increased the ROS level in HepG2 cells compared to the control group. After pretreatment with PTS, the cellular ROS levels were significantly decreased compared to those in the DOX-treated group. Moreover, after treatment with DOX, the NOX-1 and NOX-4 mRNA levels were both increased compared to the control group. After pretreatment with PTS, NOX-1 and NOX-4 mRNA levels were decreased compared to those in the DOX-treated group (Figure 5B). We next analyzed the expression of Trx-1. As shown in Figure 5C, DOX induced the downregulation of Trx-1 and PTS pretreatment was able to rescue the expression of Trx-1.

A TUNEL assay was used to detect apoptosis in DOX-treated HepG2 cells and the protective effects of PTS. For this, HepG2 cells were pretreated with PTS (10 μM) for 4 h whereafter they were treated with DOX (5 μM) for 24 h. As shown in Figure 6A, DOX induced HepG2 cell apoptosis which could be reverted by pretreatment with PTS. Compared to the control group, the NLRP3 and IL-1β mRNA levels were markedly increased after DOX treatment and this effect was inhibited by PTS. Similarly, the expression of NLRP3, Caspase-1 p20, IL-1β, and IL-18 were significantly decreased after PTS treatment (Figure 6B).