OI and ML385 were obtained from Med Chem Express (United States). Carbon tetrachloride was obtained from Acmec biochemical (Shanghai, China). Alanine transaminase (ALT), aspartate transaminase (AST), myeloperoxidase (MPO), reduced glutathione/oxidized glutathione assay kit (GSH/GSSG) and superoxide dismutase (SOD) assay kit were purchased from Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). Thiobarbituric acid reactive substances (TBARS) assay kit was obtained from Elabscience (Wuhan, China). 2,7-Dichlorofluorescein diacetate (DCFH-DA), 4’6-diamidino-2-phenylindole (DAPI) and BeyoECL Plus were bought from Beyotime Biotechnology (Shanghai, China). HMGB1 was purchased from HMGBiotech (Milano, Italy). Mouse TNF-α, IL-1β, IL-6, MCP-1 enzyme-linked immunosorbent assay (ELISA) kits were purchased from Dakewei Bioengineering (Shenzhen, China). HMGB1 ELISA kit was brought from Westang Biotech (Shanghai, China). The PrimeScript™ RT Master Mix, and TB Green Premix Ex Taq™ were obtained from Takara (Tokyo, Japan). Rabbit polyclonal antibodies against Nrf2, NF-𝜅B p65, phospho-NF-𝜅B p65, IκB-α, cleaved poly ADP ribose polymerase (PARP), F4/80 and cleavage Caspase-3 were purchased from cell signaling Technology (Beverly, MA, United States). Anti-heme oxygenase-1 (HO-1), anti-NADPH quinine oxidoreductase-1 (NQO-1), anti-lamin B1 and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies were brought from Proteintech (Wuhan, China). Fetal bovine serum and high glucose Dulbecco’s modified Eagle’s medium (DMEM) were brought from Gibco Life Technologies (Carlsbad, CA, United States). All other chemicals used were of the highest commercial grade.

All C57BL/6 mice (8–10 weeks old, weighted 24–26 g) were purchased from SPF Biotechnology Co., Ltd. (Beijing, China). Mice were kept in a specific pathogen-free and environmentally controlled room and with free access to standard laboratory food and water with 12-h light and dark cycle. All animal experiments were approved by the Animal Care and Use Committee of Tongji Medical College of Huazhong University of Science and Technology. OI was dissolved in saline with a 2 mg/ml concentration and was given via intraperitoneal injection (50 mg/kg body weight) 2 h before CCl4 treatment. ML385 was treated once a day for 2 days by intraperitoneal injection (30 mg/kg body weight). The acute hepatic injury was induced by intraperitoneal injection of CCl4 0.1 ml/kg (10 ml/kg bodyweight, CCl4/olive oil volume = 1:99). Generally, animals were divided into five groups (n = 5): 1) control group: mice were given same volume vehicle during the whole procedure; 2) OI group: mice received OI without CCl4 treatment; 3) CCl4 group: mice were treated with the same volume of saline vehicle and then were treated with CCl4; 4) CCl4 + OI group: OI was given 2 h before receiving the same dose of CCl4 given in the CCl4 group; 5) CCl4 + OI + ML385 group: ML385 was treated once a day for two days then OI and CCl4 were administrated as CCl4 + OI group. Twenty-four hours after CCl4 treatment, all animals were euthanized. Blood samples and liver tissues were collected.

Commercial assay kits measured serum ALT and AST levels according to the manufacturer’s instructions (Nanjing Jiancheng Biological Technology. Nanjing, China).

The murine macrophage cell line RAW264.7 was obtained from the Cell Bank of the Chinese Academy of Science (Shanghai, China). The murine normal hepatic cell line NCTC1469 was purchased from China Center for Type Culture Collection (Wuhan, China). RAW264.7 and NCTC1469 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM high glucose) with 10% fetal bovine serum under a humidified atmosphere of 5% CO₂ at 37°C. NCTC1469 and RAW264.7 cells were grown in six well plates (3*10⁵ per well) overnight. OI (final concentration: 125 μM) or same volume of vehicle was treated 2 h before CCl4 administration (0.5% v/v). Twenty-four hours after CCl4 treatment, NCTC1469 cells were harvested for further analysis. For RAW264.7 cells, OI (final concentration: 125 μM) or the same volume was added, and HMGB1 (final concentration: 2 μg/ml) was added 2 h later. Twenty-four hours after treatment, cells and supernatants were collected for the next analysis.

NCTC1469 cells were grown in six wells plates and treated as previously described in 2.4. Then cells were incubated with 10 uM DCFH-DA for 45 min. Images were captured and cells were harvested and recorded by FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, United States). Data were analyzed using FCS express 3 (De Novo Software) and mean fluorescence intensity (MFI) was used to represented the ROS levels.

Each mouse liver was collected and washed using cold PBS and homogenized in PBS (weight/volume = 1:10). The homogenates were centrifuged at 4,000 g for 20 min and supernatants were collected. Then level of TBARS was evaluated by using commercial kit from Elabscience (Wuhan, China) according to provider’s instruction. The SOD activity and GSH/GSSG ratio were measured by using commercial kit from Nanjing Jiancheng Institute of Biotechnology (Nanjing, China) according to provider’s instruction.

Total RNA was extracted from cells or tissues by TRIzol reagent according to the manufacturer’s instructions. According to the manufacturer’s protocol, mRNA was reversely transcribed to cDNA using PrimeScript RT Master Mix (TAKARA, Japan). Then the expression levels of mRNA in each sample was measured by TB Green Premix Ex Taq according to the supplier’s protocol in the StepOnePlus system (Applied Biosystems, United States). All tests were run for 40 cycles consisting of denaturation at 95°C for 30°s, annealing at 60°C for 30°s, and an extension at 73°C for 30°s. The relative expression of each gene was evaluated using the 2^−ΔΔCt method, and Gapdh was used as the internal control. The primer pairs were used previously (Xiao et al., 2019; Zhang J. et al., 2020) and are listed below: Tnf-α F: 5-CCT​GTA​GCC​CAC​GTC​GTA​G-3, R: 5-GGG​AGT​AGA​CAA​GGT​ACA​ACC​C-3; Il-6 F: 5-AGT​TGC​CTT​CTT​GGG​ACT​GA-3, R: 5-TCC​ACG​ATT​TCC​CAG​AGA​AC-3; Il-1β F: 5-GGG​CCT​CAA​AGG​AAA​GAA​TC-3; R: 5-TAC​CAG​TTG​GGG​AAC​TCT​GC-3; Gapdh F: 5-ATT​CAA​CGG​CAC​AGT​CAA​G-3, R: 5-CTT​CTG​GGT​GGC​AGT​GAT-3; Mcp-1 F: 5-ACT​GAA​GCC​AGC​TCT​CTC​TTC​CTC-3, R: 5-TTC​CTT​CTT​GGG​GTC​AGC​ACA​GAC-3.

According to the manufacturer’s instructions, prospective enzyme-linked immunosorbent assay kits determined the levels of IL-6, TNF-α, IL-1β, and MCP-1 in serum and supernatant. The hepatic tissue MPO activity was assessed by a detection kit according to the manufacturer’s instructions.

TUNEL was used to evaluate cell death in hepatic sections. Briefly, wax-embedded hepatic tissue sections were dewaxed and rehydrated using xylene and a graded series of ethanol. The sections were treated with 20 μl/ml proteinase K without DNase at room temperature for 30 min and washed with PBS 3 times. Then sections were incubated without light in a 50 μl TUNEL detection mixture for 1 h at 37°C. After washing with PBS three times, sections were observed under a fluorescence microscope. Five random fields (200X) were selected, and positive cells were measured by ImageJ software (National Institutes of Health, Bethesda, MD).

Hepatic tissues were fixed in 4% paraformaldehyde solution at 4°C for 24 h and embedded in paraffin. Then 4 μm thick sections were cut and stained with hematoxylin and eosin (H&E) according to standard procedure. Five random fields (200X) were selected and cellular damage was evaluated by standard morphologic criteria such as nuclear pyknosis and loss of architecture. The necrotic area rates were measured by ImageJ. Immunohistochemical analysis was performed in murine paraffin-embedded hepatic sections by incubated with Ly6G antibody (Servicebio Technology Co., Ltd., Wuhan, China) to detect inflammatory cells. Five random fields (200X) were selected and positive cells were measured by ImageJ software (National Institutes of Health, Bethesda, MD, United States).

RAW264.7 cells (2.5 × 10⁴/well) were grown on cover glass slips in 24 wells plate and treated as described previously. Then cells were washed and fixed in 4% paraformaldehyde for 20 min. Then cells were treated with 0.1% TritonX-100 for 10 min and blocked in 5% bull serum albumin for 60 min. NF-𝜅B p65 antibody (1:500) was added and incubated for 10 h at 4°C, and the second antibody was added and incubated in the dark for 60 min; then DAPI was incubated for 5 min to stain the nucleus. For F4/80 staining, briefly, liver tissue sections were dewaxed and rehydrated using xylene and a graded series of ethanol. After antigen retrieval, sections were blocked in 5% BSA for 60 min and then incubated with F4/80 antibody (1:200) overnight at 4°C. After washing with PBS three times, a second antibody was added and incubated in the dark for 60 min, then DAPI was incubated for 10 min to stain the nucleus.

The apoptosis in NCTC1469 cells were evaluated by APC Annexin V Apoptosis Detection Kit with 7-AAD (BioLegend, San Diego, United States) according to producer’s protocol. Briefly, stimulated cells were washed twice in cold PBS and resuspended in annexin V binding buffer. Add 5 ul APC marked annexin V and 5 ul 7-amino-actinomycin D (7-AAD) in 100 ul cells suspension and incubated in the dark for 20 min at room temperature. Add 400 ul annexin V binding buffer to each sample then analyzed by flow cytometry.

NCTC 1469 cells (40–50% confluent) were grown in a 6-well plate and transfected with 50 nM NRF2 specified siRNA or siRNA control using Lipofectamine 6,000 as we reported previously (Zhang et al., 2020b). The transfected cells were cultured 48 h after transfection and then used in the experiments.

According to the manufacturer’s protocol, the proteins of hepatic tissues and cell lines were extracted using the cell lysis buffer for Western blot and IP (Beyotime Biotechnology). According to the manufacturer’s protocol, cell nucleus proteins were extracted using Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology). The concentration of protein was determined by BCA Protein Assay Kit (Beyotime Biotechnology). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting were performed as we reported previously (Li et al., 2020a; Zhang et al., 2020b). Images were recorded with the Image Lab system and analyzed using ImageJ software.

The results were expressed as the mean ± standard deviation. Statistical analysis was performed by one-way analysis of variance and followed by Tukey’s multiple comparison test to evaluate significance. Analyses were performed using Prism8. p < 0.05 was considered significant.

As shown in Figure 1, the sections from single OI or control group mice showed no hepatocyte necrosis or cellular architecture damage and no abnormal hepatic function. In contrast, CCl4 induced hepatic damage in mice. In gross hepatic specimens, the liver’s appearance in the CCl4 group was markedly enlarged in size with a tight and smooth capsule, edema, lighter in color, and brittle texture. Administration of OI mitigated swelling and appearance of necrosis in the liver (Figure 1A). In the CCl4 group, H&E staining showed that CCl4-induced significant liver damage, including massive hepatocyte necrosis, loss of normal architecture, and an influx of inflammatory cells compared to the control group (Figures 1B,C). In contrast, mice that received OI showed a significantly decreasing necrotic area and histopathological damage (Figures 1B,C). Serum ALT and AST are two common indexes for evaluating the severity of hepatic injury. Our study found that ALT and AST significantly elevated in responses to CCl4 treatment. However, OI treatment markedly reduced ALT and AST activities compared to those mice that received vehicle treatment (Figures 1D,F). In addition, our results showed that the liver/body weight ratio significantly increased in the CCl4 group compared to the control group (Figure 1F), which was remarkably alleviated with OI pretreatment. Moreover, the TUNEL assay was used to evaluate OI’s effect on hepatocytes’ death in response to CCl4. As shown in Figures 1G,H, the apoptosis of hepatocytes significantly increased in the CCl4 group compared to the control group. However, pretreatment with OI markedly reduced the TUNEL-positive cells in the OI + CCl4 group compared to the CCl4 group. OI alone could not induce the death of hepatocytes.

To evaluate OI’s effect in regulating inflammatory conditions in CCl4 treated mice, we analyzed inflammatory infiltrating cells such as macrophages and neutrophils with antibodies against F4/80 and Ly6G, respectively. Our study confirmed that F4/80-positive cells could be detected by immunostaining in hepatic tissues. Single OI treatment did not increase F4/80-positive cells infiltration compared to the control group. Notably, a massive increase of F4/80-positive cells infiltration was observed after treatment with CCl4. However, pretreatment with OI significantly reduced F4/80-positive cell recruitment (Figures 2A,C). Through immunohistochemistry, we detected infiltration of Ly6G-positive cells, which is considered a marker of neutrophils. Ly6G cells were rarely observed in the control and OI treatment groups. In the CCl4 group, a massive accumulation of Ly6G cells was observed in injured hepatic tissues. Moreover, the activity of MPO, which is considered a marker of neutrophils, was elevated in liver tissues by CCl4. (Figure 2G). However, OI administration significantly mitigated this neutrophil recruitment effect induced by CCl4 (Figures 2B,D,G).

The levels of SOD, TBARS, and GSH/GSSG ratio in the liver were measured to evaluate oxidative stress and antioxidant capacity in hepatic tissue. We found that CCl4 treatment could decrease SOD activity. However, OI could increase SOD activity (Figure 2E). Moreover, administration of CCl4 significantly increased the TBARS level (Figure 2F) compared to the control group. However, OI pretreatment led to a significant reduction of TBARS in the CCl4-induced hepatic injury model. The GSH/GSSG ratio is considered a pivotal index representing redox changes. In our study, administration of CCl4 could markedly reduce the GSH/GSSH ratio in hepatic tissues. In contrast, that reduction could be partially restored by OI pretreatment (Figure 2H).

Pro-inflammatory cytokines play an essential role during hepatic damage. Our experiment measured the transcription and serum levels of pro-inflammatory mediators such as TNF-α, IL-1β, IL-6, and MCP-1. Administration of CCl4 markedly increased the levels of serum TNF-α (Figure 3A), IL-1β (Figure 3B), IL-6 (Figure 3C), and MCP-1 (Figure 3D) compared to the control group, while OI pretreatment significantly attenuated CCl4-induced production of those pro-inflammatory cytokines. Similarly, the increase in the mRNA levels of those cytokines induced by CCl4 in liver tissues was inhibited by OI pretreatment (Figures 3E,F). And an OI single treatment showed no effect compared to the control group.

NCTC 1469, a murine normal hepatic cell line, was used to evaluate the effect of CCl4 in hepatocytes. ROS level was displayed using probe DCFH-DA. As shown in Figures 4A–C, CCl4 significantly enhanced ROS production in NCTC 1469 cells compared to the control group. In contrast, OI administration could markedly reduce ROS in CCl4 treated cells. We measured the respective cleavage form of caspase 3 and PARP, both of which are considered apoptosis markers. The expression of cleavage-caspase 3 and cleavage-PARP was markedly induced by CCl4 stimulation, while OI could significantly reduce their production during CCl4 treatment (Figures 4D–F). To further validate this observation, we used annexin V and 7AAD to measure the apoptosis rates in NCTC 1469 by flow cytometry. Similarly, the results showed that CCl4 markedly induced more hepatocytes’ death compared to the control cells, and OI treatment significantly reduced cell death and exerted a protective effect in CCl4-treated hepatic cells (Figures 4G,H).

To further explore OI’s protective mechanism in the CCl4-treated mouse model, ML385, a specific NRF2 inhibitor, was used to counteract OI’s effect. As shown in Figures 5A,C, CCl4-induced significant hepatic injury and OI administration mitigated liver histopathology as described before. However, pretreatment of ML385 significantly diminished the protective effect of OI in CCl4-induced injury in mice. In addition, we detected hepatocyte apoptosis in the liver by TUNEL assay. Similarly, CCl4 treatment-induced obvious cell death in liver tissues, which was markedly improved by OI pretreatment. However, this protective effect was eliminated by pretreating ML385, as shown in Figures 5B,D. Also, we found that the CCl4-induced elevation of ALT and AST was significantly inhibited by OI, while administration of ML385 to CCl4 + OI treated mice led to a significant increase in ALT and AST activities. In addition, TBARS level and SOD activity were measured to evaluate oxidative stress and antioxidant capacity, respectively. As shown in Figures 5G,H, OI pretreatment could decrease TBARS induced by CCl4 in hepatic tissues. However, the addition of ML385 eliminated the reduction of TBARS by OI pretreatment, and SOD activity was elevated by OI, while ML385 could block this effect in CCl4-treated mice.

We used siRNA to knock down Nrf2 expression in the murine normal hepatic cell line NCTC 1469 to explore the possible mechanism of OI’s protective effect in CCl4-induced hepatocyte damage. We successfully knocked down the expression of Nrf2 in NCTC 1469 cells (Figures 6A,B). During the CCl4 administration process in vitro, the OI + CCl4 + siNC group significantly expressed more Nrf2 in the cytoplasm and nucleus than the CCl4 + siNC group. In CCl4 + siNrf2 and CCl4 + siNrf2 + OI groups, expression of Nrf2 was inhibited considerably (Figures 6A–C). In addition, we detected the expression of NQO-1 and HO-1 by immunoblotting. Similarly, the expression of NQO-1 and HO-1 was increased in the OI + CCl4 + siNC group compared to the CCl4 + siNC group (Figures 6A,C,D). However, NQO-1 and HO-1 were inhibited in CCl4 + siNrf2 and CCl4 + siNrf2 + OI groups (Figures 6A,C,D). To further explore the effect of OI and Nrf2 knockdown in CCl4 treated hepatocytes, we analyzed apoptosis by flow cytometry. We found that the OI + CCl4 + siNC group showed markedly less cell death compared to the CCl4 + siNC group, while this effect was diminished in both the CCl4 + siNrf2 and CCl4 + siNrf2 + OI groups (Figures 6F,G).

It was reported previously that HMBG1 is associated with CCl4-induced liver injury (Chen et al., 2014). We sought to explore the impact of OI in CCl4-induced expression of HMGB1 in CCl4-treated mice. To measure HMGB1 release, ELISA analysis showed that serum HMGB1 was markedly increased by administration of CCl4 compared to the control group. However, pretreatment of OI in CCl4-treated mice significantly decreased HMGB1 in serum (Figure 7A), and OI alone did not induce HMGB1 release in serum.

Macrophage activation plays important roles in CCl4-induced hepatic injury. The above results indicated that CCl4 increased HMGB1 in serum. We verified that HMGB1 could enhance NF-𝜅B nuclear translocation by promoting phosphorylation of p65 and reducing the IκB-α level. At the same time, OI administration significantly inhibited this effect by decreasing the phosphorylation of p65 and restored IκB-α expression in RAW 264.7 cells (Figures 7B–D). Furthermore, nuclear translocation of NF-𝜅B p65 was detected by immunofluorescence. NF-𝜅B p65 nuclear translocation was increased by HMGB1, while OI administration inhibited such translocation (Figure 7E) in macrophages. This indicated that OI mitigated the NF-𝜅B nuclear translocation induced by HMGB1. The hallmark of macrophage activation is the secretion of specific pro-inflammatory cytokines. Therefore, we evaluated the activities of TNF-α and IL-1β in the supernatant. We found that the levels of TNF-α and IL-1β were significantly enhanced by HMGB1, while OI treatment reduced this effect (Figures 7F,G).