Introduction

Molecular Medicine Reports

1791-2997 1791-3004

D.A. Spandidos

10.3892/mmr.2018.8849

mmr-17-06-7939

Articles

Saikosaponin-d alleviates carbon-tetrachloride induced acute hepatocellular injury by inhibiting oxidative stress and NLRP3 inflammasome activation in the HL-7702 cell line

Lin

Liubing

1 * Que

Renye

2 * Shen

Yanting

1 Chen

Yirong

1 Yan

1 Li

Yong

1Digestive Department, Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 200071, P.R. China 2Department of Gastroenterology, Shanghai Traditional Chinese Medicine Integrated Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200082, P.R. China

Correspondence to: Dr Yong Li, Digestive Department, Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, 274 Middle Zhijiang Road, Shanghai 200071, P.R. China, E-mail: liyong_sci@126.com *

Contributed equally

062018

05042018

17 6 7939 7946 30102017 02032018

2018

Saikosaponin-d (SSd) the primary active component of triterpene saponin derived from Bupleurum falcatum L., possesses anti-inflammatory and antioxidant properties. The present study aimed to examine the potential therapeutic effects of SSd on carbon tetrachloride (CCl₄)-induced acute hepatocellular injury in the HL-7702 cell line and its underlying mechanisms. HL-7702 cells were treated with SSd at different doses (0.5, 1 or 2 µmol/l). Cell viability was determined using an MTT assay. Injury was assessed by the levels of serum alanine aminotransferase (ALT) and aspartate transaminase (AST). Oxidative stress was assessed using malondialdehyde (MDA) content and total-superoxide dismutase (T-SOD) activity. The expression of nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3), apoptosis-associated speck-like protein (ASC), caspase-1 and high mobility group protein B1 (HMGB1) was assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. Interleukin (IL)-1β and IL-18 were determined by RT-qPCR and ELISA. SSd attenuated the inhibition of cell viability and the high AST and ALT levels induced by CCl₄ in HL-7702 cells. Oxidative stress was induced in HL-7702 cells by CCl₄, as demonstrated by the increase of MDA and the decrease of T-SOD activity. These changes were reversed by SSd. SSd significantly downregulated the mRNA and protein expression of NLRP3, ASC, caspase-1, IL-1β, IL-18 and HMGB1 induced by CCl₄. In conclusion SSd alleviated CCl₄-induced acute hepatocellular injury, possibly by inhibiting oxidative stress and NLRP3 inflammasome activation in the HL-7702 cell line.

saikosaponin-d carbon tetrachloride acute hepatocellular injury HL-7702 cell line nucleotide-binding domain leucine-rich-containing family pyrin domain-containing-3 inflammasome oxidative stress

Introduction

The liver is important for metabolic homeostasis, detoxification, immunity, and secretory functions (1). Sustained and progressive liver disorders will result in severe liver injury due to increasing cellular, tissue, and function disruption, and is associated with high mortality (2). Management of liver injury remains among the most challenging issues of contemporary medicine (3) and the development of new therapies for acute liver injury is needed.

Carbon tetrachloride (CCl₄) is one of the most potent hepatotoxic compounds (4). Acute liver injury models produced using CCl₄ are good models of acute chemical liver injury in humans. CCl₄ result in the generation of trichloromethyl free radicals (•CCl₃) by cytochrome P₄₅₀. The •CCl₃ causes lipid peroxidation and excessive production of reactive oxygen species (ROS), resulting in liver injury (5,6). The imbalance between ROS production and the antioxidant defense leads to oxidative stress, as shown by increased lipid peroxidation and decreased activities of antioxidant enzymes such as the superoxide dismutase (SOD), catalase, and glutathione peroxidase (7).

The nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome has been reported to be involved in the pathogenesis of acute liver injury (8). It is composed of three proteins: NLRP3, apoptosis-associated speck-like protein (ASC), and caspase-1. Once activated by stimuli, the NLRP3 binds to the adaptor protein ASC, and in turn leads to oligomerization of caspase-1, subsequently promoting the activation of caspase-1. Active caspase-1 initiates the cleavage of pro-interleukin (IL)-1β and pro-IL-18 and then promotes the secretion of IL-1β, IL-18, and high mobility group protein B1 (HMGB1) (9). These inflammatory cytokines are critical in various liver diseases such as hepatic ischemia reperfusion injury, alcoholic steatohepatitis, non-alcoholic steatohepatitis, and drug-induced liver injury (10–13).

Many herbs exhibit anti-inflammatory and anti-oxidative capacity, and are being used as adjunctive therapy for liver injury. Some compounds extracted from traditional Chinese medicine herbs possess anti-inflammatory and antioxidants effects against CCl₄-induced oxidative stress, such as cardamomum, esculentoside A, Ocimum gratissimum, resveratrol, and quercetin (14–18). Among these herbs, Radix bupleuri is one of the most commonly used for many diseases such as viral hepatitis and chronic hepatic inflammation (19). Saikosaponin-d (SSd) is the major active component of Bupleurum falcatum L. and has been reported to have anti-inflammatory, antiviral, immunomodulatory, and antioxidant activity (20,21). SSd inhibits the proliferation of rat hepatic stellate cells via decreasing lipid peroxidation (22) and restrains the proliferation of activated T lymphocyte via the NF-AT, NF-κB, and AP-1 pathways (23). In addition, SSd alleviates ventilator-induced lung injury via inhibiting inflammatory responses and oxidative stress (24). Nevertheless, the hepatoprotective effects of SSd against CCl₄-induced acute hepatocellular injury remain unclear.

Based on the known effects of SSd, we hypothesized that SSd alleviates the effects of CCl₄ on hepatocytes by reducing inflammation and oxidative stress. Therefore, the aim of this study was to investigate the protective effects of SSd on CCl₄-induced acute hepatocellular injury in the HL-7702 cell line and whether the effects were related to oxidative stress and NLRP3 inflammasome activation.

Materials and methods <sec> <title>Cell culture

HL-7702 cells (the human normal liver cell line from FuDan IBS Cell Center, Shanghai, China) were routinely cultured in RPMI-1640 medium (Wuhan Boster Biological Technology, Ltd., Wuhan, China) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in 5% CO₂ at 37°C. The cells were treated with CCl₄ (Beijing Chemical Reagent Company, Beijing, China), SSd (batch no. 110778-201409; China's Drug Supervision, Beijing, China), and N-acetyl-L-cysteine (NAC; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). SSd, CCl₄, and NAC were dissolved in dimethyl sulphoxide (DMSO) immediately before use (final concentration of DMSO: <0.1%). DMSO (<0.1%) alone was included as a control in all experiments and did not have any effect on the parameters measured.

MTT assay

Cell proliferation was analyzed by the MTT assay. HL-7702 cells were washed twice with phosphate-buffered saline solution (PBS) and counted. Then, 1×10⁴ cells were seeded on 96-well plates and cultured for 24 h. The cells were exposed to SSd at various concentrations for 24 h. Other cells were seeded onto 96-well plates, incubated for 24 h, and preincubated with NAC (100 µmol/l) or the indicated doses of SSd for 1 h. After that, CCl₄ was added at 10 mmol/l to induce acute injury for 24 h (25,26). Then, 100 µl of MTT solution (0.5 mg/ml; Sigma-Aldrich; Merck KGaA) were added to each well and incubated for 4 h. The medium was discarded and 150 µl of DMSO was added for 24 h. The absorbance of each well was measured at 570 nm with an ELx800 universal microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The relative cell viability was calculated as: (absorbance of drug treated group)/(absorbance of untreated group) × 100 (%).

Alanine aminotransferase (ALT) and aspartate transaminase (AST) in supernatants

HL-7702 cells were seeded onto 6-well plates at 8×10⁵ cells/well and cultured in RPMI-1640 with 10% FBS for 24 h. After HL-7702 cells were pretreated with NAC or the indicated doses of SSd for 1 h, CCl₄ was added at 10 mmol/l to induce acute hepatocellular injury for 24 h (25,26). The supernatants were collected and stored at −20°C. ALT and AST levels were measured using a Var10skan Flash fluorescence plate reader (Thermo Fisher Scientific, Inc.) using the ALT and AST assay kits (C009-1 and C010-1; Nanjing Jiancheng Institute of Biotechnology, Nanjing, China).

Total-superoxide dismutase (T-SOD) activity and malondialdehyde (MDA) levels

HL-7702 cells were seeded into 6-well plates and cultured in RPMI-1640 with 10% FBS for 24 h. The cells were pretreated with NAC or the indicated doses of SSd for 1 h, followed by CCl₄ treatment for another 24 h (25,26). The supernatants were collected and stored at −20°C. MDA was detected by the thiobarbituric acid (TBA) assay using a MDA assay kit (A003-1; Nanjing Jiancheng Institute of Biotechnology). T-SOD activity was determined using the hydroxylamine method with the T-SOD assay kit (A001-1-1; Nanjing Jiancheng Institute of Biotechnology).

Western blotting

NLRP3, caspase-1, ASC, and HMGB1 proteins were measured by western blot. Whole cellular proteins were extracted using the M-PER lysis buffer (Thermo Fisher Scientific, Inc.). Lysates were centrifuged at 12,000 × g for 15 min at 4°C to remove debris. Proteins (50 µg) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Immobilon-P; EMD Millipore, Billerica, MA, USA). Membranes were blocked with 5% skimmed milk and probed using NLRP3 (1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), ASC (1:500; Santa Cruz Biotechnology, Inc.), pro-caspase-1 (1:1,000; Abcam, Cambridge, MA, USA), caspase-1 (1:1,000; Abcam), HMGB1 (1:1,000; Abcam), or β-actin (1:2,000; Santa Cruz Biotechnology, Inc.) antibodies overnight at 4°C. Primary antibodies were detected with the corresponding horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology, Inc., Danvers, MA, USA) and visualized using an enhanced chemiluminescence (ECL) kit (EMD Millipore) western blotting detection system (Amersham, GE Healthcare, Waukesha, WI, USA). The band intensity was quantified using a Bio-Rad GS-690 Scanner (Bio-Rad Laboratories, Inc.). The relative expression level of each protein was normalized to β-actin or pro-caspase-1.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.). RNA (3 µg) was reverse-transcribed to cDNA with the ReverTra Ace-α-^® RT kit (Toyobo, Inc., Tokyo, Japan), according to the manufacturer's protocols. RT-qPCR was performed using the SYBR-Green PCR Master Mix (Toyobo, Inc.) in a PCR detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The primers were: NLRP3 forward 5′-TTGACTTCCGCAAGGACCTCGG-3′ and reverse 5′-GCGCCCGGGTTATGCTGCTGGT-3′; ASC forward 5′-GGCTGCTGGATGCTCTGTA-3′ and reverse 5′-AGGCTGGTGTGAAACTGAAGA-3′; caspase-1 forward 5′-CAGACAAGGGTGCTGAACAA-3′ and reverse 5′-TCGGAATAACGGAGTCAATCA-3′; IL-1β forward 5′-TGGCAATGAGGATGACTTGT-3′ and reverse 5′-TGGTGGTCGGAGATTCGTA-3′; IL-18 forward 5′-GACCTTCCAGATCGCTTCCTC-3′ and reverse 5′-GATGCAATTGTCTTCTACTGGTTC-3′; and HMGB1 forward 5′-TATCGCCCAAAAATCAAAGG-3′ and reverse 5′-TAAGGCTGCTTGTCATCTGC-3′. The amplification process was the same for all genes and was 35 cycles of denaturation at 95°C for 45 sec, annealing at 50°C for 45 sec, and elongation at 72°C for 45 sec. β-actin was used as internal control and the primers were: forward 5′-CTCCATCCTGGCCTCGCTGT-3′ and reverse 5′-GCTGTCACCTTCACCGTTCC-3′. The 2^−ΔΔCq method was used to represent the relative mRNA expression of the target genes.

Enzyme-linked immunosorbent assay (ELISA)

The levels of IL-18 and IL-1β in the supernatants of HL-7702 cells were measured by ELISA (BMS224HS and BMS267-2; eBioscience, San Diego, CA, USA), according to the manufacturer's instructions.

Statistical analysis

Statistical analysis was conducted using SPSS 21.0 (IBM Corp., Armonk, NY, USA). Results were expressed as mean ± standard deviation. The differences among groups were analyzed using one way analysis of variance (ANOVA) with the Student-Newman-Keuls test for post hoc analysis. P<0.05 was considered to indicate a statistically significant difference.

Results <sec> <title>Effects of SSd on the viability of HL-7702 cells

The MTT cell viability assay was performed to examine whether SSd produced cytotoxic effects on HL-7702 cell line. The treatment of HL-7702 cells with SSd at 0.5–2 µmol/l for 24 h did not affect cell viability (Fig. 1), while cell viability was decreased using 2–24 µmol/l (Fig. 1). Therefore, SSd at 0.5, 1, and 2 µmol/l were selected as the low-dose, moderate-dose, and high-dose groups in the subsequent experiments.

SSd alleviates CCl<sub>4</sub>-induced acute hepatocellular injury

The MTT assay was used to investigate the effects of SSd on CCl₄-induced acute hepatocellular injury. As shown in Fig. 2A, CCl₄ alone significantly decreased cell viability and SSd reversed the phenomenon in a dose-dependent manner (all P<0.01; Fig. 2A). NAC, as a positive control, also attenuated the decreased cell viability induced by CCl₄ (P<0.01; Fig. 2A).

The effects of SSd on CCl₄-induced serum AST and ALT levels were examined. CCl₄ significantly increased the levels of serum ALT and AST. SSd treatment prevented CCl₄-induced increase of AST level in a dose-dependent manner (P<0.05 for low-dose and P<0.01 for the other doses, Fig. 2B). The levels of ALT in the SSd groups were decreased significantly at the moderate and high doses compared to the CCl₄ group (P>0.05 for low-dose and P<0.01 for the other doses, Fig. 2C). NAC almost completely reversed the increases of AST and ALT induced by CCl₄ (P<0.01; Fig. 2B-C).

SSd attenuates CCl4-induced oxidative stress

To explore the mechanisms by which SSd alleviates CCl₄-induced acute hepatocellular injury, the anti-oxidative effects of SSd on CCl₄-induced acute hepatocellular injury in HL-7702 cells were explored. We examined the levels of T-SOD and MDA in the cell culture supernatants. The decreased T-SOD induced by CCl₄ was attenuated in a dose-dependent manner by SSd (all P<0.01; Fig. 3A), while MDA showed opposite changes (P<0.05 for low-dose and P<0.01 for the other doses, Fig. 3B). CCl₄-induced oxidative stress was blocked by NAC (P<0.01; Fig. 3A and B). Thus, the suppressive effect of SSd on oxidative stress could be related to the increase of T-SOD and the reduction of MDA.

SSd inhibits CCl4-induced NLRP3 inflammasome activation

The NLRP3 inflammasome is composed of the NLRP3, caspase-1, and ASC (27,28). To verify whether SSd may affect the NLRP3 inflammasome, NLRP3, ASC, caspase-1, and HMGB1 were assessed by RT-qPCR and western blotting. As shown in Fig. 4A, CCl₄ significantly increased NLRP3, caspase-1, ASC, and HMGB1 mRNA levels. Treatment with SSd decreased CCl₄-induced NLRP3, ASC, and HMGB1 mRNA expression in a dose-dependent manner (all P<0.01; Fig. 4A), and decreased the level of caspase-1 mRNA at the moderate and high doses (P>0.05 for low-dose, P<0.05 for the moderate dose, and P<0.01 for the high dose; Fig. 4C). The effects of CCl₄ injury were blocked by the positive control drug NAC (P<0.01; Fig. 4A).

Similar to the qPCR results, western blotting showed that the expressions of NLRP3, ASC, caspase-1 and HMGB1 were increased by CCl₄, but these effects were blocked by NAC (P<0.01; Fig. 4B). The protein expressions of NLRP3, ASC, caspase-1, and HMGB1 were gradually reduced as the dosage of SSd increased (P<0.05 for the low-dose group for ASC, and all other P<0.01; Fig. 4B). Therefore, the anti-inflammatory effects of SSd could be associated with the inhibition of the NLRP3 inflammasome.

SSd inhibits the secretion of proinflammatory cytokines

The production of inflammatory cytokines IL-1β and IL-18 in culture supernatant was examined by qPCR and ELISA. CCl₄ significantly increased IL-1β and IL-18 expression, which could be attenuated by NAC. More importantly, treatment with SSd gradually attenuated the expressions of IL-1β and IL-18 as the dosage of SSd increased (all P<0.01; Fig. 5).

Discussion

The CCl₄-induced acute liver injury model is frequently used to examine the efficacy of liver protective agents. SSd has been found to attenuate CCl₄-induced hepatic injury in rats by inhibiting lipid peroxidation (22). Nevertheless, the downstream molecular mechanism remains unclear. Therefore, this study aimed to examine the effects of SSd on CCl₄-induced acute injury in HL-7702 cells and whether the mechanisms could be related to oxidative stress and NLRP3 inflammasome activation. The results suggest that SSd attenuated CCl₄-induced acute injury by inhibiting oxidative stress and NLRP3 inflammasome activation in the HL-7702 cell line.

In the present study, SSd at 0.5–2 µmol/l was protective against CCL₄-induced injury, but SSd was toxic at doses >2 µmol/l. Chen et al showed that mitochondrial apoptosis was the main toxic effect of SSd at high concentration (21). Li et al found that saikosaponins at 200 µg/ml induced hepatotoxicity through oxidative stress and lipid metabolism dysregulation (29). On the other hand, Zhao et al found that SSd at 45–60 µmol/l could protect renal tubular epithelial cell against high-glucose-induced injury by regulation of SIRT3 (30). Yao et al found that SSd inhibited the proliferation of prostate cancer cells at 50 µM (31). These results suggest that SSd is toxic at high concentrations, but protective at lower doses.

Oxidative stress is known to be related to the pathogenesis of acute liver injury (32–34). CCl₄, is widely used to induce acute hepatic injury in animals, has strong hepatotoxic effects that leads to the excessive generation of free radicals associated with oxidative stress, and ultimately results in acute liver injury with functional impairment. Serum AST and ALT are widely used as makers of acute hepatic injury. Decreased levels of AST and ALT associated with fewer necrotic lesions and lipid peroxidation could imply protection against CCl₄-induced injury (35). Therefore, we first verified that SSd could attenuate CCl₄-induced AST and ALT increases. MDA and T-SOD are indicators of CCl₄-induced oxidative stress (36). The increase of MDA, a product of lipid peroxidation, is considered to be a direct indicator of abnormal peroxidation and impaired antioxidant defenses (36). As an antioxidant enzyme, T-SOD catalyzes the dismutation of superoxide anions into hydrogen peroxide and oxygen. Studies showed that CCl₄ could lead to excessive generation of free radicals and oxidative stress in the liver by increasing the levels of MDA and decreasing the levels of T-SOD, ultimately leading to acute liver injury (37–39). In the present study, SSd played an antioxidant role by inhibiting the production of MDA and improving T-SOD levels in CCl₄-induced acute injury in liver cells.

The activation of the NLRP3 inflammasome results in the maturation of the inflammatory cytokines IL-1β and IL-18, and also leads to the release of HMGB1 (9). These potent proinflammatory cytokines further aggravate the inflammatory progress initiated by oxidative stress. IL-1β and IL-18 are members of the IL-1 superfamily and contribute to inflammation (40,41). HMGB1 is a nuclear protein and proinflammatory mediator, and promotes inflammation and necrotic cell death. IL-1β, IL-18, and HMGB1 may be involved in acute hepatocellular injury (42–46). The effect of the NLRP3 inflammasome has been explored in many liver conditions such as drug-induced liver injury, non-alcoholic steatohepatitis, alcoholic steatohepatitis, hepatic ischemia reperfusion injury, and fibrosis (10–13,47,48). Kim et al (49) showed that NLRP3 inflammasome activation play a central role in GalN/LPS-induced inflammatory responses and the development of hepatic injury. Gong et al (50) showed that the activation of the NLRP3 inflammasome contributes to the induction of inflammation, which might be associated with BDL-induced fibrosis in non-alcoholic and alcoholic steatohepatitis. In the present study, SSd decreased the mRNA and protein expression of the NLRP3 inflammasome components after induction by CCl₄, which is consistent with the role of the NLRP3 inflammasome in liver injury. In addition, the levels of IL-1β and IL-18 in the culture supernatants were reduced.

Oxidative stress induced by the overproduction of ROS is an important cause of organ and tissue injury in various inflammatory diseases (33,34). In addition, ROS play crucial roles in the activation of the NLRP3 inflammasome (51,52). Recent evidence also indicate that some herb extracts have anti-inflammatory effects by suppressing ROS production (53). Moreover, it has been confirmed that oxidative stress and inflammatory cytokines are frequently identified as the main factors contributing to acute liver injury, and anti-inflammatory and antioxidant compounds are thought to prevent acute hepatocellular injury (54–56). Dang et al (57) showed that SSd attenuated CCl₄-induced liver fibrosis in rats through the downregulation of TNF-α, IL-6, and NF-κBp65, and the upregulation of I-κBα. Wu et al (55) also showed the beneficial effects of SSd against liver fibrosis in CCl₄ rats models. Taken together, ROS-induced NLRP3 inflammasome activation may be involved in CCl₄-induced acute hepatocellular injury. Furthermore, the anti-inflammatory effect of SSd may depend on the modulation the inflammation through the NLRP3 inflammasome. Nevertheless, ROS and factors such as TNF-α, IL-6, NF-κBp65, and I-κBα were not evaluated in this study, and further experiments are needed to confirm those mechanisms. In addition, NLRP3 inhibitors should be used to confirm the role of the NLRP3 inflammasome in CCl₄-induced injury and the beneficial effects of SSd.

In conclusion, this study suggests that the suppressive effects of SSd on CCl₄-induced acute hepatocellular injury may depend on the inhibition of oxidative stress and NLRP3 inflammasome activation. This study suggests new evidence for the potential efficacy of SSd for the treatment of acute hepatocellular injury.

Acknowledgements

Not applicable.

Funding

This study was supported by grants from the National Natural Science Foundation of China (grant no. 81573775), the Shanghai Natural Science Foundation (grant no. 09ZR1429700) and the Shanghai Outstanding Academic Leader of Health System (grant no. XBR2013120).

Availability of data and materials

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

LL made substantial contributions to the acquisition of the data, data analysis and interpretation of the data and writing the manuscript. RQ and YL participated in the conception and design of the study and the revision of the manuscript. LL, YS, YC and NY performed the experiments. YL was primarily responsible for the revision of the manuscript.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Abbreviations

SSd

saikosaponin-d

CCl₄

carbon tetrachloride

NLRP3

nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3

ALT

alanine aminotransferase

AST

aspartate transaminase

T-SOD

total-superoxide dismutase

MDA

malondialdehyde

HMGB1

high mobility group protein B1

interleukin

References 1

Zhao

Wang

Dai

Hou

Enrichment and purification of total flavonoids from Cortex Juglandis Mandshuricae extracts and their suppressive effect on carbon tetrachloride-induced hepatic injury in Mice

J Chromatogr B Analyt Technol Biomed Life Sci10078172015

10.1016/j.jchromb.2015.10.019

26562802

Tuñón

Miguel

San B

Crespo

Jorquera

Santamaria

Alvarez

Prieto

González-Gallego

Melatonin attenuates apoptotic liver damage in fulminant hepatic failure induced by the rabbit hemorrhagic disease virus

J Pineal Res5038452011

10.1111/j.1600-079X.2010.00807.x

20964705

Auzinger

Wendon

Intensive care management of acute liver failure

Curr Opin Crit Care141791882008

10.1097/MCC.0b013e3282f6a450

18388681

Assayed

Khalaf

Salem

Protective effects of garlic extract and vitamin C against in vivo cypermethrin-induced teratogenic effects in rat offspring

Food Chem Toxicol48315331582010

10.1016/j.fct.2010.08.011

20728494

Kodai

Takemura

Minamiyama

Hai

Yamamoto

Kubo

Yoshida

Niki

Okada

Hirohashi

Suehiro

S-allyl cysteine prevents CCl(4)-induced acute liver injury in rats

Free Radic Res414894972007

10.1080/10715760601118361

17454131

Recknagel

Glende

JrDolak

Waller

Mechanisms of carbon tetrachloride toxicity

Pharmacol Ther431391541989

10.1016/0163-7258(89)90050-8

2675128

Zelen

Djurdjevic

Popovic

Stojanovic

Jakovljevic

Radivojevic

Baskic

Arsenijevic

Antioxidant enzymes activities and plasma levels of oxidative stress markers in B-chronic lymphocytic leukemia patients

J BUON153303362010

20658731

Pan

Chen

Zhou

Lin

Jin

Mangiferin alleviates lipopolysaccharide and D-galactosamine-induced acute liver injury by activating the Nrf2 pathway and inhibiting NLRP3 inflammasome activation

Eur J Pharmacol77085912016

10.1016/j.ejphar.2015.12.006

26668000

Keyel

How is inflammation initiated? Individual influences of IL-1, IL-18 and HMGB1

Cytokine691361452014

10.1016/j.cyto.2014.03.007

24746243

Arteel

Marsano

Mendez

Bentley

McClain

Advances in alcoholic liver disease

Best Pract Res Clin Gastroenterol176256472003

10.1016/S1521-6918(03)00053-2

12828959

Neuman

Cytokines-central factors in alcoholic liver disease

Alcohol Res Health273073162003

15540802

Petrasek

Bala

Csak

Lippai

Kodys

Menashy

Barrieau

Min

Kurt-Jones

Szabo

IL-1 receptor antagonist ameliorates inflammasome-dependent alcoholic steatohepatitis in mice

J Clin Invest122347634892012

10.1172/JCI60777

22945633

3461900

Zhu

Duan

Chen

Xiong

Zhang

Zheng

Tan

Gong

Fang

Gene silencing of NALP3 protects against liver ischemia-reperfusion injury in mice

Hum Gene Ther228538642011

10.1089/hum.2010.145

21128730

Zhang

Wang

Qiu

Wang

Fang

Wang

Sun

Xia

The protective effect of Esculentoside A on experimental acute liver injury in mice

PLoS One9e1131072014

10.1371/journal.pone.0113107

25405982

4236201

Lim

Kim

Park

Kim

Moon

Park

Amomum cardamomum L. ethyl acetate fraction protects against carbon tetrachloride-induced liver injury via an antioxidant mechanism in rats

BMC Complement Altern Med161552016

10.1186/s12906-016-1121-1

27246748

4886410

Chiu

Chao

Tsai

Chiou

Liu

Hung

Shih

Lai

Liu

Ocimum gratissimum is effective in prevention against liver fibrosis in vivo and in vitro

Am J Chin Med428338522014

10.1142/S0192415X14500530

25004878

Chan

Lee

Huang

Chou

Lin

Lee

Regulation by resveratrol of the cellular factors mediating liver damage and regeneration after acute toxic liver injury

J Gastroenterol Hepatol296036132014

10.1111/jgh.12366

23981054

Zhang

Shi

Huang

Wang

Therapeutic detoxification of quercetin against carbon tetrachloride-induced acute liver injury in mice and its mechanism

J Zhejiang Univ Sci B15103910472014

10.1631/jzus.B1400104

25471833

4265558

Lee

Wang

Chen

Risk of liver injury associated with Chinese herbal products containing radix bupleuri in 639,779 patients with hepatitis B virus infection

PLoS One6e160642011

10.1371/journal.pone.0016064

21264326

3020221

Wong

Zhang

Zhou

Lam

Chan

Law

Yue

Liu

Saikosaponin-d enhances the anticancer potency of TNF-α via overcoming its undesirable response of activating NF-Kappa B signalling in cancer cells

Evid Based Complement Alternat Med20137452952013

10.1155/2013/745295

23573150

3610377

Chen

Zhang

Kong

Zhu

Chen

Wang

Zheng

Saikosaponin D disrupts platelet-derived growth factor-β receptor/p38 pathway leading to mitochondrial apoptosis in human LO2 hepatocyte cells: A potential mechanism of hepatotoxicity

Chem Biol Interact20676822013

10.1016/j.cbi.2013.08.006

23994742

Fan

Wang

Shen

Siow

Choy

Gong

Saikosaponin-d attenuates the development of liver fibrosis by preventing hepatocyte injury

Biochem Cell Biol851891952007

10.1139/O07-010

17534399

Yuan

Zhang

Yan

Zhao

Liao

Chen

Saikosaponin a and its epimer saikosaponin d exhibit anti-inflammatory activity by suppressing activation of NF-κB signaling pathway

Int Immunopharmacol141211262012

10.1016/j.intimp.2012.06.010

22728095

Wang

Liu

Zhong

Fang

Saikosaponin-d attenuates ventilator-induced lung injury in rats

Int J Clin Exp Med81513745122015

26628997

4658886

Yang

Zhao

Jiang

Protective effect of compounds from the flowers of Citrus aurantium L. var. amara Engl against carbon tetrachloride-induced hepatocyte injury

Food Chem Toxicol624324352013

10.1016/j.fct.2013.08.041

23985451

Han

Shi

Song

Ren

Duan

Chen

Augmenter of liver regeneration promotes the proliferation of HL-7702 cells in carbon tetrachloride-induced acute liver injury via increasing autophag

Zhonghua Gan Zang Bing Za Zhi247617662016(In Chinese)

27938562

DeNicola

Karreth

Humpton

Gopinathan

Wei

Frese

Mangal

Yeo

Calhoun

Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis

Nature4751061092011

10.1038/nature10189

21734707

3404470

Schroder

Tschopp

The inflammasomes

Cell1408218322010

10.1016/j.cell.2010.01.040

20303873

Huang

Luan

Liu

Sun

Saikosaponins induced hepatotoxicity in mice via lipid metabolism dysregulation and oxidative stress: A proteomic study

BMC Complement Altern Med172192017

10.1186/s12906-017-1733-0

28420359

5395759

Zhao

Zhang

Bao

Liu

Saikosaponin-d protects renal tubular epithelial cell against high glucose induced injury through modulation of SIRT3

Int J Clin Exp Med8647264812015

26131275

4483897

Yao

Yang

Cao

Zhang

Gao

Saikosaponind inhibits proliferation of DU145 human prostate cancer cells by inducing apoptosis and arresting the cell cycle at G0/G1 phase

Mol Med Rep103653722014

10.3892/mmr.2014.2153

24736800

Kadiiska

Gladen

Baird

Germolec

Graham

Parker

Nyska

Wachsman

Ames

Basu

Biomarkers of oxidative stress study II: Are oxidation products of lipids, proteins, and DNA markers of CCl4 poisoning?

Free Radic Biol Med386987102005

10.1016/j.freeradbiomed.2004.09.017

15721980

Alfadda

Sallam

Reactive oxygen species in health and disease

J Biomed Biotechnol20129364862012

10.1155/2012/936486

22927725

3424049

Brüne

Dehne

Grossmann

Jung

Namgaladze

Schmid

von Knethen

Weigert

Redox control of inflammation in macrophages

Antioxid Redox Signal195956372013

10.1089/ars.2012.4785

23311665

3718318

Kim

Flamm

Di Bisceglie

Bodenheimer

Public Policy Committee of the American Association for the Study of Liver Disease: Serum activity of alanine aminotransferase (ALT) as an indicator of health and disease

Hepatology47136313702008

10.1002/hep.22109

18366115

Hoek

Pastorino

Ethanol, oxidative stress, and cytokine-induced liver cell injury

Alcohol2763682002

10.1016/S0741-8329(02)00215-X

12062639

Weber

Boll

Stampfl

Hepatotoxicity and mechanism of action of haloalkanes: carbon tetrachloride as a toxicological model

Crit Rev Toxicol331051362003

10.1080/713611034

12708612

Cheng

Ren

Gao

Lei

Zheng

Cao

Antioxidant and hepatoprotective effects of Schisandra chinensis pollen extract on CCl4-induced acute liver damage in mice

Food Chem Toxicol552342402013

10.1016/j.fct.2012.11.022

23201450

Tirkey

Pilkhwal

Kuhad

Chopra

Hesperidin, a citrus bioflavonoid, decreases the oxidative stress produced by carbon tetrachloride in rat liver and kidney

BMC Pharmacol522005

10.1186/1471-2210-5-2

15683547

549532

Dinarello

Immunological and inflammatory functions of the interleukin-1 family

Annu Rev Immunol275195502009

10.1146/annurev.immunol.021908.132612

19302047

Davis

Wen

Ting

The inflammasome NLRs in immunity, inflammation, and associated diseases

Annu Rev Immunol297077352011

10.1146/annurev-immunol-031210-101405

21219188

4067317

Petrilli

Papin

Dostert

Mayor

Martinon

Tschopp

Activation of the NALP3 inflammasome is triggered by low intracellular potassium concentration

Cell Death Differ14158315892007

10.1038/sj.cdd.4402195

17599094

Franchi

Eigenbrod

Muñoz-Planillo

Nuñez

The inflammasome: A caspase-1-activation platform that regulates immune responses and disease pathogenesis

Nat Immunol102412472009

10.1038/ni.1703

19221555

2820724

Yang

Antoine

Andersson

Tracey

The many faces of HMGB1: Molecular structure-functional activity in inflammation, apoptosis, and chemotaxis

J Leukoc Biol938658732013

10.1189/jlb.1212662

23446148

4051189

Chen

Yin

Wei

Tong

Shen

Zhao

Chamley

Increased expression of high mobility group box 1 (HMGB1) in the cytoplasm of placental syncytiotrophoblast from preeclamptic placentae

Cytokine8530362016

10.1016/j.cyto.2016.06.001

27285673

Zhu

Zhang

Shi

Chang

Gao

Feng

Yang

HMGB1-RAGE signaling pathway in severe preeclampsia

Placenta36114811522015

10.1016/j.placenta.2015.08.006

26303759

Imaeda

Watanabe

Sohail

Mahmood

Mohamadnejad

Sutterwala

Flavell

Mehal

Acetaminophen-induced hepatotoxicity in mice is dependent on Tlr9 and the Nalp3 inflammasome

J Clin Invest1193053142009

19164858

2631294

Artlett

The Role of the NLRP3 Inflammasome in Fibrosis

Open Rheumatol J680862012

10.2174/1874312901206010080

22802905

3395884

Kim

Lee

NLRP3 inflammasome activation in D-galactosamine and lipopolysaccharide-induced acute liver failure: Role of heme oxygenase-1

Free Radic Biol Med6599710042013

10.1016/j.freeradbiomed.2013.08.178

23994575

Gong

Zhou

Zhao

Tian

Wang

Chen

Cai

Chenodeoxycholic acid activates NLRP3 inflammasome and contributes to cholestatic liver fibrosis

Oncotarget783951839632016

10.18632/oncotarget.13796

27924062

5356637

Dostert

Pétrilli

Van Bruggen

Steele

Mossman

Tschopp

Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica

Science3206746772008

10.1126/science.1156995

18403674

2396588

Sayan

Mossman

The NLRP3 inflammasome in pathogenic particle and fibre-associated lung inflammation and diseases

Part Fibre Toxicol13512016

10.1186/s12989-016-0162-4

27650313

5029018

Shen

Chou

Wang

Chen

Chou

Anti-inflammatory activity of the extracts from mycelia of Antrodia camphorata cultured with water-soluble fractions from five different Cinnamomum species

FEMS Microbiol Lett231137432004

10.1016/S0378-1097(03)00953-4

14769478

Jaeschke

Reactive oxygen and mechanisms of inflammatory liver injury

J Gastroenterol Hepatol157187242000

10.1046/j.1440-1746.2000.02207.x

10937675

Lin

Chu

Tsai

Chao

Curcumin or saikosaponin a improves hepatic antioxidant capacity and protects against CCl4-induced liver injury in rats

J Med Food112242292008

10.1089/jmf.2007.555

18598162

Zheng

Yin

Han

Liu

Peng

Protective effects of the total saponins from Dioscorea nipponica Makino against carbon tetrachloride-induced liver injury in mice through suppression of apoptosis and inflammation

Int Immunopharmacol192332442014

10.1016/j.intimp.2014.01.019

24491258

Dang

Wang

Cheng

Song

Liu

Inhibitory effects of saikosaponin-d on CCl4-induced hepatic fibrogenesis in rats

World J Gastroenterol135575632007

10.3748/wjg.v13.i4.557

17278221

4065977

Figure 1.

Effects of SSd on the viability of HL-7702 cells. HL-7702 cells were treated with various doses of SSd (0.1–24 µmol/l). The cell viability was assessed by an MTT assay. Data are shown as mean ± standard deviation for at least three independent experiments. **P<0.01 vs. the untreated group. SSd, saikosaponin-d.

Figure 2.

Effects of SSd on CCl₄-induced acute hepatocellular injury. HL-7702 cells were pretreated with 100 µmol/l NAC or different doses of SSd for 1 h, and then treated with 10 mmol/l CCl₄ for 24 h. (A) Cell viability was examined by the MTT assay. The levels of (B) AST and (C) ALT in the supernatants were determined using commercial kits. Data are shown as mean ± standard deviation for at least three independent experiments. *P<0.05, **P<0.01 vs. the CCl₄ only group. CCl_4, carbon tetrachloride; SSd, saikosaponin-d; NAC, N-acetyl-L-cysteine; L, low dose, 0.5 µmol/l; M, moderate dose, 1 µmol/l; H, high dose, 2 µmol/l; ALT, alanine aminotransferase; AST, aspartate transaminase.

Figure 3.

Effects of SSd on CCl₄-induced change of T-SOD and MDA in the culture supernatants. HL-7702 cells were pretreated with 100 µmol/l NAC or different doses of SSd for 1 h and then treated with 10 mmol/l CCl₄ for 24 h. The levels of (A) T-SOD and (B) MDA in the supernatants were determined using commercial kits. Data are shown as mean ± SD for at least three independent experiments. *P<0.05, **P<0.01 vs. the CCl₄ only group. CCl_4, carbon tetrachloride; SSd, saikosaponin-d; T-SOD, total-superoxide dismutase; MDA, malondialdehyde; NAC, N-acetyl-L-cysteine; L, low dose, 0.5 µmol/l; M, moderate dose, 1 µmol/l; H, high dose, 2 µmol/l.

Figure 4.

Effects of SSd on CCl₄-induced NLRP3, ASC, caspase-1 and HMGB1 expression. HL-7702 cells were pretreated with 100 µmol/l NAC or different doses of SSd for 1 h and then treated with 10 mmol/l CCl₄ for 24 h. (A) The mRNA levels of NLRP3, ASC, caspase-1, and HMGB1 were detected by reverse transcription-quantitative polymerase chain reaction. (B) The protein levels of NLRP3, ASC, caspase-1 and HMGB1 were determined by western blotting. Data are shown as mean ± standard deviation for at least three independent experiments. *P<0.05, **P<0.01 vs. the CCl₄ only group. CCl_4, carbon tetrachloride; SSd, saikosaponin-d; NAC, N-acetyl-L-cysteine; L, low dose, 0.5 µmol/l; M, moderate dose, 1 µmol/l; H, high dose, 2 µmol/l; NLRP3, nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3; HMGB1, high mobility group protein B1; ASC, apoptosis-associated speck-like protein.

Figure 5.

Effects of SSd on IL-1β and IL-18 production in CCl₄-induced acute hepatocellular injury. HL-7702 cells were pretreated with 100 µmol/l NAC or different doses of SSd for 1 h and then treated with 10 mmol/l CCl₄ for 24 h. (A) The mRNA expressions of IL-1β and IL-18 were determined using reverse transcription-quantitative polymerase chain reaction. (B) The protein levels of IL-1β and IL-18 were detected by ELISA assay. Data are shown as mean ± standard deviation for at least three independent experiments. *P<0.05, **P<0.01 vs. the CCl₄ only group. CCl_4, carbon tetrachloride; SSd, saikosaponin-d; NAC, N-acetyl-L-cysteine; L, low dose, 0.5 µmol/l; M, moderate dose, 1 µmol/l; H, high dose, 2 µmol/l; IL, interleukin.