2,3,5,4'-Tetrahydroxystilbene-2-O-Β-D-Glucoside Inhibiting Hepatocytes Injury Through Reducing the Expression of Tumor Necrosis Factor-a in LPSStimulated Kupffer Cells via Regulating PPAR-γ/NFκB

Research Article

Austin J Pharmacol Ther. 2022; 10(2).1165.

2,3,5,4'-Tetrahydroxystilbene-2-O-Β-D-Glucoside Inhibiting Hepatocytes Injury Through Reducing the Expression of Tumor Necrosis Factor-a in LPSStimulated Kupffer Cells via Regulating PPAR-γ/NFκB

Wei D1, Jin R1, Zhao H1 and Li Q1,2*

¹School of Public Health, Shanghai University of Traditional Chinese Medicine, China

²Shanghai Frontiers Science Center of TCM Chemical Biology; Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, China

*Corresponding author: Qian Li, School of Public Health, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai 201203, China

Received: October 20, 2022; Accepted: November 15, 2022; Published: November 22, 2022


2,3,5,4'-Tetrahydroxystilbene-2-O-Β-D-Glucoside (TSG), a main bioactive component of polygonum multiflorum Thumb, exerts anti-oxidant, antitumor, anti-inflammatory effects. However, the protective effects of TSG on hepatocytes through anti-inflammatory effects have rarely been investigated. Mouse liver macrophages (Kupffer cells, KUP5 cells) were primed with various concentrations of TSG before Lipopolysaccharide (LPS) treatment, and AML12 hepatocytes were cultured with the supernatants of KUP5 cells. The results showed that TSG inhibits the expression of iNOS in KUP5 cells. Meanwhile, Tumor Necrosis Factor-a (TNF-a) and interleukin-1Β (IL-1Β) expression in KUP5 cells were reversed after TSG treatment. Additionally, the expression of Peroxisome Proliferators-Activated Receptors-γ (PPAR-γ) was up-regulated while the phosphorylation of p65 and IκB-a were decreased in KUP5 cells after TSG treatment. Furthermore, GW9662 (PPAR-γ antagonist) treatment could eliminate the anti-inflammatory effects of TSG. In AML12 hepatocytes, the level of AST, ALT, and apoptosis was decreased after being cultured with the supernatants of KUP5 cells pretreated with TSG. Moreover, TNF-a neutralization of KUP5 cells supernatants could decrease the level of AST, ALT, and AML12 hepatocyte apoptosis. These results indicate that TSG activates PPAR-γ, thereby decreasing nuclear factor kappa-B (NFκB) activation and reducing the release of TNF-a in macrophages to protect hepatocytes from injury. Our study may help further understand the protective effects of TSG on hepatocytes by suppressing liver inflammation.

Keywords: 2,3,5,4'-tetrahydroxystilbene-2-O-Β-D-glucoside; Kupffer cells; Tumor necrosis factor-a; PPAR-γ; NFκB; Hepatocyte


TSG: 2,3,5,4'-Tetrahydroxystilbene-2-O-B-D-Glucoside; LPS: Lipopolysaccharide; Inos: Inducible Nitric Oxide Synthase; TNF-A: Tumor Necrosis Factor-A; IL-1Β: Interleukin-1Β; PPAR-Γ: Peroxisome Proliferators-Activated Receptors-G; AST: Aspartate Transaminase; ALT: Alanine Transaminase; CCK-8 Assay: Cell Counting Kit-8 Assay; MFI: Mean Fluoresce Intensity; Qpcr: Quantitative Polymerase Chain Reaction; ELISA: Enzyme-Linked Immunosorbent Assay Kcs: Kupffer Cells; Nfκb: Nuclear Factor Kappa-B; IL-6: Interleukin-6


Inflammation plays an important role in many diseases [1]. The liver is a target of inflammatory cytokines and can release a wide number of proteins to control the inflammatory response [2]. Moreover, multiple hepatic disorders may become more severe as a result of excessive inflammation, which can lead to a significant loss of hepatocytes [3]. Chronic liver inflammation leads to fibrosis and cirrhosis, which is the 12th leading cause of death in the United States [4]. Great attention and efforts have been devoted to discovering drugs against liver inflammation-induced liver injury. What is more, it has been reported that several herbal medicines can be used to treat liver injury [5].

Polygonum multiflorum Thumb could exert multiple effects, including anti-inflammation, immunomodulation, antioxidant, and anti-cancer activities [6]. 2,3,5,4'- Tetrahydroxystilbene -2-O-Β-D-Glucoside (TSG) is a main bioactive component of polygonum multiflorum Thumb and has several functions, including immunosuppressive, anti-tumor, anti-inflammatory, and anti-fatty liver effects [7]. What is more, TSG effectively protects the liver against acute alcoholic liver injury [8]. Accumulating evidence has shown that TSG decreases the production of pro-inflammatory cytokines [9]. However, the protective effects of TSG on hepatocytes through suppressing liver inflammation have rarely been investigated.

Kupffer Cells (KCs) are liver-derived macrophages and account for 80–90% of tissue macrophages in the body [10]. KCs counteract the aforementioned stimuli and play a fundamental role in the liver inflammatory response. KCs secrete pro-inflammatory cytokines including TNF-a and IL-6 after stimulation with LPS, which leads to liver inflammation [11]. Notably, TNF-a participates in the occurrence and process of liver diseases and induces hepatocyte apoptosis, which is the most important event in the mechanism and a common feature of hepatic failure [12]. Moreover, TNF-a can induce multiple mechanisms to initiate apoptosis in hepatocytes leading to subsequent liver injury [13]. It has been reported that TNF-a is involved in the pathophysiology of viral hepatitis, alcoholic liver disease, and ischemia-reperfusion injury [14].

Peroxisome Proliferation-Activated Receptors (PPARs) are nuclear receptors that include three subfamilies encoded by distinct genes: a, Β, and γ. Among PPARs, PPAR-γ matters considerably in the immune response [15]. PPAR-γ regulates the polarization of macrophages and the production of TNF-a was increased in PPAR-γ KO macrophages [16]. Moreover, PPAR-γ exerts antiinflammatory effects by reducing NFκB activation. After binding to toll-like receptors, LPS activates the NFκB signaling pathway. NFκB trans-locates into the nucleus, and then pro-inflammatory genes are activated [17].

Therefore, the protective effects of TSG on hepatocytes through anti-inflammatory effects and its mechanism were explored in the present study. KCs (KUP5 cells) were stimulated with LPS, and then the anti-inflammatory effects of TSG were tested. Moreover, AML12 hepatocytes were cultured with the supernatants of KUP5 cells to evaluate the role of TNF-a. We further explore the involvement of PPAR-γ and NFκB in LPS-induced inflammation of KUP5 cells. Our study demonstrated that TSG activates PPAR-γ, thus inhibiting the activation of NFκB, and then reduces the production of TNF-a of KCs, thereby protecting hepatocytes from injury. Our study provides the basis for the clinical application of TSG in the treatment of liver inflammation-induced liver injury.

Materials and Methods

Reagents and Chemicals

2,3,5,4'-tetrahydroxystilbene-2-O-Β-D-glucoside (analytical standard, purity 98.0%) was purchased from Yuanye Bio-technology (Shanghai, China); Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS) and penicillin/streptomycin were obtained from Thermo (Thermo Scientific, CA, USA). 1% insulin-transferrinselenium (ITS), LPS, and GW9662 (PPAR-γ antagonist) were from Sigma (St. Louis, MO, USA).

Cell Culture and Treatment

Mouse liver macrophages (KUP5 cells) and AML12 hepatocytes were obtained from RIKEN Cell Bank (Japan) and the Cell Bank of the Chinese Academy of Science (Shanghai, China), respectively. Both cell types were cultured in DMEM containing 10% FBS, and 1% penicillin/streptomycin in a 5% CO2 incubator at 37°C. AML12 hepatocytes were cultured in DMEM/F12 medium with 1% Insulin- Transferrin-Selenium (ITS), 10% FBS, 40 ng/mL dexamethasone, and 1% penicillin/streptomycin at 37°C with 5% CO2. KUP5 cells were primed with TSG before LPS stimulation, and AML12 hepatocytes were cultured with the supernatants of KUP5 cells. KUP5 cells were pretreated with GW9662 (PPAR-γ antagonist) for testing the rule of PPAR-γ, and TNF-a was neutralized in the supernatants of KUP5 cells for testing the rule of TNF-a.

Cell Viability Assay

Cell counting kit-8 assay (CCK-8, beyotime, China) was performed to evaluate the cytotoxicity of TSG and LPS. KUP5 cells were pretreated with various concentrations of TSG (0, 60, 120, 240, and 480 μM) for 1h before the treatment with LPS (1 μg/ml) for 24h. The mediums were removed, and 110μl CCK-8 detection solution (100μl DMEM containing 10μl CCK-8 solution) was added into each well. After incubation for 1h, the optical density at 450nm was measured using a microplate reader (Biotek, USA). The concentration of TSG used in this study (0, 60, 120, 240, and 480 μM) was based on the previous research [18].

RNA Isolation and qPCR Analysis

Total RNA was isolated using TRIzol regent (Invitrogen, CA, USA), and the RNA was reversely transcribed into cDNA using the cDNA Synthesis Kit (Invitrogen, CA, USA). The mRNA amplifications were conducted with a qPCR system (Thermo Fisher Scientific, Vantaa, Finland). The experimental results were analyzed using the 2-ΔΔCt method. GAPDH was employed as the internal standard. The primer sequences, iNOS, forward 5'-ATGTCCGAAGCAAACATCAC-3', reverse 5' -TAATGTCCAGGAAGTAGGTG-3', GAPDH forward 5'- TTCAACGGCACAGTCAAGGC -3', reverse 5' GACTCCACGACATACTCAGCACC -3'.

Detection of TNF-a, IL-1Β, and IL-6 (ELISA)

KUP5 cells were primed with TSG for 1h and then stimulated with LPS for 24h. The supernatants were collected after centrifuging at 10,000g for 15min. The level of TNF-a and IL-1Β was assessed by ELISA Kit according to the instructions (Biolegend, San Diego, USA).

Flow Cytometry

The antibodies included FITC-anti-NOS2 (5C1B52, Thermo Scientific, CA, USA), PPAR-γ (81B8) Rabbit mAb, p-p65 Rabbit mAb (93H1), p-IκB-a Rabbit mAb (14D4) (Cell Signaling Technology, MA, USA), and Alexa Fluor 488 Donkey anti-rabbit IgG (poly4064) (BioLegend, San Diego, USA). KUP5 cells were treated with TSG for 1h after the treatment with LPS (1μg/ml) for 1h or 24h. Then, cells were harvested and placed into the cell plate for detection. Cells were first fixed/permeabilized with nuclear staining kit (Thermo Scientific, CA, USA) for 1h. For the measurement of iNOS, cells were incubated with the diluted antibody of iNOS for 1h. In the case of detecting PPAR-γ, p-p65, and p-IκB-a, the cells were incubated with diluted primary antibodies for 1h and then incubated with secondary antibodies for another 1h. AML12 cells were cultured with supernatants of KUP5 cells. AML12 apoptosis was detected using Annexin V / PI apoptosis kit (Beyotime Biotechnology, China). After staining, cells were detected using a BD flow cytometer (BD Biosciences, San Jose, USA) and data analysis was performed with Flowjo (9.3.2, FlowJo LLC, Ashland, USA).

Biochemical Tests

AML12 cell supernatants were collected for detection after centrifugation. The level of AST and ALT in supernatants of AML12 cells was measured using the Reitman-Frankel assay (Nanjing Jiancheng Institute, China).

TNF-a Neutralization

To test the effect of TNF-a on the apoptosis of hepatocytes, 100μl anti-TNF-a (final concentration, 1μg/ml) (clone: XT3.11) or control antibody (clone: MOPC-21) (BioXcell, West Lebanon, NH BE0058) was added into the supernatants of LPS-treated KUP5 cells for TNF-a neutralization. AML12 cells were then cultured with the supernatants.

PPAR-γ Inhibition

GW9662 was used for PPAR-γ inhibition. KUP5 cells were treated with 10 μM GW9662 for 30 min and incubated with TSG for 1 h, and then cells were treated with 1μg/mL LPS.

Statistical Analysis

Data analysis was performed using GraphPad Prism 9 (GraphPad Software Inc., CA, USA). One-way ANOVA and Tukey's post hoc test were used for the comparison between groups. Data were presented as the means ± Standard Deviation (SD) of three independent experiments, and P < .05 was considered significant.


The Cytotoxicity of TSG and LPS in KUP5 Cells

The viability of KUP5 cells was not decreased after the treatment with different concentrations of TSG and LPS. Moreover, cytotoxic effects were not observed after the co-treatment with TSG and LPS, indicating that the effects of TSG on KUP5 cells were not attributed to cytotoxic effects (Figure 1). Therefore, 60, 120, and 240μM of TSG were selected for the following experiments.