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Food and Beverage Chemistry/Biochemistry
Protective effects of sesquiterpenoids from the root of Panax Ginseng on fulminant liver injury induced by lipopolysaccharide/D-galactosamine Weidong Wang, Yanguo Zhang, Haijun Li, Yan Zhao, Enbo Cai, Hongyan Zhu, Pingya Li, and Jinping Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02627 • Publication Date (Web): 05 Jul 2018 Downloaded from http://pubs.acs.org on July 7, 2018
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Journal of Agricultural and Food Chemistry
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Protective effects of sesquiterpenoids from the root of Panax Ginseng
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on fulminant liver injury induced by
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lipopolysaccharide/D-galactosamine
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Weidong Wang†,#, Yanguo Zhang‡,#, Haijun Li§,#, Yan Zhao†,*, Enbo Cai†, Hongyan
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Zhu†, Pingya Li§, Jinping Liu§,*
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†
8
Changchun, China
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‡
.
.
College of Chinese Medicinal Materials, Jilin Agricultural University,
Department of Anesthesiology, Changchun Shuangyang District Hospital,
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Changchun, China
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§
.
Jilin University, Changchun, China
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* Corresponding author:
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College of Chinese Medicinal Materials, Jilin Agricultural University, Changchun
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130118,
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[email protected] (Y. Zhao); School of Pharmaceutical Sciences, Jilin University,
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Changchun 130021, Jilin, China. Tel/Fax: +86 431 85619803, E-Mail address:
18
[email protected] (J.P. Liu).
19
#
Jilin,
China.
Tel/Fax:
+86
431
84533358,
These authors contributed equally to this work.
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Journal of Agricultural and Food Chemistry
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Protective effects of sesquiterpenoids from the root of Panax Ginseng
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on fulminant liver injury induced by
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lipopolysaccharide/D-galactosamine
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Abstract: It is reported sesquiterpenoids from Panax ginseng (SPG) possess various
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pharmacological
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antiinflammatory. The purpose of this study was to examine the hepatoprotective
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effects of SPG (2.5 and 10 mg/kg, i.g.) on fulminant liver injury induced by
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D-galactosamine (D-GalN) and lipopolysaccharide (LPS) and discuss its mechanisms
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of action. 24 h after D-GalN (400 mg/kg, i.p.) and LPS (25 µg/kg, i.p.) exposed, the
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serum levels of alanine transaminase (ALT) and aspartate transaminase (AST),
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hepatic malondialdehyde (MDA) level, hepatic activities of superoxide dismutase
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(SOD), catalase (CAT) and glutathione (GSH), and hepatic tissue histology were
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measured. Expression levels of tumor necrosis factor-α (TNF-α) and interleukin-1
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beta (IL-1β) were detected by ELISA and RT-PCR. Moreover, the nuclear factor-κB
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(NF-κB), nuclear factor erythroid 2-related factor 2 (Nrf2), sirtuin type 1 (Sirt 1) and
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heme oxygenase 1 (HO-1) were determined by western blotting. The results indicated
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that SPG evidently restrained the increasement of serum ALT and AST levels induced
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by D-GalN/LPS. SPG obviously down-regulated TNF-α and IL-1β levels and their
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mRNA expression in liver. In addition, D-GalN/LPS injection induced severe
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oxidative stress in liver by boosted MDA level as well as decreased CAT, GSH and
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SOD capacities, and SPG reversed these changes. Meanwhile, SPG inhibited NF-κB
activities,
for
example,
antidepressant,
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activation induced by D-GalN/LPS and up-regulation Sirt 1, Nrf2 and HO-1
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expression levels. Therefore, SPG might protect against the fulminant liver injury
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induced by D-GalN/LPS via inhibiting inflammation and oxidative stress. The
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protective effect of SPG on fulminant liver injury induced by D-GalN/LPS might be
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mediated by Sirt 1/Nrf2/NF-κB signaling pathway. All these results implied that SPG
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might be a promising food additives and therapeutic agent for fulminant liver injury.
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Keywords: ginseng, sesquiterpenoids, D-GalN/LPS, fulminant liver injury, NF-κB,
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Sirt 1, Nrf2
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INTRODUCTION
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Liver is a relatively important organ in human, and plays a key effect in material
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metabolism and biotransformation. In addition, the liver is susceptible to a variety of
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pathogenic factors and stimuli in vitro and vivo, causing liver damage and
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inflammatory reaction 1, 2. Fulminant liver injury is a common serious liver disease
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with rapid degeneration of liver function occurring in patients without history of
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hepatic disease, and might lead to lethal outcomes 3. Fulminant liver injury induced by
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D-galactosamine (D-GalN) and lipopolysaccharide (LPS) is a representative acute
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liver injury animal model, which has been widely used to elucidate the mechanism of
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fulminant liver injury and find new hepatoprotective agents 4-6. As we known, the
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endotoxins from Gram-negative bacteria can induce acute liver injury. LPS, as the
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main endotoxins from Gram-negative bacteria cell wall 7, 8, promotes the secretion of
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inflammatory mediators from hepatic giant salivary cells 9-11. D-GalN can directly
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deplete uridine nucleotides in liver cells and prevent the regeneration of organelles,
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architecture and function of the hepatic cells are abnormal 12. According to a large
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number of studies, D-GalN/LPS is an inflammatory mediator-induced hepatotoxicity,
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which can induced excessive production of inflammatory cytokines and lead to
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abnormally activated inflammatory responses
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D-GalN/LPS can activate the signal pathway of NF-κB/Nrf2
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erythroid 2-related factor 2 (Nrf2), as an important regulator for regulating oxidative
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stress in cell defense, can prevent oxidative damage caused liver injury by regulating
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the expression of antioxidant proteins 16, 17. Nuclear factor-κB (NF-κB) is considered
13, 14
. Recent reports displayed that
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. Nuclear factor
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to be response to oxidative stress when it is activated. Panax ginseng C.A. Mayer, as a famous nourishing medicine, has been widely
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18
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used in the treatment of various diseases, for example, tumor
, cardiovascular
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diseases 19, nervous system diseases 20 and so on in China, Japan, Korea and other
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countries 21, 22. Sesquiterpenoids from of P. Ginseng (SPG) were the active ingredients
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refined from fat-soluble part of ginseng. Our research group attached great importance
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to the SPG and had conducted a series of interesting studies. We found that SPG
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exhibited good antidepressant-like activity 23, 24, and strongly prevented the liver from
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chemical acute liver injury induced by CCl4 via its antioxidative and antiinflammatory
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capacities 25. However, the hepatoprotective effect of SPG on fulminant liver injury
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induced by D-GalN/LPS is undefined. Previous study also indicated that red ginseng
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oil showed good safety in rat at dose of 5000 mg/kg
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experiment is to explore the protective effect of SPG on fulminant liver injury
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induced by D-GalN/LPS and its potential molecular mechanisms.
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MATERIALS AND METHODS
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. Therefore, the aim of this
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Animals. Male ICR mice (18 - 22 g) were obtained from the experimental
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animal Changchun ACEE Technology Co. ltd (Changchun, China). The mice were
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fed a standard diet and controlled environment under a 12 h light/dark cycle at 23 ± 2
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o
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acclimated for at least 7 days before the experiment. All the experiments on animals
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were abided by Jilin Agricultural University guidelines for the care and use of
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laboratory animals.
C and 50 ± 10 % humidity. In order to adapt the mice to the environment, mice were
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SPG. SPG was extracted from the roots of P. ginseng (4 years, Ji’an, China) by
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the ASI Spe-ed SFE-2 supercritical CO2 extraction system (Allentown, PA, U.S.A.)
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and
separated
by
distillation.
According
spectrometry
to
our
previous
(GC−MS)
was
used
to
methods,
gas
analyze
the
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chromatography−mass
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sesquiterpenoids fraction, and there were 21 kinds of sesquiterpene compounds in
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SPG, with a total content of 66% 23, 25.
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Experiment Model and Experimental Protocol. ICR mice were randomly
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divided into four groups (n = 10 per group): the control group, D-GalN/LPS group,
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SPG (2.5 or 10 mg/kg) + D-GalN/LPS groups. The mice of the SPG (2.5 or 10 mg/kg)
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+ D-GalN/LPS groups were intragastrically (i.g.) treated with SPG (2.5 and 10 mg/kg,
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dissolved in soybean oil.) for 7 days; and the D-GalN/LPS group and the control
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group were administered with normal saline (0.1 mL/10 g body weight 0.9 % NaCl aq,
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i.g.). 1 h after the 7th treatment, the mice of the D-GalN/LPS group and SPG (2.5 or
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10 mg/kg) + D-GalN/LPS groups were attacked by intraperitoneal injection (i.p.) of
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D-GalN (400 mg/kg) and LPS (25 µg/kg). Within 24h after D-GalN/LPS injection, the
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survival analysis of mice was monitored. The amount of dead mice was counted every
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2 h after the D-GalN/LPS injection. The whole blood and liver tissue were obtained,
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and the liver tissue was placed at -80 oC for subsequent analysis.
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Biochemical analysis. The blood samples were centrifuged at 10000 rmp for 10
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min at 4 oC to get the serum. The serum ALT and AST levels were determined with
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the corresponding kits (Jiancheng Bioengineering Institute of Nanjing, Nanjing, China)
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according to the manufacturer's instructions. 6
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Analysis of oxidative stress parameters. Liver tissue was homogenized using
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an automatic homogenizer. First, nine fold (w/v) cold ice normal saline was added to
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the liver tissue for homogenization. Second, a part of liver tissue was centrifuged at
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12000 rpm for 10 min at 4 °C. Finally, the supernatant was assayed for biochemical
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indicators. Hepatic superoxide dismutase (SOD), catalase (CAT) and glutathione
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(GSH) activities, and malondialdehyde (MDA) level were detected by the enzyme
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linked immunosorbent assay (ELISA) kits (R&D, Minneapolis, MN, USA) according
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to the manufacturer’s instructions.
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Analysis of inflammatory cytokines levels. The serum tumor necrosis factor-α
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(TNF-α) and interleukin-1 beta (IL-1β) levels were determined by ELISA kits (R&D,
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Minneapolis, MN, USA) according to the manufacturer’s instructions.
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Histopathological analysis. So as to detect histopathological alterations, liver
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tissue was fixed with 10 % formalin buffer, embedded in paraffin and sliced into 5 µm
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sections. The sections were stained using hematoxylin and eosin (H&E), the
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pathological alterations were observed using light microscope.
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Quantitative real-time PCR. The hepatic mRNA levels of TNF-α and IL-1β
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was detected by Real-time-polymerase chain reaction (RT-PCR). The total RNA of
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liver tissue was extracted using TRIzol (Invitrogen, Carlsbad, CA). The RNA was
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reverse transcribed into cDNA with RevertAid First Strand cDNA Synthesis Kit
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(Thermo Fisher Scientific，San Jose，CA) according to the manufacturer's instructions.
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RT-PCR was operated with a 7500 real-time PCR system (Applied Biosystems,
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Carlsbad, CA). The multiple changes between the mRNA levels in the treatment 7
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groups and the untreated group were corrected by the level of β-actin. Primer sets for
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RNA quantification were used: β-actin: 5’-CTG AGA GGG AAA TCG TGC GT-3’
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(forward) and 5’-CCA CAG GAT TCC ATA CCC AAG A-3’ (reverse); TNF-α:
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5’-GGC AGG GAG TCT ACT TTG GAG C-3’ (forward) and 5’-ACA TTC GAG
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GCT CCA GTG AAT TCG G-3’ (reverse); IL-1β: 5’-GCA ACT GTT CCT GAA
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CTC A-3’ (forward) and 5’-CTC GGA GCC TGT AGT GCA G-3’ (reverse).
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Western blotting. According to the instructions of the protein extraction kit
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(Beyotime, Nanjing, China), the nuclear (Nrf2, NF-κB, Sirt1) and cytoplasmic (HO-1)
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proteins were extracted from the liver tissue. The protein concentration was detected
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by BCA protein determination kit (Beyotime, Nanjing, China). The protein extracts
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were separated on a 12 % sodium dodecyl sulfate polyacrylamide (SDS) gel and
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transferred to a PVDF membrane. At 4 °C, membranes were incubated with primary
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antibody overnight. After washing three times with Tris buffered saline, the
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membrane is incubated with secondary antibodies. The membrane was indicated by
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the ECL Plus Western blot system (Amersham Life Science, Amersham, the United
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Kingdom).
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Statistical analysis. The values presented are the mean ± SD. SPSS statistical
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software was used for statistical analysis. The Prism 5 software package (version 5,
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GraphPad Software, USA) software was used to generate the figures. A value of P
