The hepatoprotective effect of myricetin against lipopolysaccharide and D-galactosamine-induced fulminant hepatitis

Journal Pre-proof The hepatoprotective lipopolysaccharide and hepatitis

effect of myricetin against D-galactosamine-induced fulminant

Hongming Lv, Beiying An, Qinlei Yu, Yu Cao, Yang Liu, Shize Li PII:

S0141-8130(19)36974-0

DOI:

https://doi.org/10.1016/j.ijbiomac.2019.11.075

Reference:

BIOMAC 13856

To appear in:

International Journal of Biological Macromolecules

Received date:

29 August 2019

Revised date:

9 October 2019

Accepted date:

7 November 2019

Please cite this article as: H. Lv, B. An, Q. Yu, et al., The hepatoprotective effect of myricetin against lipopolysaccharide and D-galactosamine-induced fulminant hepatitis, International Journal of Biological Macromolecules(2019), https://doi.org/10.1016/ j.ijbiomac.2019.11.075

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© 2019 Published by Elsevier.

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The hepatoprotective effect of myricetin against lipopolysaccharide and D-galactosamine-induced fulminant hepatitis Hongming Lv 1, #, Beiying An 2, #, Qinlei Yu 3, Yu Cao 1, Yang Liu 1, Shize Li 1, * 1

College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University,

Daqing, 163319, Heilongjiang Bayi, China. 2

Department of Clinical Laboratory, the First Hospital of Jilin University, Changchun 130021, Jilin,

China. Jilin Provincial Animal Disease Control Center, 4510 Xi'an Road, Changchun 130062, China.

*

corresponding author. (SZ. Li), E-mail address: [email protected];

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3

These authors contributed equally to this work.

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#

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Tel.: +86-45-9681-9190

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Abstract

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Fulminant hepatitis (FH) is a severe liver disease characterized by extensive hepatic necrosis, oxidative stress, and inflammation. Myricetin (Myr), a botanical flavonoid glycoside, is recognized to

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exert antiapoptosis, anti-inflammatory, and antioxidant properties. In the current study, we focused on exploring the protective effects and underlying mechanisms of Myr against lipopolysaccharide (LPS) and D-galactosamine (D-GalN)-induced FH. These data indicated that Myr effectively protected from LPS/D-GalN-induced FH by lowering the mortality of mice, decreasing ALT and AST levels, and alleviating histopathological changes, oxidative stress, inflammation, and hepatic apoptosis. Moreover, Myr could efficiently mediate multiple signaling pathways, displaying not only the regulation of caspase-3/9 and P53 protein, inhibition of toll-like receptor 4 (TLR4)-nuclear factor-kappa B (NF-κB) activation, and -mitogen-activated protein kinase (MAPK), but also the increase of heme oxygenase-1 (HO-1) and nuclear factor-erythroid 2-related factor 2 (Nrf2) expression, as well as induction of

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AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) phosphorylation in mice with LPS/D-GalN-induced FH. Importantly, our further results in vitro suggested that Myr remarkably attenuated H2O2-triggered hepatotoxicity and ROS generation, activated Keap1-Nrf2/HO-1 and AMPK/ACC signaling pathway. However, Myr-enhanced the expression of HO-1 and Nrf2 protein was reversed by Keap1-overexpression, Nrf2-null and AMPK inhibitor. Meanwhile, Myr-relieved

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hepatotoxicity excited by H2O2 was blocked by Nrf2-null and AMPK inhibitor. Taken together, Myr

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exhibits a protective role against LPS/D-GalN-induced FH by suppressing hepatic apoptosis, inflammation, and oxidative stress, likely involving in the regulation of apoptosis-related protein,

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TLR4-NF-κB/-MAPK and NLRP3 inflammasome, and AMPK-Nrf2/HO-1 signaling pathway.

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Keywords: Fulminant hepatitis; myricetin; hepatic necrosis; oxidative stress; inflammation; signaling

Introduction

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pathway.

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Fulminant hepatitis (FH, also called fulminant hepatic failure, acute hepatic failure, or acute liver injury) is a severe liver disease characterized pathologically by extensive hepatic necrosis with massive loss of parenchyma, violent oxidative stress, and sustained inflammation [1, 2]. Despite great clinical efforts, it is involved in a poor prognosis and high mortality [3]. As we know, the liver has extraordinary detoxification ability to eliminate various xenobiotics including drugs and toxins which result in liver injury [4]. Administration of D-galactosamine (D-GalN, as a specific hepatotoxin) in combination with lipopolysaccharide (LPS, a key factor for pathogenesis of hepatic injury) could greatly induce an animal model of liver injury which is strongly similar to liver disease in humans and is widely accepted to study the underlying mechanism of FH and its potential therapeutic drugs [5, 6]. It is indispensable that LPS/D-GalN stimulates oxidative stress and inflammatory responses, promoting 2

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hepatic necrosis and participating in the occurrence and development of FH [7, 8]. Therefore, intervention with oxidative stress and inflammation may be very necessary for the treatment of FH. When hepatocytes are subjected to LPS/D-GalN, massive pro-inflammatory cytokines, such as interleukin (IL)-6, IL-1β, and tumor necrosis factor-alpha (TNF-α), were released to liver and serum, leading to serious inflammation damage [9]. In fact, mitogen-activated protein kinase (MAPK), which is composed of c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38,

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and nuclear factor-kappa B (NF-κB), which is composed of the p50/p65 and the inhibitor of κB (IκB) protein, are an important downstream inflammatory signaling pathway of toll-like receptor 4 (TLR4)

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that is one of a crucial regulator of pro-inflammatory cytokines and its activation [10, 11]. Apart from

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TLR4 pathway, the nucleotide-binding domain-(NOD-) like receptor protein 3 (NLRP3)

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inflammasome is also a key signal pathway, regulating inflammatory responses, which plays a crucial

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role in the pathogenesis of FH induced by LPS/D-GalN [12, 13]. Meanwhile, LPS/D-GalN can trigger excessive reactive oxygen species (ROS), participating in the requirement signaling to promote the

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NLRP3 inflammasome and leading to serious oxidative insult in liver injury [14, 15]. Importantly, nuclear erythroid 2-related factor 2 (Nrf2), as an important transcription factor, is a vital regulator of oxidative stress and inflammation through modulating the expression of various cytoprotective enzyme, including heme oxygenase (HO)-1, and thereby is a potential target to treat a variety of diseases, including liver injury [16-18]. Indeed, it is reported that Nrf2-konckout mice are more susceptible to nephrotoxicity caused cisplatin, when compared to normal mice [19]. Similar results were found higher sensitivity to acetaminophen-induced acute liver failure in Nrf2-null mice more than wild-type mice [20]. Furthermore, AMP-activated protein kinase (AMPK), a heterotrimeric serine/threonine kinase as an essential enzyme energy metabolism, has been thought to an upstream 3

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signal of Nrf2 activation [21, 22]. Once AMPK is activated, it plays an essential role in ameliorating APAP-induced acute liver failure [23]. Therefore, the regulation of these molecules related to oxidative stress and inflammation may serve as effective therapeutic strategies for LPS/D-GalN-induced FH. There is accumulating evidence that naturally occurring compounds, distributed in widespread plants, vegetables and fruits, has recognized to possess antioxidant and anti-inflammatory roles by

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regulating multiple signal molecules, and has long been thought to be potential hepatoprotective agents

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[24, 25]. Myricetin (Myr, Fig. 1A) is a botanical flavonoid glycoside extracted from the fruits, leaves, and branches of Myrica cerifera and other plants [26]. It is reported that Myr exerts pleiotropic

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biological activities, including antiapoptosis, antinociceptive, anti-inflammatory, and antioxidant

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properties [27-29]. For example, Myr could directly suppress copper-mediated LDL-oxidation and

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ROS generation by activating the STAT3 and PI3K/Akt/eNOS signaling pathways, as well as inhibit

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apoptosis by regulating pro/anti-apoptosis proteins [30]. In addition, Myr treatment improved dextran sulfate sodium-induced acute colitis via modulation of PI3K/AKT, MAPK and NF-κB signaling

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pathway [31]. In the present study, we aimed to explore whether Myr treatment could alleviate LPS/D-GalN-induced FH related to the repression of inflammatory responses and oxidative stress.

2. Materials and methods

2.1. Reagents Myricetin (Myr, purity up to 98 %), was supplied by the Chengdu Herbpurify CO., LTD (Chengdu, China). LPS (Escherichia coli 055: B5), d-Galactosamine, Compound C (CC, a specific inhibitor of the AMPK), H2O2 and dimethyl sulfoxide (DMSO) were afforded by Sigma-Aldrich (St. Louis, MO, USA). Antibodies against HO-1 (ab68477), Keap1 (ab66620), Nrf2 (ab31163), P-AMPK (ab195946), NLRP3 (ab210491), TLR4 (ab13556), caspase-9 (ab202068), and JNK (ab208035) were offered by Abcam 4

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(Cambridge, MA, USA). AMPK (50081), P-ACC (11818)/ACC (3676T), P-ERK (9101s), P-p38 (9211s)/p38 (9212), P-IκBα (9246)/IκBα (9242s), IL-1β (12242), P53 (11818), caspase-3 (9662s) and β-actin (3700s) were purchased from Cell Signaling (Boston, MA, USA). Lamin B (SGA1910) and P-JNK (ap0276) were purchased from Sungene Biotech (Shangdong, China). ASC (A1170, Abclonal), caspase-1 (A0964, Abclonal) and ERK (A10613) were provided by ABclona Technology (Wuhan,

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China). In addition, ALT, AST, MDA, GSH and SOD test kits were offered by Nanjing Jiancheng

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Bioengineering Institute (Nanjing, China). ROS staining test kit was obtained from Beyotime

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Sigma-Aldrich (St. Louis, MO, USA).

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Biotechnology (Shanghai, China). If not otherwise indicated, other reagents were supplied by

2.2. Animals

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Six to eight-week-old male C57BL/6 mice weighing 18-22 g were obtained from Liaoning

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Changsheng Technology Industrial Co., LTD (Certificate SCXK2010-0001; Liaoning, China). All

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animals were raised in a room with a temperature at 24 ± 1 °C and a 12 h light-dark cycle for 7 days prior to the experiment. The current study was conducted in accordance with the International Guiding Principles for Biomedical Research Involving Animals, which has been approved by the Institutional Animal Care and Use Committee of Heilongjiang Bayi Agricultural University. 2.3. Experimental protocol To induce fulminant hepatitis (FH) by LPS/D-GalN, mice were randomly divided into six groups: Control (PBS), Myr (100 mg/kg) alone, LPS/D-GalN (30 μg/kg and 600 mg/kg dissolved in PBS), Myr (25 mg/kg) + LPS/D-GalN, Myr (50 mg/kg) + LPS/D-GalN and Myr (100 mg/kg) + LPS/D-GalN group, were injected intraperitoneally. Briefly, the mice were subjected to Myr (25, 50, or 100 mg/kg) i.p. two times for 12 h each time. Then, at 1 h after the last dose of Myr, they were injected with LPS 5

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and D-GalN. The survival rates of mice were recorded for 24 h after LPS/D-GalN administration. For other assays, after LPS/D-GalN challenge for 6 h, the animals were euthanized by diethyl ether asphyxiation and then whole liver tissues and serum were harvested and used for enzyme-linked immunosorbent assay (ELISA) western blot, and hematoxylin and eosin (H & E) staining assay.

2.4. Biochemical indexes assay

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Fresh liver and serum from mice were subjected to biochemical analysis. The whole liver tissues

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were collected, homogenized and centrifuged at 3000 rpm/min for 10 min. The supernatant was used

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for the measurement of levels of GSH, MDA and SOD levels in accordance with the manufacturer's

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instructions. Moreover, the plasma samples was collected, stood at 37 °C for 30 min, and then centrifuged at 3000 rpm/min for 10 min. serum ALT and AST levels was assessed by using assay kits.

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In addition, serum IL-1β, IL-6, and TNF-α level were measured by ELISA kit as the manufacturer's

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2.5. H & E staining

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instructions (BioLegend, Inc., CA, USA).

The whole liver tissues were fixed with 4 % paraformaldehyde, dehydrated with a graded ethanol, embedded in paraffin wax, and then cut into 5-μm-thick sections. These sections were stained with hematoxylin-eosin (H & E) to evaluate the liver histopathological injury under a light microscopy.

2.6. Cell culture HepG2 cell line, which was obtained from the China Cell Line Bank (Beijing China), was maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 100 U/mL of streptomycin, 100 U/mL of penicillin, 10 % fetal bovine serum (FBS), and 3 mM glutamine (Invitrogen-Gibco, Grand Island, NY), in an 37 °C incubator with 5 % CO2. In our experiments, cells

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were allowed to accommodate for 24 h before any treatments.

2.7. CRISPR/Cas9 knockout of Nrf2 gene As previously described [32], HepG2 cells were grown in 12-well plates at a density of 3 × 105 cells/well for 24 h, and were co-transfected by plasmids of expressing Cas9 and puromycin resistant gene with Nrf2-sgRNA using Viafect transfection reagent (Promega). Subsequently, cells were treated with puromycin at a concentration of 2 μg/mL after transfection for 48 h, and collected for western blot

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analysis with Nrf2 antibody. After 7 days, cells were selected in a 96-well plate (1 cell/well).

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2.8. Cell viability

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HepG2 cells were grown in a 96-well plate at a density of 5 × 103 cells/well and were subjected to different dosages of Myr (5, 10 or 20 μg/mL) for 1 h and stimulated with or without H2O2 (300 μM) for

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24 h. Moreover, WT and Nrf2 −/− HepG2 cells were treated with Myr (20 μg/mL) for 1 h in presence or

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absence of H2O2 (300 μM) for 24 h. After the prescribed treatment, cells viability were detected by

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CCK-8 reagent (Dojindo Molecular Technologies, Dojindo, Japan) at 10 μL/well for 2 h at 37 °C. The

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absorbance was measured using a microplate reader at 450 nm.

2.9. Plasmid constructs and overexpression of Keap1 The primers was used to amplify the full coding region sequence of Keap1 with the total cDNA as the template were the following: 5′-GCT CTA GAG CAT GCA GCC AGA TCC CAG GC-3′ (forward) and 5′-CGC GGA TCC GCG TCA ACA GGT ACA GTT CTG CTG G-3′ (reverse). The PCR product was digested with XbaI and BamHI and cloned into the expression vector VR1012. The recombinant vector VR1012-Keap1 was transfected into HepG2 cell line by Viafect transfection reagent (Promega) for overexpressing of the Keap1, as previously described [33].

2.10. Measurement of cellular and liver ROS

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As previously reported [34], cold liver homogenization was centrifuged (12, 000 rpm, 20 min, 4 °C) to perform the determination of liver ROS production. The supernatants were subjected to 50 μM of DCFH-DA in 37 °C without light for 1 h, and then transferred into 96-well plate. Moreover, to detect cellular ROS generation, HepG2 cells were seeded into 96-well plates at a density of 5 × 103 cells/well for 24 h, and recovered in serum-free DMEM for 6 h. Subsequently, the cells were treated with three concentration of Myr (5, 10 or 20 μg/mL) for 18 h, and stained with DCFH-DA for 30 min

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before exposure to H2O2 (300 μM) for 30 min. The relative fluorescence intensity was measured by using a fluorescence spectrophotometer at emission and excitation wavelengths of 535 and 488 nm,

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respectively.

2.11. Extracts of nuclear and cytosolic protein

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According to the manufacturer’s instructions, the nuclear and cytoplasmic proteins were extracted

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by a NE-PER Cytoplasmic and Nuclear Extraction Reagents kit (Pierce Biotechnology, Rockford, IL,

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USA). All steps must been carried out on ice or at 4 °C.

2.12. Western blot analysis

Total protein from the frozen liver tissue was defrosted on ice, homogenized, and extracted with protein extraction kit (Beyotime, China) in accordance with the manufacturer's protocol. The concentration of protein was measured by a BCA protein assay kit (Pierce, USA). Samples of 40 μg protein was separated by 12 % SDS-polyacrylamide gel and then electrophoretically transferred to PVDF membrane. The membrane was block with (5 % (w/v) nonfat dry milk) for 1 h, and then incubated overnight at 4 °C with many specific primary antibodies. After that, the membrane was washed with PBST three times and incubated 1 h with HRP-conjugated secondary antibody (1: 3000). Lastly, the membranes were again washed and visualized by the ECL western blot detection system. 8

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The band intensities were quantified using Image J gel analysis software. All experiments were carried out three independent experiments.

2.13. Statistical analysis These results above except the survival data were calculated as the means ± SEM and analyzed by SPSS19.0 (IBM) software. Comparisons between each group were conducted using one-way ANOVA, followed by LSD multiple comparison test. Moreover, the survival rates were presented as a

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of <0.05 or < 0.01 was accepted as statistical significance.

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Kaplan-Meier curve and analyzed by the log-rank test (version 5, GraphPad Software, USA). P values

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3. Results

3.1. Myr treatment protected against LPS/D-GalN-induced FH in mice.

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In order to explore the preventive effect of Myr treatment on LPS/D-GalN-induced fulminant

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hepatitis (FH), the survival rate was initially observed for 24 h after LPS/D-GalN administration. As

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seen in Fig. 1B, mice of LPS/D-GalN-treated group began to die 6 h, and the survival rate reached 0 % at 24 h; however, Myr treatment could not only prolong the survival time of mice, but also increase the

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survival rate. Specifically, the dose of 25 mg/kg, 50 mg/kg and100 mg/kg Myr enhanced the survival rate 28%, 54% and 66%, respectively, in a dose-dependent manner. Moreover, the levels of ALT and AST in the serum are thought to be key indexes of hepatic injury in clinical trials. The results indicated that the levels of ALT and AST were obviously augmented in the LPS/D-GalN-treated group compared to the control group (** p < 0.01). Conversely, 50 mg/kg and 100 mg/kg Mry treatment decreased their levels and displayed evidently a significant difference compared with the LPS/D-GalN-treated group (## p < 0.01) (Fig. 1C-D). Meanwhile, histological examinations of mice liver tissues were carried out H & E staining assay. As presented in Fig. 1E, the observation of liver sections showed that the liver architecture in the control group was well preserved; LPS/D-GalN challenge could disturb architecture 9

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of liver, displaying lymphocytic infiltration, hemorrhage and hepatocyte apoptosis or confluent necrosis. In contrast, the LPS/D-GalN-caused liver change was efficiently relieved by Myr treatment, especially 100 mg/kg Myr. These investigations suggested that Myr treatment could protected from LPS/D-GalN-induced FH in Mice.

3.2. Myr treatment decreased LPS/D-GalN-induced hepatocyte apoptosis in mice.

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Hepatocyte apoptosis has been regarded as one of an important event in FH induced by

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LPS/D-GalN, we thus further determined that the effect of Myr treatment apoptosis-related protein

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measured by western blot analysis. As seen in Fig. 2, LPS/D-GalN could remarkably increase the level

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of cleaved-caspase3/9 protein, and effectively decrease pro-caspase3/9 protein and P53 protein expression. However, Myr treatment markedly reversed these apoptosis-related protein change induced

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LPS/D-GalN-caused FH.

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by LPS/D-GalN, suggesting that Myr treatment effectively alleviated hepatocyte apoptosis in

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3.3. Myr treatment reduced LPS/D-GalN-induced the secretion of IL-1β, IL-6 and TNF-α in mice.

During LPS/D-GalN-induced FH, hepatic inflammation is majorly resulted in the secretion of LPS/D-GalN-caused proinflammatory cytokines, including IL-1β, IL-6 and TNF-α. Thus, we determined the effect of Myr treatment on the release of IL-1β, IL-6 and TNF-α in the serum. ELISA assay results showed that LPS/D-GalN effectively induced the elevation of IL-1β, IL-6 and TNF-α (**p < 0.01) in the sera compared to control (Fig 3), whereas Myr treatment reduced the release of IL-1β, IL-6 and TNF-α by LPS/D-GalN (# p < 0.05 or ##p < 0.01).

3.4. Myr treatment inactivated the TLR4-NF-κB and -MAPK signaling pathway in mice with LPS/D-GalN-induced FH. 10

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To further investigate the underlying mechanism of how Myr suppressed the release of pro-inflammatory cytokines resulted from LPS/D-GalN, the key inflammation-associated TLR4-NF-κB and -MAPK signaling pathways have been measured by western blot analysis. As illustrated in Fig 4, when compared to the control group, LPS/D-GalN efficiently enhanced the expression of TLR4 protein, induced the phosphorylation and degradation of IκBα, and promoted the phosphorylation of JNK, ERK, and P38 (** p < 0.01). In contrast, the levels of these proteins change were apparently inhibited in the

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Myr-treated group, especially 100 mg/kg Myr (## p < 0.01). Hence, the suppressive effect of Myr treatment on the LPS/D-GalN-induced inflammatory responses perhaps is associated with the

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inactivation of the TLR4-NF-κB and -MAPK signaling pathway.

3.5. Myr treatment suppressed NLRP3 inflammasome in mice with LPS/D-GalN-induced FH.

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Given NLRP3 inflammasome activation is strongly involved in inflammation and oxidative stress,

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the effects of Myr on NLRP3, ASC, caspase-1 and IL-1β expressions were tested by western blot analysis. As presented in Fig 5, LPS/D-GalN significantly induced the expressions of NLPR3,

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cleaved-caspase-1 (p10), Pro-IL-1β (p45) and mature-IL-1β (p20) protein (** p < 0.01) in liver. However, Myr treatment inhibited LPS/D-GalN-induced these protein levels (## p < 0.01), indicating that Myr treatment ameliorated the NLRP3 inflammasome activation caused by LPS/D-GalN.

3.6. Myr treatment relieved LPS/D-GalN-triggered oxidative injury in mice with FH. Moreover, oxidative insult is an important pathological process in LPS/D-GalN-induced FH, we thus determined the preventive effects of Myr treatment on hepatic oxidative stress triggered by LPS/D-GalN. The results showed that Myr treatment dramatically improved LPS/D-GalN-caused oxidative status in the liver by increasing endogenous antioxidants contents, including GSH and SOD (Fig 6A-B), and decreasing oxidative indicators levels, such as MDA formation, and ROS generation 11

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(Fig 6C-D). These findings determined that Myr treatment relieved oxidative injury stimulated by LPS/D-GalN.

3.7. Myr treatment enhanced the Nrf2/HO-1 and AMPK/ACC signaling pathways in mice with LPS/D-GalN-induced FH. Importantly, Nrf2/HO-1 and AMPK/ACC signaling pathway is thought to be essential cellular

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defense mechanisms by inhibiting oxidative stress and inflammation to mitigate hepatic failure. Our

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further work explored that the effect of Myr on Nrf2/HO-1 and AMPK/ACC signaling pathway in

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LPS/D-GalN-induced FH. As shown in Fig. 7, when compared with the control group, LPS/D-GalN

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could significantly reduce the expression of Nrf2 and HO-1 protein, as well as the phosphorylation of

changes rather than 25 mg/kg.

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AMPK and ACC, whereas Myr (50 mg/kg and 100 mg/kg) treatment remarkably restored these protein

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3.8. Myr exposure upregulated the Keap1-Nrf2/HO-1 signaling pathway in HepG2 cells.

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Based on the above outcomes, we further probed into the underlying mechanisms of Myr-upragulated Nrf2/HO-1 signaling pathway in HepG2 cells. Our findings revealed that Myr exposure to cells could strikingly enhance the expression of HO-1 protein, promote the degradation of Keap1 and increase Nrf2 nuclear translocation in a dose-dependent manner (Fig. 8A-C). In addition, immunocytochemistry analysis also showed that Myr could promote Nrf2 from cytoplasm to nucleus (Fig. 8D). Next, to further investigate whether Myr-enhanced Nrf2/HO-1 signaling pathway is directly regulated by Keap1 inhibition, we used HepG2 cell lines overexpressing Keap1 to explain the potential function of Keap1. As shown in Fig. 8E-F, the overexpression of Keap1 significantly reduced the expression of HO-1 and Nrf2 protein enhanced by Myr. Furthermore, to investigate whether HO-1 is downstream factor of Nrf2 by Myr, WT and Nrf2

−/−

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HepG2 cells were used in this study. The results

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HepG2 cells, and found that Myr-induced HO-1 protein expression was increased, while

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almost inhibited in Nrf2 −/− HepG2 cells.

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3.9. Myr exposure promoted the AMPK/ACC signaling pathways in HepG2 cells.

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Meanwhile, the upstream regulator of Nrf2/HO-1 mediated by Myr is associated with the

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activation of AMPK/ACC signaling pathways. As indicated in Fig. 9A, Myr exposure to cells could effectively promoted phosphorylation of AMPK and ACC consistent with results in vivo. Accordingly,

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to further demonstrate the relationship between Myr-mediated Nrf2 and AMPK, cells were

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pre-incubated with Compound C (CC, an inhibitor of AMPK) in this study. Our results discovered that

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Myr boosted the expression of Nrf2 and HO-1 protein, as well as phosphorylation of AMPK, which were effectively blocked by CC, suggesting that AMPK may act as upstream of Nrf2/HO-1 signaling pathway (Fig. 9B).

3.10. Myr exposure improved H2O2-stimulated hepatotoxicity in HepG2 cells. Last but not least, the antioxidant mechanism of Myr in HepG2 cells was further studied. Our results suggested that Myr exposure obviously inhibited hepatotoxicity and ROS acclamation stimulated by H2O2 (Fig. 10A-C). However, Myr-relieved hepatotoxicity and ROS generation induced by H2O2 was mostly prevented in the presence of AMPK inhibitor and Nrf2-null (Fig. 10D-E). The result demonstrated that the protective effect of Myr against H2O2-excited oxidative injury, likely depending on the activation of AMPK/Nrf2 signaling pathways. 13

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4.

Discussion Liver disease is defined as a global burden of fearful health problems, with more than one million

estimated to die each year, particularly fulminant hepatitis (FH) [35, 36]. In FH disease, multiple mechanisms work simultaneously to cause liver hepatocyte apoptosis, inflammation, and oxidative stress, while LPS/D-GalN-induced liver injury parallels clinical viral hepatitis [13, 37]. Moreover, they

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also are considered to be major three pathogenic factors in LPS/D-GalN-induced FH which is a

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well-established animal model [38] Therefore, the suppression of these pathogenic factors should be an effective measure for the prevention and remedy of FH. Myricetin (Myr), a botanical flavonoid

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glycoside, has been revealed to exert various biological activities, including anti-apoptosis,

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anti-inflammatory, and antioxidant properties [39]. Here, our studies found that Myr treatment

and

oxidative

stress

through

the

via inhibiting hepatocyte apoptosis,

regulation

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apoptosis-related

signals,

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inflammation

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dose-dependently attenuated LPS/D-GalN-induced FH

TLR4-NF-κB/-MAPK, NLRP3 inflammasome, and AMPK/Nrf2-HO-1 signaling pathway.

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Accumulating evidence has revealed that FH caused by LPS/D-GalN has the characteristics of high mortality, increased of serum ALT and AST, and liver pathological alteration [40]. As mentioned earlier, the current results showed that LPS/D-GalN evidently elevated mortality, serum AST and ALT levels, as well as liver histopathological changes. In contrast, Myr treatment effectively alleviated these elevations, indicating that Myr could protect from liver injury caused by LPS/D-GalN. Considering that LPS/D-GalN-induced hepatocyte apoptosis is an important pathological manifestation of FH, it is a vital target to improve liver diseases [41, 42]. Our findings showed that Myr efficiently rescued D-GalN/LPS-induced hepatocytes apoptosis by reducing expression of cleaved-caspase-3/9, and increasing expression of pro-caspase-3/9 and P53. Moreover, massive production of harmful 14

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inflammatory cytokines is considered as the main pathological mechanism of FH [43]. In particularly, proinflammatory cytokines, IL-1β, IL-6 and TNF-α, have been recognized to be key potent contributors because of leading to hepatocyte necrosis and organ failure in LPS/D-GalN-induced liver injury [44]. In the present study, the results indicated that the secretion of serum IL-1β, IL-6, and TNF-α were significantly enhanced by LPS/D-GalN, whereas the enhancement was reduced by Myr. These

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proinflammatory cytokines are mediated by multiple upstream signaling pathways activation, including TLR4 and NLRP3 inflammasome. Indeed, previous researches noted that the TLR4 signal cascades can

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directly result in the downstream signaling pathway NF-κB and MAPK activation, promoting

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pro-inflammatory cytokine secretion and exacerbating various disorders, including liver impairment

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[45-48]. Meanwhile, NLRP3 can stimulate the assembly of apoptosis-related speck-like protein (ASC)

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and pro-caspase-1 into large cytoplasmic complexes that cause production of cleaved-caspase-1, and

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subsequently promote the maturation and secretion of IL-1β, thereby contributing to the development of liver diseases [12, 49]. Hence, to investigate the mechanism of Myr's anti-inflammatory effect, the

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NLRP3 inflammasome, TLR4-NF-κB and -MAPK signal was measured by western blot. In our studies, Myr treatment significantly not only inhibited LPS/D-GalN-induced the phosphorylation of IκBα, JNK, ERK and P38, but also reduced the expression of TLR4, NLRP3, ASC, cleaved-caspase1, pro-/mature-IL-1β protein, which were nearly associated with the inhibition of inflammation responses. It is worth noting that excessive oxidative stress could not only induce inflammation and apoptosis, it also is acknowledged as another primary pathological mechanism in FH induced by LPS/D-GalN [50, 51]. Additionally, the overproduction of ROS is thought to play an essential role in activating NF-κB and NLRP3 inflammasome signaling pathway [52]. Thus, inhibiting oxidative stress is also an effective means to relive FH. In our studies, LPS/D-GalN could promote dramatically the accumulation of 15

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malondialdehyde (MDA) and ROS, as well as the depletion of glutathione (GSH) and superoxide (SOD) in the liver, whereas these oxidative markers were effectively decreased by Myr treatment. Recent studies have shown that antioxidant enzymes, such as HO-1, are of important for alleviating LPS/D-GalN-induced hepatic injury by the inhibition of oxidative stress and inflammatory responses [53, 54]. Nevertheless, these antioxidant enzymes are mainly controlled by Nrf2 which is a key

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modulator of resisting oxidative stress and a crucial potential target to attenuate liver diseases [19, 55].

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In the basic state, Nrf2 binds to the cytoplasmic inhibitory protein kelch-ech related protein 1 (Keap1). When exposed to oxidative or electrophilic stress, Nrf2 dissociates from Keap1, enter into the nucleus

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in which it combines with the antioxidant response element (ARE) to regulate a series of transcriptional

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antioxidant proteins, including HO-1 [56]. Therefore, it is necessary to investigate the effect of Myr on

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Keap1-Nrf2/HO-1 signaling pathway. In vitro and in vivo studies have suggested that Myr treatment

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notably induced the expression of HO-1 protein, which is directly involved in the activation of Keap1-Nrf2 signaling pathway. Furthermore, Myr treatment relieved hepatotoxicity triggered by H 2O2 −/−

HepG2 cells, indicating that Myr plays a preventive role in

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that was impeded in Nrf2

hepatocytotoxicity by regulating Nrf2 pathway. To date, many signaling pathways are considered as the upstream pathway of Nrf2, among which AMPK is one of the most important pathways and has aroused extensive interest of researchers [57, 58]. Importantly, it is reported that AMPK activation played a protective role in the process of FH [59, 60]. Our further work also demonstrated the effect of Myr on AMPK signaling pathway in vivo and in vitro. The results displayed that Myr treatment could increase and restore the phosphorylation of AMPK and ACC reduced by LPS/D-GalN. Furthermore, Compound C (CC, an inhibitor of AMPK) could remarkably block the Nrf2/HO-1 pathway activation

16

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and cytoprotection mediated by Myr treatment in HepG2. In other words, Myr-mediated the Nrf2/HO-1 pathway activation and cytoprotection is directly related to the activation of AMPK. In summary, as presented in Fig. 11, the current study indicates that Myr can efficiently relieve hepatocyte necrosis, inflammation and oxidative stress, during LPS/D-GalN-induced FH, likely via mechanisms involving the regulation of apoptosis-related protein (P53 and caspase3/9),

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TLR4-NF-κB/-MAPK and NLRP3 inflammasome, and AMPK-Nrf2/HO-1 signaling pathway.

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Collectively, our study provides novel evidence of the benefit for the application of Myr in the

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prevention and treatment of FH.

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Acknowledgments

This work was funded by the National Natural Science Foundation of China (project no.

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Conflict of interest

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Province (ZD2019C004).

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31972637 and no. 31772695), and Key Program of Natural Science Foundation of Heilongjiang

All authors of the manuscript declare no conflict of interest.

17

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Journal Pre-proof Figure legends Fig.1. Myr treatment alleviated LPS/D-GalN-induced FH in mice. (A) The chemical structure of myricitrin (Myr). (B) The effect of Myr on the survival rates of the mice within 24 h after LPS/D-GalN administration. (C-D) The effect of Myr on ALT and AST levels at 6 h after LPS/D-GalN administration. (E) Representative histological sections of the livers were stained with hematoxylin and eosin (H & E)-stained (magnification × 400, blue arrows: necrotic area). Similar results were obtained from three independent experiments. All data are presented as means ± SEM (n = 5/group). *p<0.05

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and **p<0.01 vs. Control group; #p<0.05 and ##p<0.01 vs. LPS/D-GalN group.

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Journal Pre-proof Fig. 2. Myr treatment inhibited LPS/D-GalN-induced hepatic apoptosis in mice. Liver tissues of mice were collected at 6 h after LPS/D-GalN administration and measured by western blot. Effects of Myr on the expression of P53, caspase-3 and caspase-9 protein. Quantification of relative protein expression was performed by densitometric analysis. Similar results were obtained from three independent experiments. β-actin was served as an internal control. All data are presented as means ± SEM (n = 5/group). *p<0.05 and

**

p<0.01 vs. Control group; #p<0.05 and

##

p<0.01 vs. LPS/D-GalN

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Journal Pre-proof Fig. 3. Myr treatment reduced LPS/D-GalN-induced the secretion of IL-1β, IL-6 and TNF-α in mice. (A-C) Effects of Myr treatment on LPS/D-GalN-induced serum IL-1β, IL-6 and TNF-α production. Similar results were obtained from three independent experiments. All data are presented as

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means ± SEM (n = 5/group). **p<0.01 vs. Control group; #p<0.05 and ##p<0.01 vs. LPS/D-GalN group.

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Journal Pre-proof Fig. 4. Myr treatment inactivated TLR4-NF-κB and -MAPK signaling pathway in mice with LPS/D-GalN-induced FH. Liver tissues were collected from the mice 6 h after LPS/D-GalN administration and analyzed by western blot. (A and B) Effects of Myr on TLR4, P-JNK, P-ERK, P-P38, P-IκBα and IκBα expression were measured by western blot. Quantification of relative protein expression was performed by densitometric analysis. β-actin was served as an internal control. Similar results were obtained from three independent experiments. All data are presented as means ± SEM (n =

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5/group). **p <0.01 vs. Control group; #p <0.05 and ##p <0.01 vs. LPS/D-GalN group.

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Journal Pre-proof Fig. 5. Myr treatment suppressed NLRP3 inflammasome in mice with LPS/D-GalN-induced FH. Liver tissues were collected from the mice 6 h after LPS/D-GalN administration and analyzed by western blot. Effects of Myr treatment on NLRP3, ASC, caspase-1 and IL-1β expression were measured by western blot. Quantification of relative protein expression was performed by densitometric analysis.

β-actin was served as an internal control. Similar results were obtained from

three independent experiments. All data are presented as means ± SEM (n = 5/group).

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Control group; #p<0.05 and ##p<0.01 vs. LPS/D-GalN group.

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**

p<0.01 vs.

Journal Pre-proof Fig. 6 Myr treatment relieved oxidative injury in mice with LPS/D-GalN-induced FH. Effects of Myr on liver ROS and MDA production, SOD and GSH activities. Similar results were obtained from three independent experiments. All data are presented as means ± SEM (n = 5/group).

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Control group; #p<0.05 and ##p<0.01 vs. LPS/D-GalN group.

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**

p<0.01 vs.

Journal Pre-proof Fig. 7 Myr treatment enhanced AMPK/ACC and Nrf2/HO-1 signaling pathway in mice with LPS/D-GalN-induced FH. The effect of Myr on AMPK/ACC and Nrf2/HO-1 activation were measured by western blot. Quantification of relative protein expression was performed by densitometric analysis. β-actin was served as an internal control. Similar results were obtained from three independent experiments. All data are presented as means ± SEM (n = 5/group). *p<0.05 and p<0.01 vs. Control group; #p<0.05 and ##p<0.01 vs. LPS/D-GalN group.

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Journal Pre-proof Fig. 8 Myr exposures up-regulated the Keap1-Nrf2/HO-1 pathway in HepG2 cells. (A) HepG2 cells were cultured with many concentrations of Myr (2.5, 5, 10 or 20 μg/mL) for 18 h. The effect of Myr on the induction of HO-1 protein was determined by western blot. Moreover, HepG2 cells were cultured with three concentrations of Myr (5, 10 or 20 μg/mL) for 18 h. (B) The effect of Myr on the expression of Keap1 protein. (C) The effect of Myr on the nuclear and cytoplasmic levels of Nrf2 was measured by western blot. (D) Myr treatment facilitated the nuclear translocation of Nrf2 and HepG2 cells were performed by immunofluorescence (IF) staining with Nrf2 antibody (green), and the nucleus with DAPI (blue). (E-F) The overexpression of Keap1 (Ov-Keap1) regulates Myr-induced the Nrf2 and −/−

HepG2 cells were extracted from total

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HO-1 protein expression. In addition, (G) WT and Nrf2

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protein for western blot. (H) WT and Nrf2 −/− HepG2 cells were treated with or without Myr (20 μg/mL) for 18 h, and then immunoblotting was performed to detect Nrf2 and HO-1 protein expression. Relative

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protein was quantitatively expressed by densitometric analysis. Lamin B and β-actin was served as an internal control. All data were expressed as means ± SEM of three independent experiments (n =

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5/group). *p<0.05 and **p<0.01 vs the Control group; ++p<0.01 vs the Ov-Keap1 group; ^^p<0.01 vs the

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Myr group.

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Journal Pre-proof Fig. 9. Myr exposure promoted the AMPK/ACC/Nrf2 signaling pathways in HepG2 cells. (A) HepG2 cells were cultured with many concentrations of Myr (5, 10 or 20 μg/mL) for 18 h. The effect of Myr on the phosphorylation of AMPK and ACC expression was determined by western blot. (B) HepG2 cells were treated with compound C (CC, an AMPK inhibitor, 3 µM) for 1 h and then incubated with Myr (20 μg/mL) for another 18 h. The effect of Myr on the phosphorylation of AMPK, Nrf2 and HO-1 expression was determined by western blot. Relative protein was quantitatively expressed by densitometric analysis. β-actin was served as an internal control. Similar results were obtained from three independent experiments. All data are presented as means ± SEM (n = 5/group). *p<0.05 and

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p<0.01 vs the Control group; ^^p<0.01 vs the Myr group.

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**

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Journal Pre-proof Fig. 10. The alleviation effect of Myr on H 2O2-triggered hepatotoxicity in HepG2 cells. (A) HepG2 cells were cultured with many concentrations of Myr (5, 10 or 20 μg/mL) for 1 h, and then were exposed to H2O2 (300 µM) for additional 18 h. Cell viability was detected by CCK8 assay. In addition, (B-C) HepG2 cells were cultured with many concentrations of Myr (5, 10 or 20 μg/mL) for 18 h, and stained with 50 μM of DCFH-DA for 30 min and then exposed to H2O2 (300 µM) for additional 30 min to excite the ROS generation. DCF fluorescence intensities were detected by a fluorescent spectrophotometer and microscope, respectively. (D) HepG2 cells were cultured with Myr (20 μg/mL) for 1 h before exposure to CC (3 μM) for 1 h, and then were stimulated with H2O2 (300 µM) for −/−

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additional 18 h. Cell viability was detected by CCK8 assay. (E) WT and Nrf2

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treated with and without Myr (20 μg/mL) for 1 h, and then exposed to H2O2 (300 µM) for 18 h, cell viability was determined by CCK8 assay. All data were expressed as means ± SEM of three

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independent experiments (n = 5/group). **p<0.01 vs the Control group; $p<0.05, $$p<0.01 vs the H2O2

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Fig. 11. Scheme summarizing the protective effects of Myricitrin on LPS/D-GalN-induced fulminant hepatits. Myricitrin (Myr) exhibits a protective role against LPS/D-GalN-induced FH by relieving hepatic apoptosis, inflammation, and oxidative stress, which might be involved in the inhibition of cleaved-caspase3/9, TLR4-NF-κB/-MAPK and NLRP3 inflammasome, and the

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upregulation of pro-caspase3/9, and P53, AMPK-Nrf2/HO-1 signal.

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