Isoliquiritigenin protects against sepsis-induced lung and liver injury by reducing inflammatory responses

Accepted Manuscript Isoliquiritigenin protects against sepsis-induced lung and liver injury by reducing inflammatory responses Xiong Chen, Xueding Cai, Rongrong Le, Man Zhang, Xuemei Gu, Feixia Shen, Guangliang Hong, Zimiao Chen PII:

S0006-291X(17)32336-7

DOI:

10.1016/j.bbrc.2017.11.159

Reference:

YBBRC 38952

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 21 November 2017 Accepted Date: 23 November 2017

Please cite this article as: X. Chen, X. Cai, R. Le, M. Zhang, X. Gu, F. Shen, G. Hong, Z. Chen, Isoliquiritigenin protects against sepsis-induced lung and liver injury by reducing inflammatory responses, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/ j.bbrc.2017.11.159. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Isoliquiritigenin protects against sepsis-induced lung and liver injury by reducing inflammatory responses

Xiong Chen1, #, Xueding Cai 2, #, Rongrong Le3, #, Man Zhang4, Xuemei Gu1, Feixia

1

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Shen1, Guangliang Hong5, *, Zimiao Chen1, *

Department of Endocrinology, the First Affiliated Hospital, Wenzhou Medical

University, Wenzhou, Zhejiang, China;

Department of Respiratory Medicine, the First Affiliated Hospital, Wenzhou Medical

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2

University, Wenzhou, Zhejiang, China;

the Affiliated Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China;

4

Department of Emergency, the Second Affiliated Hospital, Wenzhou Medical

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3

University, Wenzhou, Zhejiang, China; 5

Department of Emergency, the First Affiliated Hospital, Wenzhou Medical University,

#

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Wenzhou, Zhejiang, China.

, These authors contribute equally to this paper.

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To whom correspondence should be addressed: Zimiao Chen, MD, at the the First Affiliated Hospital, Wenzhou Medical University, Wenzhou 325035, China. Telephone:

+86-577-55579381;

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[email protected];

and

[email protected]

Fax:

Guangliang

+86-577-55579329; Hong,

MD,

PhD,

E-mail: E-mail:

ACCEPTED MANUSCRIPT

Abstract Sepsis, one of the most fatal diseases worldwide, often leads to multiple organ failure, mainly due to uncontrolled inflammatory responses. Despite accumulating

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knowledge obtained in recent years, effective drugs to treat sepsis in the clinic are still urgently needed. Isoliquiritigenin (ISL), a chalcone compound, has been reported to exert anti-inflammatory properties. However, little is known about the effects of ISL on sepsis and its related complications. In this study, we investigated the potential

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protective effects of ISL on lipopolysaccharide (LPS)-induced injuries and identified the mechanisms underlying these effects. ISL inhibited inflammatory cytokine

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expression in mouse primary peritoneal macrophages (MPMs) exposed to LPS. In an acute lung injury (ALI) mouse model, ISL prevented LPS-induced structural damage and inflammatory cell infiltration. Additionally, pretreatment with ISL attenuated sepsis-induced lung and liver injury, accompanied by a reduction in inflammatory responses. Moreover, these protective effects were mediated by the nuclear factor

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kappa B (NF-κB) pathway-mediated inhibition of inflammatory responses in vitro and in vivo. Our study suggests that ISL may be a potential therapeutic agent for

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sepsis-induced injuries.

Key words

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lipopolysaccharide, Isoliquiritigenin, sepsis, acute lung injury, inflammation

Introduction

Sepsis, a systemic response to severe infection, leads to life-threatening

multi-organ dysfunction, or even failure, and especially induces acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Despite the optimization of clinical therapeutic strategies, ALI and ARDS occur in approximately 50% of septic patients 4]

[1, 2]

and account for approximately 30% of septic mortality in recent years [3,

. Therefore, a profound understanding of the precise mechanisms underlying

ACCEPTED MANUSCRIPT sepsis-induced injuries and discovery of new pharmacological interventions are needed. Lipopolysaccharide (LPS), a major glycolipid in the outer membranes of Gram-negative bacteria, is a known endotoxin that induces pro-inflammatory cytokine

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expression and is involved in the development and progression of ALI and sepsis [5]. After engagement of membrane Toll-like receptors (TLRs) and cytokine receptors, the nuclear factor kappa B (NF-κB) signaling pathway is activated by LPS

[6, 7]

.

NF-κB, comprising IκBα and p50/p65, is an essential pro-inflammatory transcription

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factor that triggers the expression of pro-inflammatory interleukins (ILs), tumor necrosis factor (TNF), interferons (IFN) and cyclooxygenases upon responses to . Thus, targeting NF-κB is an attractive strategy for

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[8, 9]

inflammatory stimuli

identifying novel anti-inflammatory compounds. Chalcone

compounds

are

recognized

as

potential

flavonoids

with

pharmacological activity and are widely distributed in fruits, vegetables and herbs [10]. Isoliquiritigenin (ISL), which belongs to the chalcone series, is found in Glycyrrhiza [11]

, Sinofranchetia chinensis [12] and Dalbergia odorifera

[13]

and

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uralensis (licorice)

exhibits a variety of biological and pharmacological activities. ISL was recently found to exert potent anti-inflammatory effects, as Jin XY et al. demonstrated that ISL

expression

[14]

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significantly attenuated TNF-α-mediated inflammation via regulating PPARγ . Additionally, ISL was demonstrated to reduce lung inflammation and

morbidity in mice infected with the PR8/H1N1 virus

[15]

. Together, these results

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support the natural compound ISL as a new anti-inflammatory treatment candidate. Increasing evidence indicates that the NF-κB signaling pathway plays a role in modulating the gene expression of pro-inflammatory cytokines; however, whether this pathway is involved in the inhibition of ALI and sepsis by ISL remains unclear. In the present study, we evaluated whether ISL exerted protective effects against the LPS-induced inflammatory response in mouse primary peritoneal macrophages (MPMs) and in lung and liver injuries. We also examined modulation of the NF-κB signaling pathway by ISL in vitro and in vivo. ISL inhibited inflammatory cytokine expression in MPMs exposed to LPS by inhibiting activation of the NF-κB signaling

ACCEPTED MANUSCRIPT pathway. Furthermore, ISL efficiently attenuated inflammatory responses in ALI and sepsis-induced lung and liver injuries by inhibiting activation of the NF-κB signaling pathway.

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Materials and Methods Reagents and chemicals

LPS was purchased from Sigma-Aldrich (St Louis, MO, USA), and ISL (98% purity) was purchased from Aladdine Company (Shanghai, China). ISL was dissolved

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in dimethyl sulfoxide (DMSO) for in vitro experiments or PEG400 (20%) for in vivo experiments. Antibodies against inhibitor of κB (IκB) and GAPDH were purchased

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from Cell Signaling Technology (Danvers, MA, USA), and the CD68 antibody was purchased from Abcam (Cambridge, MA, USA). Horseradish peroxidase-conjugated anti-rabbit secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Murine IL-6 and TNF-α enzyme-linked immunosorbent assay (ELISA) kits were purchased from eBioscience (San Diego, CA, USA). TRIzol

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reagent and the two-step M-MLV Platinum SYBR Green qPCR SuperMix-UDG kit were purchased from Invitrogen (Carlsbad, CA, USA). Animals

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Male C57BL/6 mice and Institute of Cancer Research (ICR) mice weighing between 18 and 22 g were obtained from the Animal Center of Wenzhou Medical University (Wenzhou, China). The animals were housed at a constant room

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temperature with a 12:12 hour light-dark cycle and fed a standard rodent diet and water for at least 7 days before being used. Animal study protocols were approved by the Wenzhou Medical University Animal Policy and Welfare Committee (approval document no. wydw2014-0058). Primary cell preparation and culture MPMs from ICR mice were prepared and cultured using a previously described method [16]. Cell cytotoxicity assay

ACCEPTED MANUSCRIPT HepG2 cells were seeded in 96-well plates at 5,000 cells per well and incubated at 37°C for 24 hours. The cells were then cultured with 1.25, 2.5, 5, 10, 20, or 40 µM ISL or an equal volume of DMSO for 24 hours and subjected to the methyl thiazolyl tetrazolium (MTT) assay.

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Quantification of TNF-α and IL-6 levels

TNF-6 and IL-6 levels in medium, serum, and bronchoalveolar lavage fluid (BALF) were quantitated using ELISA kits. MPMs were seeded in 6-well plates at 5×105 cells per well and incubated at 37°C and 5% CO2 for 24 hours. Cells were

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cultured with ISL in different concentrations (10 and 40 µM) or an equal volume of DMSO for 30 min and then treated with 0.5 µg/ml LPS for an additional 24 hours.

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Media were harvested to evaluate TNF-α and IL-6 expression. Total cytokines in the cell medium were normalized to the total protein content in viable cells. Experiments were performed at least three times in vitro.

Real-time quantitative polymerase chain reaction

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Total RNA was isolated from cells or tissues (20 mg) using TRIzol according to the manufacturer’s protocol. Reverse transcription and quantitative PCR (RT-qPCR) were performed using the M-MLV Platinum RT-qPCR kit and an Eppendorf Real

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Plex 4 instrument (Eppendorf, Hamburg, Germany). Primers for amplification of the TNF-α, IL-6, MCP-1 and β-actin genes, shown below, were synthesized by Invitrogen

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(Shanghai, China): mouse TNF-α sense primer: 5′- TGATCCGCGACGTGGAA-3′, mouse TNF-α antisense primer: 5′- ACCGCCTGGAGTTCTGGAA-3′; mouse IL-6 sense primer: 5′-GAGGATACCACTCCCAACAGACC-3′, mouse IL-6 antisense primer: 5′-AAGTGCATCATCGTTGTTCATACA-3′; mouse MCP-1 sense primer: 5′-TCACCTGCTGCTACTCATTCACCA-3′,

mouse

MCP-1

antisense

primer:

5′-TACAGCTTCTTTGGGACACCTGCT-3′; and mouse β-actin sense primer: 5′-CCGTGAAAAGATGACCCAGA-3′,

mouse

β-actin

antisense

primer:

5′-TACGACCAGAGGCATACAG -3′. The relative amount of each gene was normalized to that of β-actin.

ACCEPTED MANUSCRIPT Western blotting Cell protein samples (50 µg) or tissues (80 µg) were subjected to 10% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories). After blocking (5% milk in tris-buffered saline containing

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0.05% Tween 20) for 1.5 h at room temperature, the membranes were incubated with the appropriate primary antibody overnight at 4°C. The membranes were then washed with TBST and incubated with a secondary horseradish peroxidase-conjugated antibody for 1 h at room temperature. Blots were then visualized using enhanced

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chemiluminescence reagents (Bio-Rad Laboratories), and the densities of the immunoreactive bands were analyzed using ImageJ software (NIH, Bethesda, MD,

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USA).

LPS-induced acute lung injury (ALI)

Male C57BL/6 mice weighing 18-22 g were treated with an ISL solution (20 mg/kg) via tail vein injection 15 minutes prior to LPS administration. After the mice

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were anesthetized, their necks were dissected to visualize the trachea, and LPS (5 mg/kg) was administered intratracheally. The mice were then intubated with a 25-gauge catheter. Control mice were administered an equal volume of saline

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intratracheally. Six hours after LPS administration, the animals were euthanized, and their BALF, serum and lung tissues were harvested. BALF collection was performed

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three times using a tracheal cannula with autoclaved physiological saline in a total volume of 800 µL.

LPS-induced systematic inflammatory responses in C57BL/6 mice Mice in the LPS+ISL group were injected with 200 µL of LPS (20 mg·kg−1, i.v. through the tail vein) 15 min after ISL administration (20 mg·kg−1, i.v. through the tail vein). Mice in both the vehicle control group and the LPS alone group received 100 µL of the vehicle, and mice in the vehicle control group also received 100 µL of saline. Six hours after the LPS injection, the mice were anaesthetized and euthanized.

ACCEPTED MANUSCRIPT Blood samples were collected from the right ventricle using a needle and syringe containing heparin. The lung and liver tissues of the mice were harvested and homogenized for RNA extraction, real-time qPCR analysis and western blot. Lung and liver histopathology and immunohistochemistry analysis

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Lung and liver tissues were fixed in a 4% paraformaldehyde solution and embedded in paraffin. After dehydration, the paraffin sections (5 µm) were stained with hematoxylin and eosin (H&E). To evaluate histopathological damage, each

Immunofluorescence for CD68 detection

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section was imaged using a light microscope (200× amplification; Nikon).

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After deparaffinization and rehydration, slides containing 5-µm-thick lung sections were treated with 1% bovine serum albumin (BSA) in PBS for 30 min. The slides were incubated with anti-CD68 (1:100) at 4°C overnight and then with a TRITC-labeled secondary antibody for 1 h at room temperature; nuclei were counterstained with DAPI for 5 min. The slides were viewed with a fluorescence

Statistical analysis

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microscope (400×; Nikon, Tokyo, Japan).

Data are presented as the mean ± SEM. Statistically significant differences

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between two groups were determined by Student’s t-tests or ANOVA for multiple comparisons using GraphPad Pro 5.0 (San Diego, CA, USA). P-values less than 0.05

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were considered significant.

Results

Cytotoxicity

MTT assays were performed in HepG2 cells to test the activities of different concentrations of ISL (1.25, 2.5, 5, 10, 20, and 40 µM) administered for 24 hours. As shown in Fig. 1B, ISL treatment exerted no significant toxicity on HepG2 cells even at the 40 µM concentration.

ACCEPTED MANUSCRIPT ISL inhibits LPS-stimulated inflammatory responses in MPMs After pre-treatment with different concentrations of ISL (10 and 40 µM), MPMs were stimulated with LPS for 24 hours. The mRNA expression levels of the pro-inflammatory cytokines TNF-a and IL-6 were dramatically induced by LPS,

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while pretreatment with ISL dose-dependently decreased the cytokine overproduction (Fig. 1C and D). To investigate the mechanism underlying the observed protective effect against LPS-induced inflammatory responses in MPMs, the effects of ISL on LPS-induced NF-κB activation were evaluated, revealing that ISL significantly

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inhibited LPS-induced IκBα degradation (Fig. 1E and F).

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ISL effectively protects mice from LPS-induced ALI

To determine the protective effect of ISL on ALI in vivo, lung wet/dry ratios (Fig. 2A) and protein concentrations in BALF (Fig. 2B) were evaluated to assess alveolar-capillary barrier damage. Administration of ISL dramatically reduced LPS-induced

pulmonary

edema

and leakage of proteins

from

the

capillary.

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Additionally, the number of neutrophils per 200 total cells (Fig. 2C) and the number of total cells (Fig. 2D) in BALF were significantly increased by LPS stimulation, whereas ISL treatment reversed these changes. H&E staining showed structural

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changes and inflammatory cell infiltration in LPS-induced lung tissues compared with lung tissues of the control group, while subjects pre-treated with ISL exhibited very few histopathological changes and little macrophage infiltration (Fig. 2E).

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Semiquantitative lung injury scores assessed by a blinded pathologist further revealed the protective effects of ISL (Fig. 2F, compared with the LPS group).

ISL suppresses LPS-induced inflammatory responses in ALI Previous studies have shown that LPS-induced ALI is associated with significant macrophage infiltration and inflammatory cytokine production [17, 18]. As shown in Fig. 3A-D, administration of ISL significantly down-regulated the TNF-a and IL-6 protein levels in BALF and serum harvested from mice with ALI induced by LPS. Fig. 3E and F show the potency of ISL on inhibiting the inflammatory genes TNF-a and IL-6

ACCEPTED MANUSCRIPT at the mRNA level, further confirming its anti-inflammatory activity. The expression of CD68, a transmembrane glycoprotein highly expressed in macrophages, was detected in lung tissues. Immunofluorescence staining and quantification of CD68 confirmed increased macrophage infiltration in ALI lung tissues (Fig. 3G and H). As

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expected, ISL treatment decreased CD68-positive macrophage infiltration in lung tissues. IκB masks the nuclear localization of NF-κB, and its degradation plays an important role in mediating NF-κB activation

[19]

. Indeed, LPS-induced reductions in

IκB levels were not observed in mice treated with ISL, as the levels of this inhibitory

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protein in the LPS+ISL group were comparable to those in the control groups (Fig.

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3I).

ISL protects against liver and lung injuries resulting from LPS-induced sepsis To further characterize the protective effects of ISL on organ injuries resulting from LPS-induced sepsis, we assessed the attenuation of sepsis-related lung and liver injuries induced by LPS injection through the tail vein. The serum levels of aspartate

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aminotransferase (AST) (Fig. 4A), alanine aminotransferase (ALT) (Fig. 4B) and LDH (Fig. 4C) in the model group were significantly higher than those in the control group. However, the serum levels of these liver enzymes in the ISL pretreatment

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group were significantly reduced compared to those of the model group (Fig. 4A-C). LPS injection induced histopathological change and inflammatory cell infiltration in liver and lung tissues, whereas the administration of ISL reversed these changes (Fig.

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4D and E, compared with the control group). Semiquantitative lung injury scores also revealed the protective effect of ISL (Fig. 4F, compared with the LPS group).

ISL attenuates sepsis-induced inflammatory responses in liver and lung tissues by inhibiting IκB degradation To assess the anti-inflammatory effects of ISL on LPS-induced MPMs and ALI, we examined the expression of inflammatory cytokines in liver and lung tissues injured by LPS-induced sepsis. Real-time qPCR analysis showed marked increases in expression of the pro-inflammatory genes TNF-α (Fig. 4G), IL-6 (Fig. 4H) and

ACCEPTED MANUSCRIPT MCP-1 (Fig. 4I) in liver tissues exposed to sepsis, and the same results were observed in lung tissues (Fig. 4J-L). Furthermore, these effects were significantly attenuated by pretreatment with ISL. The reversed tissue injuries and decreased inflammatory cytokine expression suggest that NF-κB activity is reduced in ISL-treated injuries.

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Thus, we examined the effects of ISL on NF-κB activation in both liver and lung tissues. Similar to the results in MPMs and ALI, administration of ISL markedly inhibited the LPS-induced degradation of IκBα in liver (Fig. 4M) and lung (Fig. 4N)

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tissues when compared with the sepsis group.

Discussion

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Sepsis can be caused by infection, burns, or trauma and can easily progress to septic shock and life-threatening organ dysfunction. Due to uncontrolled systematic inflammatory responses to infection, sepsis has high morbidity and mortality rates worldwide

[20, 21]

. While researchers have developed numerous potential agents

targeting sepsis and its related complications in recent years, it remains a leading [22]

. ISL, a constituent of licorice and other food products, has been

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cause of death

shown to possess anti-inflammatory activity in vivo and in vitro

[14, 15]

. This study

showed that ISL protected against LPS-induced inflammatory response by inhibiting

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NF-κB activation in MPMs; ISL also protected against LPS-induced lung and liver injuries in vivo.

To clarify the protective effects of ISL in vitro, LPS-stimulated inflammatory

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responses in MPMs were evaluated. Increasing evidence has revealed that macrophages are necessary for regulating inflammatory responses, and activated macrophages induce the secretion of pro-inflammatory cytokines, including TNF-α and IL-6, which are primarily involved in promoting inflammatory processes and play important roles in sepsis

[23-25]

. Similarly, LPS increased TNF-α and IL-6 expression

in macrophages. As expected, ISL exhibited anti-inflammatory effects by inhibiting the secretion of these pro-inflammatory cytokines in LPS-stimulated macrophages. As a key transcription factor that modulates the production of inflammatory cytokines, NF-κB was demonstrated to play a pivotal role in controlling the macrophage

ACCEPTED MANUSCRIPT expression of inflammation-related genes localization

of

NF-kB

by

[19]

retaining

. IκB molecules prohibit the nuclear the

protein

in

the

cytosol

(http://www.bu.edu/nf-kb/). Orlikova B elucidated the inhibitory effect on ISL on TNFα-induced NF-κB activity among a number of natural chalcones [26]. Chi JH et al.

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also demonstrated that ISL inhibited TNF-α-induced NF-κB activation in human intestinal epithelial HT-29 cells [27]. In our study, NF-κB activation was observed in LPS-stimulated MPMs by detecting IκB degradation, while ISL reversed these changes.

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Furthermore, we also examined the anti-inflammatory activities of ISL in vivo. Among sepsis-related complications, pneumonia is the most common, accounting for

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approximately half of all cases [28]. Induced by intratracheal LPS administration, ALI is characterized by pulmonary edema, impairment of the alveolar-capillary barrier, enhanced cell recruitment and inflammatory response cascades

[29]

. Consistent with

previous reports, lung injury development in an LPS-induced mouse model was characterized by a series of histopathological changes (Fig. 2), accompanied by

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increased inflammatory cytokine expression and macrophage infiltration (Fig. 3). Animals receiving ISL were protected from the LPS-induced overproduction of inflammatory cytokines and macrophage infiltration, which ultimately protected

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against lung injury. (Fig. 2 and 3). Lung tissue damage resulting from sepsis induced by LPS administered via the tail vein was similarly protected by ISL (Fig. 4E and F), accompanied by a reduction in inflammatory responses (Fig. 4G-I). Additionally,

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numerous clinical studies have recognized septic liver dysfunction as another major component of multiple organ dysfunction syndrome (MODS) [30, 31], which contributes to septic severity and poor outcomes. In this study, we demonstrated the protective role of ISL against sepsis-induced liver injury in vivo (Fig. 4A-D), which was accompanied by a reduced inflammatory response. Consistent with MPMs induced by LPS in vitro, substantial in vivo evidence supports that the NF-κB signaling pathway is activated during sepsis

[32]

. Our results agree with those of previous studies

demonstrating enhanced NF-κB activation in ALI models and septic injuries in mice (Fig. 3I, 4M and N). Restoring abnormal NF-κB activity reversed the inflammatory

ACCEPTED MANUSCRIPT response, accompanied by the attenuation of LPS-induced injuries [17, 33, 34]. Together with the findings in MPMs, these results show that pharmacological inhibition of NF-κB by ISL might greatly impact the inhibition of inflammatory cytokine overexpression, resulting in the reversal of sepsis-related injuries (Fig. 3I, 4M and N).

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These results strongly suggest that targeting NF-κB might be a good therapeutic strategy for the treatment of sepsis.

In conclusion, this study demonstrated that ISL treatment not only effectively reduced TNF-α and IL-6 gene expression in LPS-induced MPMs but also attenuated

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lung and liver injuries induced by LPS, accompanied by decreased inflammatory responses. The underlying mechanisms of these phenomena may be strongly related

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to NF-κB inhibition. Together, these results indicate that ISL may be a potential therapeutic agent for the prevention and treatment of inflammatory diseases.

Conflict of interest

Acknowledgement

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Authors declare no conflicts of interest.

The project supported by Public Welfare Science and Technology Program of

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Wenzhou City (Y20130238) and Natural Science Funding of Zhejiang Province

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(LY13H150006).

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[29] G.D. Rubenfeld, E. Caldwell, E. Peabody, J. Weaver, D.P. Martin, M. Neff, E.J.

(2005) 1685-1693.

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Stern, L.D. Hudson, Incidence and outcomes of acute lung injury, N Engl J Med, 353

[30] K.E. Sands, D.W. Bates, P.N. Lanken, P.S. Graman, P.L. Hibberd, K.L. Kahn, J.

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Parsonnet, R. Panzer, E.J. Orav, D.R. Snydman, E. Black, J.S. Schwartz, R. Moore, B.L. Johnson, Jr., R. Platt, G. Academic Medical Center Consortium Sepsis Project Working, Epidemiology of sepsis syndrome in 8 academic medical centers, JAMA,

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[31] J. Bakker, R. Grover, A. McLuckie, L. Holzapfel, J. Andersson, R. Lodato, D. Watson, S. Grossman, J. Donaldson, J. Takala, G. Glaxo Wellcome International Septic Shock Study, Administration of the nitric oxide synthase inhibitor NG-methyl-L-arginine hydrochloride (546C88) by intravenous infusion for up to 72 hours can promote the resolution of shock in patients with severe sepsis: results of a randomized, double-blind, placebo-controlled multicenter study (study no. 144-002), Crit Care Med, 32 (2004) 1-12. [32] J. Hu, J. Liu, Licochalcone A Attenuates Lipopolysaccharide-Induced Acute

ACCEPTED MANUSCRIPT Kidney Injury by Inhibiting NF-kappaB Activation, Inflammation, 39 (2016) 569-574. [33] S. Yang, Z. Yu, L. Wang, T. Yuan, X. Wang, X. Zhang, J. Wang, Y. Lv, G. Du, The natural product bergenin ameliorates lipopolysaccharide-induced acute lung injury by inhibiting NF-kappaB activition, J Ethnopharmacol, 200 (2017) 147-155.

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[34] K.C. Lan, S.C. Chao, H.Y. Wu, C.L. Chiang, C.C. Wang, S.H. Liu, T.I. Weng, Salidroside ameliorates sepsis-induced acute lung injury and mortality via downregulating NF-kappaB and HMGB1 pathways through the upregulation of SIRT1, Sci Rep, 7 (2017) 12026.

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Figure Legends

isoliquiritigenin (ISL).

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Figure 1: The chemical structure, MTT assay and anti-inflammation effects of

(A) The chemical structure of X22. (B) HepG2 cells (50,000 cells/mL) were seeded in 96-well plates and treated with the indicated concentration of ISL for 24 hours. Mouse primary macrophages (MPMs) were subjected to LPS for indicated times with or

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without 1 h pretreatment with LPS. (C-D) Detection of TNF-α and IL-1β protein in the condition medium from MPMs stimulated with PA for 24 h. (E) Representative western blot analysis of IκB-α degradation in MPMs stimulated with LPS for 30 min.

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(F) The densitometric quantifications for western blot results in Figure 1E was performed via normalizing the band density of the indicated protein to the loading control protein, respectively. Data are presented as means ± SEM (n=3 per group; P<0.05,

##

P<0.01, compared to control group; *P<0.05,

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#

**

P<0.01 compared to LPS

group).

Figure 2 ISL suppresses lung injuries induced by intratracheal instillation of LPS. Mice were treated by intratracheal instillation of LPS (5 mg/kg) 15 minutes before ISL (tail vein injection, 20 mg/kg) injection. Six hours later after mice were anesthetized and killed, BALF and lung tissues were collected for further tests. (A) The lung Wet/Dry weight ratio. (B) Protein concentration in BALF. (C) Neutrophils

ACCEPTED MANUSCRIPT of 200 total cells in BALF. (D) Total cells in BALF. (E) Representative light micrographs for the histochemical assessment of lung tissues (H&E staining). (F) The lung injury score was determined. Data are presented as means ± SEM (n=7 per group; #

P<0.05,

##

P<0.01, compared to control group; * P<0.05,

**

P<0.01 compared to LPS

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group).

Figure 3 ISL suppresses overexpression of inflammatory response in lung tissue induced by intratracheal instillation of LPS.

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(A) The effect of ISL on the amount of TNF-a in BALF. (B) The effect of ISL on the amount of IL-6 in BALF. (C) The effect of ISL on the amount of TNF-a in serum. (D)

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The effect of ISL on the amount of IL-6 in serum. (E, F) The mRNA level of TNF-a and IL-6 in lung tissue was determined by RT-qPCR using β-actin mRNA as the internal

control.

(G)

Macrophages

infiltration

was

determined

by CD68

immunofluorescence staining. (H) The number of CD68-positive cells in 10 fields based on G. (I)The protein levels of IκBα in the lung tissues were determined by ##

P<0.01, compared

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western blotting. Data are presented as means ± SEM; #P<0.05, to control group; * P<0.05, ** P<0.01 compared to LPS group).

tissues.

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Figure 4 ISL suppresses sepsis-induced inflammatory response in liver and lung

Mice were treated by tail vein injection of LPS (20 mg/kg) 15 minutes before ISL (tail

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vein injection, 20 mg/kg) injection. Six hours later after mice were anesthetized and killed, liver and lung tissues were collected for further tests. (A-C) Liver function was determined by measuring serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH). (D) Representative light micrographs for the histochemical assessment of liver tissues (H&E staining). (E) Representative light micrographs for the histochemical assessment of lung tissues (H&E staining). (F) The lung injury score was determined.

(G-I) TNF-α, IL-6 and

MCP-1 mRNA levels in liver was analyzed by real-time qPCR using β-actin mRNA as the internal control. (J-L) TNF-α, IL-6 and MCP-1 mRNA levels in lung was

ACCEPTED MANUSCRIPT analyzed by real-time qPCR using β-actin mRNA as the internal control. (M, N) The protein levels of IκBα in the liver (M) and lung (N) tissues were determined by western blotting. Data are presented as means ± SEM; #P<0.05, ##P<0.01,

###

P<0.01,

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compared to control group; * P<0.05, ** P<0.01 compared to LPS group).

Figure 1 ACCEPTED MANUSCRIPT B

A

HepG2

Survival ratio (%)

100

50

10

40

ISL (μM) LPS LPS

ISL (μM) con

AC C

EP

GAPDH

10

40

TE D

IκB

50

0

** **

con

10

40

ISL (μM) LPS

2.5

F

**

2.0

**

1.5

##

1.0 0.5 0.0

40

20

10

5

2. 5

## IL-6

SC

**

E

RI PT

co n

*

con

ISL (μM)

100

50

0

Relative amount of cytokines (compared to LPS%)

100

D

TNF-α

IκB/GAPDH

##

M AN U

C

Relative amount of cytokines (compared to LPS%)

Isoliquiritigenin

1. 25

0

Molecular Weight(MW): 256.25

con

10

40

ISL (μM) LPS

Figure 2

E

LPS

LPS+ISL

0

con

LPS+ISL

LPS

100μm

** 50 0

con

LPS

LPS+ISL

EP

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100μm

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H&E staining

CON

LPS

Total cell in BALF(10^6/ml)

**

D

##

15

100

1

4.2

20

#

10

LPS+ISL

*

5 0

6

100μm

Lung injury scores

*

C

con

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4.4

con

150

##

2

4.6

4.0

B

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#

wet/dry ratio

4.8

3

A

Neutrophils of 200 total cell in BALF

5.0

Total protein concentration in BALF

ACCEPTED MANUSCRIPT

F

LPS

LPS+ISL

##

4

*

2 0

con

LPS

LPS+ISL

40 40

LPS

20

EP TE D

G

SO

P

S

20 20

50 50

30 30

10 10

LPS+ISL

con

##

**

IkB

GAPDH

502

LPS

con

*

LPS+ISL

LPS

LPS

100 100

00

con

LPS 20

150 150

L

P

S

+

IS

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O

O

L

C

P

S

N

LPS 20

TNF-α

SO

1004

con

PS

#

00

L

TNF-α

N

F

O

55

44

33

22

11

Relative R e l a t i v eamount a m o u n t o fofc ycytokines to k in e s i n s e r u(pg/ml) m in serum

20

LPS+ISL

C

1506

SO

50 50

Relative amount of mRNA R e la tiv e a m o u n t o f m R N A

+I

*

+I

con

PS

##

PS

00

LPS

SC

TNF L

N

ACCEPTED MANUSCRIPT C IL-6

L

** con

20

## PS

E L

100 100

O

D 00

S

LPS+ISL O

**

C

100

Relative amount of cytokines R e la tiv e a m o u n t o f c y to k in e s in BALF in B A L(pg/ml) F 150 150

P

2008

R e la tiv e a m o u n t o f m R N A

##

IS

N

IL-6

Relative amount of mRNA

LPS+ISL

O

20

TNF-α

B

L

C

SO

50 50

+

+I

LPS

L

PS

50

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L

100 100

+I

con PS

LPS

L

N

con

O

C

150 150

PS

CD68 staining con

L

AC C

DAPI

0

PS

Merge

Relative amount of cytokines in BALF (pg/ml) 150

L

0

0

N

H 00

O

Relative R e l a t i v eamount a m o u n t oof f cytokines c y to k in e s i n s e r(pg/ml) um in serum

A

C

CD68-positive cells C D 6 8 -p o s itiv e c e lls (10 (c e llsfields) /1 0 fie ld s )

Figure 3

#

*

LPS+ISL

IL-6

##

50 50

**

LPS+ISL

LPS+ISL

I

LPS+ISL

Figure 4

0.4

**

0.15 0.10 0.05

0.2 0.1

con

LPS LPS+ISL

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H&E staining 75 50

LPS

TNF-α ##

25 0

M

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LPS+ISL

con

con

LPS

** LPS+ISL

LPS

K

0

con

**

LPS

LPS+ISL

L

IL-6

125

#

100

150

MCP-1 ##

50 0

con

LPS

** LPS+ISL

MCP-1

150

#

100

75 50 25 0

100μm

100

50

EP

con

AC C

100

###

100

*

50

125

Relative amount of mRNA

J

Relative amount of mRNA

100

0

IL-6

150

Relative amount of mRNA

Relative amount of mRNA

150

I

200

#

100μm

100μm

H

TNF-α

200

LPS LPS+ISL

100μm

100μm

G

con

LPS+ISL

100μm

E

* 2

LPS LPS+ISL

LPS

H&E staining

con

## 4

0

con

LPS LPS+ISL

Relative amount of mRNA

D

0.3

0.0

0.00

con

*

0.4

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*

F6

Relative amount of mRNA

0.5

#

0.20

LDH(OD450nm)

##

##

0.5

0.25

Lung injury scores

B AST(OD450nm)

ALT(OD 450nm)

A 0.6

ACCEPTED MANUSCRIPT C

con

LPS+ISL

* LPS+ISL

LPS

N

IkB

IkB

GAPDH

GAPDH

con

50 **

0

con

LPS

LPS

LPS+ISL

LPS+ISL

ACCEPTED MANUSCRIPT Isoliquiritigenin protects against sepsis-induced lung and liver injury by reducing inflammatory responses

Xiong Chen1, #, Xueding Cai 2, #, Rongrong Le3, #, Man Zhang4, Xuemei Gu1, Feixia

Department of Endocrinology, the First Affiliated Hospital, Wenzhou Medical

University, Wenzhou, Zhejiang, China; 2

Department of Respiratory Medicine, the First Affiliated Hospital, Wenzhou Medical

University, Wenzhou, Zhejiang, China;

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1

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Shen1, Guangliang Hong5, *, Zimiao Chen1, *

the Affiliated Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China;

4

Department of Emergency, the Second Affiliated Hospital, Wenzhou Medical

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3

University, Wenzhou, Zhejiang, China; 5

Department of Emergency, the First Affiliated Hospital, Wenzhou Medical University,

Highlights

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Wenzhou, Zhejiang, China.

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1. Inflammation plays a key role in sepsis-induced lung and liver injury. 2. Isoliquiritigenin reduced the LPS-induced inflammation in MPMs. 3. Isoliquiritigenin attenuated the inflammation-mediated liver and injury.

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4. Isoliquiritigenin exhibited anti-inflammatory effects via inactivation of NF-κB. 5. Isoliquiritigenin is a potential agent for inflammatory injuries in sepsis.