Isoorientin exerts a urate-lowering effect through inhibition of xanthine oxidase and regulation of the TLR4-NLRP3 inflammasome signaling pathway

Abstract

Isoorientin (ISO), a natural flavonoid compound, has been identified in several plants and its biological activity is determined and the study on lowering uric acid has not been reported. In view of the current status of treatment of hyperuricemia, we evaluated the hypouricemic effects of ISO in vivo and in vitro, and explored the underlying mechanisms. Yeast extract-induced hyperuricemia animal model as well as hypoxanthine and xanthine oxidase (XOD) co-induced high uric acid L-O2 cell model and enzymatic experiments in vitro were selected. The XOD activity and uric acid (UA) level were inhibited after the treatment of ISO in vitro and in vivo. Furthermore, serum creatinine (CRE) and blood urea nitrogen (BUN) levels were also significantly reduced and liver damage was recovered in pathological histology after the ISO administration in hyperuricemia animal model. The results of mechanism illustrated that protein expressions such as XOD, toll-like receptor 4 (TLR4), cathepsin B (CTSB), NLRP3, and its downstream caspase-1 as well as interleukin-18 (IL-18) were markedly downregulated by ISO intervention in vitro and in vivo. Our results suggest that ISO exerts a urate-lowering effect through inhibiting XOD activity and regulating TLR4-NLRP3 inflammasome signal pathway, thus representing a promising candidate therapeutic agent for hyperuricemia. Both animal models and in vitro experiments suggested that ISO may effectively lower uric acid produce. The mechanism might be the inhibition of XOD activity and NLRP3 inflammasome of upregulation.

Full text

Vol.:(0123456789)
1 3
Journal of Natural Medicines
https://doi.org/10.1007/s11418-020-01464-z
ORIGINAL PAPER
Isoorientin exerts aurate‑lowering eect throughinhibition
ofxanthine oxidase andregulation oftheTLR4‑NLRP3 inammasome
signaling pathway
Meng‑FeiAn1,3· Ming‑YueWang1,3· ChangShen1,3· Ze‑RuiSun1,3· Yun‑LiZhao1,2,4· Xuan‑JunWang1,2,5·
JunSheng1,2,5
Received: 21 May 2020 / Accepted: 29 October 2020
© The Japanese Society of Pharmacognosy 2020
Abstract
Isoorientin (ISO), a natural flavonoid compound, has been identified in several plants and its biological activity is determined
and the study on lowering uric acid has not been reported. In view of the current status of treatment of hyperuricemia, we
evaluated the hypouricemic effects of ISO invivo and invitro, and explored the underlying mechanisms. Yeast extract-
induced hyperuricemia animal model as well as hypoxanthine and xanthine oxidase (XOD) co-induced high uric acid L-O2
cell model and enzymatic experiments invitro were selected. The XOD activity and uric acid (UA) level were inhibited
after the treatment of ISO invitro and invivo. Furthermore, serum creatinine (CRE) and blood urea nitrogen (BUN) levels
were also significantly reduced and liver damage was recovered in pathological histology after the ISO administration in
hyperuricemia animal model. The results of mechanism illustrated that protein expressions such as XOD, toll-like receptor
4 (TLR4), cathepsin B (CTSB), NLRP3, and its downstream caspase-1 as well as interleukin-18 (IL-18) were markedly
downregulated by ISO intervention invitro and invivo. Our results suggest that ISO exerts a urate-lowering effect through
inhibiting XOD activity and regulating TLR4-NLRP3 inflammasome signal pathway, thus representing a promising candidate
therapeutic agent for hyperuricemia.
Meng-Fei An and Ming-Yue Wang have contributed equally to this
work.
* Yun-Li Zhao
zhaoyunli123@163.com
* Xuan-Jun Wang
wangxuanjun@gmail.com
* Jun Sheng
shengj@ynau.edu.cn
1 Key Laboratory ofPu-erh Tea Science, Ministry
ofEducation, Yunnan Agricultural University,
Kunming650224, People’sRepublicofChina
2 College ofScience, Yunnan Agricultural University,
Kunming650224, People’sRepublicofChina
3 College ofFood Science andTechnology, Yunnan
Agricultural University, Kunming650224,
People’sRepublicofChina
4 Key Laboratory ofMedicinal Chemistry forNatural
Resource, Ministry ofEducation andYunnan Province,
School ofChemical Science andTechnology, Yunnan
University, Kunming650091, People’sRepublicofChina
5 State Key Laboratory forConservation andUtilization
ofBio-Resources inYunnan, Kunming650224,
People’sRepublicofChina

Journal of Natural Medicines
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Graphic abstract
Both animal models and invitro experiments suggested that ISO may effectively lower uric acid produce. The mechanism
might be the inhibition of XOD activity and NLRP3 inflammasome of upregulation.
Keywords Isoorientin· Hyperuricemia· Xanthine oxidase· NLRP3 inflammasome· Uric acid
Abbreviations
ISO Isoorientin
XOD Xanthine oxidase
UA Uric acid
CRE Creatinine
BUN Blood urea nitrogen
TLR 4 Toll-like receptor 4
NF-κB Nuclear factor-κB
CTSB Cathepsin B
NLRP NOD-like receptor superfamily pyrin
PRRs Pattern recognition receptors
NLRC NOD-like receptor subfamily C
NAIP NLR family, apoptosis inhibitory protein
CARD Caspase activation and recruitment domain
PYHIN Pyrin and HIN domain-containing protein
URAT1 Urate anion transporter 1
GLUT9 Glucose transporter 9
OAT Organic cation transporter
ABCG2 ATP-binding cassette transporter ABCG2/
BCRP
ASC Apoptosis-associated speck-like protein
IL-1β Interleukin-1β
IL-18 Interleukin-18
IR Insulin resistance
LPS Lipopolysaccharide
K Potassium
Ca Calcium
FDA Food and Drug Administration
BCA Bicinchoninic acid
SPF Specific pathogen free
PBS Phosphate-buffered saline
PMSF Phenylmethylsulfonyl fluoride
DMSO Dimethyl sulfoxide
FBS Fetal bovine serum
MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide
DMEM Dulbecco’s modified Eagle’s medium
ROS Reactive oxygen species
H&E Hematoxylin and eosin
TGF-β Transforming growth factor beta
RIPA Radioimmunoprecipitation
PVDF Polyvinylidene fluoride
Introduction
Hyperuricemia is characterized by increased level of
serum uric acid exceeding 339μmol/L for women and
416μmol/L for men [1, 2]. The prevalence of hyperurice-
mia has increased annually worldwide over the past 2 dec-
ades [3], recorded as > 21% in USA [4], > 10% in Korea [5],
and > 13% in China [6].

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Uric acid, the end product of purine metabolism in
humans, is generated by oxidation of xanthine oxidase.
About 90% urate is filtered by the kidney every day and
uric acid is reabsorbed through the sequential activities of
various transporters in the proximal tubules of the kidney
while the remainder is broken down by the small intestine
[7]. These transporters include the urate anion transporter 1
(URAT1) [8], glucose transporter 9 (GLUT9), organic cation
transporter (OAT) [9], and ATP-binding cassette transporter
ABCG2/BCRP (ABCG2) [10]. The majority of studies to
date have focused on URAT1, ABCG2, and GLUT9. In
rodents (rats and mice), uricase (uric acid oxidase) further
oxidizes uric acid to allantoin, which is more water soluble
with higher excretion efficiency from urine [11]. However,
humans and advanced primates lack a functional uricase
gene owing to gene mutations during the evolutionary pro-
cess [1]. Therefore, excessive production and limited excre-
tion of uric acid play an important role in the strong associa-
tions between hyperuricemia and disease.
Xanthine oxidase, mainly found in the liver, is the key
rate-limiting enzyme in the uric acid synthesis pathway,
which oxidizes hypoxanthine to xanthine and xanthine
further to uric acid. Accumulating evidence suggests that
high expression of XOD is correlated with blocking the
metabolism of thiopurine in Crohn’s disease [12], age in
vascular aging studies [13], pro-inflammatory responses in
heart failure [14], and insulin resistance in diabetes [15].
Elevated uric acid triggers the production and release of
various inflammatory mediators and cytokines in the body.
Moreover, increased xanthine oxidase activity and uric acid
promote activation of the NLRP3 inflammasome, in turn,
leading to stimulation of the downstream inflammatory fac-
tor, interleukin-1β (IL-1β), and release of interleukin-18
(IL-18) [16].
As part of the innate immune system, inflammasomes are
macromolecular polyprotein complexes recruited by pattern
recognition receptors to identify pathogenic microorganisms
and endogenous risk signals, i.e., pathogen-associated and
risk-related molecular patterns, which can activate cas-
pase-1, cleave IL-1β and IL-18 precursors to generate the
corresponding mature cytokines, and participate in clear-
ance of pathogens and adaptive immune responses of the
body [16]. In particular, the NLRP3 inflammasome, which
recognizes numerous pathogen types, has been extensively
characterized. The NLRP3 inflammasome corpuscle is
composed of NLRP3, apoptosis-associated speck-like
protein (ASC) containing a caspase activation and recruit-
ment domain (CARD), and pro-caspase-1 [17]. Three main
NLRP3 inflammasome activation pathways have been iden-
tified to date: (1) potassium ion (K+) efflux [18]; (2) lyso-
somal destruction [19, 20]; and (3) mitochondrial reactive
oxygen species (ROS) production [19]. Hyperuricemia is a
contributory factor for gout. Clinical studies have shown that
gout occurs due to deposition and stimulation of monoso-
dium urate (MSU) [21]. MSU is reported to bind the ubiq-
uitin domains of the three inflammasomes of the caspase-
1-activated NOD-like receptor superfamily, which triggers
the inflammatory response, producing active IL-1β and
IL-18 [22]. However, increasing evidence over recent years
suggests that elevated serum uric acid is accompanied by
inflammation, even in the absence of gout [23, 24]. Soluble
uric acid increases TLR-induced inflammatory factor acti-
vation in human primary cells [25] and amplifies human
intestinal cell inflammation through the activation of TLR4-
NLRP3 signaling pathway [26]. Furthermore, hyperuricemia
is accompanied by numerous health complications.
Hyperuricemia is regarded as a risk factor for several dis-
eases including gout, obesity, chronic kidney disease, and
diabetes [3, 27, 28]. At present, the treatment of hyperurice-
mia is mainly to inhibit uric acid synthesis drugs, promote
excretion of drugs, while inhibiting uric acid synthesis and
promoting excretion of drugs. Xanthine oxidase is the key
enzyme in uric acid synthesis. Inhibition of uric acid syn-
thesis has been successfully achieved through suppression
of xanthine oxidase activity. The xanthine oxidase inhibi-
tor, allopurinol, was approved by the US Food and Drug
Administration (FDA) in 1966 for treatment of recurrent
gouty arthritis, tophi, uric acid kidney stones, and other dis-
orders [1]. Importantly, however, allopurinol is commonly
associated with hypersensitivity reactions, including hepa-
totoxicity, Stevens–Johnson syndrome (SJS), toxic epider-
mal necrosis, eosinophilia, and systemic rash manifestations
[29–31]. Febuxostat was subsequently approved by the Euro-
pean Medicines Agency and US FDA in 2008 for patients
intolerant to allopurinol [1]. Clinically, febuxostat has higher
urate-lowering activity and better safety than allopurinol
[32]. Initial studies have shown that the main adverse reac-
tions include joint pain, rash, diarrhea, and liver damage
[33, 34], most of which are mild to moderate and disappear
after termination of treatment [33, 35]. A series of complica-
tions caused by hyperuricemia, such as gout, the incidence is
increasing year by year; for the majority of patients, hyper-
uricemia is associated with extremely high costs of treat-
ment and poses a significant economic burden along with the
added burden of several side effects. Thus, identification of
active ingredients that lower uric acid level in natural plants
would not only prevent the occurrence of hyperuricemia
but also reduce the economic burden. Accordingly, natural
compounds that facilitate reduction of uric acid level have
become a hotspot of hyperuricemia research.
ISO, luteolin 6C-glucoside, is a flavonoid derived from
numerous plants, such as Commelina communis, Crataegus
pentagyna, Cymbopogon citratus, Cucumis satirus, Passi-
flora edulis, and Oxalis corniculata [36], with confirmed
biological activity [49]. The chemical structure of ISO was
shown in Fig.1a. Studies have shown that luteolin possess

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significantly XOD inhibitory activity, antioxidant activity,
anti-inflammatory activity and so on [37]. And ISO is one
of the most common C-glycosides in luteolin [38]. Fur-
thermore, multiple biological properties of ISO have been
reported, including anti-cancer [39–42], anti-oxidation [43,
44], anti-inflammatory [45–47], anti-bacterial, anti-noci-
ceptive activity, and liver protection effects [45, 48]. For
instance, ISO has been shown to inhibit carbon tetrachloride
(CCl4)-induced hepatic fibrosis via free radical scavenging
and antioxidant activity, and regulation of nuclear factor-κB
(NF-κB) and transforming growth factor beta (TGF-β)/Smad
signaling pathways, and attenuate the activity of pro-inflam-
matory cytokines [49]. However, the study on lowering uric
acid has not been reported. Therefore, we hypothesized ISO
could regulate uric acid synthesis invivo and invitro, and
explored its mechanism primarily.
Materials andmethods
Reagents
Isoorientin (95–99%, BP0733/CAS No.: 4261-42-1) was
purchased from Chengdu Purifa Technology Development
Co., Ltd (Chengdu, China). In this study, xanthine oxidase
(CAS-9002-17-9), allopurinol (CAS-315-30-0), uric acid
(CAS-69-93-2), xanthine (CAS-69-89-6), and yeast extract
(2340951-02) were obtained from Sigma-Aldrich (St. Louis,
MO, USA), and hypoxanthine (H108384) was purchased
from Aladdin (Shanghai, China). The xanthine oxidase
(XOD) (A002) and uric acid (C012) test kits were purchased
from Nanjing Jiancheng Bioengineering Institute (Nanjing,
China), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide (MTT) from Solarbio (Beijing, China).
Anti-NLRP3 antibody [EPR20425] (ab210491), anti-cas-
pase-1 antibody [EPR19672]-human, anti-IL-18 antibody
[EPR19954]-human, anti-IL-18 antibody [EPR19956]-
mouse, and anti-xanthine oxidase antibody [EPR4605]
(ab109235) were purchased from Abcam (Shanghai, China).
TLR4 (25): sc-293072-mouse was purchased from Santa
Cruz Biotechnology (Santa Cruz, CA, USA) and NF-κB
p65 (C22B4) rabbit mab was purchased from Cell Signaling
Technology (Beverly, MA, USA). Caspase-1 polyclonal anti-
body (Catalog No A0964) and cathepsin B polyclonal anti-
body (Catalog No A0967) were purchased from ABclonal
(Wuhan, China). In addition, anti-β-tubulin was purchased
from Proteintech Group, Inc. (Rosemont, IL, USA) while
horseradish peroxidase (HRP)-conjugated goat anti-mouse
IgG was purchased from R&D Systems (Minneapolis, MN,
USA).
In vitro assay ofxanthine oxidase activity
According to the related literatures [50], different con-
centrations of uric acid (i.e. 0mmol/L, 0.002 mmol/L,
0.004mmol/L, 0.006mmol/L, 0.01mmol/L, 0.02mmol/L,
0.04mmol/L, 0.08mmol/L, 0.10mmol/L) were diluted with
distilled water. The optical density (OD) values were meas-
ured at 295nm and adjusted to zero with distilled water to
obtain a standard curve of uric acid concentrations. Uric
acid (maximum absorption at the wavelength of 295nm)
is an indicator of xanthine oxidase activity. The uric acid
Fig. 1 Effect of ISO on XOD
activity in enzymatic analyses
invitro. a The chemical struc-
ture of ISO. b Establishment of
enzymatic experiments invitro.
c Effect of ISO on XOD activ-
ity. XOD, xanthine oxidase. The
applied concentration of allopu-
rinol was 80μmol/L invitro
enzymatic experiments. Data
were expressed as mean ± SEM
(n = 3) and representative of
three independent experiments.
Statistics: ###p < 0.001 versus
the control group; **p < 0.01
and ***p < 0.001 versus the
DMSO group

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standard curve equation is y = Ax + B (y is the uric acid
content of the sample; x is the concentration of the sample
measured; A and B are coefficients of the equation). The
reaction system (300μL) was composed of 50μL test sam-
ple of various concentrations, 0.1mol/L Tris–HCL 170μL
(pH 7.6), 0.5mmol/L xanthine 30 μL, and 40 U/L xan-
thine oxidase (XOD) 50μL. All reaction components were
mixed in 96-well plate. At last, xanthine oxidase (XOD) was
added to initiate the reaction. The optical density at 295nm
(OD295) of the reaction system was monitored dynamically
for 20min at 25°C on a SpectraMax M3 microplate reader.
Enzyme activity (U) unit definition is the amount of
enzyme required to catalyze the conversion of 1μmol xan-
thine to uric acid in 1min at pH 7.6 and 25°C. XOD activity
and inhibition rate were calculated according to the follow-
ing formulae:
(A1 is the OD value measured before the reaction; A2 is the
OD value measured after the reaction; A and B are coeffi-
cients of the standard uric acid curve equation).
Urate‑lowering eect ofISO onhyperuricemic mice
induced byyeast extract
Specific pathogen-free (SPF) male ICR mice weighing
approximately 20–22g were purchased from Kunming
Medical University (license number SCXK 2015-0004). All
animals were housed at room temperature (20–25°C) and
constant humidity (40–70%) under a 12h light–dark cycle
in an SPF grade laboratory. Food and water were supplied
adlibitum. Animals were acclimated for 1week before the
experiment period. Animal studies were performed accord-
ing to the international guidelines of animal experiments
and internationally accepted ethical principles for labora-
tory animal use and care. The experiment was reviewed and
approved by the Institutional Animal Care and Use Commit-
tee of the Yunnan Agricultural University. (Animal ethics
approval code number is YUAN2019LLWYH003-2).
Mice were randomly divided into five groups: normal,
model, allopurinol (10mg/kg) treatment, ISO (10mg/kg)
treatment, and ISO (5mg/kg) treatment. The normal group
was intragastrically administered distilled water and the
other groups intragastrically administered yeast extract at a
dose of 15g/kg every day with reference to previous meth-
ods [51]. On the day of the experiment, the positive control
was administered intragastrically at 10mg/kg according to
the previous report [52], while treatments in the remaining
groups were administered via continuous intraperitoneal
Enzyme activity =(
A
2−
A
1−
B
)∕(
A
×min (20)) (U∕L),
Inhibition rate (%)=(standard tube enzyme activity −test tube enzyme activity)∕standard tube enzyme activity ×100%,
injection. Treatments (allopurinol and ISO) were adminis-
tered after 6h. After 14days, mice were subjected to fast-
ing for 10h and blood collected via the orbital vein. Serum
samples were obtained by centrifuging blood at 4°C and
4000 × g for 10min, and serum UA, CRE, and BUN levels
determined using the Beckman AU680 automatic biochem-
istry analyzer (Kraemer Boulevard Brea, USA). Liver XOD
activity was measured using the xanthine oxidase (XOD)
test kit.
Histopathological examination
Tissues of the left upper lobe liver were embedded in paraf-
fin, cut into sections of 5μm thickness, and stained with
Hematoxylin and Eosin (H&E) for evaluation of general
morphology under a light microscope, as reported previ-
ously [53].
Determination ofuric acid level andXOD activity
incells
Normal liver cell line (L-O2 cells; MXC207) was purchased
from Shanghai Meixuan Biotechnology Co. Human normal
liver cell line (L-O2) was cultured in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10% fetal
bovine serum (FBS) and antibiotics (1% penicillin–strepto-
mycin) in 5% CO2 at 37°C.
To assess the inhibitory effects of ISO, L-O2 cells
were seeded in 96-well plates at a density of 5 × 104cells
per well and treated with different concentrations of ISO
(i.e. 25μmol/L, 50μmol/L, 100μmol/L, 200μmol/L, and
400μmol/L) for 24h, followed by addition of 20μL MTT.
After incubation for 4h in the dark, 200μL dimethyl sulfox-
ide (DMSO) was added to dissolve the remaining formazan
crystals of MTT. Following oscillation for 15min, cell via-
bility was measured at 492nm using a Flex Station 3 multi-
mode microplate reader (molecular devices).
L-O2 cells in the logarithmic growth phase were resus-
pended with DMEM and seeded in 6-well plates at a density
of 3 × 105cells per well. After a 24h inoculation period, the
supernatant in each well was discarded and gently washed
with phosphate-buffered saline (PBS) one time, followed by
the addition of 2mL ISO (i.e. 25μmol/L, 50μmol/L, and
100μmol/L) (ISO groups). The positive control allopurinol
concentration was set at 100μmol/L (prepared with complete
medium). After 24h of dosing, the supernatant in each well
was discarded. Cells were washed gently with PBS three
times and added 2mL hypoxanthine (800μmol/L). After
incubation for 24h at 37°C, xanthine oxidase was added at

Journal of Natural Medicines
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a concentration of 4μL/well (final concentration is 0.3U/g),
incubation for 1h in dark, followed by extraction of superna-
tant or protein. Experiments were repeated at least three times.
The uric acid content of supernatant was measured using
a specific uric acid test kit (Nanjing, China). XOD activity
was measured using xanthine oxidase (XOD) test kit (Nanjing,
China).
Western blot analysis ofprotein contents incells
andliver tissues
After pre-treatment, cells were rinsed once with pre-cooled
PBS and treated with high-efficiency radioimmunoprecipita-
tion (RIPA) tissue/cell lysate (lysate: phenylmethylsulfonyl
fluoride (PMSF) = 100:1; 150μL/well). Cells were placed on
ice for 20min, transferred to 1.5mL centrifuge tubes, and
centrifuged at 4°C and 15,000 × g for 10min. Proteins from
liver tissues were extracted using RIPA tissue/cell lysates,
homogenized for 2min and centrifuged at 1036 × g for 10min
at 4°C. The supernatant fractions from cells and liver tissues
were transferred to fully labeled 1.5mL centrifuge tubes. The
bicinchoninic acid (BCA) assay kit was employed to determine
protein concentrations for preparing samples for western blot
analysis. Protein samples were separated using 8–12% poly-
acrylamide gels and transferred to a solid-phase carrier poly-
vinylidene fluoride (PVDF) membrane for 1h. Next, the mem-
brane was blocked in Tris-buffered saline with 0.1% Tween-20
(TBST) + 5% milk for 1h to block non-binding sites. Then, the
membrane were incubated overnight with specific antibod-
ies in PBS, including XOD (1:1000), NLRP3 (1:1000), IL-18
(1:200), CTSB (1: 1000), caspase-1 (1:1000), TLR4 (1:200),
NF-κB p65 (1:1000), and β-tubulin (1:1000), washed three
times with TBST for 5min each, and subsequently incubated
with horseradish peroxidase (HRP)-conjugated goat anti-rabbit
IgG (1:5000) diluted with TBST for 1h at room temperature.
After three 5min washes with TBST, images were acquired
using a Pro-light HRP Chemiluminescence Kit (Tiangen Bio-
tech, Beijing, China) on a FluorChem E System (Protein Sim-
ple, Santa Clara, CA, USA).
Statistical analysis
Analyses were performed with GraphPad Prism 5 (Graph-
Pad Software, Inc., La Jolla, CA, USA) and data presented as
mean ± standard error (SEM). p values < 0.05 were considered
statistically significant in all cases.
Results
Inhibition ofXOD byISO invitro
Xanthine oxidase is a key rate-limiting enzyme in uric acid
synthesis. As shown in Fig.1c, ISO inhibited XOD activ-
ity in a dose-dependent manner by 42μmol/L, 83μmol/L,
167μmol/L, 333μmol/L, and 667μmol/L ISO induced
12%, 25%, 51%, 78%, and 96% inhibition ratio, respec-
tively. The inhibitory effect of the highest concentrations
of ISO (667μmol/L) examined was similar to that of
allopurinol (80μmol/L), with a calculated inhibition ratio
of 97% (Fig.1b). These results indicated that ISO was a
potential XOD inhibitor.
Eects ofISO onbiochemical indicators
inhyperuricemia model induced byyeast extract
As shown in Fig.2, serum UA, CRE, and BUN levels
and liver XOD activity of the yeast extract-induced hyper-
uricemia model were significantly higher than the corre-
sponding measurements in the normal group (p < 0.01).
The positive control group (allopurinol) showed a marked
decrease in serum UA and CRE levels and liver XOD
activity, compared with the model group (p < 0.05/0.01).
As expected, serum UA, CRE, and BUN contents were
markedly reduced after ISO treatment at doses of 5mg/
kg and 10mg/kg (p < 0.05/0.01) while liver XOD activity
was inhibited to a limited extent (p > 0.05). Our findings
suggested that ISO not only reduced the uric acid level
and XOD activity but also protected kidneys from damage
caused by high-purine diets.
Liver histopathological changes
As shown in Fig.3, the normal group showed no obvious
degeneration or necrosis. The liver was rosy, shiny, and elas-
tic in appearance and tissue structure was normal. Hepato-
cytes in the model group displayed obvious pathological
changes. The structure of the hepatic lobule was unclear
and significant hepatocyte vacuolar degeneration, accompa-
nied by inflammatory cell infiltration, was observed. Upon
treatment with ISO, hepatocyte vacuolar degeneration and
inflammatory cell infiltration were significantly improved
in a dose-dependent manner. A similar improvement in the
inflammatory cell infiltration and hepatocyte vacuolation of
liver tissues was observed in the positive control—allopu-
rinol group although it can inhibit the activity of liver drug
enzyme. Our findings collectively demonstrated that ISO
exerted a protective effect on livers of hyperuricemic mice.

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ISO suppressed XOD protein expression
anddownregulated activation oftheNLRP3
inammasome invivo
As shown in Fig.4, the expressions of XOD (Fig.4b) and
NLRP3 inflammatory signaling proteins such as TLR4
(Fig.4c), NF-κB (Fig.4d), CTSB (Fig.4e), NLRP3 (Fig.4f),
caspase-1 (Fig.4g) and IL-18 (Fig.4h) were significantly
increased in liver tissues of mice induced by yeast extract
compared with the normal group (p < 0.05). Notably, all of
them were downregulated after the treatment of ISO for suc-
cessive 14days compared to the model group (p < 0.05).
The similar results were observed in allopurinol group, of
them, the downregulation of NLRP3 inflammatory signaling
protein was inferior to that of ISO-10mg/kg group.
ISO induced signicant decrease onuric acid level
andXOD activity inhigh uric acid cell model
In this study, we established a liver cell model of high
uric acid co-induced by hypoxanthine and XOD. The uric
acid level was significantly increased by 30-fold in the
model, compared to control (Fig.5a). The inhibitory effect
of ISO on L-O2 cells was presented in Fig.5b. Interest-
ingly, ISO exerted no obvious inhibition at a concentra-
tion of 200μmol/L while the uric acid level was signifi-
cantly decreased at both 50μmol/L and 100μmol/L ISO
(p < 0.01) with inhibitory ratios 9% and 14%, respectively.
The inhibitory effect of 100μmol/L ISO was comparable
to that of allopurinol at the same concentration.
ISO inhibited XOD andNLRP3 inammasome
proteins expression inhigh uric acid cell model
The expressions of related proteins were examined to
explore the mechanism of lowering uric acid of ISO
invitro. Expectedly, the upregulated expression of these
proteins including XOD, NF-κB p65, CTSB, NLRP3, cas-
pase-1, IL-18 were observed in L-O2 high uric acid cell
model compared the control group (p < 0.05, Fig.6). How-
ever, the treatment with ISO could suppress the upregu-
lated expressions of them compared with those of model
group (p < 0.05/0.01), which is superior to that of the posi-
tive control (allopurinol) and consistent with the results
of invivo.
Fig. 2 Effects of ISO on serum
parameters and liver XOD activ-
ity in hyperuricemic mice. a
Uric acid (UA). b Serum blood
urea nitrogen (BUN). c Serum
creatinine (CRE). d Liver XOD
activity. The dose of allopurinol
was 10mg/kg invivo experi-
ments. Data are expressed as
mean ± SEM (n = 10). Statistics:
##p < 0.01 versus the normal
group; *p < 0.05 and **p < 0.01
versus the model group

Journal of Natural Medicines
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Discussion
Currently available treatment agents for hyperuricemia, such
as allopurinol, febuxostat and other drugs, are associated
with a number of side effects. Accumulating studies have
highlighted the utility of natural components of plant materi-
als in effectively reducing uric acid [54], which may provide
further potential treatment options for hyperuricemia.
Hyperuricemia and its complications have considerable
detrimental effects on quality of life. Several factors, such
as high-purine foods and high-fructose corn syrup bever-
ages, trigger uric acid imbalance in the body. XOD, the key
to producing uric acid, is an indicator of hyperuricemia.
XOD oxidation of xanthine to uric acid is accompanied by
the production of superoxide anions [55]. In patients with
hyperuricemia, excessive ROS leads to oxidative stress,
further increasing the risk of disease secondary to hyper-
uricemia including cardiovascular events [56]. In addition,
the increase of uric acid is accompanied by inflammatory
disease such as gout and nephritis [16]. Therefore, ingredi-
ents with anti-inflammatory activity or antioxidant activity
may have therapeutic value in delaying these pathogenic
processes. Data from invitro enzymatic experiments in
the current study demonstrated that ISO inhibited XOD
activity in a dose-dependent manner (Fig.1c). Therefore,
the experimental models used in this paper were mainly
referred to the previous literatures. The main innovation of
this study was to explore the mechanisms of ISO on uric
acid synthesis.
Several types of hyperuricemic mouse models have been
developed to date. For instance, hyperuricemia could be co-
induced by hypoxanthine/xanthine and potassium oxonate
[57, 58], potassium oxonate [59] or yeast extract alone.
High protein and purine levels are responsible for purine
metabolism disorders. Yeasts rich in protein, nucleotides,
and B vitamins are fully decomposed in the body increase
nitrogen-containing organic bases and the content of uric
acid precursors such as phosphoric acid, promoting the pro-
duction of uric acid, is the best inducer for the study of uric
acid anabolism [60]. Moreover, hyperuricemia can cause
liver and kidney dysfunction [61], and liver disease or even
liver failure has been reported in patients treated with allopu-
rinol [62]. In uric acid-related excretory disorders that lead
to a decline in renal function, serum creatinine and blood
urea nitrogen are important indicators reflecting glomerular
filtration function and renal failure.
The hyperuricemia model induced by yeast extract and
high uric acid cell model co-induced by hypoxanthine and
XOD were selected to examine the effects of ISO on the uric
acid synthesis pathway. An animal model of hyperuricemia
Fig. 3 Hepatoprotective effects of ISO on liver morphology in hyper-
uricemic mice. Liver sections subjected to H&E staining (n = 10) are
presented at a magnification of × 200. Scale bar, 100μm. ISO (10mg/
kg and 5mg/kg) and allopurinol (10mg/kg) were respectively admin-
istered to mice in addition to yeast extract treatment

Journal of Natural Medicines
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was successfully generated via intragastric administration of
yeast extract for 14days. Following intraperitoneal injection
of ISO for 14days, a significant reduction in serum UA,
BUN, and CRE levels were observed. ISO clearly exerted
a urate-lowering effect and improved glomerular filtration
function, reduced the possibility of kidney failure (Fig.2).
The protective effects of ISO were further examined via his-
topathological examination of the liver. ISO induced a sig-
nificant improvement in patchy necrosis and inflammatory
cell infiltration, compared with the model group (Fig.3).
Our data confirmed a hepatoprotective effect of ISO in the
hyperuricemia model, consistent with previous reports of
beneficial effects of it on liver fibrosis caused by alcohol
[48].
To date, studies on hyperuricemia mainly focused on
XOD activity [57] and XOD protein expression not deter-
mined [63]. In this study, both the activity and protein
expression of XOD were suppressed by ISO treatment
in animal and cells model confirming uric-acid-lowering
effect of ISO primarily by inhibiting XOD. Uric acid is
known to promote activation of the NLRP3 inflamma-
some [64]. Inflammation is an immune response in the
body that accompanied by inflammatory processes under
conditions of hyperuricemia. In our studies, ISO effec-
tively induced downregulation of NLRP3 inflammasome
as well as the downstream signal, caspase-1, and inflam-
matory factor, IL-18 invivo (Fig.4) and invitro (Fig.6).
A variety of endogenous and exogenous stimuli promote
assembly of NLRP3 inflammasome corpuscles, such as
uric acid, ROS, lysosomal leakage, lipopolysaccharide
(LPS), K+ efflux, and calcium ion (Ca2+) signals [65].
In most cases, the NLRP3 inflammasome is activated
by a dual signal mode. In resting cells, the components
of NLRP3 inflammasome corpuscles (NLRP3, ASC,
Fig. 4 Effects of ISO on protein levels in liver tissues. This figure
includes immunotbloting strips of protein expressions (a) levels of
XOD (b), TLR4 (c), NF-κB p65 (d), cathepsin B (e), NLRP3 (f),
caspase-1 (g), IL-18 (h) and β-tubulin were determined via Western
Blotting quantified using the software Image J Launcher. The dose of
allopurinol was 10mg/kg invivo experiments. These data were from
three separate experiments and expressed as the mean ± SEM. Statis-
tics: #p < 0.05 and ##p < 0.01 versus the normal group; *p < 0.05 and
**p < 0.01 versus the model group

Journal of Natural Medicines
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and pro-caspase-1) and their substrates (pro-IL-1β and
pro-IL-18) are undetectable level [66]. Binding of pat-
tern recognition receptors, such as toll-like receptor, to
their corresponding ligands, act as a preliminary signal
that mediates nuclear translocation of NF-κB, trigger-
ing NLRP3, pro-IL-1β, and pro-IL-18 transcription and
thereby upregulating expression [66]. The second signal
mediates assembly of the NLRP3 inflammasome, activa-
tion of caspase-1, and processing of pro-IL-1β and pro-
IL-18, ultimately resulting in secretion of mature IL-1β
and IL-18 [66]. Notably, the expression levels of TLR4,
NF-κB p65, CTSB and the downstream regulation pro-
tein NLRP3 inflammasome were downregulated by ISO
invivo (Fig.4) and invitro (Fig.6), demonstrating that
UA production was inhibited by interference on NLRP3
inflammasome signal pathway. Based on the findings of
the study, we will explore the effect of ISO on uric acid
excretion and complications such as gout and nephritis
syndrome. Moreover, a new botanical drug rich in ISO
used in lowering uric acid is worth developing in our
future study.
Conclusions
In summary, ISO not only inhibited XOD activity and uric
acid level invitro and invivo, but also protected kidney
and liver from damage induced by high-purine diet invivo.
It was by this dual mechanism of action primarily by inhib-
iting XOD and regulating of the TLR4-NLRP3 inflamma-
some signaling pathway, which directly led to urate low-
ering. This study validates the therapeutic effects of ISO
against hyperuricemia and provides valuable insights into
the underlying biological mechanisms.
Fig. 5 Establishment of the
high uric acid cell model (a)
and effects of ISO on L-O2
cell toxicity (b), XOD activity
(c) and uric acid (UA) level
(d). L-O2 cells were seeded
in six-well plates overnight,
incubated with 2mL of dif-
ferent concentrations of ISO
(i.e. 25μmol/L, 50μmol/L,
and 100μmol/L) or allopu-
rinol (100μmol/L) for 24h,
followed by incubation with
hypoxanthine (800μmol/L) for
24h and addition of xanthine
oxidase (4μL/well). Data were
expressed as means ± SEM
(n = 3) and representative of
three independent experiments.
Statistics: ###p < 0.001 versus
the control group; *p < 0.05
versus the DMSO group in B,
***p < 0.001 versus the model
group in a, c and d

Journal of Natural Medicines
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Funding This work was supported by the Yunnan provincial key
programs of Yunnan Eco-friendly Food International Cooperation
Research Center project under Grant (2019ZG00904, 2019ZG00909),
and the Science and Technology Plan Project of Yunnan Province
(2018IA060).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
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