Article Exposé TD stress Oxydatif

Journal of Functional Foods 90 (2022) 104954
Available online 3 February 2022
1756-4646/© 2022 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Fisetin represses oxidative stress and mitochondrial dysfunction in NAFLD
through suppressing GRP78-mediated endoplasmic reticulum (ER) stress
Xianling Dai
a , b , 1
, Qin Kuang
a , b , 1
, Yan Sun
a , b , 1
, Minxuan Xu
a , b , c
, Liancai Zhu
a , *
,
Chenxu Ge
a , b , c , *
, Jun Tan
b , c , *
, Bochu Wang
a , *
a
Key Laboratory of Biorheological Science and Technology (Chongqing University), Ministry of Education, College of Bioengineering, Chongqing University, Chongqing
400030, PR China
b
Chongqing Key Laboratory of Medicinal Resources in the Three Gorges Reservoir Region, School of Biological and Chemical Engineering, Chongqing University of
Education, Chongqing 400067, PR China
c
Research Center of Brain Intellectual Promotion and Development for Children Aged 0-6 Years, Chongqing University of Education, Chongqing 400067, PR China
ARTICLE INFO
Keywords:
NAFLD
Fisetin
ROS
Mitochondrial impairment
ER stress
ABSTRACT
Fisetin (FisT) is a bioactive avonoid polyphenol with antioxidant, anti-inammatory and anti-tumor activities.
Although the effects of FisT to meliorate non-alcoholic fatty liver disease (NAFLD) have been investigated, the
underlying mechanisms are not fully understood. In the present study, we found that FisT remarkably suppressed
cellular and mitochondrial reactive oxygen species (ROS) generation in human and murine hepatocytes with
palmitate (PA) stimulation. Additionally, mitochondrial impairment and dysfunction induced by PA were
considerably abrogated in hepatocytes with FisT co-incubation. Furthermore, Cyto-c releases and mitochondrial
apoptosis were detected in PA-treated hepatocytes, while being greatly repressed by FisT. PA-induced inam-
mation and lipid deposition were also strongly reduced by FisT in hepatocytes. Importantly, our in vitro exper-
iments showed that promoting ROS by nuclear factor erythroid 2-related factor 2 (Nrf2) deletion signicantly
abolished the function of FisT to meliorate apoptosis, inammation and lipid accumulation in PA-incubated
hepatocytes. What’s more, ER stress was strongly induced by PA via increasing 78-kDa glucose-regulated pro-
tein (GRP78) and C/EBP-homologous protein (CHOP), which were, however, dramatically repressed after FisT
co-exposure. Intriguingly, we found that strengthening ER stress by GRP78 over-expression considerably abol-
ished the capacity of FisT to retard ROS generation and mitochondrial impairment in PA-stimulated hepatocytes,
but GRP78 knockdown exhibited totally opposite effects. Thus, ER stress blockage was required for FisT to
ameliorate NAFLD development in vitro. Consistently, our in vivo studies using high fat diet (HFD)-fed mice
conrmed that FisT administration exerted inhibitory and therapeutic potential on fatty liver progression via the
same mechanisms monitored in vitro. Collectively, all our ndings disclosed that FisT can efciently attenuate
NAFLD through restraining ROS generation and mitochondrial impairment mediated by ER stress.
1. Introduction
Excessive fat deposition contributes to non-alcoholic fatty liver dis-
ease (NAFLD) progression. Although advances in NAFLD treatment have
been achieved, a large number of patients still has worse clinical
outcome (Im et al., 2021; Zhou et al., 2020). The consequential surplus
of lipids in hepatocytes leads to oxidative stress and lipotoxicity and
promotes mitochondrial dysfunction through different molecular
mechanisms (Friedman et al., 2018). Mitochondria play irreplaceable
role in adenosine triphosphate (ATP) production and modulate various
Abbreviations: HO-1, heme oxygenase-1; NQO1, NAD(P)H dehydrogenase (quinone 1); GCLM, glutamate-cysteine-ligase modier; NOX1, NADPH oxidase 1;
NOX2, NADPH oxidase 2; NOX4, NADPH oxidase 4; TNF-
α , tumor necrosis factor- α ; IL-1β, interleukin-1β; IL-6, interleukin-6; FASN, fatty acid synthase; SCD1,
stearoyl-CoA desaturase 1; PPARγ, peroxisome proliferator-activated receptor-γ.
* Corresponding authors at: Chongqing Key Laboratory of Medicinal Resources in the Three Gorges Reservoir Region, School of Biological and Chemical Engi-
neering, Chongqing University of Education, Chongqing 400067, PR China (C. Ge and J. Tan).
E-mail addresses: [email protected] (L. Zhu), [email protected] (C. Ge), [email protected], [email protected] (J. Tan), [email protected]
(B. Wang).
1
These authors contributed equally to this work.
Contents lists available at ScienceDirect
Journal of Functional Foods
journal homepage: www.elsevier.com/locate/jff
https://doi.org/10.1016/j.jff.2022.104954
Received 20 October 2021; Received in revised form 3 January 2022; Accepted 10 January 2022

Journal of Functional Foods 90 (2022) 104954
2
cellular processes in both physiological conditions and pathological
stress insults (Fanta et al., 2021). Mitochondrial impairment can induce
hepatocyte death and associated abnormal cellular events, which is a
key factor for NAFLD development and hepatic dysfunction (Eduardo
et al., 2021; Sanda et al., 2021). Emerging evidence has suggested that
defects in mitochondrial hemostasis are common characteristics of fatty
liver and maintaining healthy mitochondria is pivotal for the survival of
hepatocytes, contributing to NAFLD treatment (Lee, Park, & Roh, 2019).
Therefore, methods to suppress oxidative stress and mitochondrial
dysfunction may be effective for NAFLD management.
Recently, endoplasmic reticulum (ER) stress has attracted increasing
attention. ER is a crucial organelle where secreted and transmembrane
proteins are synthesized and correctly folded into three-dimensional
conformations. These above dynamic events need highly precise mod-
ulation to sustain normal protein homeostasis (Marciniak & Ron, 2006;
Schr¨ oder, 2008). Numerous stimuli including HFD can break the above
equilibrium, subsequently inducing the unfolded protein response
(UPR) and ER stress occurrence (Ghemrawi, Battaglia-Hsu, & Arnold,
2018; Iurlaro & Mu˜ noz-Pinedo, 2016). Upon ER stress initiation, GRP78,
as a key ER stress sensor, is released. Severe ER stress can facilitate the
expression of pro-apoptotic proteins, such as CHOP. Finally, cells un-
dergo apoptotic cell death under stimulated conditions (Lee, 2005). In
addition, ER stress may result in the activation of multiple different
intracellular stress pathways, which can induce or accelerate insulin
resistance, dyslipidemia, inammation and, in some cases, culminate in
hepatocyte cell death. All these processes are involved in the patho-
genesis of fatty liver (Zhang et al., 2014). Herein, nding therapeutic
approaches to reduce ER stress may be effective for NAFLD treatment.
Fisetin (FisT; 3,3′,4′,7-tetrahydroxyavone) is an important dietary
avonoid and presents in numerous fruits and vegetables. FisT has
antioxidant, anti-inammatory, anti-cancer and neuroprotective prop-
erties (Grynkiewicz & Demchuk, 2019; Khan et al., 2013; Zhang et al.,
2018). We previously showed that FisT could ameliorate HFD-induced
fatty liver in mice through suppressing hepatic inammation and lipid
deposition by depressing TNF-
α /RIPK3 signaling pathway (Xu, Ge, &
Qin et al., 2019; Xu, Sun, & Dai et al., 2019). Recently, FisT was
demonstrated to prohibit ROS/ER stress-mediated inammatory
response, contributing to the amelioration of metabolic stimuli-induced
cardiac injury both in vivo and in vitro (Ge et al., 2019). Besides, FisT can
ameliorate oxidative stress, inammation and apoptosis in HFD- or
streptozotocin (STZ)-induced diabetic cardiomyopathy, exerting car-
dioprotective action (Althunibat et al., 2019; Hu et al., 2020). Moreover,
the potential of FisT to regulate mitochondrial function and apoptosis
was shown to suppress tumor growth and protect against myocardial
ischemia–reperfusion (I/R) injury (Pal et al., 2013; Sabarwal, Agarwal,
& Singh, 2017; Shanmugam et al., 2021). Although these protective
effects of FisT against metabolic stresses-triggered liver and heart injury
have been reported, the underlying molecular mechanisms and partic-
ularly the possible crosstalk between them are still not fully understood.
Here in the present study, we performed in vitro and in vivo experi-
ments to deeply explore and conrm the regulatory role of FisT on PA-
and HFD-induced hepatic injury, respectively. We newly found that FisT
could restrain cellular and mitochondrial ROS generation, contributing
to the inhibition of inammatory response and lipid accumulation in PA-
incubated hepatocytes. More importantly, all these effects mediated by
FisT were largely attributed to the suppression of GRP78-mediated ER
stress. The function of FisT to depress ER stress was validated in HFD-
challenged mice, consequently ameliorating NAFLD progression.
2. Materials and methods
2.1. Cells and culture
Human hepatocyte cell line L02 and murine hepatocyte AML12 were
purchased from the Type Culture Collection of the Chinese Academy of
Sciences (Shanghai, China) and American Type Culture Collection
(ATCC; Manassas, VA, USA), respectively. All cells were cultured in
Dulbecco’s Modied Eagle Medium (DMEM) (#22320030, Gibco®)
supplemented with 10% fetal bovine serum (FBS; #10100147, Gibco®)
and 1% penicillin-streptomycin in a humidied incubator (Thermo
Fisher Scientic; USA) with 5% CO
2
at 37 ◦C. To imitate the in vivo liver
lipid deposition, cells were incubated with PA (Cat#P9767) obtained
from Sigma-Aldrich (St. Louis, USA).
2.2. Cell viability
Cell viability was measured using a Cell Counting Kit-8 assay kit
according to the manufacturer’s instructions (#C0039, Beyotime,
Shanghai, China). Briey, cells were planted into 96-well plates. After
treatments, 10
μ L of Cell Counting Kit-8 reagent was added into each
well. After incubation for 4 h at 37 ◦C, the absorbance of each well at
450 nm was measured using a microplate reader (SpectraMax iD3,
Molecular Devices, USA) to examine the number of viable cells.
2.3. Transfection in vitro
Nrf2 si-RNAs (si-Nrf2), GRP78 si-RNAs (si-GRP78) and the corre-
sponding negative control si-RNAs (si-Con) were obtained from Generay
Biotechnology (Shanghai, China). The GRP78 plasmid based on
pcDNA3.1 vector and empty vector (EV) were constructed by GeneChem
Technology (Shanghai, China) using the standard molecular biology
techniques. Transfection for gene knockdown or over-expression was
performed using Lipofectamine 3000 reagent (Invitrogen Life Technol-
ogies, Carlsbad, USA) according to the provider’s instructions.
2.4. Animals and treatments
The animal study protocols were permitted and authorized by the
Institutional Animal Care and Use Committee in College of Bioengi-
neering, Chongqing University (Chongqing, China). The procedures
used in this study were in accordance with the Regulations of Experi-
mental Animal Administration issued by the Ministry of Science and
Technology of the People’s Republic of China. All male, C57BL/6N mice
(6- to 8-week-old; 22–25 g body weight) used in the study were pur-
chased from Beijing Vital River Laboratory Animal Technology Co., Ltd.
(Beijing, China). Mice were allowed to adapt to their living environment
for 1 week before all experiment proper starts. All animals were fed in a
constant temperature (23 ±25 ◦C), humidity (50–60%) and pathogen-
free-controlled environment cage with a standard 12 h light/12 h dark
cycle, plenty of water and food (pathogen-free) in their cages.
Animal experimental design 1: Mice were then randomly divided
into 4 groups, including (i) the normal chow diet/Vehicle group (NCD/
Veh); (ii) NCD/FisT group; (iii) HFD/Veh group; and (iv) HFD/FisT
group. At the beginning of the experiments, HFD (60 kcal% fat; D12492;
Research Diets, New Brunswick, USA) was subjected to experimental
mice to induce NAFLD murine model. Mice fed with NCD (10.2% fat and
71.5% carbohydrates; D12450B; Research Diets) were served as the
normal controls. For pharmacological analysis of FisT, FisT (80 mg/kg)
as drug (HPLC purity ≥98%, Xi’an Ruiying Biotechnology Co. Ltd,
Xi’an, China) was given to mice by gavage daily with HFD for 16 weeks,
continuously (Xu, Ge, & Qin et al., 2019; Xu, Sun, & Dai et al., 2019).
Vehicle group of mice received the same volume of saline. Body weights
were measured during HFD and FisT treatments weekly.
Animal experimental design 2: Male C57BL/6N mice (6–8 weeks
old; 22–25 g) were fed HFD (D12492, Research Diets) for 16 weeks to
induce NASH. And after 8 weeks of HFD, mice were randomly assigned
to four experimental groups: (i) NCD/Veh; (ii) NCD/FisT group; (iii)
HFD/Veh group; and (iv) HFD/FisT group. FisT (80 mg/kg) was
administrated to mice every day by gavage for 8 weeks. Vehicle group of
mice received the same volume of saline.
After 16 weeks, blood was obtained through retro-orbital bleeding,
and the serum was separated. Liver samples from each mouse were
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Journal of Functional Foods 90 (2022) 104954
3
removed, weighed and then embedded in parafn for histological
analysis or stored at −80 ◦C for further analysis.
2.5. Liver function and lipid contents analysis
The concentration of serum alanine transaminase (ALT) (#MAK052,
Sigma-Aldrich), aspartate aminotransferase (AST) (#MAK055, Sigma-
Aldrich), serum or hepatic triglyceride (TG) (#MAK266, Sigma-
Aldrich) and total cholesterol (TC) (#ab65359, Abcam) were
measured using commercially available detection kits according to the
manufacturers’ protocols.
2.6. ROS measurements
After treatments, cellular ROS was stained using dichlorouorescin
diacetate (DCFH-DA) dye (#S0033M; Beyotime). The viable cells were
then incubated with 10
μ M of DCFH-DA for 20 min at 37 ◦C. Mito-
chondrial ROS generation in cells following treatments was examined
using a MitoSOX Red Mitochondrial Superoxide Indicator Kit (YEASEN
Biotechnology, Shanghai, China) according to the provider’s in-
structions. Images indicating cellular and mitochondrial ROS production
were captured under a uorescence microscope. Total ROS levels in the
liver sections were detected using the uorescent probe DHE (dihy-
droethidium). In brief, cryosections from snap-frozen liver (5
μ m thick)
tissues were prepared. In situ ROS was measured using DHE (#D23107;
Invitrogen) as the provider described. Cryosections were stained with
5
μ M DHE for 30 min, mounted by anti-uorescence quenching sealing
tablets in dark at room temperature and monitored under a uorescence
microscope.
2.7. Mitochondrial function assays
JC-1 dye (#C2006; Beyotime) accumulates in mitochondria of cells
with mitochondrial depolarization to form monomers and emits green
uorescence and was used to measure mitochondrial membrane po-
tential (MMP, Δ
ψ
m
) (Perelman et al., 2012). All cells were planted into
six-well plates and were subjected to drug treatments. Cells were then
incubated with 500
μ L of JC-1 dye following the provider’s instructions.
The JC-1 red/green images were captured under a uorescent micro-
scope. After different treatments, cells were incubated with 20 nM tet-
ramethyl rhodamine methyl ester (TMRM) (#T5428; Sigma Aldrich) in
Tyrode’s buffer for 45 min in dark for MMP assessment. After incuba-
tion, the cells were visualized with a uorescence microscope. Fluo-
rescence intensity was measured with Image-J software (Version 1.52v,
Image J, National Institutes of Health, USA)).
2.8. Biochemical parameters determination
Assay kits for the measurements of malondialdehyde (MDA; #A003-
1-2), superoxide dismutase (SOD; #A001-3-2) and glutathione (GSH;
#A006-2-1) were obtained from Nanjing Jiancheng Bioengineering
Institute (Nanjing, China). ATP Assay Kit (#S0026, Beyotime) was used
to examine ATP contents in accordance with the provider’s instructions.
Caspase-3 Activity Assay Kit (#ab252897, Abcam) and Caspase 9 Ac-
tivity Assay Kit (#1158, Beyotime) were used to examine Caspase-3 and
Casaspe-9 activities, respectively, following the providers’ instructions.
The contents of cytokines in serum were measured using corresponding
commercial enzyme-linked immuno sorbent assay (ELISA) kits,
including the mouse TNF-
α (#MTA00B), IL-1β (#MLB00C) and IL-6
(#M6000B) ELISA kits that were all from R&D system (USA) accord-
ing to the manufacturer’s protocols.
2.9. Real time-quantitative PCR (RT-qPCR)
Total RNA in cells or liver tissues were extracted using TRIzol™ re-
agent (#15596-018, Thermo Fisher Scientic) following the
manufacturer’s protocols. Then, total RNA extraction was reverse
transcribed using the M-MLV-RT system (Invitrogen). Subsequently,
PCR were performed with SYBR Green (Bio-Rad) on an ABI PRISM
7900HT system (Applied Biosystems, USA). The specic primer se-
quences were produced by Invitrogen or Generay Biotech (Shanghai,
China), which were listed in Supplementary Table 1. Fold induction
values were evaluated according the 2
(−ΔΔCt)
expression. ΔCt represents
the differences in cycle threshold number between the target gene and
GAPDH, and ΔΔCt represents the relative change in the differences
between the control and treatment groups.
2.10. Western blotting analysis
Cells or liver samples were homogenized using RIPA Lysis and
Extraction Buffer (#89900, Thermo Fisher Scientic) to yield a ho-
mogenate for total protein extraction. The Nuclear and Cytoplasmic
Protein Extraction Kit (#P0028; Beyotime) was used to extract nuclear
and cytosol protein from cells according to the manufacturer’s in-
structions. Protein concentrations were measured using a BCA Protein
Assay Kit (#P0012; Beyotime) with BSA as a standard following the
supplier’s protocols. Equal amounts of the obtained protein (20–50
μ g)
were subjected to 10–12% sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) system and then transferred to PVDF
membranes (GE Healthcare Life Science, Germany). Next, the mem-
branes were blocked using 5% skim milk (DifcoTM Skim Milk, USA) for
1 h and incubated with the primary antibodies (Supplementary Table 2;
diluted at 1:1000 or 1:500) at 4 ◦C overnight. Then, the PVDF mem-
branes were washed and incubated with horseradish peroxidase (HRP)-
conjugated anti-rabbit (#ab6721) or anti-mouse (#ab6789) secondary
antibodies (Abcam, dilution 1:5000). Immunoblotting bands were
visualized with a PierceTM ECL Plus Western Blotting Substrate
(#32134, Thermo Fisher Scientic) and exposed to Kodak (Eastman
Kodak Company, USA) Xray lm. Protein expression levels were nally
determined as grey value (Version 1.52v, Image J) and standardized to
housekeeping gene (GAPDH) and expressed as a fold of control.
2.11. Histological analysis
To explore histopathologic changes, the liver tissues were xed with
10% neutral formalin, embedded in parafn, and then sectioned trans-
versely (5-µm-thick). The liver tissue sections were then stained with
hematoxylin and eosin (H&E) to visualize the pattern of hepatic
inammation. NAS score following H&E analysis was quantied as
previously described (McPherson et al., 2015). To further indicate lipid
accumulation in livers, the sections were stained with Oil Red O Stain Kit
(#ab150678, Abcam). After washing with 60% isopropyl alcohol, the
liver sections were re-stained with haematoxylin. Images were captured
under a light microscope.
2.12. Immunouorescence (IF) staining
For IF analysis, the cells after treatments were washed with PBS, and
were then blocked in 10% goat serum (#C0265, Beyotime) containing
0.3% Triton X-100 (#ST797, Beyotime) for 1 h at room temperature and
incubated with primary antibodies against Cyto-c (#PA5-19462, dilu-
tion 1:200) at 4 ◦C overnight. Cells were then washed, and anti-rabbit
IgG H&L (Alexa Fluor® 594) (#ab150080) secondary uorescent anti-
body (Abcam, dilution 1:300) were prepared for cell incubation at room
temperature in dark. After washing, 2-(4-Amidinophenyl)-6-indole-
carbamidine dihydrochloride solution (DAPI; #C1006, Beyotime) was
added to the cells for nuclei staining. Images were visualized and
captured under a uorescence microscopy.
2.13. Apoptosis analysis
Apoptosis of cells in the treated cells or the prepared liver sections
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Journal of Functional Foods 90 (2022) 104954
4
was measured by terminal-deoxynucleotidyl transferase-mediated nick
end labeling (TUNEL) staining (Beyotime) according to the manufac-
turer’s instructions. Representative images were captured under a
uorescent microscope. The apoptosis was calculated as TUNEL-positive
cells (red)/DAPI (blue), and the number of TUNEL-positive staining cells
was quantied.
2.14. Statistical analysis
Data represented as mean ±standard error of the mean (SEM) unless
otherwise indicated. All analysis were repeated independently with
similar results at least three times. Statistical analysis was conducted
using GraphPad Prism 8.0 (San Diego, CA, USA). Differences between
two groups were analyzed by Student’s t test. One-way analysis of
variance (ANOVA) with Tukey’s post hoc tests were performed for
comparisons between multiple groups. P value <0.05 was considered
indicative of statistical signicance.
3. Results
3.1. Fisetin restrains ROS production and improves Nrf2 activation in PA-
incubated hepatocytes
Oxidative stress is crucial for NAFLD progression, and the regulatory
role of FisT on ROS production has been reported though without fully
understanding (Friedman et al., 2018; Ge et al., 2019), and thus was
further explored. First, CCK-8 analysis suggested that FisT treatments at
different concentrations and time points showed no signicant inuence
on the changes of L02 and AML12 cell viability (Supplementary Fig. 1A-
C), indicating the non-cytotoxicity of FisT. DCF-DA staining subse-
quently indicated that FisT treatments signicantly reduced ROS pro-
duction in human and murine hepatocyte lines L02 and AML12 via a
dose-dependent manner (Fig. 1A and B). Given that 20
μ M of FisT
exhibited the most efcient capacity to restrain ROS generation, and
thereafter was chosen for subsequent in vitro analysis. Additionally, we
found that 20
μ M of FisT treatment for 24 or 48 h did not inuence the
morphology of L02 and AML12 cells (Supplementary Fig. 1D), con-
rming the safe use of FisT. The contents of oxidative stress marker MDA
were highly increased in PA-incubated L02 and AML12 cells, while
being greatly ameliorated upon FisT exposure (Fig. 1C). On the contrary,
the activities of antioxidant enzyme including SOD, CAT and GSH were
signicantly downregulated in L02 and AML12 cells after PA stimula-
tion, which were, however, remarkably rescued in hepatocytes co-
treated with FisT (Fig. 1D-F). NADPH oxidases (NOXs) including
NOX1, NOX2 and NOX4 that contribute to ROS production were
strongly upregulated in PA-incubated cells, whereas being efciently
abolished by FisT (Fig. 1G). However, FisT remarkably restored the
expression of antioxidants HO-1, NQO1 and GCLM in L02 and AML12
cells after PA stimulation (Fig. 1H). As expected, both nuclear and total
Nrf2 protein expression levels were evidently decreased in PA-treated
hepatocytes, while being considerably restored by FisT (Fig. 1I and J).
Together, these ndings conrmed that FisT exerted antioxidant
bioactivity in PA-treated hepatocytes.
3.2. Fisetin improves mitochondrial dysfunction and impairment in PA-
treated hepatocytes
In this regard, MitoSOX staining further showed that besides cellular
ROS, FisT incubation also remarkably decreased the generation of
mitochondrial ROS in PA-stimulated L02 and AML12 cells (Fig. 2A).
Both TMRM and JC-1 staining suggested that PA exposure evidently
resulted in MMP impairments in hepatocytes, and these events were
signicantly mitigated upon FisT co-incubation (Fig. 2B-D). Consis-
tently, ATP levels downregulated by PA were also strongly increased in
L02 and AML12 cells after FisT treatment (Fig. 2E). These ndings
illustrated that FisT could meliorate mitochondrial impairment and
dysfunction in PA-incubated hepatocytes.
3.3. Fisetin mitigates mitochondrial apoptosis, inammation and lipid
deposition in PA-incubated hepatocytes
Mitochondrial impairment contributes to apoptotic cell death under
different stimuli (Maharjan et al., 2014). Thereafter, apoptosis in PA-
treated hepatocytes was examined. The Cyto-c liberation from mito-
chondria to cytoplasm/nucleus is reported as the molecular feature of
mitochondrial apoptosis (Chang, Xing, & Yu, 2014). As shown in Fig. 3A
and B, IF staining showed that nuclear Cyto-c was markedly promoted in
response to PA, whereas being signicantly meliorated by FisT co-
treatment. Furthermore, Caspase-9 and Caspase-3 activities were
considerably down-regulated by FisT in PA-stimulated L02 and AML12
cells (Fig. 3C and D). Western blotting results conrmed that Caspase-3
cleavage induced by PA was highly abrogated by FisT (Fig. 3E). TUNEL
staining subsequently indicated that PA stimulation clearly led to
apoptosis in hepatocytes, while being dramatically ameliorated after
FisT incubation (Fig. 3F and G). Additionally, RT-qPCR results indicated
that FisT treatment remarkably decreased the gene expression of pro-
inammatory cytokines including TNF-
α , IL-1β and IL-6 in PA-treated
cells (Fig. 3H). ORO staining showed that PA-caused increases in lipid
accumulation and cellular TG contents were remarkably abolished upon
FisT co-incubation (Fig. 3I and J). Consistently, the mRNA expression
levels of fatty acid synthesis markers including FASN, SCD1 and PPARγ
were considerably upregulated by PA, which were, however, strongly
depressed by FisT (Fig. 3K). Together, these data suggested that FisT
could meliorate mitochondrial apoptosis, inammation and lipid
deposition in PA-treated hepatocytes, contributing to NAFLD
amelioration.
3.4. Fisetin represses apoptosis, lipid accumulation and inammatory
response via ROS suppression in PA-stimulated hepatocytes
Due to the crucial role of ROS production in mediating cell death,
inammatory response and lipid metabolism (Oyinloye, Adenowo, &
Kappo, 2015), deeper molecular mechanism by which FisT regulated
these cellular events was then explored. Nrf2 increase plays a key role in
the suppression of ROS generation (De Vries et al., 2008), and was then
knocked down in L02 and AML12 cells by transfecting with the con-
structed si-Nrf2. RT-qPCR and western blotting results conrmed the
successful Nrf2 knockdown in cells, particularly si-Nrf2-2# (Supple-
mentary Fig. 2A and B), which was thus selected for subsequent analysis.
As expected, DCF-DA staining showed that FisT-restrained ROS gener-
ation was signicantly diminished upon Nrf2 deletion in PA-treated
hepatocytes (Fig. 4A). TUNEL staining demonstrated that Nrf2 silence
signicantly abolished the function of FisT to reduce apoptotic cell
death in PA-stimulated cells (Fig. 4B). Similarly, lipid accumulation and
cellular TG levels restrained by FisT in PA-exposed cells were markedly
abrogated when Nrf2 was deleted (Fig. 4C and D). What’s more,
Caspase-3 activity, the gene expression levels of fatty acid synthesis
markers and pro-inammatory cytokines downregulated by FisT were
markedly restored upon Nrf2 knockdown in PA-exposed hepatocytes
(Fig. 4E-G). Together, these results indicated that FisT-meliorated
apoptosis, lipid deposition and inammatory response induced by PA
were largely associated with the suppression of ROS regulated by Nrf2.
3.5. Fisetin-mediated cellular events is mainly through ER stress inhibition
In this part, we found that the mRNA and protein expression levels of
ER stress hallmarks GRP78 and CHOP were signicantly promoted in
PA-exposed hepatocytes, while being greatly meliorated upon FisT co-
incubation (Fig. 5A and B). ER stress plays an essential role in regu-
lating several cellular events, including ROS generation, cell death and
inammatory response under different stresses (Chaudhari et al., 2014).
To further explore the underlying mechanisms by which FisT mediated
X. Dai et al.

Journal of Functional Foods 90 (2022) 104954
5
Fig. 1. Fisetin restrains ROS production and improves Nrf2 activation in PA-incubated hepatocytes. (A&B) DCF-DA staining to examine ROS production in PA
(250
μ M)-incubated L02 and AML12 cells in the presence or absence of FisT at the shown concentrations for 24 h. Scale bar was 75 μ m. (C-J) L02 and AML12 cells
were treated with PA (250
μ M) for 24 h with or without FisT (20 μ M). Then, all cells were collected for studies as follows. Quantication for (C) MDA levels, (D) SOD
activities, (E) CAT activities and (F) GSH levels. (G) RT-qPCR results for oxidative stress markers including NOX1, NOX2, and NOX4. (H) RT-qPCR analysis for the
gene expression levels of antioxidants HO-1, NQO1 and GCLM in cells. (I&J) Western blotting results for nuclear and total Nrf2 protein expression levels. Data were
expressed as the mean ±SEM (n =4 per group). *p <0.05,
**
p <0.01 and
***
p <0.001 versus the Con/Veh group;
+
p <0.05 and
++
p <0.01 versus the PA/
Veh group.
X. Dai et al.