Journal of Functional Foods 90 (2022) 104954 Contents lists available at ScienceDirect Journal of Functional Foods journal homepage: www.elsevier.com/locate/jff 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 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 b A R T I C L E I N F O A B S T R A C T Keywords: NAFLD Fisetin ROS Mitochondrial impairment ER stress Fisetin (FisT) is a bioactive flavonoid polyphenol with antioxidant, anti-inflammatory 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 inflam­ 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 significantly abolished the function of FisT to meliorate apoptosis, inflammation 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 confirmed that FisT administration exerted inhibitory and therapeutic potential on fatty liver progression via the same mechanisms monitored in vitro. Collectively, all our findings disclosed that FisT can efficiently 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 modifier; 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. https://doi.org/10.1016/j.jff.2022.104954 Received 20 October 2021; Received in revised form 3 January 2022; Accepted 10 January 2022 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/). X. Dai et al. Journal of Functional Foods 90 (2022) 104954 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; Schröder, 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 & Muñoz-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, inflammation 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, finding therapeutic approaches to reduce ER stress may be effective for NAFLD treatment. Fisetin (FisT; 3,3′ ,4′ ,7-tetrahydroxyflavone) is an important dietary flavonoid and presents in numerous fruits and vegetables. FisT has antioxidant, anti-inflammatory, 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 inflammation 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 inflammatory 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, inflammation 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 confirm the regulatory role of FisT on PAand HFD-induced hepatic injury, respectively. We newly found that FisT could restrain cellular and mitochondrial ROS generation, contributing to the inhibition of inflammatory response and lipid accumulation in PAincubated 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 HFDchallenged mice, consequently ameliorating NAFLD progression. (ATCC; Manassas, VA, USA), respectively. All cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (#22320030, Gibco®) supplemented with 10% fetal bovine serum (FBS; #10100147, Gibco®) and 1% penicillin-streptomycin in a humidified incubator (Thermo Fisher Scientific; USA) with 5% CO2 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). Briefly, cells were planted into 96-well plates. After treatments, 10 μL 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 pathogenfree-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 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 2 X. Dai et al. Journal of Functional Foods 90 (2022) 104954 removed, weighed and then embedded in paraffin for histological analysis or stored at − 80 ◦ C for further analysis. 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 specific 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. 2.5. Liver function and lipid contents analysis The concentration of serum alanine transaminase (ALT) (#MAK052, Sigma-Aldrich), aspartate aminotransferase (AST) (#MAK055, SigmaAldrich), serum or hepatic triglyceride (TG) (#MAK266, SigmaAldrich) and total cholesterol (TC) (#ab65359, Abcam) were measured using commercially available detection kits according to the manufacturers’ protocols. 2.10. Western blotting analysis 2.6. ROS measurements Cells or liver samples were homogenized using RIPA Lysis and Extraction Buffer (#89900, Thermo Fisher Scientific) 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 μg) 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 Scientific) and exposed to Kodak (Eastman Kodak Company, USA) Xray film. Protein expression levels were finally determined as grey value (Version 1.52v, Image J) and standardized to housekeeping gene (GAPDH) and expressed as a fold of control. After treatments, cellular ROS was stained using dichlorofluorescin diacetate (DCFH-DA) dye (#S0033M; Beyotime). The viable cells were then incubated with 10 μM 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 fluorescence microscope. Total ROS levels in the liver sections were detected using the fluorescent probe DHE (dihy­ droethidium). In brief, cryosections from snap-frozen liver (5 μm thick) tissues were prepared. In situ ROS was measured using DHE (#D23107; Invitrogen) as the provider described. Cryosections were stained with 5 μM DHE for 30 min, mounted by anti-fluorescence quenching sealing tablets in dark at room temperature and monitored under a fluorescence 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 fluorescence and was used to measure mitochondrial membrane po­ tential (MMP, Δψm) (Perelman et al., 2012). All cells were planted into six-well plates and were subjected to drug treatments. Cells were then incubated with 500 μL of JC-1 dye following the provider’s instructions. The JC-1 red/green images were captured under a fluorescent 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 fluorescence microscope. Fluo­ rescence intensity was measured with Image-J software (Version 1.52v, Image J, National Institutes of Health, USA)). 2.11. Histological analysis To explore histopathologic changes, the liver tissues were fixed with 10% neutral formalin, embedded in paraffin, 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 inflammation. NAS score following H&E analysis was quantified 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. 2.8. Biochemical parameters determination Assay kits for the measurements of malondialdehyde (MDA; #A0031-2), 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. 2.12. Immunofluorescence (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 fluorescent 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 fluorescence microscopy. 2.9. Real time-quantitative PCR (RT-qPCR) 2.13. Apoptosis analysis Total RNA in cells or liver tissues were extracted using TRIzol™ re­ agent (#15596-018, Thermo Fisher Scientific) following the Apoptosis of cells in the treated cells or the prepared liver sections 3 X. Dai et al. Journal of Functional Foods 90 (2022) 104954 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 fluorescent microscope. The apoptosis was calculated as TUNEL-positive cells (red)/DAPI (blue), and the number of TUNEL-positive staining cells was quantified. dysfunction in PA-incubated hepatocytes. 3.3. Fisetin mitigates mitochondrial apoptosis, inflammation and lipid deposition in PA-incubated hepatocytes Mitochondrial impairment contributes to apoptotic cell death under different stimuli (Maharjan et al., 2014). Thereafter, apoptosis in PAtreated 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 significantly meliorated by FisT cotreatment. 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 confirmed 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 proinflammatory 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, inflammation and lipid deposition in PA-treated hepatocytes, contributing to NAFLD amelioration. 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 significance. 3. Results 3.1. Fisetin restrains ROS production and improves Nrf2 activation in PAincubated 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 significant influence on the changes of L02 and AML12 cell viability (Supplementary Fig. 1AC), indicating the non-cytotoxicity of FisT. DCF-DA staining subse­ quently indicated that FisT treatments significantly 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 μM of FisT exhibited the most efficient capacity to restrain ROS generation, and thereafter was chosen for subsequent in vitro analysis. Additionally, we found that 20 μM of FisT treatment for 24 or 48 h did not influence the morphology of L02 and AML12 cells (Supplementary Fig. 1D), con­ firming 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 significantly downregulated in L02 and AML12 cells after PA stimula­ tion, which were, however, remarkably rescued in hepatocytes cotreated 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 efficiently 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 findings confirmed that FisT exerted antioxidant bioactivity in PA-treated hepatocytes. 3.4. Fisetin represses apoptosis, lipid accumulation and inflammatory response via ROS suppression in PA-stimulated hepatocytes Due to the crucial role of ROS production in mediating cell death, inflammatory 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 confirmed 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 significantly diminished upon Nrf2 deletion in PA-treated hepatocytes (Fig. 4A). TUNEL staining demonstrated that Nrf2 silence significantly 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-inflammatory 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 inflammatory response induced by PA were largely associated with the suppression of ROS regulated by Nrf2. 3.2. Fisetin improves mitochondrial dysfunction and impairment in PAtreated hepatocytes 3.5. Fisetin-mediated cellular events is mainly through ER stress inhibition 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 significantly 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 findings illustrated that FisT could meliorate mitochondrial impairment and In this part, we found that the mRNA and protein expression levels of ER stress hallmarks GRP78 and CHOP were significantly promoted in PA-exposed hepatocytes, while being greatly meliorated upon FisT coincubation (Fig. 5A and B). ER stress plays an essential role in regu­ lating several cellular events, including ROS generation, cell death and inflammatory response under different stresses (Chaudhari et al., 2014). To further explore the underlying mechanisms by which FisT mediated 4 X. Dai et al. Journal of Functional Foods 90 (2022) 104954 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 μM)-incubated L02 and AML12 cells in the presence or absence of FisT at the shown concentrations for 24 h. Scale bar was 75 μm. (C-J) L02 and AML12 cells were treated with PA (250 μM) for 24 h with or without FisT (20 μM). Then, all cells were collected for studies as follows. Quantification 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. 5 X. Dai et al. Journal of Functional Foods 90 (2022) 104954 Fig. 2. Fisetin improves mitochondrial dysfunction and impairment in PA-treated hepatocytes. After incubation with PA (250 μM) for 24 h in the absence or presence of FisT (20 μM), L02 and AML12 cells were harvested for the following studies. (A) Mitochondrial ROS production was measured using MitoSOX staining. Scale bar was 75 μm. (B) TMRM staining of cells to monitor mitochondrial function. Scale bar was 75 μm. (C&D) MMP examination using JC-1 staining. Scale bar was 75 μm. (E) Assessments of cellular ATP levels. Data were expressed as the mean ± SEM (n = 4 per group). *p < 0.05 and **p < 0.01 versus the Con/Veh group; + p < 0.05 and ++p < 0.01 versus the PA/Veh group. NAFLD in vitro, GRP78 was then silenced or promoted by transfection with GRP78 siRNAs or plasmids, respectively. Transfection efficacy was validated by RT-qPCR analysis (Supplementary Fig. 3A and B). Because the most inhibitory effect of si-GRP78-1# on GRP78 expression, and thus was used for the following studies. Firstly, DCF-DA and MitoSOX staining indicated that GRP78 knockdown markedly reduced cellular and mitochondrial ROS generation in PA-incubated hepatocytes. Surprisingly, FisT-restrained ROS production was significantly restored by GRP78 over-expression in PA-treated cells (Fig. 5C-F). Consistently, ER stress suppression by si-GRP78 markedly improved MMP in L02 and AML12 cells with PA stimulation; however, facilitating GRP78 expres­ sion remarkably abrogated the function of FisT to promote MMP in PAincubated hepatocytes (Fig. 5G). The effects of ER stress on the media­ tion of lipid metabolism have been increasingly demonstrated (Basseri & 6 X. Dai et al. Journal of Functional Foods 90 (2022) 104954 Fig. 3. Fisetin mitigates mitochondrial apoptosis, inflammation and lipid deposition in PA-incubated hepatocytes. L02 and AML12 cells were exposed to PA (250 μM) for 24 h with or without FisT (20 μM) treatment and were then harvested for studies as follows. (A&B) IF staining for Cyto-c expression in cells. Scale bar was 25 μm. Measurements of (C) Caspase-9 and (D) Caspase-3 activity in cells. (E) Western blotting results for cleaved Caspase-3 protein expression levels in cells. (F&G) TUNEL staining for apoptotic cell death in cells. Scale bar was 50 μm. (H) RT-qPCR analysis for inflammatory cytokines including TNF-α, IL-1β and IL-6 in cells. (I) ORO staining to monitor lipid deposition in cells. (J) Cellular TG contents were examined. (K) RT-qPCR analysis for FASN, SCD1 and PPARγ mRNA expression levels in cells. Data were expressed as the mean ± SEM (n = 4 per group). **p < 0.01 and ***p < 0.001 versus the Con/Veh group; +p < 0.05 and ++ p < 0.01 versus the PA/Veh group. Austin, 2012; Lebeaupin et al., 2018). We then attempted to explore whether GRP78-regulated ER stress was involved in FisT-ameliorated lipid deposition under metabolic stresses. As expected, both FisT and GRP78 knockdown significantly reduced lipid accumulation in PAstimulated L02 and AML12 cells; however, promoting GRP78 expres­ sion completely abolished the function of FisT to restrain cellular lipid deposition after PA exposure (Supplementary Fig. 4A-C). Consistently, in FisT-treated hepatocytes, the mRNA expression levels of FASN, SCD1 and PPARγ were almost re-strengthened by GRP78 over-expression after PA stimulation (Supplementary Fig. 4D). Collectively, these results revealed that FisT could repress PA-induced ER stress through inhibiting GRP78/CHOP signaling, contributing to the suppression of ROS pro­ duction, mitochondrial dysfunction and lipid deposition. 3.6. Effects of fisetin on the crosstalk between Nrf2 and GRP78 signaling in PA-stimulated hepatocytes Cross talk between oxidative stress and ER stress by Nrf2 and GRP78 signaling pathways under physiological and pathological conditions has been widely demonstrated (Ge et al., 2019; Xu et al., 2020; Zhang et al., 2019). As shown in Fig. 6A and B, we found that FisT-reduced gene and protein expression levels of GRP78 a CHOP were significantly abrogated upon Nrf2 silence both in L02 and AML12 cells under PA stimulation. However, under normal status, Nrf2 knockdown showed no significant influence on GRP78 and CHOP expression changes. These findings suggested that Nrf2 might not directly interact with GRP78, but could indeed affect GRP78-mediated ER stress in FisT-treated hepatocytes under metabolic pressure. Furthermore, we found that in hepatocytes without PA treatment, GRP78 knockdown or over-expression had no significant influences on Nrf2 activation, confirming that there might not be a direct correlation between Nrf2 and GRP78. Under PA7 X. Dai et al. Journal of Functional Foods 90 (2022) 104954 Fig. 4. Fisetin represses apoptosis, lipid accumulation and inflammatory response via ROS suppression in PA-stimulated hepatocytes. L02 and AML12 cells were transfected with si-Nrf2 for 24 h, and were then incubated with PA (250 μM) for another 24 h in the presence or absence of FisT (20 μM). Subsequently, all cells were collected for the following studies. (A) ROS production was examined using DCF-DA staining. Scale bar was 75 μm. (B) TUNEL staining was used to monitor apoptosis. Scale bar was 50 μm. (C) ORO staining to investigate lipid accumulation. Scale bar was 40 μm. (D) Quantification for cellular TG contents. (E) Caspase-3 activity was measured. The gene expression levels of (F) FASN, SCD1, PPARγ, (G) TNF-α, IL-1β and IL-6 in cells were quantified by RT-qPCR analysis. Data were expressed as the mean ± SEM (n = 4 per group). **p < 0.01 and ***p < 0.001 versus the Con/Veh group; +p < 0.05 and ++p < 0.01 versus the PA/Veh group; # p < 0.05 and ##p < 0.01 versus the PA/FisT group. stimulated status, similar with FisT biological function, si-GRP78 significantly rescued nuclei and total Nrf2 expression levels; however, FisT-improved Nrf2 expression was almost abolished upon GRP78 overexpression in PA-stimulated hepatocytes (Fig. 6C). These findings further suggested that under metabolic stresses, there may be a crosstalk between oxidative stress and ER stress mediated by Nrf2 and GRP78 signaling pathways, respectively, which is a key metabolism for FisT to perform its protective function against NAFLD. 3.7. Fisetin ameliorates NAFLD development in HFD-fed mice Animal studies were then performed using a 16-week HFD-induced 8 X. Dai et al. Journal of Functional Foods 90 (2022) 104954 Fig. 5. Fisetin-mediated cellular events is mainly through ER stress inhibition. (A) Rt-qPCR and (B) western blotting analysis for GRP78 and CHOP gene and protein expression levels in L02 and AML12 cells after 24 h incubation with PA (250 μM) in the presence or absence of FisT (20 μM). (C-G) L02 and AML12 cells were transfected with si-GRP78 or GRP78 plasmids for 24 h to reduce or promote GRP78 expression, respectively, and were then exposed to PA (250 μM) for another 24 h with or without FisT (20 μM) co-incubation. Finally, all cells were collected for studies as follows. (C) DCF-DA and (D) MitoSOX staining for cellular and mito­ chondrial ROS production, respectively. Scale bar was 75 μm. Quantification for (E) cellular ROS and (F) mitochondrial ROS was shown. (G) MMP of cells was assessed using JC-1 staining. Scale bar was 75 μm. Data were expressed as the mean ± SEM (n = 4 per group). **p < 0.01 and ***p < 0.001 versus the Con/Veh group; ++ p < 0.01 versus the PA/Veh group; #p < 0.05 versus the PA/FisT group. 9 X. Dai et al. Journal of Functional Foods 90 (2022) 104954 Fig. 6. Effects of fisetin on the crosstalk between Nrf2 and GRP78 signaling in PA-stimulated hepatocytes. (A&B) L02 and AML12 cells were transfected with si-Nrf2 for 24 h, and were then exposed to PA (250 μM) for an additional 24 h with or without FisT (20 μM). Next, all cells were harvested for RT-qPCR and western blotting assays of GRP78 and CHOP. (C) L02 and AML12 cells were transfected with si-GRP78 or GRP78 plasmids. After 24 h, cells were subjected to PA (250 μM) for another 24 h with or without FisT (20 μM) co-incubation, followed by western blotting analysis of nuclei and total Nrf2 protein expression levels. Data were expressed as the mean ± SEM (n = 4 per group). **p < 0.01 and ***p < 0.001 versus the Con/Veh group; +p < 0.05 and ++p < 0.01 versus the PA/Veh group; # p < 0.05 and ##p < 0.01 versus the PA/FisT group; ns, no significant difference. NAFLD, and FisT was subjected to mice for a total of 16 weeks to confirm its inhibitory potential on fatty liver. At first, we found that HFD feedingincreased body weights of mice were remarkably decreased by FisT administration, along with significantly reduced fasting blood glucose and insulin levels (Fig. 6A-C), indicating the effects of FisT to improve metabolic disorders in vivo. Additionally, higher LW/BW was observed in HFD-challenged mice, while being greatly meliorated by FisT (Fig. 6D). Hepatic dysfunction triggered by HFD was also considerably alleviated by FisT, as evidenced by the decreased serum ALT and AST levels (Fig. 6E). Histological staining by H&E and ORO showed that FisT treatment markedly reduced the NAFLD characteristics such as inflam­ matory cell infiltration and lipid deposition in HFD-fed mice, along with decreased NAS scores (Fig. 6F and G). Moreover, TC and TG contents in serum and liver were strongly upregulated by HFD feeding, while being significantly meliorated after FisT treatment (Fig. 6H and I). We also found that serum and hepatic pro-inflammatory cytokines including TNF-α, IL-1β and IL-6 enhanced by HFD were dramatically restrained by FisT co-administration (Fig. 6J and K). As expected, hepatic FASN, SCD1 and PPARγ gene expression levels boosted by HFD feeding were highly diminished in mice co-treated with FisT (Fig. 6L). These data demon­ strated that FisT could ameliorate NAFLD characteristics in HFD-fed mice. 10 X. Dai et al. Journal of Functional Foods 90 (2022) 104954 3.8. Fisetin suppresses oxidative stress and mitochondrial apoptosis in liver of HFD-challenged NAFLD mice However, antioxidants HO-1, NQO1 and GCLM mRNA levels were considerably rescued in liver of HFD-challenged mice with FisT administration (Fig. 7G). Consistent with in vitro results, nuclear and total Nrf2 protein expression levels were significantly decreased in liver of HFD-fed mice, while being dramatically restored by FisT (Fig. 7H). TUNEL staining indicated that FisT administration remarkably decreased the number of apoptotic cells in liver of HFD-challenged mice (Fig. 7I and J), accompanied by reduced expression of cleaved Caspase-9 and Caspase-3 (Fig. 7K). These data disclosed that FisT could restrain hepatic ROS and apoptosis in HFD-fed mice. In this section, DHE staining in Fig. 7A and B showed that HFD feeding led to ROS production in liver sections of mice, while being significantly ameliorated by FisT. Hepatic MDA levels elevated by HFD were also abolished in mice co-treated with FisT (Fig. 7C). On the con­ trary, HFD-reduced SOD activity and GSH levels in liver tissues were markedly upregulated after FisT administration (Fig. 7D and E). As ex­ pected, liver NOX1, NOX2 and NOX4 gene expression levels enhanced by HFD were strongly downregulated by FisT treatment (Fig. 7F). Fig. 7. Fisetin ameliorates NAFLD development in HFD-fed mice. (A) Initial and final body weights of mice. (B) Fasting blood glucose and (C) insulin levels were measured. (D) Quantification for LW/BW. (E) Serum ALT and AST levels were examined. (F) H&E and ORO staining of liver sections from the shown groups of mice. Scale bar was 200 μm. (G) NAS score was quantified. (H) Serum TC and TG levels were tested. (I) Liver TG and TC contents were examined. (J) Serum TNF-α, IL-1β and IL-6 levels were examined using ELISA analysis. RT-qPCR analysis for (K) TNF-α, IL-1β, IL-6, (L) FASN, SCD1 and PPARγ gene expression levels in liver samples. Data were expressed as the mean ± SEM (n = 5 or 8 per group). **p < 0.01 and ***p < 0.001 versus the NCD/Veh group; +p < 0.05 and ++p < 0.01 versus the HFD/ Veh group. 11 X. Dai et al. Journal of Functional Foods 90 (2022) 104954 3.9. Fisetin prohibits ER stress in liver of HFD-fed mice hepatocyte damage and immune cell activation (Ghemrawi, BattagliaHsu, & Arnold, 2018; Zhang et al., 2014). Approaches to suppress mitochondrial dysfunction and ER stress are likely to be effective for NAFLD treatment. FisT is a dietary flavonoid and can be found in many fruits and vegetables. FisT could prohibit inflammatory response to ameliorate fatty liver and nephropathy progression induced by HFD through various signaling pathways (Chenxu et al., 2021; Xu, Ge, & Qin et al., 2019; Xu, Sun, & Dai et al., 2019). The regulatory effects of FisT to mediate ER stress and mitochondrial dysfunction have also been re­ ported to control the progression of cardiovascular diseases and different types of tumors (Althunibat et al., 2019; Sabarwal, Agarwal, & Singh, 2017). However, whether these mechanisms, especially the possible interrelation between them can be regulated by FisT to effec­ tively ameliorate NAFLD, it’s not been fully investigated. In the present study, we provided deeper insights into the thera­ peutic potential of FisT on NAFLD progression, particularly the under­ lying mechanisms. Our in vitro studies showed that FisT markedly reduced cellular ROS production in PA-incubated hepatocytes through increasing Nrf2 expression levels. In addition, mitochondrial ROS, impairment and dysfunction triggered by PA were also strongly miti­ gated by FisT. Furthermore, PA exposure led to mitochondrial apoptosis, inflammatory response and lipid deposition in L02 and AML12 cells; however, these events were remarkably ameliorated after FisT treat­ ment. Importantly, we found that promoting ROS by Nrf2 knockdown strongly abolished the anti-apoptotic, anti-inflammatory and antilipogenic biological functions of FisT in PA-treated hepatocytes. Addi­ tionally, FisT significantly suppressed ER stress induced by PA in he­ patocytes by decreasing GRP78 and CHOP expression. Notably, we showed that the capacity of FisT to prohibit ROS production, mito­ chondrial damage and lipid deposition were dramatically abrogated upon ER stress facilitation induced by GRP78 over-expression. We further found that under PA-stimulated status, Nrf2 silence abolished the inhibitory effects of FisT on GRP78 expression. Meanwhile, pro­ moting GRP78 markedly eliminated the function of FisT to improve Nrf2 in PA-treated hepatocytes. These results demonstrated that crosstalk between oxidative stress and ER stress mediated by Nrf2 and GRP78 signaling pathways, respectively, might be involved in FisT-ameliorated fatty liver progression (Fig. 9C). Our in vivo experiments finally sup­ ported that FisT administration both for 16 weeks and 8 weeks could efficiently mitigate NAFLD pathologies in HFD-fed mice through the molecular mechanisms as we observed in vitro. Collectively, all our studies confirmed that FisT exerted suppressive effects on GRP78mediated ER stress, contributing to the inhibition of ROS production and mitochondrial dysfunction, which ameliorated NAFLD development ultimately. Oxidative stress is a key factor involved in NAFLD pathogenesis. • During oxidative stress, excessive ROS production, including H2O2, O2 − − and ONOO , leads to damage to virtually any cellular component, and elevated ROS has been detected in patients with NAFLD (Jianhua et al., 2021). NOXs are major source of ROS production and have the precise • role of directly catalyzing O2 − production from O2 by transferring electrons from NADPH across biological membranes. NOX-produced ROS in particular are toxic in non-neoplastic conditions, since they are associated with the establishment of an inflammatory vicious cycle determining and sustaining the transition of NAFLD to NASH (Chen et al., 2020; Gabbia, Cannella, & De Martin, 2021). Nrf2 is served as a master redox switch that turns on the expression of endogenous anti­ oxidant genes and ultimately promotes the cellular redox potential. Cells adapt to ROS through this potent feedback mechanism, strengthening the protection against oxidative damage (Oyinloye, Adenowo, & Kappo, 2015). The effects of FisT to restrain oxidative stress and ROS production through improving Nrf2 signaling pathway have been demonstrated in metabolic stress-induced cardiac injury (Ge et al., 2019). Here, we confirmed that PA caused aberrant ROS production in hepatocytes by decreasing Nrf2 nuclear expression, along with markedly increased expression of NOX1, NOX2 and NOX4. Meanwhile, antioxidants such as Finally, ER stress stimulated by HFD was also detected in liver of mice, as proved by the markedly increased expression of GRP78 and CHOP both from mRNA and protein levels; however, these effects were considerably abolished after FisT administration (Fig. 8A and B). These findings indicated the suppressive effects of FisT on ER stress, contrib­ uting to the inhibition of NAFLD development. 3.10. Therapeutic efficiency examination of 8-week fisetin administration in NAFLD mice after a 16-week HFD feeding To further examine the therapeutic potential of FisT on NAFLD context, another animal experiment design was performed in 16 weeks of HFD-challenged mice with FisT supplementation for 8 weeks (Sup­ plementary Fig. 5A). We found that HFD-increased body weights of mice were also significantly reduced after an 8-week FisT therapy (Supple­ mentary Fig. 5B). Serum fasting blood glucose and insulin levels induced by HFD were strongly ameliorated by an 8-week FisT supplementation (Supplementary Fig. 5C and D). HFD-triggered higher LW/BW was also significantly alleviated after FisT treatment for 8 weeks (Supplementary Fig. 5E). Moreover, HFD-induced mice suffered higher levels of serum ALT and AST, and treatment of 8-week FisT remarkably decreased ALT and AST contents in serum (Supplementary Fig. 5F). H&E and ORO staining indicated that 8-week FisT effectively alleviated NAFLD symptoms, reflected by the descent of lipid accumulation and inflam­ matory cells infiltration in hepatic sections, accompanied by the decreased NAS scores (Supplementary Fig. 5G and H). After HFD feeding, higher TC and TG contents both in serum and liver were significantly mitigated after FisT administration for 8 weeks (Supple­ mentary Fig. 5I and J). Furthermore, FisT treatment remarkably decreased sera and hepatic TNF-α, IL-1β and IL-6 levels in HFD-fed mice (Supplementary Fig. 5K and L). Liver FASN, SCD1 and PPARγ gene expression levels were also downregulated by 8-week FisT consumption in HFD-treated mice (Supplementary Fig. 5M). These findings suggested that an 8-week FisT therapeutic approach could ameliorate NAFLD progression in HFD-fed mice. DHE staining further showed that FisT administration for 8 weeks had antioxidant functions in liver of NAFLD mice, proved by the evidently weakened fluorescent intensity, which was along with the decreased MDA contents (Supplementary Fig. 6A-C). However, mark­ edly rescued SOD and GSH activities were detected in liver of HFD-fed mice with FisT treatment for 8 weeks (Supplementary Fig. 6D and E). Hepatic NOX1, NOX2 and NOX4 mRNA expression levels provoked by HFD were highly diminished in mice after an 8-week FisT administration (Supplementary Fig. 6F), accompanied with improved nuclei and total Nrf2 protein expression levels (Supplementary Fig. 6G). Western blot­ ting results confirmed that 8-week FisT supplementation remarkably downregulated Caspase-9 and Caspase-3 cleavage in liver of NAFLD mice (Supplementary Fig. 6H). Consistently, GRP78 and CHOP expres­ sion levels induced by HFD were strongly decreased in HFD-fed mice after FisT treatment for 8 weeks (Supplementary Fig. 6I and J). These findings supported that FisT exerted therapeutic potential against HFDtriggered NAFLD by suppressing oxidative stress and ER stress. 4. Discussion NAFLD includes a broad spectrum of hepatic dysfunctions, and it is predicted to become the primary cause of liver failure and even hepa­ tocellular carcinoma (HCC) (Im et al., 2021; Zhou et al., 2020). Mito­ chondria is a type of highly dynamic organelles associated with multiple metabolic/bioenergetic pathways in liver (Fanta et al., 2021). During NAFLD progression, mitochondrial dysfunction may precede insulin resistance and participate to fat accumulation (Eduardo et al., 2021; Lee, Park, & Roh, 2019; Sanda et al., 2021). ER stress is also attributed to multi-hits of NAFLD development in various different aspects such as 12 X. Dai et al. Journal of Functional Foods 90 (2022) 104954 Fig. 8. Fisetin suppresses oxidative stress and mitochondrial apoptosis in liver of HFD-challenged NAFLD mice. (A&B) DHE staining for ROS production in liver samples. Scale bar was 200 μm. (C) MDA levels, (D) SOD activity and (E) GSH contents in liver of mice were measured. (F) RT-qPCR analysis for NOX1, NOX2 and NOX4 gene expression levels in liver tissues of mice. (G) RT-qPCR results for HO-1, NQO1 and GCLM mRNA levels in liver samples. (H) Nuclear and total Nrf2 protein expression levels measured by western blotting analysis. (I&J) TUNEL staining for apoptosis in hepatic sections. Scale bar was 200 μm. (K) Cleaved Caspase-9 and Caspase-3 protein expression levels in liver samples by western blotting assays. Data were expressed as the mean ± SEM (n = 5 per group). *p < 0.05, **p < 0.01 and ***p < 0.001 versus the NCD/Veh group; +p < 0.05 and ++p < 0.01 versus the HFD/Veh group. 13 X. Dai et al. Journal of Functional Foods 90 (2022) 104954 Fig. 9. Fisetin prohibits ER stress in liver of HFD-fed mice. (A) RT-qPCR and (B) western blotting analysis for hepatic GRP78 and CHOP gene and protein expression levels, respectively. (C) Working model illustrating the regulatory role of FisT on NAFLD progression through the suppression of GRP78-mediated ER stress to subsequently inhibit ROS accumulation and mitochondrial dysfunction that contributed to inflammation, lipid deposition and apoptosis. Data were expressed as the mean ± SEM (n = 5 per group). ***p < 0.001 versus the NCD/Veh group; ++p < 0.001 and +++p < 0.001 versus the HFD/Veh group. HO-1, NQO1 and GCLM were also found to be decreased in PAincubated hepatocytes, causing oxidative damage to cells. However, these effects were consistently mitigated by FisT through enforcing Nrf2 nuclear translocation. Excessive ROS production can cause lipid accu­ mulation, inflammation, and cell apoptosis (Oyinloye, Adenowo, & Kappo, 2015; Turillazzi et al., 2016). FisT has been shown to restrain inflammatory response and dyslipidemia under metabolic stresses (Ge et al., 2019; Xu, Ge, & Qin et al., 2019; Xu, Sun, & Dai et al., 2019). In our present study, we confirmed that FisT exposure could suppress inflammation and lipid deposition in PA-stimulated hepatocytes. Intriguingly, we found that promoting ROS by Nrf2 deletion signifi­ cantly abolished the effects of FisT to suppress PA-induced inflammation and lipid accumulation in L02 and AML12 cells. Therefore, we concluded that the inhibitory effects of FisT on inflammation and lipid deposition caused by metabolic stress were at least in part attributed to ROS suppression. These events mediated by FisT were validated in liver of NAFLD mice induced by HFD. Mitochondria are considered as primary contributors to oxidative stress since the mitochondrial single-electron transport chain can generate most of ROS (Fanta et al., 2021). Fatty liver occurs when fatty acid availability exceeds the demand for ATP in hepatocytes (Zhang et al., 2011). In addition, the reduction of mitochondrial ATPsynthesizing respiration has been observed in murine model with sim­ ple steatosis, indicating that fat accumulates in liver because the mito­ chondria fail to oxidize enough fatty acids (Wang et al., 2009). Mitochondrial dysfunction leads to a vicious cycle of hepatocyte damage with ROS over-production, releases of inflammatory factors and cell death (Eduardo et al., 2021; Lee, Park, & Roh, 2019; Sanda et al., 2021; Wei et al., 2008). Consistently, here in our study, we also found that PA stimulation led to mitochondrial ROS generation and impaired MMP, contributing to the dysfunction of mitochondria in hepatocytes. More­ over, TUNEL staining indicated that PA exposure caused severe apoptosis in hepatocytes. Cyto-c locates in mitochondria under normal conditions; however, in response to stimuli such as PA, Cyto-c will release from mitochondria into nuclear or cytoplasm, thereafter, inducing Caspase-9 and Caspase-3 cleavage to trigger mitochondrial apoptosis (Brentnall et al., 2013). In our study, we further found that PA treatment markedly increased nuclear Cyto-c expression, along with elevated activation of Caspase-9 and Casapse-3. Notably, all these pro­ cesses including mitochondrial ROS production, dysfunction and apoptosis were dramatically abrogated by FisT. Our in vivo experiments confirmed that FisT administration restrained hepatic apoptosis and Caspase-9/-3 activation in HFD-induced NAFLD mice. Therefore, we concluded that FisT could ameliorate fatty liver progression partially through improving mitochondrial function. The ER is an intracellular membranous organelle and responsible for numerous critical cellular events such as protein synthesis, lipid syn­ thesis, carbohydrate metabolism, oxidative damage and cell death (Ghemrawi, Battaglia-Hsu, & Arnold, 2018; Iurlaro & Muñoz-Pinedo, 2016; Marciniak & Ron, 2006; Zhang & Kaufman, 2008). ER in hepa­ tocytes has an important capacity to adapt to extracellular and intra­ cellular alterations, which ensure that normal and crucial hepatic metabolic functions are preserved. ER stress is clinically associated with NAFLD progression (Gonzalez-Rodriguez et al., 2014; Lebeaupin et al., 2015). It has been demonstrated that experimental ER stress inducers affect hepatocyte survival and the intrinsic functions of hepatocytes, including lipogenesis and inflammatory response (Parafati et al., 2018). GRP78, a molecular Chaperone, monitors ER protein activities. Under resting status, GRP78 binds to the luminal part of ER membrane pro­ teins. However, under stimulated conditions, GRP78 will be released from ER membrane proteins, enabling the homo-oligomerization of protein kinase RNA-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1) or both and resulting in the autophosphorylation of these en­ zymes and the activation of their substrates, such as CHOP (Lee, 2005). CHOP plays an essential role in ER stress-induced apoptosis, including mitochondrial apoptosis (Hu et al., 2019). In a study with a small cohort 14 X. Dai et al. Journal of Functional Foods 90 (2022) 104954 of patients, GRP78 and CHOP mRNA and protein levels were markedly upregulated in liver of patients with steatohepatitis (Gonzalez-Rodri­ guez et al., 2014; Lebeaupin et al., 2015; Toriguchi et al., 2014). Simi­ larly, in our present study, we also found that PA exposure led to significant increases in GRP78 and CHOP expression both from mRNA and protein levels, which were, however, remarkably downregulated in hepatocytes with FisT treatment, indicating that FisT could suppress ER stress caused by metabolic stimuli. Intriguingly, we found that pro­ moting ER stress by GRP78 over-expression remarkably abolished the capacities of FisT to suppress ROS production and mitochondrial impairment in PA-treated hepatocytes. Increasing studies have intro­ duced the crosstalk between ER stress and oxidative stress under numerous physiological conditions (Xu et al., 2020; Zhang et al., 2019). Here, our in vitro studies showed that GRP78 knockdown upregulated Nrf2 nuclei expression in PA-incubated hepatocytes, while its overexpression significantly diminished the capacity of FisT to improve Nrf2 signaling. Nrf2, as a central role orchestrating the antioxidant response, is a direct substrate of PERK and an effector of PERKdependent cell survival (Cullinan et al., 2003; Lebeaupin et al., 2018), which explained why the GRP78 knockdown cells expressed a large amount of nuclear Nrf2, and over-expression hepatocytes showed evidently decreased Nrf2 expression. There are also studies suggesting that Nrf2-mediated antioxidant signaling may contribute to the inhibi­ tion of ER stress by restraining GRP78 expression, exerting protective effects against cell death and metabolic disorders (Niazpour et al., 2021; Xu et al., 2020). Similarly, here we also found that Nrf2-silent hepato­ cytes abrogated the inhibitory effect of FisT on GRP78 signaling, exhibiting significantly elevated GRP78 and CHOP expression after PA stimulation. However, under normal status without PA exposure, neither GRP78 nor Nrf2 knockdown had no significant influences on the expression changes of Nrf2 and GRP78, respectively, revealing that there may be no direct interaction between the two signals. These in vitro findings suggested that the crosstalk between Nrf2-regulated oxidative stress and GRP78-mediated ER stress under pathological conditions was involved in FisT-ameliorated fatty liver context (Fig. 9C). Finally, the inhibitory and therapeutic potential of FisT to prohibit ER stress in liver were confirmed in HFD-fed mice with NAFLD, contributing to the amelioration of fatty liver. In summary, our results demonstrated that FisT significantly restrained cellular and mitochondrial ROS production through improving Nrf2 in PA-incubated hepatocytes, subsequently contributing to the inhibition of inflammatory response, lipid deposition and apoptotic cell death. Moreover, ER stress stimulated by PA was also highly prohibited by FisT in hepatocytes. Notably, FisT-inhibited ROS generation and mitochondrial dysfunction were largely attributed to GRP78-mediated ER stress inhibition. These cellular events mediated by FisT were also detected in HFD-induced murine model with NAFLD (Fig. 9C). Therefore, we provided deeper and further insights for the molecular mechanisms of FisT in NAFLD treatment. Herein, FisT is ex­ pected to become a promising drug for the management of NAFLD. CRediT authorship contribution statement Xianling Dai: Conceptualization, Methodology, Software, Visuali­ zation, Investigation. Qin Kuang: Conceptualization, Methodology, Software, Data curation, Writing – original draft, Visualization, Inves­ tigation. Yan Sun: Conceptualization, Methodology, Software, Data curation, Writing – original draft, Visualization, Investigation. Minxuan Xu: Data curation, Writing – original draft. Liancai Zhu: Data curation, Writing – original draft, Supervision, Software, Validation, Writing – review & editing. Chenxu Ge: Conceptualization, Methodology, Soft­ ware, Data curation, Writing – original draft, Supervision, Software, Validation, Writing – review & editing. Jun Tan: Conceptualization, Methodology, Software, Supervision, Software, Validation, Writing – review & editing. Bochu Wang: Conceptualization, Methodology, Software, Supervision, Software, Validation, Writing – review & editing. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgement This work was supported by (1) Graduate Student Research Inno­ vation Project of Chongqing University (grant number: CYS21070); (2) Chinese Medicine Technology Innovation and Application Development Project (grant number: 2020ZY013802) funded by Chongqing Munic­ ipal Health Commission and Chongqing Science and Technology Bu­ reau; (3) Chongqing Professional Talents Plan for Innovation and Entrepreneurship Demonstration Team (grant number: CQCY201903258, cstc2021ycjh-bgzxm0202); (4) Advanced Programs of Post-doctor of Chongqing (grant number: 2017LY39); (5) Supported by Youth Project of Science and Technology Research Program of Chongqing Education Commission of China (grant number: KJQN201901606) and (6) Chongqing Research Program of Basic Research and Frontier Technology(grant number: cstc2018jcyjAX0393). Appendix A. Supplementary material Supplementary data to this article can be found online at https://doi. org/10.1016/j.jff.2022.104954. References Althunibat, O. 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Ethics statement All research procedures associating with mice were approved by the Institutional Animal Care and Use Committee in Chongqing Key Labo­ ratory of Medicinal Resources in the Three Gorges Reservoir Region, School of Biological and Chemical Engineering, Chongqing University of Education, and were performed in accordance with the Guide for the Care and Use of Laboratory Animals, issued by the National Institutes of Health in 1996. The protocols used in this study were in accordance with the Regulations of Experimental Animal Administration issued by the Ministry of Science and Technology of the People’s Republic of China (http://www.most.gov.cn). 15 X. Dai et al. Journal of Functional Foods 90 (2022) 104954 Pal, H. C., Sharma, S., Elmets, C. A., et al. (2013). Fisetin inhibits growth, induces G 2/M arrest and apoptosis of human epidermoid carcinoma A 431 cells: Role of mitochondrial membrane potential disruption and consequent caspases activation. 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