Effects of resveratrol and its derivative pterostilbene on hepatic injury and immunological stress of weaned piglets challenged with lipopolysaccharide

Abstract

The present study was to investigate the protective effects of resveratrol (RSV) and its 3,5-dimethylether derivative pterostilbene (PT) against liver injury and immunological stress of weaned piglets upon lipopolysaccharide (LPS) challenge. Seventy-two weaned piglets were divided into the following groups: control group, LPS-challenged group, and LPS-challenged groups pretreated with either RSV or PT for 14 d (n = 6 pens, three pigs per pen). At the end of the feeding trial, piglets were intraperitoneally injected with either LPS or an equivalent amount of sterile saline. After 6 h of sterile saline or LPS injection, plasma and liver samples were collected. LPS stimulation caused massive apoptosis, activated inflammatory responses, and incited severe oxidative stress in the piglet livers while also promoting the nuclear translocation of nuclear factor kappa B (NF-κB) p65 (P < 0.001) and the protein expression of Nod-like receptor pyrin domain containing 3 (NLRP3; P = 0.001) and cleaved caspase 1 (P < 0.001). PT was more effective than RSV in alleviating LPS-induced hepatic damage by decreasing the apoptotic rate of liver cells (P = 0.045), inhibiting the transcriptional expression of interleukin 1 beta (P < 0.001) and interleukin 6 (P = 0.008), and reducing myeloperoxidase activity (P = 0.010). The LPS-induced increase in hepatic lipid peroxidation accumulation was also reversed by PT (P = 0.024). Importantly, inhibiting protein phosphatase 2A (PP2A) activity in a hepatocellular model largely blocked the ability of PT to prevent tumor necrosis factor alpha-induced increases in NF-κB p65 protein phosphorylation (P = 0.043) and its nuclear translocation (P = 0.029). In summary, PT is a promising agent that may alleviate liver injury and immunological stress of weaned piglets via the PP2A/NF-κB/NLRP3 signaling pathway.

This study indicates the beneficial roles of resveratrol and its derivative pterostilbene in alleviating liver injury and immunological stress, which may broaden our understanding of stilbene-mediated antiinflammatory mechanisms and promote the development of novel feeding strategies for young piglets under stressful conditions.

Introduction

Modern swine production is becoming increasingly intensive to enhance productive efficiency, but it also increases the exposure risk of young piglets to various stressful events, especially immunological stress (Lee et al., 2016; Zhang et al., 2020a). This stress occurs frequently in the early period after weaning, which is characterized by infections, diarrhea, and other production losses (Campbell et al., 2013). Considerable attention has been focused on developing appropriate nutrition strategies to minimize the adverse effects of intestinal disorders (Campbell et al., 2013; Xiong et al., 2019). However, the liver may be another important target to be considered, since it extensively communicates with the gut through the biliary tract, portal vein, and systemic circulation and is continuously exposed to gut-derived toxins such as bacteria and bacterial products (Wang et al., 2021).

Physiologically, the liver serves as the final barrier against the entry of the gut microbiome-associated and immunologically active molecules into the systemic circulation. Among them, lipopolysaccharide (LPS), the major outer membrane component of Gram-negative bacteria, is a representative activator that incites inflammatory responses related to the host defense against infection (Nakao et al., 1994). A well-controlled inflammatory response is beneficial for the clearance of bacteria and bacterial LPS, but this process may be a source of additional damage to the liver when circulatory LPS become abundant, as this can result in parenchymal tissue injury, liver dysfunction, and multiple-system organ failure (Cohen et al., 2002). Hence, it is important to identify an effective approach to prevent uncontrolled inflammatory processes in the liver, which may help improve the growth performance and health status of young piglets under stressful situations.

Previous studies have implicated the beneficial roles of several bioactive natural products in inhibiting inflammation, and these compounds are gaining increasingly more interest as an auxiliary therapeutic treatment for inflammatory liver injury (Yang and Lim, 2014; Tsai et al., 2017; Cao et al., 2020; Olcum et al., 2020; Soukhtanloo et al., 2020). Among the most promising candidates are the stilbenes, a series of phytoalexins present in Vitis species to resist disease (Tsai et al., 2017). The best-studied stilbene is resveratrol (RSV) because of its known benefits to liver function. More importantly, studies on different cell types and rodent organs have shown that RSV confers protection against the activation of several signaling pathways involved in inflammatory response (Yang and Lim, 2014; Cao et al., 2020; Olcum et al., 2020). Unfortunately, RSV has a low bioavailability owing to its short half-life and rapid metabolism (Tsai et al., 2017), and this may limit its effectiveness in practice. Thus, identifying RSV derivatives with better bioavailability and greater potency is important in practice.

Pterostilbene (PT), a natural dimethylether analog of RSV, has been reported to possess preferable bioavailability compared with its parent compound. The replacement of two hydroxyl groups in the RSV structure with methoxy groups imparts greater lipophilicity and membrane permeability for PT, while also reducing its susceptibility to phase II metabolism, thereby promoting its resistance to degradation and elimination from the body (Yeo et al., 2013; Elango et al., 2016). In this regard, PT may be superior to its parent compound for further development as a hepatoprotective substance. Recently, we have found that PT is more effective than RSV in protecting the liver of young piglets from oxidative stress (Zhang et al., 2020b). Similarly, Choo et al. (2014) assessed the antiinflammatory activities of PT in vitro using RSV as a comparator and showed that PT has greater antiinflammatory potency than its parent. However, further study is needed to determine whether PT has a therapeutic advantage over RSV in alleviating the liver inflammation. The underlying mechanisms, and especially the signaling pathway involved, also need further clarification. Thus, this study was to investigate the potential of RSV and PT to protect the liver from LPS-induced immunological stress in weaned piglets.

Materials and Methods

Experimental design

All animal procedures performed in the present study were fully reviewed and specifically approved by the Jiangsu Academy of Agricultural Sciences Institutional Animal Care and Use Committee (Permit number SYXK-2020-0023). Seventy-two of Duroc × Landrace × Yorkshire weaned piglets with an average body weight (6.30 ± 0.34 kg) were randomly divided into four groups, with six replicates and three piglets per replicate (n = 6): (1) CS group (piglets were received a basal diet for 2 wk before intraperitoneal injection of 0.86% sterile saline); (2) CL group (piglets were received a basal diet for 2 wk before intraperitoneal injection of LPS [Escherichia coli O55:B5; Sigma–Aldrich, St. Louis, MO, USA] at a dosage of 25 μg/kg body weight); (3) RL group (piglets were received a basal diet supplemented with RSV [BOC Sciences, Shirley, NY, USA; purity ≥ 99.5%] at a dosage of 300 mg/kg for 2 wk before intraperitoneal injection of LPS); (4) PL group (piglets were received a basal diet supplemented with PT [BOC Sciences; purity ≥ 99.0%] at a dosage of 300 mg/kg for 2 wk before LPS injection). The dosage of PT used in this investigation was selected based on results from our previous study, which had shown that feeding piglets with a diet containing 300 mg/kg of PT was efficient in preventing diquat-induced hepatic oxidative stress and mitochondrial injury (Zhang et al., 2020b). LPS was dissolved in sterile saline and injected at a dosage determined as previously described (Hou et al., 2017). The basal diet was formulated as recommended by the National Research Council (NRC 2012; Supplementary Table S1). During the feeding experiment, no differences were observed among the groups in terms of body weight gain, feed intake, or feed efficiency before the LPS challenge (Supplementary Table S2).

Sample collection

After completion of the feeding period, six piglets in each treatment group (one piglet per replicate) were euthanized by exsanguination after electrical stunning at 6 h postinjection (Hou et al., 2017). Blood samples were taken by aspiration from the vena jugularis and collected into heparinized polyethylene tubes. Thereafter, a part of tissue samples from the left lobe of the liver were immediately cryopreserved in optimal cutting temperature medium (Servicebio Technology, Wuhan, Hubei, China) for dihydroethidium (DHE) staining or fixed in 4% paraformaldehyde solution (Servicebio Technology) for hematoxylin and eosin (H&E), terminal deoxynucleotidyl transferase-mediated 2ʹ-deoxyuridine-5ʹ-triphosphate nick end labeling (TUNEL), and myeloperoxidase (MPO) protein staining. The remainder of liver samples was stored at 4 °C for the extraction of nuclear protein or stored at −80 °C for metabolite contents, enzyme activities, and protein and mRNA expression analyses.

Determination of plasma aminotransferase activities

Plasma was obtained by centrifugation of the blood (2,500 × g, 10 min, 4 °C). Then, plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were detected using appropriate colorimetric kits obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China).

Hepatic histological examination

Liver tissues were fixed in 4% paraformaldehyde solution, dehydrated, and embedded in paraffin based on the methods described by Li et al. (2018a, b). Paraffin-embedded tissue sections were sliced at a thickness of 5 μm according to Leica’s protocol. The slides were stained with H&E for the examination of overall morphology, and images were taken at 400× magnification using a light microscope (Nikon Eclipse 80i; Nikon, Tokyo, Japan).

Hepatic apoptosis analysis

Hepatic apoptotic cells were quantified by TUNEL staining. The liver slices (5 μm) were prepared as described above and then deparaffinized by ethanol solutions. After that, the slices were stained with a TUNEL BrightGreen Apoptosis Detection Kit (Yeasen, Shanghai, China) according to the manufacturer’s instructions. This was followed by the 4ʹ-6-diamidino-2-phenylindole (DAPI) staining to label the nuclei. Positive staining of apoptotic cells showed a green fluorescent signal emitted by fluorescence dye. Specimen were observed and photographed under a fluorescence microscope (Nikon Eclipse C1; Nikon, Tokyo, Japan). Excitation/emission filters used for visualizing the green fluorescence of fluorescein dye and the blue fluorescence of DAPI were 465–495/515–555 nm and 330–380/420 nm, respectively. The apoptosis of liver cells was calculated by dividing the number of TUNEL-positive cells by the total number of nuclei.

Hepatic caspase 3 activity

Hepatic caspase 3 (Casp-3) activity was determined using a colorimetric detection kit (Beyotime, Haimen, Jiangsu, China). Hepatic samples were lysed with the lysis buffer, homogenized in ice. The supernatant samples were obtained by centrifugation (16,000 × g, 15 min, 4 °C), and the concentrations of total protein were quantified using a Bicinchoninic Acid Protein Quantitation Kit (Beyotime). After that, 40 μL of supernatant samples were incubated with 140 μL of detection buffer and 20 μL of reactive substrates (Ac-DEVD-pNA) at 37 °C for 1 h. The release of pNA was monitored at 405 nm to calculate the activity of Casp-3.

Analysis of plasma inflammatory cytokine contents

The contents of tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β), interleukin 6 (IL-6), and interleukin 18 (IL-18) in the plasma samples were determined by enzyme linked immunosorbent assay (ELISA) using porcine-specific detection kits obtained from CUSABIO Biotech (Wuhan, China).

Immunofluorescent staining of hepatic myeloperoxidase protein

The expression levels of hepatic MPO protein were measured via immunofluorescent staining. Five-micrometer liver paraffin-embedded sections were prepared, deparaffinized, rehydrated, and incubated with the Antigen Retrieval Solution according to the manufacturer’s instructions (Servicebio Technology). Then, the sections were treated with phosphate-buffered saline (PBS) containing 1% hydrogen peroxide to inhibit endogenous peroxidase. The primary antibody against MPO (1:100; Bioss, Beijing, China) was applied for 2 h after blocking with 10% fetal calf serum (Gibco-BRL, Grand Island, NY, USA). A Cy3–conjugated goat-anti-mouse IgG was applied as a secondary antibody. Nuclei were counterstained with DAPI before the slides were observed under a fluorescence microscope (Nikon Eclipse C1). The Cy3 emitted red fluorescence at an excitation wavelength of 510–560 and an emission wavelength of 590 nm. The fluorescence intensity of MPO protein was quantified using the software Image J (NIH, Bethesda, MD, USA). Analysis was performed on 10 randomly chosen fields in each section.

Measurement of hepatic superoxide anion radicals

The accumulation of superoxide anions in piglet liver tissues was determined with a specific fluorescent probe DHE (Sigma–Aldrich) according to the method of Yueh et al. (2014). Frozen sections of liver tissues were incubated with 10 μM DHE in a light-protected humidified chamber at 37 °C for 30 min. The fluorescent signals produced from DHE staining were observed and quantified as the methods described above.

Determination of hepatic enzyme activities and metabolite contents

The activities of hepatic superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and MPO and the concentrations of reduced glutathione (GSH), oxidized glutathione (GSSG), and malondialdehyde (MDA) were detected by using colorimetric kits (Nanjing Jiancheng Bioengineering Institute), as the recommended procedures of respective instructions.

Analysis of hepatic 8-hydroxy-2-deoxyguanosine contents

Deoxyribonucleic acid was isolated from frozen liver samples with an Animal Tissues Genomic DNA Extraction Kit purchased from Solarbio (Beijing, China), and the levels of 8-hydroxy-2-deoxyguanosine (8-OHdG) in the hepatic DNA samples were measured by ELISA using a commercial kit (Cayman Chemical, Ann Arbor, MI, USA).

Cell culture and treatment

Alpha mouse liver 12 (AML-12) hepatocytes were purchased from American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium/F-12 medium supplemented with 10% fetal bovine serum (Gibco-BRL), 100 U/mL penicillin and streptomycin (Gibco-BRL), insulin-transferrin-selenium Liquid Media Supplement (Gibco-BRL), and 40 ng/mL dexamethasone (Gibco-BRL) at 37 °C in a humidified O₂/CO₂ (19:1) atmosphere. For the experiments, AML-12 cells were treated with different levels of PT (0−50 μM) with or without TNF-α (10 ng/mL) for 24 h. The activities of protein phosphatases protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) were inhibited by pretreating the cells with tautomycetin (TA; 1 μg/mL; MedChemExpress, Shanghai, China) and fostriecin sodium (FO, 10 nM; Sigma–Aldrich) for 5 h, respectively, before incubation with TNF-α and PT.

Cell viability assay

The AML-12 cells were planted in 96-well microplates at a density of 8,000–10,000 cells per well. After completion of the various treatments, the viability of AML-12 cells was detected by treating the cells with Cell Counting Kit-8 solution (10 μL/well; Yeasen, Shanghai, China) at 37 °C for 1.5 h. The optical density at 450 nm was determined using a microplate reader.

Immunofluorescence staining of nuclear factor kappa B p65 protein

The AML-12 cells were inoculated into 35 mm confocal dishes and treated as indicated. The cells were then rinsed with PBS, fixed with 4% paraformaldehyde solution (Servicebio Technology) at 37 °C for 30 min, incubated with 0.1% Triton×100-PBS (Servicebio Technology) at 37 °C for 30 min, and blocked with 3% fetal calf serum at 37 °C for 40 min. After that, the cells were treated with the primary antibody against nuclear factor kappa B (NF-κB) p65 (1:200; Proteintech, Chicago, IL, USA) overnight at 4 °C, followed by the incubation of CoraLite594-labeled secondary antibody (1:200; Proteintech) in a light-protected humidified chamber at room temperature for 1 h. The nuclei were counterstained with DAPI. The fluorescent signals were captured at excitation/emission wavelengths of 593/614 nm for CoraLite594 (red) and 340/488 nm for DAPI (blue) using a laser confocal microscope (SP8; Leica Microsystems, Buffalo Grove, IL, USA).

Immunoblotting Analysis

Total protein and nucleoprotein were isolated from liver tissues or AML-12 cells using corresponding protein extraction reagents following the manufacturer’s instructions (Beyotime). Immunoblot analysis for the protein expression of NF-κB p65 (dilution 1:750; Proteintech; catalog number: 10745-1-AP), histone H3 (dilution 1:1000; Cell Signaling Technology [CST], Danvers, MA, USA; catalog number: 5192S), Nod-like receptor pyrin domain containing 3 (NLRP3; dilution 1:750; Proteintech; catalog number: 19771-1-AP), cleaved caspase 1 (Casp-1; dilution 1:750; CST; catalog number: 89332S), phosphorylated NF-κB p65 (Ser⁵³⁶; dilution 1:1000; CST; catalog number: 3033S), and beta actin (ACTB; dilution 1:1000; CST; catalog number: 4970S) was performed based on the procedures of Zhang et al. (2020c). Blots were incubated with an HRP-conjugated goat antirabbit IgG antibody (dilution 1:10000; Proteintech; catalog number: SA00001-2), exposed to BeyoECL Star substrate (Beyotime), and then visualized with a Tanon 6200 chemiluminescence imaging workstation (Tanon Science & Technology Inc., Shanghai, China).

Quantitative real-time PCR analysis

Total RNA from frozen liver samples was isolated with Trizol (TaKaRa, Dalian, Liaoning, China). The first-strand complementary DNA was synthesized with the PrimeScript^TM RT Master Mix (TaKaRa). Real-time PCR was performed with the SYBR Green Real-time PCR Kit (TaKaRa) using the PCR primers listed in Supplementary Table S3 on an ABI StepOnePlus connect system (Applied Biosystems, Life Technologies, CA, USA). Fold change in the expression of specific mRNA was calculated using the 2^−ΔΔCt method.

Statistical analysis

Statistical tests were completed using IBM SPSS Statistics (Version 26.0, IBM, Armonk, NY, USA). The Shapiro–Wilk test was used to evaluate the normality of data. Normal data were analyzed by the one-way analysis of variance (ANOVA). When overall differences were significant, the Tukey’s post hoc test for pairwise comparisons was performed to compare the differences between the two groups that only differed in one factor (i.e., immunological stress, stilbene treatment, or inhibitor administration factor). Additionally, the nonnormally distributed data were assessed by the Kruskal–Wallis one-way ANOVA on ranks to verify the null hypothesis that the mean ranks of the groups were the same. When the null hypothesis was rejected, pairwise comparisons were conducted to identify underlying differences between the specific groups as above. Significance level was set at P < 0.05. The results are expressed as mean values and standard errors.

Results

Hepatic injury

LPS-induced a clearly discernible pattern of hepatic damage, as indicated by the presence of severe vacuolation of hepatocytes, massive infiltration of neutrophils and inflammatory macrophages, and hemorrhage (Figure 1A). Consistently, statistically significant increases were observed for activities of plasma ALT (P = 0.027; Figure 1C) and AST (P = 0.001; Figure 1D), numbers of hepatic apoptotic cells (P = 0.001; Figure 1B and E), and activity of Casp-3 (P = 0.021; Figure 1F) in the LPS-exposed piglets compared with their nonchallenged counterparts, further demonstrating the severity of liver injury caused by LPS challenge. In contrast, both stilbenes significantly prevented the increase in plasma AST activity (P = 0.047 for RSV; P = 0.015 for PT) and hepatic histopathological alterations caused by LPS, but PT showed significantly better efficacy compared to RSV. Furthermore, PT, but not RSV, significantly decreased plasma ALT activity (P = 0.043) and hepatic apoptosis rate (P = 0.045).

Figure 1.

Effects of resveratrol and pterostilbene on hepatic injury of lipopolysaccharide-challenged piglets. (A and B) Representative photographs of H&E and TUNEL staining from liver sections. (C and D) Plasma ALT and AST activities. (E) Quantitative analysis of the TUNEL-positive cells in the liver slices. (F) Hepatic Casp-3 activity. The results are expressed as mean values and standard errors represented by a vertical bar, n = 6. ¹One-way ANOVA test. ^aSignificant difference compared with CS group. ^bSignificant difference compared with CL group.

Plasma cytokine contents

The effectiveness of stilbenes in inhibiting inflammation in piglets was tested by measuring the levels of several proinflammatory cytokines. Compared with the CS piglets, significantly greater contents of circulating TNF-α (P = 0.031) and IL-1β (P = 0.001; Table 1) were observed in the CL piglets. In contrast, the plasma IL-1β content was significantly lower in the RL piglets (P = 0.023) than in the CL piglets, and this effect was further reduced in the PL piglets (P = 0.001).

Table 1.

Effects of resveratrol and pterostilbene on plasma cytokine contents in weaned piglets challenged with lipopolysaccharide¹

Items2	CS	CL	RL	PL
TNF-α3 (pg/mL)	315.58 ± 44.68	520.54 ± 69.16a	447.93 ± 38.02	360.82 ± 30.99
IL-1β3 (pg/mL)	129.13 ± 15.00	426.52 ± 82.28a	226.22 ± 16.01b	141.49 ± 26.75b
IL-63 (pg/mL)	158.94 ± 42.28	308.58 ± 50.16	256.79 ± 34.48	283.50 ± 31.48
IL-183 (pg/mL)	69.62 ± 14.26	107.19 ± 10.11	113.63 ± 28.71	73.15 ± 11.90

¹Values are means ± SE, n = 6.

²CL = piglets received a basal diet and injected with lipopolysaccharide; CS = piglets received a basal diet and injected with sterile saline; IL-1β = interleukin 1 beta; IL-6 = interleukin 6; IL-18 = interleukin 18; PL = piglets received a pterostilbene-supplemented diet and injected with lipopolysaccharide; RL = piglets received a resveratrol-supplemented diet and injected with lipopolysaccharide; TNF-α = tumor necrosis factor alpha.

³One-way ANOVA test.

^a P < 0.05 compared with CS group.

^b P < 0.05 compared with CL group.

Hepatic gene expression

Relative to CS group, the CL group exhibited obvious increases in the expression levels of hepatic TNF-α (P = 0.005), IL-1β (P < 0.001), and IL-6 (P < 0.001) mRNA (Table 2). Conversely, PT attenuated the LPS-induced increases in hepatic IL-1β (P < 0.001) and IL-6 (P = 0.008) mRNA expression. A decrease in the mRNA abundance of hepatic IL-1β (P = 0.046) was also observed in the PL piglets compared with the RL piglets. Additionally, LPS significantly increased hepatic toll-like receptor 4 (TLR4; P = 0.015) expression, whereas neither stilbenes had any effect on this parameter (P > 0.05).

Table 2.

Effects of resveratrol and pterostilbene on the expression levels of hepatic genes related to inflammatory response in weaned piglets challenged with lipopolysaccharide¹

Items2	CS	CL	RL	PL
TLR43	1.00 ± 0.20	2.69 ± 0.39a	2.02 ± 0.49	1.55 ± 0.27
MyD884	1.00 ± 0.15	1.93 ± 0.30	1.34 ± 0.33	1.00 ± 0.15
TRAF63	1.00 ± 0.16	1.47 ± 0.36	0.74 ± 0.16	0.89 ± 0.23
IRAK-13	1.00 ± 0.20	1.60 ± 0.25	1.26 ± 0.22	0.99 ± 0.17
TNF-α4	1.00 ± 0.20	3.79 ± 0.82a	1.79 ± 0.30	1.40 ± 0.25
IL-1β3	1.00 ± 0.25	7.64 ± 0.83a	5.29 ± 0.91	2.73 ± 0.24b,c
IL-63	1.00 ± 0.12	3.56 ± 0.45a	2.79 ± 0.24	1.98 ± 0.31b
IL-103	1.00 ± 0.16	1.63 ± 0.39	1.77 ± 0.37	1.89 ± 0.35
IL-184	1.00 ± 0.53	1.57 ± 0.60	1.15 ± 0.39	1.17 ± 0.41

¹Values are means ± SE, n = 6.

²CL = piglets received a basal diet and injected with lipopolysaccharide; CS = piglets received a basal diet and injected with sterile saline; IL-1β = interleukin 1 beta; IL-6 = interleukin 6; IL-10 = interleukin 10; IL-18 = interleukin 18; IRAK-1 = interleukin 1 receptor associated kinase 1; MyD88 = myeloid differentiation factor 88; PL = piglets received a pterostilbene-supplemented diet and injected with lipopolysaccharide; RL = piglets received a resveratrol-supplemented diet and injected with lipopolysaccharide; TLR4 = toll-like receptor 4; TNF-α = tumor necrosis factor alpha.

³One-way ANOVA test.

⁴Nonparametric Kruskal–Wallis test.

^a P < 0.05 compared with CS group.

^b P < 0.05 compared with CL group.

^c P < 0.05 compared with RL group.

Hepatic myeloperoxidase activity and its protein expression

The activity and expression of MPO in the liver were determined to monitor the severity of neutrophil infiltration and inflammation (Rensen et al., 2009). Administration of LPS elevated hepatic MPO at both the protein expression (P = 0.020; Figure 2B) and enzyme activity levels (P = 0.001; Figure 2C). However, PT had a lowered effect on hepatic MPO activity (P = 0.010), and this effect was not detected in the RL group (P > 0.05).

Figure 2.

Effects of resveratrol and pterostilbene on hepatic myeloperoxidase expression and its activity of lipopolysaccharide-challenged piglets. (A) Representative photographs of MPO protein expression in the liver slices. (B) Hepatic MPO protein level. (C) Hepatic MPO protein activity. The expression of MPO protein was determined by quantifying its fluorescence intensity with the Image J software. The results are expressed as mean values and standard errors represented by a vertical bar, n = 6. ¹Nonparametric Kruskal–Wallis test. ²One-way ANOVA test. ^aSignificant difference compared with CS group. ^bSignificant difference compared with CL group.

Hepatic redox status

In addition to inflammation, oxidative stress is considered a major factor that promotes the development of inflammatory liver injury (Matsuzawa et al., 2007). The enhanced levels of MPO activity and its protein expression implied the occurrence of oxidative stress in the LPS-exposed livers, since MPO activity is linked to both inflammation and oxidative stress (Pulli et al., 2015). Compared with the CS piglets, the CL piglets exhibited a pronounced increase (P < 0.001) in hepatic superoxide anion accumulation (Table 3; Supplementary Figure S1), while piglets treated with either stilbene (P = 0.029 for RSV; P = 0.004 for PT) displayed significantly less superoxide anion production in their livers. In comparison with the CS group, the CL group exhibited substantially increased levels of MDA (P = 0.042) and 8-OHdG (P = 0.018) in their livers. Stilbene treatment inhibited the LPS-induced increase in hepatic MDA content, but PT (P = 0.024) was more effective than RSV (P > 0.05).

Table 3.

Effects of resveratrol and pterostilbene on hepatic redox metabolite levels and antioxidant enzyme activities in weaned piglets challenged with lipopolysaccharide¹

Items2	CS	CL	RL	PL
Redox metabolites
DHE3 (fold to CS group)	1.00 ± 0.06	2.24 ± 0.27a	1.49 ± 0.17b	1.28 ± 0.10b
MDA3 (nmol/mg protein)	0.57 ± 0.05	0.85 ± 0.09a	0.66 ± 0.06	0.54 ± 0.07b
8-OHdG3 (ng/mg DNA)	1.86 ± 0.23	3.50 ± 0.40a	2.70 ± 0.40	2.28 ± 0.36
GSH3 (nmol/100 mg wet weight)	116.94 ± 8.42	92.89 ± 7.04	106.17 ± 7.19	117.11 ± 7.35
GSSG3 (nmol/100 mg wet weight)	5.20 ± 0.60	9.60 ± 1.10a	8.77 ± 1.25	4.68 ± 0.60b,c
GSH:GSSG4 (nmol/nmol)	24.15 ± 3.80	10.54 ± 1.77a	13.07 ± 1.65	27.10 ± 3.55b,c
Antioxidant enzymes
SOD3 (U/mg protein)	140.95 ± 7.92	134.17 ± 5.23	145.00 ± 6.99	155.24 ± 4.39
CAT3 (U/mg protein)	140.43 ± 15.52	165.57 ± 7.76	182.94 ± 13.04	222.10 ± 17.02b
GPx3 (U/mg protein)	53.11 ± 4.66	56.58 ± 6.54	76.67 ± 5.42	63.78 ± 3.40
GR4 (U/g protein)	18.31 ± 2.53	13.61 ± 1.07	15.01 ± 1.89	16.55 ± 2.17

¹Values are means ± SE, n = 6.

²CAT = catalase; CL = piglets received a basal diet and injected with lipopolysaccharide; CS = piglets received a basal diet and injected with sterile saline; DHE = dihydroethidium; DNA = deoxyribonucleic acid; GPx = glutathione peroxidase; GR = glutathione reductase; GSH = reduced glutathione; GSSG = oxidized glutathione; MDA = malondialdehyde; 8-OHdG = 8-hydroxy-2-deoxyguanosine; PL = piglets received a pterostilbene-supplemented diet and injected with lipopolysaccharide; RL = piglets received a resveratrol-supplemented diet and injected with lipopolysaccharide; SOD = superoxide dismutase.

³One-way ANOVA test.

⁴Nonparametric Kruskal–Wallis test.

^a P < 0.05 compared with CS group.

^b P < 0.05 compared with CL group.

^c P < 0.05 compared with RL group.

Sensitive biomarkers of enzymatic and nonenzymatic antioxidant defense were then measured to determine the mechanism by which stilbenes alleviated hepatic oxidative stress (Table 3). LPS treatment significantly increased GSSG (P = 0.016) content in the liver, resulting in an obvious decrease in the hepatic GSH:GSSG (P = 0.026) ratio in the CL group. However, stilbene treatment prevented the excessive accumulation of hepatic GSSG induced by LPS, but PT (P = 0.007) was more effective than RSV (P > 0.05), which may explain the concurrent increase in GSH:GSSG (P = 0.007) ratio in the PL livers. Compared with the RL group, the PL group exhibited a decreased GSSG (P = 0.027) content and an increased GSH:GSSG (P = 0.042) ratio in the liver. RSV treatment also tended to enhance hepatic GPx (P = 0.053) activity, whereas PT significantly promoted hepatic CAT (P = 0.041) activity when compared with the LPS-treated piglets not supplemented with stilbenes.

Hepatic inflammatory signal activities

The NF-κB pathway is a crucial inflammatory signaling required for the release of inflammatory mediators. Thus, we examined the nuclear accumulation of NF-κB p65 protein, a sensitive and reliable indicator of the active levels of NF-κB signals (Pradere et al., 2016). Compared with the CS piglets, the protein expression of nuclear NF-κB p65 (P < 0.001; Figure 3B) was markedly higher in the livers of CL piglets; however, this increase was reversed by substituting PT (P < 0.001) for RSV (P > 0.05). LPS challenge induced a robust increase in the accumulation of hepatic NLRP3 (P = 0.001; Figure 3C) and cleaved Casp-1 (P < 0.001; Figure 3D) proteins, but this effect were prevented by PT (P = 0.006 for NLRP3; P < 0.001 for cleaved Casp-1). RSV decreased hepatic cleaved Casp-1 (P = 0.025) expression, but it failed to affect the expression of NLRP3 (P > 0.05). Furthermore, the PL group showed decreased expression levels of hepatic nuclear NF-κB p65 (P = 0.001) and cleaved Casp-1 (P = 0.011) in comparison with the RL group.

Figure 3.

Effects of resveratrol and pterostilbene on hepatic inflammatory signal activities of lipopolysaccharide-challenged piglets. (A) Representative bands of western blotting analysis. (B–D) Hepatic protein expression of NF-κB p65, NLRP3, and cleaved Casp-1 was measured by western blotting. The results are expressed as mean values and standard errors represented by a vertical bar, n = 4. ¹One-way ANOVA test. ^aSignificant difference compared with CS group. ^bSignificant difference compared with CL group. ^cSignificant difference compared with RL group. ACTB = beta actin.

Inflammatory response of tumor necrosis factor alpha-stimulated alpha mouse liver 12 cells

We explored how PT exerts its antiinflammatory action by extending our analysis to an in vitro model system. First, the effect of PT on cell viability was determined. The addition of PT at concentrations of 50 μM or lower for 24 h had no adverse effect on cell viability (P > 0.05; Supplementary Figure S2A). Subsequently, the proinflammatory cytokine TNF-α was included to evaluate the potential of PT against inflammatory response. The AML-12 cells treated with TNF-α alone or plus varying amounts of PT (1, 2.5, 5, 10, 25, and 50 μM, safe concentrations) showed similar (P > 0.05) viabilities (Supplementary Figure S2B) to the vehicle-treated control cells. PT (2.5 and 5 μM) effectively inhibited the increase in mRNA abundance of TNF-α (P < 0.05; Supplementary Figure S2C), which occurred upon stimulation of the control cells by TNF-α. Notably, the upregulations of IL-1β (P = 0.026; Supplementary Figure S2D) and IL-18 (P = 0.019; Supplementary Figure S2F) mRNA by TNF-α treatment were prevented by PT at 5 μM. Therefore, 5 μM PT was selected for further investigation of its potential to alleviate the activation of NF-κB signals and the NLRP3 inflammasome.

Protein phosphatase 2A mediates the inhibition of pterostilbene on nuclear factor kappa B signals in tumor necrosis factor alpha-stimulated alpha mouse liver 12 cells

Recent evidence indicates that stilbenes can activate protein phosphatases, such as PP1 and PP2A (Shati and Alfaifi, 2019), which are known to negatively regulate the NF-κB signaling pathway via dephosphorylation (Zhang et al., 2019). We tested the potential role of protein phosphatases in the PT-induced inhibition of NF-κB signals by coincubating AML-12 cells with PT and specific phosphatase inhibitors. TA is a specific PP1 inhibitor, whereas FO is a highly selective inhibitor of PP2A and PP2A-like phosphatases (Yan et al., 2008). PT significantly inhibited the TNF-α-mediated increase in the phosphorylation of NF-κB p65 protein (P = 0.048; Figure 4D), an essential event in nuclear translocation of that protein (Jiang et al., 2003). PT treatment, therefore, reduced the nuclear accumulation of NF-κB p65 protein (P = 0.015; Figure 4A and C), in agreement with the findings shown in Figure 3B. Interestingly, this beneficial action by PT was abrogated after FO treatment (P = 0.029 for the nuclear accumulation of NF-κB p65 protein; P = 0.043 for the phosphorylation of NF-κB p65 protein), whereas TA treatment did not suppress the antiinflammatory effect of PT (P > 0.05). These findings suggested that PP2A, rather than PP1, maybe a critical regulator that mediates the inhibitory action of PT on NF-κB signals.

Figure 4.

Inhibiting protein phosphatase 2A activity affects the effect of pterostilbene on nuclear factor kappa B signals in alpha mouse liver 12 cells. (A) Representative photographs of the immunofluorescent staining of NF-κB p65 protein in AML-12 cells. (B) Representative bands of western blotting analysis. (C and D) Nuclear NF-κB p65 protein accumulation and cellular NF-κB p65 phosphorylated levels were measured by western blotting. The results are expressed as mean values and standard errors represented by a vertical bar, n = 3. ¹One-way ANOVA test. ^aSignificant difference compared with CON group. ^bSignificant difference compared with TNF group. ^cSignificant difference compared with TNF-PT group. ^dSignificant difference compared with TNF-PT-TA group. CON = control treatment.

Discussion

This study presents novel evidence that PT is an effective agent for protecting piglets from inflammatory liver injury. In particular, PT ameliorates LPS-induced liver inflammation and resultant oxidative damage, possibly by inhibiting the sustained activation of NF-κB signals and the NLRP3 inflammasome. In vitro cell studies further indicate that PT may inhibit NF-κB activation in a PP2A-dependent manner. Thus, these findings support the possibility that PT could serve as a potent agent against inflammatory liver disorders.

Stilbenes, including RSV, have antiinflammatory roles in various diseases (Yang and Lim, 2014; Tsai et al., 2017; Cao et al., 2020; Olcum et al., 2020); however, some evidence indicates that PT, the dimethylether analog of RSV, appears to be a preferred candidate for the treatment of acute liver inflammation. The present study indicated that PT at a dose æmediator expression and activity resulting from LPS exposure. The overproduction of inflammatory mediators is a critical event in the progression of liver diseases (Fang et al., 2018; Xue et al., 2021). Decreases in inflammatory mediator accumulation in the liver can inhibit intrahepatic immune cell activation and their recruitment in inflamed tissues, thereby preventing the sustained activation of specific intracellular pathways involved in the inflammatory response and cell death (Tacke et al., 2009). However, the present study found that supplementation with RSV at a dose of 300 mg/kg failed to inhibit the overproduction of inflammatory mediators in the LPS-exposed piglets. In a rodent study investigating the protective effects of RSV and PT against steatohepatitis, oral administration of PT at a low dose of 15 mg/kg/d was capable of alleviating liver inflammation, an effect comparable with RSV at 30 mg/kg/d (Gómez-Zorita et al., 2020), which may be due to the superior intestinal absorption and enhanced hepatic stability of methylated stilbenes (Wen and Walle, 2006). These benefits of PT, together with its better pharmacokinetic characteristics, may explain its protective action to alleviate inflammatory liver injury in LPS-challenged piglets.

The PT response appears to involve an interruption of NF-κB signals as a potential mechanism underlying the antiinflammatory action. Activation of the NF-κB signals requires a series of phosphorylation steps; however, PT could inhibit the hyperphosphorylation of the p65 subunit and its regulators, including inhibitor of κB (IκB) and IκB kinase (IKK; Yao et al., 2018; Liu et al., 2019). Under basal conditions, NF-κB (consisting of subunits p50 and p65) is bound to the cytoplasmic IκB in an inactive state. Upon stimulation, IKK induces a rapid phosphorylation and proteolytic degradation of IκB, which frees the p65 subunit to translocate to the nucleus (Hansberger et al., 2007). Of note, the phosphorylation of p65 is also essential for its nuclear translocation and subsequent transcriptional regulation (Moreno et al., 2010). Therefore, we inferred from previous observations that PT may blunt NF-κB signals through a dephosphorylation mechanism mediated by protein phosphatases (Shati and Alfaifi, 2019).

We showed, using specific inhibitors of the protein phosphatases, that PT inhibited the activation of NF-κB signals in TNF-α-exposed cells in essentially a PP2A-dependent manner. This finding adds novel insight into the beneficial roles of PT in alleviating inflammatory liver diseases. First, PP2A is a negative regulator of the NF-κB feedback loop, as it dephosphorylates IKK, IκBα, and p65 directly (DiDonato et al., 1997; Hsieh et al., 2011). Second, PP2A can form a complex with transforming growth factor-β-activated kinase 1 (TAK1), which is an upstream-activating kinase of IKK (Su et al., 2011). Formation of this complex disrupts the interaction between TAK1 and IKK, thereby indirectly inactivating NF-κB signals. For this reason, PP2A is now considered an important therapeutic target for inflammatory diseases (Clark and Ohlmeyer, 2019). Thus, the activation of PP2A by PT may serve to mitigate other inflammation-related diseases, which deserves further investigation.

Nuclear factor kappa B signaling is a prerequisite for the activation of the NLRP3 inflammasome; therefore, the mechanism by which PT inhibits the upregulation of NLRP3 and cleaved Casp-1 may involve its inhibitory action on the NF-κB pathway (Szabo and Petrasek, 2015). PT has been reported to inactivate the NLRP3 inflammasome in several different disease models, including early brain injury (Liu et al., 2017), diabetic cardiomyopathy (Kosuru et al., 2018), neuroinflammation (Li et al., 2018), acute liver failure (Zhang et al., 2020d), and pulmonary fibrosis (Yang et al., 2020). PT has also been reported to ameliorate NLRP3 inflammasome activation by suppressing the expression of microRNA-377, which drives inflammation and oxidative stress in fructose-induced podocyte injury (Wang et al., 2015). The results obtained here substantiate these previous observations and provide further evidence to support the powerful antiinflammatory properties of PT.

In addition to their antiinflammatory roles, stilbenes show antioxidant properties that contribute to the mitigation of liver injury. Excessive free radicals can trigger leukocyte accumulation in the involved tissues and ultimately potentiate hepatocyte death and liver damage (Maresh et al., 2020). In this study, both RSV and PT inhibited superoxide anion production in LPS-treated livers. Stilbenes contain hydroxyl groups, so they can act as scavengers of free radicals. The 4ʹ-hydroxyl group determines the radical-scavenging efficiency of stilbenes because it is the preferred reaction site due to the resonance effects induced by aromatic rings (Ovesna and Horvathova-Kozics, 2005).

PT treatment essentially prevented the lipid peroxidation caused by LPS, but RSV treatment did not induce a similar suppression. This difference may be associated with the higher lipophilicity imparted by the 3,5-dimethoxy structure of PT (Yeo et al., 2013). Mikstacka et al. (2010) have reported that PT is more favorable than RSV as a scavenger of lipid peroxyl radicals, and this could explain why PT exhibited comparable or even better antioxidant properties than its parent compound, despite having only one hydroxyl group in the 4ʹ position.

We found that stilbenes could activate hepatic antioxidant defenses under conditions of LPS exposure. PT significantly promoted hepatic CAT activity in the LPS-treated piglets, while a tendency toward increased GPx activity was observed in the livers of the RL piglets. Both GPx and CAT degrade hydrogen peroxide, but GPx has a greater affinity for hydrogen peroxide than CAT under normal conditions (Jones et al., 1981). However, as the accumulation of hydrogen peroxide increases, CAT makes a greater contribution to hydrogen peroxide degradation under the conditions of oxidative stress (Verkerk and Jongkind, 1992). An increase in CAT activity can decrease the formation of GSSG and preserve glutathione in its reduced state, thereby possibly explaining the higher GSH:GSSG ratio in the livers of the PL piglets. PT failed to promote significant hepatic SOD activity when compared with the CL group, although the data showed a strong trend toward significance. As an important detoxification enzyme, SOD catalyzes the conversion of the highly reactive, unstable superoxide anion to less hazardous hydrogen peroxide, which is then eliminated by either GPx or CAT. PT treatment also prevented the LPS-induced increase in hepatic MPO activity in the piglets. MPO is an important neutrophil enzyme that can generate aggressive oxidants (Rensen et al., 2009). The decrease in MPO activity by PT may also assist in the recovery from oxidative stress by preventing the overproduction of oxygen radicals.

In summary, the findings presented here provide new insights into the beneficial roles of stilbenes in alleviating liver injury and immunological stress in piglets challenged with LPS. In particular, PT, the natural dimethyl ether analog of RSV, exhibits significantly better efficacy than its parent comæof stilbene-mediated antiinflammatory mechanisms and promote the development of novel feeding strategies for young piglets under stressful conditions.

Glossary

Abbreviations

ACTB

beta actin

ALT

alanine aminotransferase

AST

aspartate aminotransferase

Casp-1

caspase 1

Casp-3

caspase 3

CAT

catalase

CL

piglets received a basal diet and injected with lipopolysaccharide

CS

piglets received a basal diet and injected with sterile saline

DAPI

4ʹ-6-diamidino-2-phenylindole

ELISA

enzyme linked immunosorbent assay

FO

fostriecin

GPx

glutathione peroxidase

GR

glutathione reductase

GSH

reduced glutathione

GSSG

oxidized glutathione

H&E

hematoxylin and eosin

IκB

inhibitor of kappa B

IKK

inhibitor of kappa B kinase

IL-1β

interleukin 1 beta

IL-6

interleukin 6

IL-18

interleukin 18

LPS

lipopolysaccharide

MDA

malondialdehyde

MPO

myeloperoxidase

NF-κB

nuclear factor kappa B

NLRP3

Nod-like receptor pyrin domain containing 3

8-OHdG

8-hydroxy-2-deoxyguanosine

PL

piglets received a pterostilbene-supplemented diet and injected with lipopolysaccharide

PP2A

protein phosphatase 2A

PT

pterostilbene

RL

piglets received a resveratrol-supplemented diet and injected with lipopolysaccharide

RSV

resveratrol

SOD

superoxide dismutase

TA

tautomycetin

TAK1

transforming growth factor-β-activated kinase 1

TNF-α

tumor necrosis factor alpha

TUNEL

terminal deoxynucleotidyl transferase-mediated 2ʹ-deoxyuridine-5ʹ-triphosphate nick end labeling

Conflict of Interest Statement

The authors declare no conflict of interest.