Use of Germination to Enhance Resveratrol Content and Its Anti-Inflammatory Activity in Lipopolysaccharide-Stimulated RAW264.7 Cells

Inflammation is triggered by a variety of danger signals and is now a worldwide concern. Resveratrol, a natural nonflavonoid polyphenol found in naturally consumed plants and foods, has a wide spectrum of bioactive potency. We successfully generated resveratrol-enriched rice by introducing the resveratrol biosynthesis gene into Dongjin rice. In this study, resveratrol- and piceid-enriched rice (DJ526) was investigated for its anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated RAW264.7 cells compared to normal rice (DJ). In addition, the 5-day-old germinated DJ526 (DJ526_5) was tested for its anti-inflammatory effects. The piceid and resveratrol amounts increased in DJ526_5 by germination. Treatment of LPS-stimulated RAW264.7 cells with resveratrol-enriched rice seed extracts (DJ526_0 and DJ526_5) significantly decreased the production of nitric oxide (NO) and the inflammatory mediator prostaglandin E2 (PGE2), downregulated proinflammatory gene expression, and inhibited nuclear factor kappa B (NF-κB) p65, p38 mitogen-activated protein kinase, and extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation. These findings demonstrated the anti-inflammatory mechanisms of resveratrol-enriched rice in LPS-stimulated RAW264.7 cells. Furthermore, resveratrol-enriched rice could be a potential source of anti-inflammatory agents.


Introduction
Inflammation is a complex set of interactions between soluble factors and cells that can occur in any tissue in response to a traumatic, infectious, postischemic, toxic, or autoimmune injury [1]. It is a protective biological response to harmful stimuli, pathogens, or irritants in vascular tissues that attempts to eliminate infectious agents [2]. Immune protection against pathogens also involves the activation of various signaling pathways, including the mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) pathways [3]. During infection, the host produces several proinflammatory cytokines that are implicated in disease pathogenesis [4]. Macrophages play essential roles in inflammation [5]. Interleukin-6 (IL-6) is a multifunctional cytokine that promotes B-cell differentiation [6] and has a wide range of biological activities in immune regulation, hematopoiesis, inflammation, and oncogenesis [7]. Furthermore, IL-1β and tumor necrosis factor-alpha (TNF-α) also play crucial roles in inflammation and osteolysis [8]. IL-1β, a proprotein produced by activated macrophages, is an important mediator of the inflammatory response and is involved in a variety of cellular activities, such as cell proliferation, differentiation, and apoptosis [9].
Cytokines are necessary, but their constitutive overproduction or prolonged or chronic inflammation is often involved in a variety of diseases, such as IL-17-driven inflammatory responses in human colon adenocarcinomas [10]. Inflammation promotes tumor development and increases the prevalence of chronic inflammatory lung diseases [11], and proinflammatory factors are associated with type 2 diabetes [12]. Therefore, inhibiting the expression and production of powerful mediators such as IL-6 and TNF-α by anti-inflammatory components could be a preventive or therapeutic target and could be used to develop anti-inflammatory agents for health promotion and disease prevention [13].
Resveratrol-enriched rice was generated through genetic engineering by transferring the Arachis hypogaea stilbene synthase (STS), a resveratrol biosynthesis gene, to Dongjin (DJ) rice [31]. Rodboon et al. [32] demonstrated that germinated riceberry rice exhibited the higher antioxidations and tyrosinase-related melanogenic inhibition effects than the ungerminated riceberry rice. Cho and Lim [33] reported that the residual phenolic acid content in brown rice increased via germination and significantly increased the antioxidant activities when comparison with nongermination brown rice. Therefore, the present study aimed to investigate the effect of germination on the change in resveratrol and piceid contents in DJ526 seeds, as well as the anti-inflammatory activities of germinated and non-germinated DJ526 seed extracts.

The Quantity of Resveratrol Content in Rice Grains
The resveratrol and piceid contents were determined using high-performance liquid chromatography (HPLC) and compared to a calibration standard mixture of resveratrol and piceid. The piceid signal peaked at a retention time of 16.947 min, while the resveratrol signal peaked at a retention time of 27.462 min. Figure 1a shows that the peaks at retention times of 16.947 and 27.462 min were not detected in the chromatograms of the normal rice seed extracts (both DJ_0 and DJ_5). However, piceid and resveratrol peaks were observed in the chromatograms of DJ526_0 and DJ526_5. The piceid and resveratrol amounts in DJ526_0 were 4.724 ± 0.02 and 2.605 ± 0.001 µg/g dry weight (dw), respectively ( Figure 1b). However, the piceid and resveratrol amounts in DJ526_5 seeds (5-day-old germinated seeds) were 16.879 ± 0.024 and 3.230 ± 0.060 µg/g dw, respectively. This finding indicates that piceid and resveratrol production sequentially increased during germination. The piceid and resveratrol amounts in DJ526_5 increased 2.573 ± 0.005-and 0.240 ± 0.023-fold, respectively, when compared to a non-germinated seed (DJ526_0).  The extract's cytotoxicity was assessed in LPS-stimulated RAW264.7 cells by comparing the cell viability of the treated groups to that of the untreated group (RPMI). Figure 2a shows that 1 µg/mL LPS treatment significantly enhanced cell proliferation (the dimethyl sulfoxide [DMSO] group). At concentrations up to 100 µg/mL, none of the extracts were cytotoxic to LPS-stimulated RAW264.7 cells. In addition, cells treated with extracts at concentrations of 25, 50, and 100 µg/mL significantly increased proliferative activity on LPS-stimulated RAW264.7 cells when compared to the untreated group. The inflammation environment was mocked up using the LPS model. The cells treated with 1 µg/mL LPS produced significantly more NO than the untreated group ( Figure 2b). Treatment with both normal and transgenic rice seed extracts significantly reduced NO production. Furthermore, increasing the extract concentrations significantly enhanced the inhibition of LPS-induced NO production. Treatment with 10−100 µg/mL of the resveratrol-enriched rice seed extracts (DJ526_0 and DJ526_5) remarkably reduced NO production when compared to the normal rice (DJ_0 and DJ_5). However, 5-day-old germination of the resveratrol-enriched rice seed extracts (DJ526_5) significantly promoted the inhibition of LPS-induced NO production when compared to the normal transgenic rice seed extract (DJ526_0). These findings indicate that increasing the treatment concentration up to 100 µg/mL inhibited LPS-induced NO production while causing no cell cytotoxicity. Additionally, germination of resveratrol-enriched rice seeds (5 days old) declined NO production significantly when compared to non-germinated seeds.

mRNA Expression Levels of the Proinflammatory Genes in LPS-Stimulated RAW264.7 Cells Treated with Rice Seed Extracts
The mRNA expression levels of LPS-induced proinflammatory genes were determined after six hours of LPS induction. Treatment with 1 µg/mL LPS significantly upregulated inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), IL-1β, IL-6, and TNF-α expressions ( Figure 3). Pretreatment with rice seed extracts (DJ-0, DJ_5, DJ526_0, and DJ526_5) significantly reduced LPS-induced proinflammatory gene expression levels. Compared to DJ_0, treatment with DJ_5 had no effect on proinflammatory gene expression levels, but treatment with DJ526_0 and DJ526_5 powerfully inhibited proinflammatory genes. In addition, the suppressive effect of DJ526_5 on LPS-induced proinflammatory gene expression was significantly greater than that of DJ526_0. These findings indicate that higher piceid and resveratrol amounts exerted a higher inhibitory effect on LPS-induced proinflammatory gene expression.

LPS-Induced Prostaglandin E2 (PGE 2 ) Production in RAW264.7 Cells Treated with Rice Seed Extracts
PGE 2 is a well-known inflammatory mediator [34]. Therefore, PGE 2 production in LPS-stimulated RAW264.7 cells was investigated. Treatment with LPS significantly induced PGE 2 production ( Figure 4). However, pretreatment with rice seed extracts inhibited PGE 2 production. There was no significant difference in PGE 2 production between DJ_0 and DJ_5. Treatment with resveratrol-enriched rice remarkably decreased LPS-induced PGE 2 production compared to normal rice (DJ_0 and DJ_5). Furthermore, DJ526_5-treated cells showed the highest suppression of LPS-induced PGE 2 production. These findings indicate that increasing resveratrol and piceid amounts significantly inhibited PGE 2 in LPS-stimulated RAW264.7 cells.

Inflammation-Related Pathway Activation in LPS-Stimulated RAW264.7 Cells Treated with Rice Seed Extracts
Treatment with LPS (DMSO group) activated the NF-κB and MAPK signaling pathways ( Figure 5) via increasing p-NF-κB p65 (Figure 5a), p-p38 MAPK (Figure 5b), and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK 1/2; Figure 5c) expression levels. Treatment with rice seed extracts significantly reduced these LPS-induced proteins. When compared to normal rice (DJ_0 and DJ_5), transgenic rice seed extracts remarkably decreased p-NF-κB p65, p-p38 MAPK, and p-ERK 1/2 levels. In addition, DJ526_5-treated cells powerfully downregulated p-NF-κB p65, p-p38 MAPK, and p-ERK 1/2 expression levels when compared to DJ526_0-treated cells. These findings indicate that treatment with rice seed extracts potentially suppressed the NF-κB and MAPK signaling pathways. In particular, the inhibition level increased as the piceid and resveratrol amounts in the transgenic rice seeds increased.

Discussion
The piceid and resveratrol contents of rice seeds were measured using HPLC. The chromatograms revealed that the retention time peaks at 16.947 (piceid) and 27.462 (resveratrol) minutes can only be detected in transgenic rice seeds, indicating that piceid and resveratrol cannot be found in normal rice seeds. For transgenic rice, DJ526_0 contained 4.727 ± 0.023 µg/g dw of piceid and 2.605 ± 0.001 µg/g dw of resveratrol, while the piceid and resveratrol contents in DJ526_5 were 16.879 ± 0.024 and 3.230 ± 0.06 µg/g dw, respectively. Piceid amounts were 1.813 ± 0.009 times higher than resveratrol amounts in DJ526_0, and 5.225 ± 0.007 times higher in DJ526_5. Our findings are consistent with those of Kuo et al. [27] and Su et al. [35], who found that piceid amounts are usually much higher than resveratrol amounts. Furthermore, Kuo et al. [36] reported that piceid amounts are 2.82-fold higher than resveratrol amounts in the root of Polygonum cuspidatum. The piceid and resveratrol contents tend to increase in germinated DJ526 (DJ526_5) when compared to nongerminated DJ526. Similarly, Hung and Chen [37] suggested that the amounts of resveratrol and γ-aminobutyric acid increased with germination time. Wang et al. [38] demonstrated that the resveratrol content in the whole sprout of peanuts increased during germination. On Day 9, the resveratrol contents in the whole sprout of Tainan Selected 9 (TNS 9), Tainan 11 (TN 11), and Tainan 14 (TN 14) were 5.38-, 5.71-, and 5.09-fold higher than those on Day 0, respectively. Macrophages can be activated by LPS via multiple signaling pathways, including NF-κB and MAPK [39][40][41]; upregulating proinflammatory cytokines, such as IL-1β, IL-6, and TNF-α; and inflammatory mediators, such as NO, PGE 2 , and iNOS [20,42]. Therefore, LPS is often used to mimic the inflammatory environment in macrophages [43][44][45]. Our findings demonstrated that resveratrol-enriched rice inhibited NF-κB and MAPK signaling pathways by lowering NF-κB p65, ERK 1/2, and p38 MAPK phosphorylation. This reduced the LPS-induced NO production (Figure 2b), PGE 2 production (Figure 4), and proinflammatory genes (iNOS, COX-2, IL-1β, IL-6, and TNF-α). The increase in piceid and resveratrol contents was correlated with an increase in anti-inflammatory properties. Treatment with DJ526 significantly suppressed NO production in LPS-stimulated cells. Compared to DJ526_0, treatment with DJ526_5, which contains a higher amount of piceid and resver-atrol, significantly reduced NO production in a concentration-dependent manner. Furthermore, NO production was found to be significantly correlated with the amount of piceid (Pearson's correlation, r = −0.710, p = 0.01) and resveratrol (Pearson's correlation, r = −0.890, p = 0.01). The findings are consistent with those from various experiments that found resveratrol to have an inhibitory effect on NO production [46][47][48]. Similarly, Zong et al. [20] found that resveratrol at 1-10 µM significantly reduced ERK 1/2 and p38 MAPK phosphorylation, reducing NO and PGE 2 production, TNF-α, and IL-1β levels, as well as iNOS and COX-2 mRNA and protein expression levels. Zimmermann-Franco et al. [49] reported the inhibition effect of resveratrol on the production of pro-inflammatory mediators such as NO, IL-1β, IL-6, and TNF-α. The anti-inflammatory potential of resveratrol was also demonstrated in an animal model. According to Simão et al. [50], resveratrol reduces neuroinflammation in rats via downregulating NF-κB-related proteins, COX-2, and iNOS. In addition, Zimmermann-Franco et al. [49] demonstrated the in vivo anti-inflammatory effect of resveratrol in a mouse model of croton-oil-induced ear edema. Furthermore, Su et al. [51] found that high-dose resveratrol (150 mg/kg body weight) reduced inflammatory responses in C57BL/6J mice. Consequently, treatment with resveratrol-enriched rice (DJ526_0 and DJ526_5) inhibited LPS-induced inflammatory responses, which correlated with resveratrol content accumulation in transgenic rice.

Plant Materials
DJ and DJ526 rice seeds were received from the Rural Development Administration (Jeonju, Republic of Korea). They were unpeeled and sterilized in a cleaner solution (70% [v/v] of ethanol and 5% [v/v] of hypochlorite) for one hour. The sterilized seeds were divided into two groups. The first group was ground into a fine powder and designated as Day 0 (DJ_0 and DJ526_0). The second group was allowed to germinate in autoclaved water for five days (DJ_5 and DJ526_5). The 5-day-old seeds were collected and ground into a fine powder. The samples were extracted as previously described [52], with the exception that the extraction buffer was changed from 100% methanol to 80% methanol. The extracts were prepared at concentrations of 10, 25, 50, and 100 mg/mL in DMSO and were diluted with cell culture medium at the concentrations of 10, 25, 50, and 100 µg/mL for in vitro experiments.

Piceid and Resveratrol Content Quantification
To determine the piceid and resveratrol content in rice grains, a fine powder of each sample was mixed with 80% methanol (300 mg fine powder: 900 µL of 80% methanol). The mixture was sonicated for 30 min at room temperature. The tube was centrifuged at 10,000× g at 4 • C for 5 min. After centrifugation, the supernatant was collected and filtered through a 0.2 m nylon membrane filter. The filtered supernatant (1 µL) was used for HPLC analysis of piceid and resveratrol amounts on a Waters e2695 (Waters, Milford, MA, USA). HPLC was performed as previously described [53]. Piceid and resveratrol contents were quantified by comparing them to the calibration standard curve ( Figure 6).

RAW264.7 Cell Viability and NO Production
RAW264.7 cells (Korean Cell Line Bank, Seoul, Republic of Korea) were seeded in a 96-well plate at a concentration of 1 × 10 5 cells/well. The plate was maintained in an environment-controlled incubator (37 • C and 5% CO 2 ) for 24 h. The culture medium was replaced with various concentrations of rice seed extracts or 200 µg/mL of aspirin (positive control) [54,55] prepared in the nonphenol red Roswell Park Memorial Institute 1640 medium. After one hour of incubation, cells were stimulated with or without 1 µg/mL LPS. The plate was incubated at 37 • C with 5% CO 2 for another 24 h. The culture medium (100 µL) was transferred to a new 96-well plate. The same volume of Griess reagent (100 µL; Sigma-Aldrich, St. Louis, MO, USA) was added to each well, and the plate was incubated at room temperature (light-protected) for 15 min. The NO production was evaluated by measuring the absorbance at 540 nm and quantified using a standard curve of sodium nitrite [NaNO 2 ; Figure S1; (1)]. For the original plate, an EZ-Cytox Cell Viability Assay Kit (10 µL; DoGenBio, Seoul, Republic of Korea) was added to each well. The plate was incubated at 37 • C for four hours. The cell viability was calculated according to the following Formula (2).
where "x" represents the absorbance value at 540 nm.
Cell viability ratio (%) = Absorbance at 450 nm for the treatment Absorbance at 450 nm for the control × 100 where "control" represents the nontreatment group.

RNA Extraction and cDNA Synthesis
RAW264.7 cells were seeded in a 24-well plate (5 × 10 5 cells/well) and incubated at 37 • C with 5% CO 2 for 24 h. The culture medium was replaced with 100 µg/mL of each treatment. After one hour of incubation, LPS was added to each well to achieve a final concentration of 1 µg/mL, except for the untreated group (RPMI). The treated cells were harvested after six hours of LPS stimulation. They were washed twice with ice-cold 1× phosphate-buffered saline. The total RNA was extracted using Tri reagent™ (Invitrogen, Waltham, MA, USA) at room temperature and precipitated using 100% isopropanol at 4 • C. The RNA pellet was washed in 7% ethanol. The total RNA was quantified and qualified using a SpectraMax ® ABS Plus Microplate Reader (Molecular Devices, San Jose, CA, USA). The extracted RNA (1000 ng) was transcribed into cDNA using a Power cDNA Synthesis Kit (Intron Biotechnology, Seongnam-si, Republic of Korea).  (Table 1) and 5 ng of cDNA template. The PCR condition was conducted as previously described [52]. The gene expression levels (fold changes) were analyzed using the CFX Maestro software (accessed on: 19 May 2023), with β-actin serving as a reference gene.

PGE 2 Production Measurement
The supernatant of the culture medium was collected from each treatment in a 1.5 mL microtube. The tubes were centrifuged at 3000 rpm for 10 min at room temperature. PGE 2 production was measured using a PGE 2 enzyme-linked immunosorbent assay kit (ADI900-001: Enzo Life Sciences, Farmingdale, NY, USA) according to the manufacturer's instructions. The PGE 2 production was calculated using a standard curve [ Figure S2; (3)].
where "x" represents the percent-bound value.

Western Blot Analysis
The treated cells were collected in a 1.5 mL microtube and lysed on ice for 30 min in the lysis buffer (radioimmunoprecipitation assay buffer; Geneall Biotechnology, Seoul, Republic of Korea) supplemented with 1× Protease Inhibitor Cocktail Kit 5 (Bio-Medical Science Co., Ltd., Seoul, Republic of Korea). The tubes were centrifuged at 13,000 rpm and 4 • C for 30 min. The supernatant was transferred to the new microtubes. The protein concentration was measured using the Bradford reagent (Sigma-Aldrich, St. Louis, MO, USA). Proteins from each treatment (30 µg) were separated using 10% sodium dodecylsulfate polyacrylamide gel electrophoresis. The separated proteins were transferred onto a nitrocellulose membrane. The primary antibodies specific to p-NF-κB p65 (Cell Signaling, Danvers, MA, USA), NF-κB p65 (Santa Cruz Biotechnology, Dallas, TX, USA), p-p38 MAPK (Cell Signaling, Danvers, MA, USA), p38 MAPK (Santa Cruz Biotechnology, Dallas, TX, USA), p-ERK 1/2 (Cell Signaling, Danvers, MA, USA), ERK 1/2 (Cell Signaling, Danvers, MA, USA), and glyceraldehyde-3-phosphate dehydrogenase (Santa Cruz Biotechnology, Dallas, TX, USA) were applied and incubated at 4 • C for overnight. After that, the secondary antibodies, goat antirabbit IgG (H + L)-horseradish peroxidase (GenDEPOT, Barker, TX, USA) or m-IgGκ BP-horseradish peroxidase (Santa Cruz Biotechnology, Dallas, TX, USA), were applied onto the membrane and incubated at room temperature for one hour. Protein signaling was detected using Clarity™ Western ECL Substrate (Bio-Rad, Hercules, CA, USA), and the detected signals were imaged and quantified in terms of intensity using a ChemiDoc Imaging system (Bio-Rad, Hercules, CA, USA).

Statistical Analysis
The data were expressed as means and standard deviations. All statistical analyses were performed using Statistix (version 8.1; Statistix, Tallahassee, FL, USA) (accessed on 19 May 2023). The data were analyzed using a one-way analysis of variance, followed by post hoc Duncan's multiple range tests. The two groups were compared using Student's t-test (p < 0.05).

Conclusions
In this study, the anti-inflammatory effects of resveratrol-enriched rice (DJ526_0 and DJ526_5) were investigated in LPS-stimulated RAW264.7 cells. The NF-κB and MAPK pathways were inactivated in the DJ526_0-and DJ526_5-treated cells via decreasing p-NF-κB p65, p-ERK 1/2, and p-p38 MAPK, suppressing NO and PGE 2 production, as well as proinflammatory gene expression levels. Furthermore, piceid and resveratrol contents can rise during germination and significantly enhance anti-inflammatory activities in LPSstimulated RAW264.7 cells. We suggest that resveratrol-enriched rice could be developed and used as an anti-inflammatory agent following further research.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules28134898/s1, Figure S1: Standard curve of sodium nitrite over the concentration range of 0-100 µM, Figure S2: Standard curve of prostaglandin E2 (PGE 2 ) over the concentration range of 39.0625-2500 pg/mL.  Data Availability Statement: All applicable data have been provided in the manuscript. The authors will provide additional details if necessary.

Conflicts of Interest:
The authors declare no conflict of interest.
Sample Availability: Samples of the compounds DJ_0, DJ_5, DJ526_0, and DJ526_5 are available from the authors.