Anti-Inflammatory Efficacy of Resveratrol-Enriched Rice Callus Extract on Lipopolysaccharide-Stimulated RAW264.7 Macrophages

: Resveratrol and its derivative piceid exhibit a wide spectrum of health-promoting bioactivities. A resveratrol-enriched variety of Dongjin rice (DJ526) has been developed by transfection of a resveratrol biosynthesis gene, and increased resveratrol content has been confirmed in seeds following germination. In the current study, these resveratrol-enriched seeds were induced to produce callus, and callus extracts were evaluated for in vitro anti-inflammatory activity. Callus cultures contained greater amounts of resveratrol and piceid than DJ526 seeds, and treatment with DJ526 callus extract significantly reduced the lipopolysaccharide (LPS)-induced production of proinflammatory mediators nitric oxide and prostaglandin E2 by RAW264.7 macrophages. The inflammation-related nuclear factor kappa B and mitogen-activated protein kinase pathways were also inhibited in DJ526 callus extract-treated RAW264.7 cells, resulting in downregulation of proinflammatory factor genes COX-2 , iNOS , IL-1 β , IL-6 , and TNF-α . Expression of the LPS-binding toll-like receptor-4 was also markedly reduced in DJ526 callus extract-treated cells compared to DJ callus extract-treated cells. These findings demonstrate increased resveratrol and piceid content by callus culture of DJ526 rice seeds and the potent anti-inflammatory activity of resveratrol-enriched callus extract.

Resveratrol is a natural polyphenol found in grapes [13], various berries [14], peanuts [15], and plums [16].Resveratrol and its derivative piceid have been demonstrated to promote multiple processes beneficial to health, including antioxidant [17,18], antifungal, antibacterial [19], anticancer [20,21], and anti-inflammatory activities [22][23][24].Moreover, these effects contribute to the observed efficacy of these compounds in models Immuno 2024, 4 132 of Alzheimer's disease [25,26] and Parkinson's disease [27].Resveratrol has been demonstrated to be in vitro and in vivo anti-inflammatory in several studies.Zhong et al. [28] reported that treatments with resveratrol significantly decreased the production of NO and PGE 2 in LPS-stimulated BV-2 cells.In addition, the expression of inflammatory-associated cytokines (iNOS, IL-1β, COX-2, and TNF-α) was also suppressed in LPS-stimulated BV-2 cells when supplemented with resveratrol [28].In LPS-stimulated monocytes, treatment with resveratrol led to inhibition of LPS-induced inflammatory mediators such as TNF-α, IL-8, and monocyte chemoattractant protein-1 (MCP-1) [29].Moreover, an in vivo mouse model exhibited that the long-term treatment of resveratrol in aged mice is able to decrease acute inflammatory stimuli by LPS [30].In 2013, the resveratrol biosynthesis gene Arachis hypogaea stilbene synthase (STS) from the pods of the peanut cultivar Palkwang was introduced into Dongjin rice (DJ) to create resveratrol-enriched rice [31].The AhSTS1 cDNA was inserted between the BamHI and SacI sites under the control of the Ubi1 promoter of the binary vector pSB22.The seed extract from this genetically modified rice (DJ526) was subsequently demonstrated to exert potent and dose-dependent anti-inflammatory activities in LPS-stimulated RAW264.7 macrophages [32].In addition, increasing the resveratrol content in rice seed via germination enhanced anti-inflammatory activities compared to nongerminated DJ526 rice seed [32].Cho and Lim [33] reported that the change in phenylalanine ammonia-lyase and cell wall peroxidase during the gemination of brown rice led to an increase in phenolic acid composition.The increase in phenolic acid content resulted in an enhancement in the antioxidant activity of brown rice.Interestingly, the antioxidant activity of phenolic acid in the shoot fraction was significantly higher than the remaining kernel fraction.From this information, we hypothesized that the resveratrol content would increase in DJ526 rice callus in comparison to the DJ526 rice seed (both germinated and nongerminated seeds).However, the anti-inflammatory activity of resveratrol from the DJ526 rice callus extract needs to be investigated.The increase in resveratrol content in DJ526 rice callus must remain an inflammatory defense activity as well.The resveratrol content of rice seeds may vary annually depending on the growing area or growing environment (light conditions, temperature, rainfall, etc.).Therefore, we have developed the DJ526 rice callus for plant factories to develop a biomaterial with a stable resveratrol content.The current study aimed to further enhance the resveratrol content of DJ526 rice seed by callus induction and evaluate the anti-inflammatory activity of callus extracts on LPS-stimulated RAW264.7 cells.

Callus Culture
Wild-type DJ and DJ526 calluses were generated from the corresponding rice seeds according to the method of Khan et al. [34].Briefly, seeds were sterilized with 70% (v/v) ethanol followed by 2% sodium hypochlorite, washed several times with sterilized distilled water, and induced by inoculation in 2N6 medium at 25 • C for 3 weeks under darkness.Calluses were then cultured in 2MS-NO 3 -free liquid medium for 10 days before collection.

Extraction of Resveratrol-Enrich Compound from Rice Callus
Dried calluses were ground, and a resveratrol-enriched extract was prepared as previously described [32,35].Briefly, callus samples were incubated in 80% methanol, filtered through 5 µm filter paper, concentrated by rotary evaporation, and lyophilized using a freeze-dry system.The lysophilized samples were dissolved in dimethyl sulfoxide (DMSO) at 10, 25, 50, and 100 mg/mL for experiments.

Quantification of Piceid and Resveratrol Content Using High-Performance Liquid Chromatography (HPLC) Analysis
Piceid and resveratrol contents in rice callus extracts were determined according to a previously described method [32,36].Briefly, sample powder was mixed with 80% methanol and sonicated at room temperature for 30 min.The mixture was then centrifuged at 10,000× g and 4 • C for 5 min, and the supernatant was collected, passed through a 0.2 µm filter, and analyzed for piceid and resveratrol content using a Waters e2695 HPLC system (Waters, Milford, MA, USA).Contents were quantified by comparison to a standard curve (Figure S1) generated from known concentrations using Empower software (Empower ® 3; Waters).

Cell Viability and Nitric Oxide Production Analysis
Cells were counted using a hemacytometer and seeded in 96-well plates at 10 5 cells/well, then cultured at 37 • C under a 5% CO 2 atmosphere for 24 h prior to treatment.Extracts were prepared at the indicated concentrations (10,25,50, and 100 µg/mL) in the treatment medium and applied to the indicated wells.Aspirin (Sigma-Aldrich, St. Louis, MO, USA) was prepared in treatment medium at 200 µg/mL and applied as the positive control [37,38].After the indicated treatment for 1 h, the cells were stimulated with LPS at the final concentration of 1 µg/mL (excluding the nontreatment group, which received the same volume of treatment medium).The plate was incubated at 37 • C under a 5% CO 2 atmosphere for 24 h.The culture medium was collected, and nitric oxide (NO) production was measured using the Griess reagent (Sigma-Aldrich), which was prepared in the deionized water at a concentration of 40 mg/mL (working solution).Briefly, the culture medium was mixed with Griess reagent working solution at 1:1 (v/v) in 96-well plates, and the mixtures were incubated at room temperature for 15 min in the dark.Absorbance at 540 nm was measured, and NO production was quantified using a standard curve for sodium nitrite solution (Figure S2).
Viable cells were then counted by adding 110 µL of the EZ-Cytox Cell Viability Assay Kit working solution (10-fold dilution in 1× PBS; DoGenBio, Seoul, Republic of Korea) to each well.After 4 h of incubation at 37 • C, 100 µL of the solution was transferred to new 96-well plates, and absorbance was measured at 450 nm.Cell viability was calculated by comparing the absorbance value of each treatment group to that of parallel control cultures incubated in treatment medium without extract.
2.6.RNA Isolation, RNA Quantification, and cDNA Synthesis Cells were seeded as described in 24-well plates at 500,000/well, incubated for 24 h in maintenance medium, and then incubated in treatment medium with the indicated concentration of extract for 1 h prior to stimulation with 1 µg/mL LPS (or vehicle as a control).After 6 h of stimulation, cells were washed twice with 1× PBS and then treated with TriZol reagent™ (Invitrogen, Waltham, MA, USA) at 500 µL/well for RNA extraction.Chloroform (200 µL) was added to each tube.The tubes were centrifuged at 13,000 rpm at 4 • C for 10 min.The upper phrase of the solution was collected.Total RNA was precipitated with 100% isopropanol at 4 • C for 30 min, and the pellet was washed three times with icecold 70% ethanol.The RNA pellet was then dissolved in nuclease-free water and stored at −80 • C until analysis.Total RNA was quantified using a SpectraMax ® ABS Plus Microplate Reader (Molecular Devices, San Jose, CA, USA) by measuring the 260 nm absorbance, and quality was checked by measuring the 260 nm to 280 nm (A260:A280) absorbance ratio and the A260:A230 ratio.Only samples with ratios of 1.800-2.000for A260:A280 and A260:A230 were processed further (Table 1).First-strand cDNA was synthesized using 1000 ng of total RNA and a Power cDNA Synthesis Kit (Intron Biotechnology, Seongnam-si, Republic of Korea).The cDNA was prepared at 5 ng/µL in nuclease-free water for measurement of inflammation-related mRNA expression levels by real-time quantitative polymerase chain reaction (RT-qPCR).* The coefficient of variation (CV) is the ratio of the standard deviation to the mean.The lower the value of the coefficient of variation, the more precise the estimate.

Measurement of mRNA from Inflammatory Genes by RT-qPCR
The mRNA levels of proinflammatory mediators COX-2 and iNOS, cytokines IL-1β, IL-6, and TNF-α, and the LPS receptor toll-like receptor-4 (TLR-4) were measured using RealMOD™ Green W 2 2× qPCR Mix (Intron Biotechnology, Seongnam-si, Republic of Korea) and a CFX Connect Real-Time PCR System (Bio-Rad, Hercules, CA, USA).Each 20-µL PCR reaction mixture consisted of 5 ng cDNA template and 0.375 M of each primer (Table 2).The thermocycle conditions for PCR are presented in Table 3. β-actin was used as the reference gene.Expression levels were calculated as fold-changes relative to parallel control groups treated with treatment medium alone using CFX Maestro software (Bio-Rad CFX Maestro 1.1).

Prostaglandin E2 (PGE 2 ) Production
The culture medium was collected following the indicated treatment and centrifuged at 3000 rpm for 10 min at room temperature.The PGE 2 concentration in the supernatant was measured using an enzyme-linked immunosorbent assay kit (ADI900-001; Enzo Life Sciences, Farmingdale, NY, USA), and PGE 2 production was calculated using a standard curve provided with the kit (Figure S3).

Statistical Analysis
All data are presented as the mean ± standard deviation.Treatment group means were compared by one-way analysis of variance followed by post hoc Duncan's multiple range tests.A p < 0.05 was considered significant for all tests, and all calculations were performed using Statistix 8.1 (Statistix, Tallahassee, FL, USA).

Piceid and Resveratrol Contents in Rice Callus Extracts
The piceid and resveratrol contents in the extracts of DJ and DJ526 callus extracts were measured by HPLC (Figure S1).The peak retention time for piceid was 16.688 min, while that of resveratrol was 27.182 min.Neither peak was detected in extract samples from DJ callus (Figure 1).In DJ526 extract, the mean (±SD) piceid content was 85.43 ± 3.44 µg/g dry weight, and that of resveratrol was 3.94 ± 0.02 µg/g dry weight.Thus, substantial amounts of these compounds remain in DJ526 rice callus after seed induction.

Piceid and Resveratrol Contents in Rice Callus Extracts
The piceid and resveratrol contents in the extracts of DJ and DJ526 callus extracts were measured by HPLC (Figure S1).The peak retention time for piceid was 16.688 min, while that of resveratrol was 27.182 min.Neither peak was detected in extract samples from DJ callus (Figure 1).In DJ526 extract, the mean (±SD) piceid content was 85.43 ± 3.44 µg/g dry weight, and that of resveratrol was 3.94 ± 0.02 µg/g dry weight.Thus, substantial amounts of these compounds remain in DJ526 rice callus after seed induction.

Effects of Resveratrol-Enriched Rice Callus Extract on the Viability of LPS-Stimulated RAW264.7 Cells
The potential cytotoxic effects of rice callus extracts on LPS-stimulated RAW264.7 cells were examined at extract concentrations ranging from 10 to 100 µg/mL (Figure 2).Treatment with LPS alone modestly but significantly increased the number of viable cells (p < 0.05) compared to parallel cultures of untreated cells (set to 100%), and these increases were maintained in cultures additionally treated with 10 µg/mL DJ callus extract and both 10 and 25 µg/mL DJ526 callus extract (p < 0.05).Further, cell viability did not fall below control (the baseline) in the presence of 100 µg/mL DJ or DJ526 extract, indicating no toxicity within the tested range.In addition, cell viability was not reduced by cotreatment with 200 µg/mL aspirin plus LPS compared to 1 µg/mL LPS alone and was still above baseline (p < 0.05).These findings indicate that extract concentrations of 10, 25, 50, and 100

Effects of Resveratrol-Enriched Rice Callus Extract on the Viability of LPS-Stimulated RAW264.7 Cells
The potential cytotoxic effects of rice callus extracts on LPS-stimulated RAW264.7 cells were examined at extract concentrations ranging from 10 to 100 µg/mL (Figure 2).Treatment with LPS alone modestly but significantly increased the number of viable cells (p < 0.05) compared to parallel cultures of untreated cells (set to 100%), and these increases were maintained in cultures additionally treated with 10 µg/mL DJ callus extract and both 10 and 25 µg/mL DJ526 callus extract (p < 0.05).Further, cell viability did not fall below control (the baseline) in the presence of 100 µg/mL DJ or DJ526 extract, indicating no toxicity within the tested range.In addition, cell viability was not reduced by cotreatment with 200 µg/mL aspirin plus LPS compared to 1 µg/mL LPS alone and was still above baseline (p < 0.05).These findings indicate that extract concentrations of 10, 25, 50, and 100 µg/mL can be safely used for potential suppression of LPS-induced inflammatory activities in RAW264.7 cells.
µg/mL can be safely used for potential suppression of LPS-induced inflammatory activities in RAW264.7 cells.

Resveratrol-Enriched Rice Callus Extract Inhibited LPS-Induced NO Production by RAW264.7 Cells
Treatment of RAW264.7 cells with 1 µg/mL LPS markedly enhanced NO production (Figure 3), and this inflammatory response was substantially and dose-dependently reduced by resveratrol-enriched rice callus extract (DJ526) (all p < 0.05).In fact, the maximum inhibition at 100 µg/mL was comparable to that of 200 µg/mL aspirin (administered as a positive control).Moreover, this dose-dependent anti-inflammatory effect of DJ526 callus extract was markedly greater than that of DJ callus extract at all equivalent doses.These results strongly suggest that piceid and resveratrol enrichment contribute to the anti-inflammatory efficacy of rice callus extract.

Resveratrol-Enriched Rice Callus Extract Inhibited LPS-Induced NO Production by RAW264.7 Cells
Treatment of RAW264.7 cells with 1 µg/mL LPS markedly enhanced NO production (Figure 3), and this inflammatory response was substantially and dose-dependently reduced by resveratrol-enriched rice callus extract (DJ526) (all p < 0.05).In fact, the maximum inhibition at 100 µg/mL was comparable to that of 200 µg/mL aspirin (administered as a positive control).Moreover, this dose-dependent anti-inflammatory effect of DJ526 callus extract was markedly greater than that of DJ callus extract at all equivalent doses.These results strongly suggest that piceid and resveratrol enrichment contribute to the anti-inflammatory efficacy of rice callus extract.

Resveratrol-Enriched Rice Callus Extract Suppressed LPS-Induced Upregulation of Multiple Proinflammatory Genes in RAW264.7 Cells
Treatment with DJ rice callus extract also dose-dependently reduced LPS-induced upregulation of multiple proinflammatory genes (all p < 0.05), including the inflammatory mediator genes iNOS and COX-2, proinflammatory cytokine genes IL-1β, IL-6, and TNFα, and the LPS receptor gene TLR-4 compared to untreated controls (Figure 4).Consistent with effects on NO, DJ526 callus extract evoked markedly greater downregulation of these genes, including iNOS, at all concentrations compared to DJ callus extract (p < 0.05).Moreover, this anti-inflammatory effect was equivalent to or greater than that of aspirin.Thus, enrichment of resveratrol and piceid in rice callus extract substantially enhanced the suppression of LPS-induced inflammatory responses by RAW264.7 cells.

Resveratrol-Enriched Rice Callus Extract Suppressed LPS-Induced Upregulation of Multiple Proinflammatory Genes in RAW264.7 Cells
Treatment with DJ rice callus extract also dose-dependently reduced LPS-induced upregulation of multiple proinflammatory genes (all p < 0.05), including the inflammatory mediator genes iNOS and COX-2, proinflammatory cytokine genes IL-1β, IL-6, and TNF-α, and the LPS receptor gene TLR-4 compared to untreated controls (Figure 4).Consistent with effects on NO, DJ526 callus extract evoked markedly greater downregulation of these genes, including iNOS, at all concentrations compared to DJ callus extract (p < 0.05).Moreover, this anti-inflammatory effect was equivalent to or greater than that of aspirin.Thus, enrichment of resveratrol and piceid in rice callus extract substantially enhanced the suppression of LPS-induced inflammatory responses by RAW264.7 cells.

Resveratrol-Enriched Rice Callus Extract Reduced the Production of PGE2 by LPS-Stimulated RAW264.7 Cells
Treatment with these extracts also significantly reduced LPS-evoked PGE2 production (p < 0.05) in RAW264.7 cells (Figure 5), consistent with downregulation of COX-2, an enzyme required for PGE2 synthesis.Again, the DJ526 callus extract was markedly more

Resveratrol-Enriched Rice Callus Extract Reduced the Production of PGE 2 by LPS-Stimulated RAW264.7 Cells
Treatment with these extracts also significantly reduced LPS-evoked PGE 2 production (p < 0.05) in RAW264.7 cells (Figure 5), consistent with downregulation of COX-2, an enzyme required for PGE 2 synthesis.Again, the DJ526 callus extract was markedly more potent than the DJ callus extract at equivalent concentrations, and maximum effect was comparable to that of aspirin.
Immuno 2024, 4, FOR PEER REVIEW 10 potent than the DJ callus extract at equivalent concentrations, and the maximum effect was comparable to that of aspirin.

Resveratrol-Enriched Rice Callus Extract Inhibited MAPK and NF-κB Pathway Activation in LPS-Stimulated RAW264.7 Cells
Stimulation of RAW264.7 cells with LPS also activated inflammation-associated MAPK pathways, as evidenced by increased phosphorylation of MAPK isoforms ERK-1/2 (p-ERK-1/2) and p38 (p-p38) (Figure 6).Further, LPS stimulation also increased phosphorylation of the NF-κB active unit p65 (p-NF-κB p65).Consistent with downregulation of inflammation-associated genes (Figure 4), many of which are known targets of MAPK and NF-κB signaling, these phosphorylation events were dose-dependently reduced by DJ rice callus extract and more potently by DJ526 rice callus extract.Also consistent with downregulation of target genes, the maximum effect of DJ526 rice callus extract was comparable to that of aspirin.Indeed, 10-100 µg/mL DJ526 rice callus extract suppressed LPS-induced activation of MAPK and NF-κB signaling pathways and the downstream upregulation of multiple proinflammatory factors.Moreover, peak effects at the highest concentration (100 µg/mL) were comparable to those of aspirin and were not associated with any substantial reduction in cell viability (Figure 2), while the higher concentrations (125 and 150 µg/mL) caused a reduction in cell viability (Table S1).Stimulation of RAW264.7 cells with LPS also activated inflammation-associated MAPK pathways, as evidenced by increased phosphorylation of MAPK isoforms ERK-1/2 (p-ERK-1/2) and p38 (p-p38) (Figure 6).Further, LPS stimulation also increased phosphorylation of the NF-κB active unit p65 (p-NF-κB p65).Consistent with downregulation of inflammationassociated genes (Figure 4), many of which are known targets of MAPK and NF-κB signaling, these phosphorylation events were dose-dependently reduced by DJ rice callus extract and more potently by DJ526 rice callus extract.Also consistent with downregulation of target genes, the maximum effect of DJ526 rice callus extract was comparable to that of aspirin.Indeed, 10-100 µg/mL DJ526 rice callus extract suppressed LPS-induced activation of MAPK and NF-κB signaling pathways and the downstream upregulation of multiple proinflammatory factors.Moreover, peak effects at the highest concentration (100 µg/mL) were comparable to those of aspirin and were not associated with any substantial reduction in cell viability (Figure 2), while the higher concentrations (125 and 150 µg/mL) caused a reduction in cell viability (Table S1).

Discussion
Resveratrol exerts anti-inflammatory effects through several signaling pathways, such as NF-κB, MAPK, and the arachidonic acid pathway [39][40][41].Arachidonic acid pathway inhibition plays a major role in the anti-inflammatory activity of resveratrol [41,42].Resveratrol inhibits the activity of COX-1, leading to a reduction in prostaglandin production [43].In PMA-treated human mammary epithelial cells, resveratrol directly inhibits the activity of COX-2, leading to the inhibition of PGE2 production [44].Therefore, resveratrol inhibits inflammatory responses through the arachidonic acid pathway by suppressing the activity of COX-1 and COX-2.The activation of NF-κB by LPS leads to the release of inflammatory mediators such as pro-inflammatory cytokines and NO [45].Resveratrol decreased the expression of TLR-4 (the LPS-associated receptor), leading to the reduction of IL-6, iNOS, and NO by preventing the translocation of NF-κB p65 to the nucleus [46].Resveratrol is able to suppress the inflammatory response by blocking the phosphorylation protein expression of p65 and IκB from the NF-κB signaling as well as phosphorylation of p38 and ERK from MAPK signaling under mastitis conditions [47].
Introduction of the resveratrol synthesis enzyme gene STS from peanut into the genome of Dongjin rice (creating the DJ526 line) markedly enhanced the concentrations of resveratrol and the resveratrol metabolite piceid in both seeds and calluses induced and expanded using 2N6 and 2MS-NO3-free liquid media, respectively.Callus induction dramatically enhanced piceid content to 85.43 ± 3.44 µg/g dry weight from 4.72 ± 0.02 µg/g dry weight in DJ526 rice seeds or 17.10 ± 0.73-fold, and resveratrol content to 3.94 ± 0.02 µg/g dry weight from 2.605 ± 0.001 µg/g dry weight in DJ526 seeds or 1.52 ± 0.01-fold [32].This enrichment markedly enhanced the anti-inflammatory activity of DJ526 callus extract compared to DJ callus extract.Thus, these DJ526-derived calluses are an excellent source

Discussion
Resveratrol exerts anti-inflammatory effects through several signaling pathways, such as NF-κB, MAPK, and the arachidonic acid pathway [39][40][41].Arachidonic acid pathway inhibition plays a major role in the anti-inflammatory activity of resveratrol [41,42].Resveratrol inhibits the activity of COX-1, leading to a reduction in prostaglandin production [43].In PMA-treated human mammary epithelial cells, resveratrol directly inhibits the activity of COX-2, leading to the inhibition of PGE 2 production [44].Therefore, resveratrol inhibits inflammatory responses through the arachidonic acid pathway by suppressing the activity of COX-1 and COX-2.The activation of NF-κB by LPS leads to the release of inflammatory mediators such as pro-inflammatory cytokines and NO [45].Resveratrol decreased the expression of TLR-4 (the LPS-associated receptor), leading to the reduction of IL-6, iNOS, and NO by preventing the translocation of NF-κB p65 to the nucleus [46].Resveratrol is able to suppress the inflammatory response by blocking the phosphorylation protein expression of p65 and IκB from the NF-κB signaling as well as phosphorylation of p38 and ERK from MAPK signaling under mastitis conditions [47].
Introduction of the resveratrol synthesis enzyme gene STS from peanut into the genome of Dongjin rice (creating the DJ526 line) markedly enhanced the concentrations of resveratrol and the resveratrol metabolite piceid in both seeds and calluses induced and expanded using 2N6 and 2MS-NO 3 -free liquid media, respectively.Callus induction dramatically enhanced piceid content to 85.43 ± 3.44 µg/g dry weight from 4.72 ± 0.02 µg/g dry weight in DJ526 rice seeds or 17.10 ± 0.73-fold, and resveratrol content to 3.94 ± 0.02 µg/g dry weight from 2.605 ± 0.001 µg/g dry weight in DJ526 seeds or 1.52 ± 0.01-fold [32].This enrichment markedly enhanced the anti-inflammatory activity of DJ526 callus extract compared to DJ callus extract.Thus, these DJ526-derived calluses are an excellent source of anti-inflammatory resveratrol and piceid for the treatment or prevention of inflammatory diseases.
Based on the demonstrated efficacy of DJ526 rice seed extract enriched in resveratrol and piceid [32] and the further enrichment observed in the extract from callus, we predicted that the DJ526 callus extract would suppress LPS-induced inflammatory activity with high potency.Thus, DJ526 rice callus extract is essentially noncytotoxic within the effective anti-inflammatory range.Further, these anti-inflammatory effects were substantially greater than those of DJ extracts, suggesting that resveratrol and piceid enrichment augment the anti-inflammatory activity of endogenous rice phytochemicals.
Macrophages exhibit different phenotypes at different stages of the inflammatory response [48].Macrophages have at least two different polarizations, the classical (M1) and alternative (M2) [49,50].M1 and M2 macrophages can provide for their biological activities by secreting different cytokines and effector molecules [51].The activation of M1 macrophages is associated with cytokine secretion for antigen defense, including antibacterial, anti-viral, and anti-tumor functions [52].Treatment of DJ526 (without LPS) on macrophage cells significantly increased the expression of pro-inflammatory cytokines such as COX-2, IL-1β, IL-6, and TNF-α (Figure S4 and Table S2).The activation of M2 macrophages relates to the natural inflammation resolution.Therefore, M2 macrophages are usually mentioned as having repair or anti-inflammatory functions [53].Numerous studies have reported that LPS activates the macrophage inflammatory response [54][55][56].Lipopolysaccharide is recognized by TLR-4 and MD-2, which are abundantly expressed by macrophages and other innate immune cells [57][58][59], and stimulation of these receptors activates intracellular cascades such as MAPK and NF-κB signaling pathways.These activated pathways in turn upregulate the expression levels of enzymes that generate proinflammatory factors, such as iNOS, the enzyme generating NO, and COX-2, an enzyme producing prostaglandins such as PGE 2 , as well as proinflammatory cytokines like IL-1β.All of these proinflammatory changes were dose-dependently suppressed by DJ526 rice callus extract and less potently by DJ rice callus extract.Moreover, the reductions evoked by DJ526 callus extract were significantly (p = 0.01) correlated with piceid and resveratrol contents according to Pearson's correlation analysis [TLR-4 (r = −0.92830),iNOS (r = −0.91994),COX-2 (r = −0.91493),IL-1β (r = −0.94282),IL-6 (r = −0.90036),and TNF-α (r = −0.96397)](Figure S5).
These results are consistent with the report by [60] that LPS activates macrophages via cell-surface TLR-4, leading to enhanced production and release of inflammatory cytokines such as TNF-α and IL-6, while resveratrol at 25 µM significantly downregulated the expression of TLR-4, TNF-α, and IL-6 at both mRNA and protein levels [60].Zong et al. [61] also reported that 10 µM resveratrol significantly suppressed TNF-α, COX-2, IL-1β, and iNOS protein and mRNA expression levels, as well as the production of NO and PGE 2 .Similarly, Bigagli et al. [62] reported that 5 and 10 µM resveratrol significantly reduced the production of NO and PGE 2 by LPS-stimulated RAW264.7 cells.Here, we show that a rich natural source of resveratrol (DJ526 rice callus) can suppress LPS-induced macrophage activation without inherent cytotoxicity.Moreover, the decreases in NO and PGE 2 production were again significantly (p = 0.01) correlated with the amount of piceid and resveratrol contained in DJ526 callus extract [r = −0.94071for NO production (Figure S6) and r = −0.97022for PGE 2 production (Figure S7)].

Figure 1 .
Figure 1.Piceid and resveratrol enrichment in DJ526 rice callus extract as measured by high-performance liquid chromatography (HPLC).Sample chromatograms show that piceid and resveratrol are undetectable in DJ rice callus extract (upper left panel) but are abundant in DJ526 rice callus extract (lower left panel).

Figure 1 .
Figure 1.Piceid and resveratrol enrichment in DJ526 rice callus extract as measured by highperformance liquid chromatography (HPLC).Sample chromatograms show that piceid and resveratrol are undetectable in DJ rice callus extract (upper left panel) but are abundant in DJ526 rice callus extract (lower left panel).

Figure 2 .
Figure 2. Low toxicities of DJ and DJ526 callus extracts.Concentrations up to 100 mg/mL had no effect on viable RAW264.7 cell numbers compared to untreated controls.The experiment was performed in triplicate (n = 3 for each replicate).Data are presented as mean ± standard deviation.*p < 0.05 vs. untreated controls; # p < 0.05 vs. cells treated with LPS alone.

Figure 2 .
Figure 2. Low toxicities of DJ and DJ526 callus extracts.Concentrations up to 100 mg/mL had no effect on viable RAW264.7 cell numbers compared to untreated controls.The experiment was performed in triplicate (n = 3 for each replicate).Data are presented as mean ± standard deviation.* p < 0.05 vs. untreated controls; # p < 0.05 vs. cells treated with LPS alone.

Figure 3 .
Figure 3. Suppression of LPS-induced NO production in RAW264.7 cells by DJ and DJ526 callus extracts.The experiment was performed in triplicate (n = 3 for each replicate).Data are presented as mean ± standard deviation.The increase in NO production induced by LPS was dose-dependently inhibited by DJ callus extract and more substantially by DJ526 callus extract enriched in resveratrol and piceid.The NO production by cells treated with LPS alone ("a") is the reference value for statistical comparison.Letters (a-h) indicate significant differences (p < 0.05) between treatments (where a > b > c > d > e > f > g > h).

Figure 3 .
Figure 3. Suppression of LPS-induced NO production in RAW264.7 cells by DJ and DJ526 callus extracts.The experiment was performed in triplicate (n = 3 for each replicate).Data are presented as mean ± standard deviation.The increase in NO production induced by LPS was dose-dependently inhibited by DJ callus extract and more substantially by DJ526 callus extract enriched in resveratrol and piceid.The NO production by cells treated with LPS alone ("a") is the reference value for statistical comparison.Letters (a-h) indicate significant differences (p < 0.05) between treatments (where a > b > c > d > e > f > g > h).

Figure 5 .
Figure 5.The DJ and DJ526 rice callus extracts dose-dependently inhibited LPS-induced PGE2 production by RAW264.7 cells.The experiment was performed in triplicate (n = 2 for each replicate).Data are presented as mean ± standard deviation.The PGE2 production by the LPS-treated cell group ("a") is the reference value for statistical comparison.Letters (a-i) indicate significant differences (p < 0.05) between the treatments (where a > b > c > d > e > f > g > h > i).

Figure 5 .
Figure 5.The DJ and DJ526 rice callus extracts dose-dependently inhibited LPS-induced PGE 2 production by RAW264.7 cells.The experiment was performed in triplicate (n = 2 for each replicate).Data are presented as mean ± standard deviation.The PGE 2 production by the LPS-treated cell group ("a") is the reference value for statistical comparison.Letters (a-i) indicate significant differences (p < 0.05) between the treatments (where a > b > c > d > e > f > g > h > i).

Figure 6 .
Figure 6.Both DJ DJ526 rice callus extracts inhibited LPS-induced activation of MAPK and NF-κB signaling pathways in RAW264.7 cells.(a) Representative western blots.(b-d) Densitometric analyses of (b) p-NF-κB p65 protein expression, (c) p-ERK 1/2 protein expression, and (d) p-p38 protein expression.The experiment was performed in duplicate.Data are presented as mean ± standard deviation.Protein expression levels in cells treated with LPS alone ("a") are the reference values for statistical comparison.Letters (a-c) indicate significant differences (p < 0.05) between treatments (where a > b > c).

Figure 6 .
Figure 6.Both DJ and DJ526 rice callus extracts inhibited LPS-induced activation of MAPK and NF-κB signaling pathways in RAW264.7 cells.(a) Representative western blots.(b-d) Densitometric analyses of (b) p-NF-κB p65 protein expression, (c) p-ERK 1/2 protein expression, and (d) p-p38 protein expression.The experiment was performed in duplicate.Data are presented as mean ± standard deviation.Protein expression levels in cells treated with LPS alone ("a") are the reference values for statistical comparison.Letters (a-c) indicate significant differences (p < 0.05) between treatments (where a > b > c).

Table 1 .
The quantity and quality of extracted RNA from each treatment.

Table 2 .
The primer sets used for RT-qPCR.
Based on the nucleotide position of coding sequences.Immuno 2024, 4

Table 3 .
PCR conditions for estimating mRNA expression levels.