Protopanaxadiol-Enriched Rice Exerted Antiadipogenic Activity during 3T3-L1 Differentiation and Anti-Inflammatory Activity in 3T3-L1 Adipocytes

Ginseng is a traditional medicine with health benefits for humans. Protopanaxadiol (PPD) is an important bioactive compound found in ginseng. Transgenic rice containing PPD has been generated previously. In the present study, extracts of this transgenic rice were evaluated to assess their antiadipogenic and anti-inflammatory activities. During adipogenesis, cells were treated with transgenic rice seed extracts. The results revealed that the concentrations of the rice seed extracts tested in this study did not affect cell viability at 3 days post-treatment. However, the rice seed extracts significantly reduced the accumulation of lipids in cells and suppressed the activation of CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ), which in turn inhibited the expression of adipogenesis-related mRNAs, such as adiponectin, PPARγ, C/EBPα, sterol regulatory element-binding protein 1, glucose transport member 4, and fatty acid synthase. In adipocytes, the extracts significantly reduced the mRNA expression of inflammation-related factors following LPS treatment. The activation of NF-κB p65 and ERK 1/2 was inhibited in extract-treated adipocytes. Moreover, treatment with extract #8 markedly reduced the cell population of the G2/M phase. Collectively, these results indicate that transgenic rice containing PPD may act as an obesity-reducing and/or -preventing agent.


Introduction
Excess energy can be stored in the form of lipids in adipocytes; however, the excessive accumulation of fat in the body is characterized as obesity, which is a major medical problem [1,2] and a serious global health concern [3]. Obesity contributes to several chronic diseases [4] and increases the risk of diabetes [5], cardiovascular disease [6], prostate disease [7], and respiratory disease [8].

Figure 1.
Overview of the experimental procedure and the timeline of adipogenesis (dpt, days posttreatment). Cells were collected for RNA extraction at 3, 5, 7, and 9 dpt (cell collection).

Cell Viability Assay
After differentiation for 3 days, the cells were collected for use in the analysis of the cytotoxic effects of extracts. Cell viability was analyzed using the 10% EZ-Cytox Cell Viability Assay Kit (DoGenBio, Seoul, Republic of Korea) in accordance with the manufacturer's instructions.

Oil Red O Assay
The cells were fixed with 3.7% formalin (Sigma-Aldrich) at room temperature for 30 min. After discarding the formalin solution, the remaining solution was removed using distilled water. Subsequently, 100 µL of 60% isopropanol was added to the cells, which were then incubated at room temperature in the absence of light for 5 min. The cells were then stained with Oil Red O solution (0.3% (v/v), Sigma-Aldrich), incubated at room temperature (in the absence of light) for 15 min, washed with distilled water to remove the excess dye, and allowed to dry at room temperature by opening the plate cover. The stained lipid droplets were imaged under an IM-3 series microscope. The intracellular lipid content was measured by eluting the dye with 60% isopropanol using a SpectraMax ® ABS Plus (Molecular Devices, San Jose, CA, USA) at 500 nm. Intracellular lipid accumulation was calculated in reference to the medium group as follows: Lipid accumulation (%) = A 500 of the test group -A 500 of the blank A 500 of the control -A 500 of the blank × 100 (1) where A500 is the absorbance at 500 nm.

RNA Extraction and Real-Time PCR Analysis
Differentiated 3T3-L1 cells were collected at 3, 5, 7, and 9 dpt and washed with icecold 1× PBS. Total RNA was isolated using TRI Reagent™ solution (Invitrogen, Waltham, MA, USA). The RNA pellet was dissolved in nuclease-free water and kept at −80 °C. The concentration and purity of the extracted RNA were measured using a SpectraDrop™ microvolume microplate (Molecular Devices). cDNA was synthesized from 1 µg of total RNA using a Power cDNA Synthesis Kit (iNtRON Biotechnology, Inc., Gyeonggi, Republic of Korea). The PCR reactions and conditions used were similar to those described in a previous study [29]. The specific primer sets used in this study are shown in Table S1; βactin was used as the reference gene. Overview of the experimental procedure and the timeline of adipogenesis (dpt, days post-treatment). Cells were collected for RNA extraction at 3, 5, 7, and 9 dpt (cell collection).

Cell Viability Assay
After differentiation for 3 days, the cells were collected for use in the analysis of the cytotoxic effects of extracts. Cell viability was analyzed using the 10% EZ-Cytox Cell Viability Assay Kit (DoGenBio, Seoul, Republic of Korea) in accordance with the manufacturer's instructions.

Oil Red O Assay
The cells were fixed with 3.7% formalin (Sigma-Aldrich) at room temperature for 30 min. After discarding the formalin solution, the remaining solution was removed using distilled water. Subsequently, 100 µL of 60% isopropanol was added to the cells, which were then incubated at room temperature in the absence of light for 5 min. The cells were then stained with Oil Red O solution (0.3% (v/v), Sigma-Aldrich), incubated at room temperature (in the absence of light) for 15 min, washed with distilled water to remove the excess dye, and allowed to dry at room temperature by opening the plate cover. The stained lipid droplets were imaged under an IM-3 series microscope. The intracellular lipid content was measured by eluting the dye with 60% isopropanol using a SpectraMax ® ABS Plus (Molecular Devices, San Jose, CA, USA) at 500 nm. Intracellular lipid accumulation was calculated in reference to the medium group as follows: Lipid accumulation (%) = A 500 of the test group − A 500 of the blank A 500 of the control − A 500 of the blank × 100 (1) where A 500 is the absorbance at 500 nm.

RNA Extraction and Real-Time PCR Analysis
Differentiated 3T3-L1 cells were collected at 3, 5, 7, and 9 dpt and washed with icecold 1× PBS. Total RNA was isolated using TRI Reagent™ solution (Invitrogen, Waltham, MA, USA). The RNA pellet was dissolved in nuclease-free water and kept at −80 • C. The concentration and purity of the extracted RNA were measured using a SpectraDrop™ microvolume microplate (Molecular Devices). cDNA was synthesized from 1 µg of total RNA using a Power cDNA Synthesis Kit (iNtRON Biotechnology, Inc., Gyeonggi, Republic of Korea). The PCR reactions and conditions used were similar to those described in a previous study [29]. The specific primer sets used in this study are shown in Table S1; β-actin was used as the reference gene.

Western Blotting Analysis
Western blotting analysis was performed according to the method of Monmai et al. [28]. The membranes were incubated at 4 • C overnight with the following antibodies: PPARγ (Santa Cruz Biotechnology, Dallas, TX, USA), C/EBPα (Santa Cruz Biotechnology), phos-Pharmaceutics 2023, 15, 2123 4 of 16 phorylated (p)-ERK 1/2 (Cell Signaling Technology, Danvers, MA, USA), ERK 1/2 (Cell Signaling Technology), p-Akt (Cell Signaling Technology), and Akt (Santa Cruz Biotechnology). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Santa Cruz Biotechnology) was used as the protein loading control. Protein signaling was detected using Clarity Max™ Western ECL Substrate (Bio-Rad, Hercules, CA, USA). ChemiDoc Imaging System (Bio-Rad) was used for detecting, imaging, and quantifying signals in terms of intensity. Protein expression was presented in terms of an expression ratio to the medium group (% fold).

3T3-L1 Cell Differentiation and LPS-Stimulated Adipocyte Viability Assay
The anti-inflammatory effects of rice seed extracts in 3T3-L1 adipocytes were measured, as shown in Figure 2, in the absence of treatment. On day 9, adipocytes were treated with treatments prepared in a cell culture medium. At 1 h after treatment, the cells were stimulated with 1 µg/mL LPS. Cell viability was determined as described in Section 2.4.

Western Blo ing Analysis
Western blo ing analysis was performed according to the method of Monmai et al. [28]. The membranes were incubated at 4 °C overnight with the following antibodies: PPARγ (Santa Cruz Biotechnology, Dallas, TX, USA), C/EBPα (Santa Cruz Biotechnology), phosphorylated (p)-ERK 1/2 (Cell Signaling Technology, Danvers, MA, USA), ERK 1/2 (Cell Signaling Technology), p-Akt (Cell Signaling Technology), and Akt (Santa Cruz Biotechnology). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Santa Cruz Biotechnology) was used as the protein loading control. Protein signaling was detected using Clarity Max™ Western ECL Substrate (Bio-Rad, Hercules, CA, USA). ChemiDoc Imaging System (Bio-Rad) was used for detecting, imaging, and quantifying signals in terms of intensity. Protein expression was presented in terms of an expression ratio to the medium group (% fold).

3T3-L1 Cell Differentiation and LPS-Stimulated Adipocyte Viability Assay
The anti-inflammatory effects of rice seed extracts in 3T3-L1 adipocytes were measured, as shown in Figure 2, in the absence of treatment. On day 9, adipocytes were treated with treatments prepared in a cell culture medium. At 1 h after treatment, the cells were stimulated with 1 µg/mL LPS. Cell viability was determined as described in Section 2.4.

Real-Time PCR and Western Blo ing in LPS-Stimulated Adipocytes
Total RNA and protein were extracted from LPS-stimulated adipocytes at 6 and 24 h after LPS stimulation, respectively, using the method described in Section 2.6. The sequences of the primer sets used are shown in Table S1. Western blo ing was performed as described in Section 2.7. The specific antibodies used for western blo ing were p-ERK 1/2 (Cell Signaling Technology), ERK 1/2 (Cell Signaling Technology), p-NF-κB p65 (Cell Signaling Technology), NF-κB p65 (Santa Cruz Biotechnology), and GAPDH (Santa Cruz Biotechnology).

Cell Cycle Assay
The cells were treated with MDI and rice seed extract #8 (100 µg/mL) in 48-well plates (6 × 10 4 cells/well). After 20 h, the cells were harvested in a 5 mL tube and stained with propidium iodide (PI). In brief, the cells were washed with PBS and fixed with 70% EtOH at 4 °C for 2 h. Then, the fixed cells were washed with cold PBS and stained using the FxCycle TM PI/RNase staining solution (Invitrogen, MA, USA). The stained cells were quantified via flow cytometry (FACS canto II, BD Biosciences, San Jose, CA, USA). Data analyses were conducted using FlowJo software version 10.9.0.

Statistical Analysis
Experimental data are presented as mean ± standard deviation. Statistix8.1 software (Statistix, Tallahassee, FL, USA) was used for statistical analysis. Significant differences

Real-Time PCR and Western Blotting in LPS-Stimulated Adipocytes
Total RNA and protein were extracted from LPS-stimulated adipocytes at 6 and 24 h after LPS stimulation, respectively, using the method described in Section 2.6. The sequences of the primer sets used are shown in Table S1. Western blotting was performed as described in Section 2.7. The specific antibodies used for western blotting were p-ERK 1/2 (Cell Signaling Technology), ERK 1/2 (Cell Signaling Technology), p-NF-κB p65 (Cell Signaling Technology), NF-κB p65 (Santa Cruz Biotechnology), and GAPDH (Santa Cruz Biotechnology).

Cell Cycle Assay
The cells were treated with MDI and rice seed extract #8 (100 µg/mL) in 48-well plates (6 × 10 4 cells/well). After 20 h, the cells were harvested in a 5 mL tube and stained with propidium iodide (PI). In brief, the cells were washed with PBS and fixed with 70% EtOH at 4 • C for 2 h. Then, the fixed cells were washed with cold PBS and stained using the FxCycle TM PI/RNase staining solution (Invitrogen, MA, USA). The stained cells were quantified via flow cytometry (FACS canto II, BD Biosciences, San Jose, CA, USA). Data analyses were conducted using FlowJo software version 10.9.0.

Statistical Analysis
Experimental data are presented as mean ± standard deviation. Statistix8.1 software (Statistix, Tallahassee, FL, USA) was used for statistical analysis. Significant differences were determined using one-way analysis of variance (ANOVA), followed by the application of Duncan's multiple-range tests. Differences were considered significant if p < 0.05.
Statistical analysis of the cell cycle was performed using SPSS ver. 22 (SPSS, Chicago, IL, USA). Student's t test was used to make comparisons between the two groups, and one-way ANOVA with Duncan's multiple-range test was used to make comparisons among more than two groups.

Effects of PPD-Containing Rice Seed Extracts on Cell Viability
The toxicity exerted by PPD-containing rice seed extracts on 3T3-L1 cells was evaluated at 3 dpt. The cells were incubated with the EZ-Cytox Cell Viability Assay Kit (DoGenBio, Seoul, Republic of Korea) in accordance with the manufacturer's instructions. An MDI medium served as the control in the untreated group (medium). Treatment with 0.1% dimethyl sulfoxide (DMSO) resulted in cell viability equivalent to that of untreated cells ( Figure 3). Compared with treatment with the medium, all concentrations of transgenic rice seed extracts and normal rice seed extracts (DJ) significantly enhanced cell proliferation. PPD-containing rice seed extracts at concentrations up to 100 µg/mL had no negative effects on cell viability. were determined using one-way analysis of variance (ANOVA), followed by the application of Duncan's multiple-range tests. Differences were considered significant if p < 0.05. Statistical analysis of the cell cycle was performed using SPSS ver. 22 (SPSS, Chicago, IL, USA). Student's t test was used to make comparisons between the two groups, and one-way ANOVA with Duncan's multiple-range test was used to make comparisons among more than two groups.

Effects of PPD-Containing Rice Seed Extracts on Cell Viability
The toxicity exerted by PPD-containing rice seed extracts on 3T3-L1 cells was evaluated at 3 dpt. The cells were incubated with the EZ-Cytox Cell Viability Assay Kit (Do-GenBio, Seoul, Republic of Korea) in accordance with the manufacturer's instructions. An MDI medium served as the control in the untreated group (medium). Treatment with 0.1% dimethyl sulfoxide (DMSO) resulted in cell viability equivalent to that of untreated cells ( Figure 3). Compared with treatment with the medium, all concentrations of transgenic rice seed extracts and normal rice seed extracts (DJ) significantly enhanced cell proliferation. PPD-containing rice seed extracts at concentrations up to 100 µg/mL had no negative effects on cell viability.

Effects of PPD-Containing Rice Seed Extracts on Lipid Accumulation
Cells treated with the adipogenic medium were assigned to the positive control group (medium). Lipid accumulation in 0.1% DMSO-treated cells was similar to that in medium-treated cells ( Figure 4). However, treatment with either normal (DJ) or transgenic rice seed extracts at the lowest concentration (25 µg/mL) significantly reduced lipid accumulation, and lipid accumulation gradually decreased further as the extract concentration increased. Lipid accumulation in cells treated with 100 µg/mL of extracts #8, #503, #557, #564, and #595 was markedly lower than that in cells treated with the DJ extract. The PPD

Effects of PPD-Containing Rice Seed Extracts on Lipid Accumulation
Cells treated with the adipogenic medium were assigned to the positive control group (medium). Lipid accumulation in 0.1% DMSO-treated cells was similar to that in mediumtreated cells (Figure 4). However, treatment with either normal (DJ) or transgenic rice seed extracts at the lowest concentration (25 µg/mL) significantly reduced lipid accumulation, and lipid accumulation gradually decreased further as the extract concentration increased. Lipid accumulation in cells treated with 100 µg/mL of extracts #8, #503, #557, #564, and #595 was markedly lower than that in cells treated with the DJ extract. The PPD content in the seeds was significantly correlated with the reduction in lipid accumulation in the cells (Pearson's correlation coefficient = 0.752). Therefore, PPD-containing rice seed extracts appeared to suppress adipogenesis by reducing lipid accumulation in a dose-dependent manner.
Pharmaceutics 2023, 15, 2123 6 of 16 content in the seeds was significantly correlated with the reduction in lipid accumulation in the cells (Pearson's correlation coefficient = 0.752). Therefore, PPD-containing rice seed extracts appeared to suppress adipogenesis by reducing lipid accumulation in a dose-dependent manner.

Effects of PPD-Containing Rice Seed Extracts on Cell Differentiation
After preadipocytes reached confluence (designated as 0 dpt), change was chemically induced in the cells to obtain mature adipocytes. Before the induction of differentiation using MDI medium, the preadipocytes appeared as fibroblasts. At 3 dpt, the cells were visibly rounder. A small number of lipid droplets was detected in medium-, DMSO-, and DJ-treated cells at 5 dpt, and lipid droplets were clearly visible at 7 dpt ( Figure 5). However, lipid accumulation was slightly delayed due to treatment with PPD-producing rice seed extracts, i.e., it was clearly visible at 7 dpt.

Effects of PPD-Containing Rice Seed Extracts on Cell Differentiation
After preadipocytes reached confluence (designated as 0 dpt), change was chemically induced in the cells to obtain mature adipocytes. Before the induction of differentiation using MDI medium, the preadipocytes appeared as fibroblasts. At 3 dpt, the cells were visibly rounder. A small number of lipid droplets was detected in medium-, DMSO-, and DJ-treated cells at 5 dpt, and lipid droplets were clearly visible at 7 dpt ( Figure 5). However, lipid accumulation was slightly delayed due to treatment with PPD-producing rice seed extracts, i.e., it was clearly visible at 7 dpt.

Effects of PPD-Containing Rice Seed Extracts on Adipogenic Gene Expression
The expression levels of PPARγ, SREBP-1, and C/EBPα were evaluated. Treatment with 100 µg/mL of rice seed extracts (either normal or transgenic rice) markedly decreased MDI-induced PPARγ, C/EBPα, and SREBP-1 mRNA expression in the early stage of

Effects of PPD-Containing Rice Seed Extracts on Adipogenic Gene Expression
The expression levels of PPARγ, SREBP-1, and C/EBPα were evaluated. Treatment with 100 µg/mL of rice seed extracts (either normal or transgenic rice) markedly decreased MDI- induced PPARγ, C/EBPα, and SREBP-1 mRNA expression in the early stage of adipogenesis (3 dpt), and the expression remained suppressed at 9 dpt (Figure 6a-c). Because the expression of transcription factors was reduced, the expression of adipocyte markers and fat-forming genes was also reduced (adiponectin, Glut4, and FAS) (Figure 6d-f). Compared with DJ, treatment with PPD-containing rice seed extracts had the potential to reduce the mRNA expression of adipogenesis-related transcription factors and biomarkers from 3 to 9 dpt. In particular, 100 µg/mL of extract #8 (with the highest PPD content) suppressed the expression of adipogenic transcription factor genes, adiponectin, Glut4, and FAS, most potently out of those examined. adipogenesis (3 dpt), and the expression remained suppressed at 9 dpt (Figure 6a-c). Because the expression of transcription factors was reduced, the expression of adipocyte markers and fat-forming genes was also reduced (adiponectin, Glut4, and FAS) ( Figure  6d-f). Compared with DJ, treatment with PPD-containing rice seed extracts had the potential to reduce the mRNA expression of adipogenesis-related transcription factors and biomarkers from 3 to 9 dpt. In particular, 100 µg/mL of extract #8 (with the highest PPD content) suppressed the expression of adipogenic transcription factor genes, adiponectin, Glut4, and FAS, most potently out of those examined.

Effects of PPD-Containing Rice Seed Extracts on Protein Expression
The protein expression of PPARγ and C/EBPα was upregulated in adipogenic-induced cells (medium group) and 0.1% DMSO-treated cells (Figure 7a). Conversely, treatment with either normal (DJ) or transgenic rice seed extracts significantly reduced the protein expression of PPARγ and C/EBPα. Furthermore, the protein expression of PPARγ and C/EBPα in cells treated with PPD-containing rice seed extracts was markedly lower than that seen in cells treated with normal rice. The cells treated with extract #8 had the lowest PPARγ expression (75.24% and 67.14% decreases compared with the medium and DJ groups, respectively) and the lowest C/EBPα expression (68.30% and 48.00% decreases compared with the medium and DJ groups, respectively).

Effects of PPD-Containing Rice Seed Extracts on Protein Expression
The protein expression of PPARγ and C/EBPα was upregulated in adipogenic-induced cells (medium group) and 0.1% DMSO-treated cells (Figure 7a). Conversely, treatment with either normal (DJ) or transgenic rice seed extracts significantly reduced the protein expression of PPARγ and C/EBPα. Furthermore, the protein expression of PPARγ and C/EBPα in cells treated with PPD-containing rice seed extracts was markedly lower than that seen in cells treated with normal rice. The cells treated with extract #8 had the lowest PPARγ expression (75.24% and 67.14% decreases compared with the medium and DJ groups, respectively) and the lowest C/EBPα expression (68.30% and 48.00% decreases compared with the medium and DJ groups, respectively).
Cells treated with PPD-containing rice seed extracts had markedly suppressed levels of phosphorylated(p)-ERK 1/2, and p-Akt (Figure 7b). The largest downregulation of p-ERK 1/2 and p-Akt protein expression was observed following treatment with extract #8, which had the highest PPD content.  Cells treated with PPD-containing rice seed extracts had markedly suppressed levels of phosphorylated(p)-ERK 1/2, and p-Akt (Figure 7b). The largest downregulation of p-ERK 1/2 and p-Akt protein expression was observed following treatment with extract #8, which had the highest PPD content.

Effects of PPD-Containing Rice Seed Extracts on the Viability of LPS-Stimulated Adipocytes
As shown in Figure 8, treatment with 1 µg/mL LPS significantly increased cell proliferation compared with the medium-treated group (p < 0.05). However, no significant difference (p < 0.05) was observed in terms of the cell viability of LPS-stimulated adipocytes between the medium and DMSO groups. Treatment with rice seed extracts (100 µg/mL) markedly enhanced the proliferation of LPS-stimulated adipocytes compared with the DMSO group (p < 0.05). Therefore, a concentration of 100 µg/mL was selected for the subsequent experiments.

Effects of PPD-Containing Rice Seed Extracts on the Viability of LPS-Stimulated Adipocytes
As shown in Figure 8, treatment with 1 µg/mL LPS significantly increased cell proliferation compared with the medium-treated group (p < 0.05). However, no significant difference (p < 0.05) was observed in terms of the cell viability of LPS-stimulated adipocytes between the medium and DMSO groups. Treatment with rice seed extracts (100 µg/mL) markedly enhanced the proliferation of LPS-stimulated adipocytes compared with the DMSO group (p < 0.05). Therefore, a concentration of 100 µg/mL was selected for the subsequent experiments.

Effects of PPD-Containing Rice Seed Extracts on Immune-Related mRNA in LPS-Stimulated Adipocytes
Treatment with LPS significantly increased the expression level of inflammation-involved genes in medium-treated cells (Figure 9). The pretreatment of cells with PPD-enriched rice seed extracts (100 µg/mL) caused a statistically significant decrease in the expression levels of LPS-induced inflammatory genes. The mRNA expression of TNF-α, COX-2, IL-6, and IL-1β was reduced by 59.19%, 59.06%, 55.20%, and 66.37%, respectively, in the cells treated with #8 and LPS compared with the level seen in cells treated with LPS treatment alone.

Effects of PPD-Containing Rice Seed Extracts on Immune-Related mRNA in LPS-Stimulated Adipocytes
Treatment with LPS significantly increased the expression level of inflammationinvolved genes in medium-treated cells (Figure 9). The pretreatment of cells with PPDenriched rice seed extracts (100 µg/mL) caused a statistically significant decrease in the expression levels of LPS-induced inflammatory genes. The mRNA expression of TNF-α, COX-2, IL-6, and IL-1β was reduced by 59.19%, 59.06%, 55.20%, and 66.37%, respectively, in the cells treated with #8 and LPS compared with the level seen in cells treated with LPS treatment alone.

Effects of PPD-Containing Rice Seed Extracts on Cell Cycle
Differentiated adipocytes display cell cycle inhibition in the G2/M phase [31]. Collectively, the data indicated that extract #8 exhibited the strongest antiadipogenic and anti-inflammatory activities. The population of cells in the G2/M phase was evaluated in cells treated with #8. We checked each cell cycle phase, i.e., G1, S, and G2/M, by performing staining with PI. MDI-treated differentiated cells had a high proportion of cells in the G2/M phase, as expected ( Figure 11). Interestingly, the induction of the G2/M phase was inhibited by treatment with PPD-producing rice seed extracts, especially extract #8. Important factors related to the induction of adipocyte differentiation, such as cell cycle progression to the G2/M phase, were clearly regulated by PPD-producing transgenic rice seed extract #8.

Effects of PPD-Containing Rice Seed Extracts on Cell Cycle
Differentiated adipocytes display cell cycle inhibition in the G2/M phase [31]. Collectively, the data indicated that extract #8 exhibited the strongest antiadipogenic and antiinflammatory activities. The population of cells in the G2/M phase was evaluated in cells treated with #8. We checked each cell cycle phase, i.e., G1, S, and G2/M, by performing staining with PI. MDI-treated differentiated cells had a high proportion of cells in the G2/M phase, as expected ( Figure 11). Interestingly, the induction of the G2/M phase was inhibited by treatment with PPD-producing rice seed extracts, especially extract #8. Important factors related to the induction of adipocyte differentiation, such as cell cycle progression to the G2/M phase, were clearly regulated by PPD-producing transgenic rice seed extract #8.

Discussion
The antiadipogenic activities of PPD-containing rice seed extracts were evaluated in 3T3-L1 cells. The cytotoxicity of PPD-containing rice seed extracts in 3T3-L1 cells was assessed to determine a suitable concentration for use in our antiadipogenic investigation. Low concentrations (25 and 50 µg/mL) of PPD-containing rice seed extracts promoted cell proliferation (Figure 3), whereas high concentrations (150 and 250 µg/mL) reduced cell viability. Therefore, the antiadipogenic effects of PPD-containing rice seed extracts were assessed using extract concentrations up to 100 µg/mL. Cotreatment with IBMX and dexamethasone induces the differentiation of preadipocytes by regulating the expression of PPARγ [32]. The expression of PPARγ is induced by insulin in the final stage of adipogenesis [33]. Rosen et al. [34] reported that PPARγ promoted adipogenesis when combined with C/EBPα; however, adipogenesis was not induced in C/EBPα-deficient cells in the absence of PPARγ. In the present study, the expression of PPARγ and C/EBPα (mRNA and protein) was promoted in preadipocytes cultured with MDI medium (Figures 6a,b and 7), whereas treatment with PPD-producing rice seed extracts significantly reduced the expression levels of PPARγ and C/EBPα. These findings

Discussion
The antiadipogenic activities of PPD-containing rice seed extracts were evaluated in 3T3-L1 cells. The cytotoxicity of PPD-containing rice seed extracts in 3T3-L1 cells was assessed to determine a suitable concentration for use in our antiadipogenic investigation. Low concentrations (25 and 50 µg/mL) of PPD-containing rice seed extracts promoted cell proliferation (Figure 3), whereas high concentrations (150 and 250 µg/mL) reduced cell viability. Therefore, the antiadipogenic effects of PPD-containing rice seed extracts were assessed using extract concentrations up to 100 µg/mL. Cotreatment with IBMX and dexamethasone induces the differentiation of preadipocytes by regulating the expression of PPARγ [32]. The expression of PPARγ is induced by insulin in the final stage of adipogenesis [33]. Rosen et al. [34] reported that PPARγ promoted adipogenesis when combined with C/EBPα; however, adipogenesis was not induced in C/EBPα-deficient cells in the absence of PPARγ. In the present study, the expression of PPARγ and C/EBPα (mRNA and protein) was promoted in preadipocytes cultured with MDI medium (Figures 6a,b and 7), whereas treatment with PPD-producing rice seed extracts significantly reduced the expression levels of PPARγ and C/EBPα. These findings are consistent with those of Zhang et al. [35], who found that the expression of the PPARγ and C/EBPα proteins was suppressed by heated PPD ginsenoside (ginsenoside Rg3). In the present study, the suppression of PPARγ and C/EBPα protein expression affected the accumulation of lipid droplets in the cells, as shown in Figures 7a and 4. Similarly, in a previous study, one of the PPD ginsenosides (ginsenoside Rc) was found to decrease lipid accumulation in cells by suppressing PPARγ and C/EBPα expression [36].
The treatment of cells with PPD-containing rice seed extracts also led to the regulation of the ERK 1/2 pathway (Figure 7b), which is an important pathway for adipogenesis [37]. Our results showed that the expression levels of p-ERK 1/2 were significantly suppressed in the cells treated with PPD-containing rice seed extracts compared with the normal rice group (DJ). These results were similar to those of Belmonte et al. [38], who suggested that the activation of PPARγ and C/EBPα is associated with p-ERK 1/2. Prusty et al. [39] reported that the activation of ERK 1/2 enhanced adipogenesis by increasing the expression of PPARγ and C/EBPα. In addition, Donzelli et al. [40] highlighted the importance of ERK 1/2 in the adipogenic differentiation of human mesenchymal stem cells. In addition to p-ERK 1/2 (the MAPK pathway), Akt induces adipocyte differentiation [41]. As shown in Figure 7b, the activation of Akt (p-Akt) was significantly decreased in the PPD-producing rice seed extract treatment groups, similar to the findings of Li et al. [42]. Li et al. [42] reported that their sample exhibited antiadipogenic activity via the downregulation of major adipogenic differentiation-related genes and Akt protein expression.
Adipokines and adipocytokines are induced during the regulation of adipogenesis [43]. We demonstrated that the expression of Glut4 and Fas was upregulated in PPARγ-and C/EBPα-activated cells. Adiponectin levels are known to be increased in differentiated adipocytes depending on the triglyceride content of the adipocytes [44,45]. We found that PPD-containing rice seed extracts inhibited the expression of adipogenic-related mRNAs, including adiponectin, Glut4, and FAS ( Figure 6). These results were similar to those of Zhang et al. [35], who found that ginsenoside Rg3 suppressed the expression of adipogenic markers. Lee et al. [46] studied the in vitro and in vivo antiadipogenic effects of ginsenoside Rg3 and found that ginsenoside Rg3 downregulated the expression levels of adipogenesisassociated mRNA by suppressing the expression of PPARγ.
Obesity has been reported to induce inflammatory cytokine secretion in adipose tissue [47,48], leading to increased risks of type 2 diabetes, hypertension, metabolic syndrome, and hypertriglyceridemia [49]. The activation of the MAPK and NF-κB signaling pathways is the key to the regulation of acute inflammatory responses [50][51][52]. Our study demonstrated that the expression of p-ERK 1/2 (MAPK signaling pathway) and p-NF-κB p65 (NF-κB signaling pathway) was significantly suppressed by cotreatment with LPS and PPD-containing rice seed extracts (100 µg/mL). Further, we showed that this led to the inhibition of the mRNA expression of LPS-induced inflammatory cytokines. The expression of IL-1β and IL-6, which are involved in chronic inflammation associated with obesity [53], was also significantly decreased in cells treated with transgenic rice seed extracts.
Mitotic clonal expansion is one of the most relevant stages of adipocyte differentiation [54]. During the differentiation process, the cell population of the G0/G1 phase decreases, whereas the cell population of the G2/M phase increases [55]. Our result showed that treatment with extract #8 at 100 µg/mL markedly decreased the cell population of the G2/M phase ( Figure 11). Thus, the content of PPD in rice seeds can be considered to cause the inhibition of adipogenesis factors during 3T3-L1 cell differentiation.

Conclusions
In summary, we investigated the antiadipogenic effects of PPD-enriched rice seed extracts on 3T3-L1 cells. The MDI-induced activation of PPARγ and C/EBPα transcription factors was inhibited by treatment with these extracts, and the suppression of these transcription factors led to reductions in several adipogenic markers, including lipid accumulation and the expression of PPARγ, C/EBPα, SREBP-1, adiponectin, Glut4, and FAS. In addition, transgenic rice seed extracts suppressed the LPS-induced activation of p-ERK 1/2 protein expression, p-NF-κB p65 protein expression, and proinflammatory cytokine production. Treatment with extract #8 significantly reduced the cell population of the G2/M phase in MDI-induced cells. The findings of this study indicate that transgenic rice containing PPD may be a potential obesity-reducing and/or -preventing agent.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/pharmaceutics15082123/s1, Table S1: Sequences of the specific primers used for real-time PCR analysis. Data Availability Statement: All applicable data have been provided in the manuscript. The authors will provide additional details if necessary.