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

Figures

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  Figure 1. 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).

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  Figure 2. Overview of the anti-inflammatory experimental procedure in LPS-stimulated adipocytes.

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  Figure 3. Effects of PPD-containing rice seed extracts on the viability of 3T3-L1 cells. Medium: untreated group (MDI medium); DMSO: 0.1% DMSO; DJ: normal rice seed extract; #8: transgenic rice line 8 seed extract; #503: transgenic rice line 503 seed extract; #557: transgenic rice line 557 seed extract; #564: transgenic rice line 564 seed extract; #595: transgenic rice line 595 seed extract. Data are presented as mean ± standard deviation. * Significant difference at p < 0.05 vs. DMSO.

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  Figure 4. Effects of PPD-containing rice seed extracts on lipid accumulation in 3T3-L1 cells. (a) Lipid accumulation ratio following various treatments. Medium: untreated group (adipogenic medium); DMSO: 0.1% DMSO; DJ: normal rice seed extract; #8: transgenic rice line 8 seed extract; #503: transgenic rice line 503 seed extract; #557: transgenic rice line 557 seed extract; #564: transgenic rice line 564 seed extract; #595: transgenic rice line 595 seed extract. (b) Representative images of Oil Red O staining of each treatment at the concentration of 100 µg/mL. Data are presented as mean ± standard deviation. * Significant difference at p < 0.05 vs. DMSO.

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  Figure 5. Representative images of 3T3-L1 cells from induction (Day 0) to 9 days post-induction (Day 9). Medium: untreated group (adipogenic medium); DMSO: 0.1% DMSO; DJ: normal rice seed extract; #8: transgenic rice line 8 seed extract; #503: transgenic rice line 503 seed extract; #557: transgenic rice line 557 seed extract; #564: transgenic rice line 564 seed extract; #595: transgenic rice line 595 seed extract. Blue arrows represent the fibroblast-like cell shape during the differentiation process. Yellow arrows represent the round shape of cells. Red arrows represent the lipid-containing cells.

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  Figure 6. Effects of PPD-containing rice seed extracts (100 µg/mL) on adipogenic-related mRNA expression levels. Expression of (a) PPARγ, (b) C/EBPα, (c) SREBP-1, (d) adiponectin, (e) Glut4, and (f) FAS mRNA. Medium: untreated group; DMSO: 0.1% DMSO; DJ: normal rice seed extract; #8: transgenic rice line 8 seed extract; #503: transgenic rice line 503 seed extract; #557: transgenic rice line 557 seed extract; #564: transgenic rice line 564 seed extract; #595: transgenic rice line 595 seed extract. Data are presented as mean ± standard deviation. Different letters (a–g) indicate significant differences at p < 0.05 among the medium, DMSO, DJ, #8, #503, #557, #564, and #595 groups on the same differentiation day.

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  Figure 7. Effects of PPD-containing rice seed extracts on protein expression in adipocytes. (a) Adipogenic proteins (PPARγ and C/EBPα) and (b) p-ERK 1/2 and p-Akt proteins. Medium: untreated group (adipogenic medium); DMSO: 0.1% DMSO; DJ: normal rice seed extract; #8: transgenic rice line 8 seed extract; #503: transgenic rice line 503 seed extract; #557: transgenic rice line 557 seed extract; #564: transgenic rice line 564 seed extract; #595: transgenic rice line 595 seed extract. Lowercase letters (a–e) indicate significant differences at p < 0.05 among the medium, DMSO, DJ, #8, #503, #557, #564, and #595 groups.

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  Figure 8. Effects of PPD-containing rice seed extracts on the cell viability of LPS-stimulated adipocytes. Medium: untreated group (DMEM); DMSO: 0.1% DMSO; DJ: normal rice seed extract; #8: transgenic rice line 8 seed extract; #503: transgenic rice line 503 seed extracts; #557: transgenic rice line 557 seed extracts; #564: transgenic rice line 564 seed extract; #595: transgenic rice line 595 seed extract. Data are presented as mean ± standard deviation. Significant differences at p < 0.05 were determined compared with the medium (no LPS stimulation; *) and DMSO (#) groups.

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  Figure 9. Effects of PPD-containing rice seed extracts on inflammation-related mRNA expression levels in LPS-stimulated adipocytes. Medium: untreated group (DMEM); DMSO: 0.1% DMSO; DJ: normal rice seed extract; #8: transgenic rice line 8 seed extract; #503: transgenic rice line 503 seed extract; #557: transgenic rice line 557 seed extract; #564: transgenic rice line 564 seed extract; #595: transgenic rice line 595 seed extract. Data are presented as mean ± standard deviation. Difference letters (a–f) indicate significant differences at p < 0.05 among the medium, DMSO, DJ, #8, #503, #557, #564, and #595 groups.

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  Figure 10. Effects of 100 µg/mL of PPD-containing rice seed extracts on the protein expression of p-ERK 1/2 and p-NF-κB p65 in LPS-stimulated adipocytes. Medium: untreated group (DMEM); DMSO: 0.1% DMSO; DJ: normal rice seed extract; #8: transgenic rice line 8 seed extract; #503: transgenic rice line 503 seed extract; #557: transgenic rice line 557 seed extract; #564: transgenic rice line 564 seed extract; #595: transgenic rice line 595 seed extract. Data are presented as mean ± standard deviation. Different letters (a–e) indicate significant differences at p < 0.05 among the medium, DMSO, DJ, #8, #503, #557, #564, and #595 groups.

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  Figure 11. Assessment of cell cycle. The cell cycle was analyzed via flow cytometry. Data are presented as mean ± standard deviation. Medium (−): untreated group (normal medium); Medium (+): untreated group (adipogenic medium); DMSO: 0.1% DMSO; DJ: normal rice seed extract; #8: transgenic rice line 8 seed extract. Lowercase letters (a–c) indicate significant differences at p < 0.05 among the medium (−), medium (+), DMSO, DJ, and #8.