Inhibition of nitric oxide and inflammatory cytokines in LPS-stimulated murine macrophages by resveratrol, a potent proteasome inhibitor

Table 5 δ-Tocotrienol and riboflavin inhibit expression of TNF-α, IL-1β, IL-6, and iNOS genes in LPS-stimulate RAW 264.7 cells, and peritoneal macrophages derived from C57BL/6 or BALB/c mice ¹

NO	Treatments	RT-PCR data (Ratios of digital values of optical density of gene expression of cytokines/β-actin).
NO	Treatments	TNF-α	IL-1β	IL-6	iNOS
RAW 264.7 cells.
1	Media + Cells = A	0.05	0.05	0.1	0.2
2	A + LPS (10 ng/well) = B	0.83	1.35	0.98	1.12
3	B + 0.2 % DMSO = C	0.80 ± 0.03^a (100)²	1.32 ± 0.03^a (100)²	0.96 ± 0.03^a (100)²	1.04 ± 0.02^a (100)²
4	C + δ-Tocotrienol (16.0 μM)	0.32 ± 0.03^c (40)	0.33 ± 0.03^c (25)	0.41 ± 0.04^c (43)	0.42 ± 0.04^c (40)
5	C + Riboflavin (16.0 μM)	0.64 ± 0.04^b (80)	0.95 ± 0.03^b (72)	0.82 ± 0.04^b (85)	0.76 ± 0.03^b (73)
8-week-old C57BL/6
1	Media + Cells (macrophages) = A	0	0	0	0
2	A + LPS (10 ng/well) = B	0.35	5.32	11.67	4.97
3	B + 0.4 % DMSO = C	0.36 ± 0.02^a (100)²	6.89 ± 0.86^a (100)²	12.34 ± 1.1^a (100)²	5.67 ± 0.67^a (100)²
4	C + δ-Tocotrienol (40.0 μM)	0.16 ± 0.08^c (45)	2.34 ± 0.42^c (34)	1.98 ± 0.24^c (16)	2.12 ± 0.56^c (56)
5	C + Riboflavin (40.0 μM)	0.22 ± 0.02^b (61)	4.15 ± 0.68^b (60)	7.23 ± 0.98^b (59)	4.16 ± 0.53^b (73)
8-week-old BALB/c
1	Media + Cells (macrophages) = A	0	0	0	0
2	A + LPS (10 ng/well) = B	0.62	8.27	14.45	7.76
3	B + 0.4 % DMSO = C	0.74 ± 0.05^a (100)²	10.67 ± 1.24^a (100)²	15.67 ± 1.45^a (100)²	9.85 ± 0.88^a (100)²
4	C + δ-Tocotrienol (40.0 μM)	0.39 ± 0.01^c (53)	4.16 ± 0.36^c (39)	3.45 ± 0.28^c (22)	6.64 ± 0.86^b (67)
5	C + Riboflavin (40.0 μM)	0.54 ± 0.03^b (73)	6.45 ± 0.91^b (60)	4.73 ± 0.22^b (30)	7.64 ± 0.67^b (78)

¹RAW 264.7 cells or thioglycolate-elicited peritoneal macrophages from 8-week-old C57BL/6 or BALB/c mice were treated with δ-tocotrienol or riboflavin (16 μM or 40 μM) for 1 h, followed by treatment with LPS (10 ng/well) for 4 h. Total RNA was extracted, reverse transcribed, and the resulting DNA was amplified and analyzed by real time PCR to quantitate expression of TNF-α, IL-1β, IL-6, and iNOS genes [5, 21].
Cell viability was >95 % in all the treatments. Data are means ± SD, n = 3 (triplicate analysis of each sample).
²Percentages of digital values of relative optical density of each cytokine/β-actin of each treatment are in parenthesis. ^a-cValues in a column not sharing a common superscript letter are significantly different at P < 0.05.

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