VA inhibits LPS-induced oxidative stress via modulating Nrf2/NF-κB-signalling pathways in bovine mammary epithelial cells

Figures & data

Table 1. Sequences of primers used in real-time polymerase chain reaction (real-time PCR).

Genes^a	GenBank no.	Primer sequences (5′–3′)^b		Length (bp)	Annealing temperature
GAPDH	XM_001252479	F:	GCGCTCTAATGTTCACCTTCC	314	60 °C
		R:	AGCATCACCCAACTTTATGTT
UXT	NM_001037471	F:	CATTGAGCGACTCCAGGAAG	112	60 °C
		R:	GGCCACATAGATCCGTGAAG
18S rRNA	Robinson et al. (2007)	F:	GTAACCCGTTGAACCCCATT	152	60 °C
		R:	CCATCCAATCGGTAGTAGCG
GPx1	NM_174076.3	F:	AGTGCGAGGTGAATGGCGAGAA	328	60 °C
		R:	TGGGCAAAATCCCTGGAGAGCA
GPx4	NM_174770.3	F:	ATCAAAGAGTTCGCCGCTGGCT	295	60 °C
		R:	TCGGAACACAGGCAACAGGCTT
iNOS	NM_001076799.1	F:	TGTCAGCGGCAAGCACCACATT	289	60 °C
		R：	CGGCTGGTTGCATGGGAAAACT
IL-1β	NM_174093.1	F：	GCCTTGGGTATCAAGGACAA	90	60 °C
		R：	TTTGGGGTCTACTTCCTCCA
IL-6	NM_173923.2	F：	ACAAGCGCCTTCACTCCATTCG	242	60 °C
		R：	GCCAGTGTCTCCTTGCTGCTTT
TNF-α	NM_173966.2	F：	TGCTTGTGCCTCAGCCTCTTCT	254	60 °C
		R：	ACGAGGGCATTGGCATACGAGT
NF-κBp50	NM_001076409.1	F:	CAGATGGGCTACACTGAGGC	179	60 °C
		R:	AAGGAGGTATCTACACCGCT
NF-κBp65	NM 001080242	F:	TCACAGACCTGGCATCTGTG	123	60 °C
		R:	CCAGGCGAGTTATAGCCTCA
Nrf2	NM_0010111678.2	F:	GCAGAGACATTCCCGTTTGT	115	60 °C
		R:	CCTGAGGAGGAGCAGTGAAG

^a GAPDH: glyceraldehyde-3-phosphate dehydrogenase; UXT: ubiquitously expressed transcript; 18S rRNA: ribosomal 18S rRNA; GPx1: glutathione peroxidase1; GPx4: glutathione peroxidase4; iNOS: inducible nitric oxide synthase; IL-1β: interleukin-1β; IL-6: interleukin-6; TNF-α: tumour necrosis factor-alpha; NF-κBp50: nuclear factor κBp50; NF-κBp65: nuclear factor κBp65; Nrf2: nuclear factor E2-related factor 2.

^b F: forward primer; R: reverse primer.

Figure 1. Effect of VA on LPS-induced BMECs proliferation. Bovine mammary epithelial cells (BMECs) were randomly divided into 10 groups with six replicates. The first group was used as a control: without vitamin A (VA) and lipopolysaccharide (LPS) for 30 h. Group 2 was the LPS-treated group: without VA for 24 h before treated with LPS (1 μg/mL) alone for an additional 6 h. Groups 3–10 were eight doses of VA plus LPS-treated groups: pre-treated BMECs with 0.05, 0.1, 0.2, 0.5, 1, 2, 3, and 4 μg/mL of VA for 24 h and then incubated in the presence of 1 μg/mL of LPS and VA for a further 6 h. The cell proliferation was evaluated by methyl thiazolyl tetrazo-lium (MTT) cytotoxicity assay. Values are means with their standard deviations depicted by vertical bars (from triplicate experiments). ^a–cMeans in the treatment groups not followed by the same letter differ significantly (p<.05) whereas the differences were considered to be a statistical trend when .05<p<.10.

Table 2. Effect of VA on the antioxidant enzyme activity and inflammatory cytokines content in BMECs upon LPS damage^a.

Index^b	Treatment										SEM^c	p-Value
	Control	LPS	0.05VA + LPS	0.1VA + LPS	0.2VA + LPS	0.5VA + LPS	1VA + LPS	2VA + LPS	3VA + LPS	4VA + LPS		Treatment	Linear	Quadratic
GPx activity (IU/mgPr)	41.20^a	20.40^c	22.00^c	22.60^c	23.40^c	25.70^b,c	30.60^b	27.20^b,c	23.50^c	22.98^c	1.984	<.0001	.7968	0.0106
T-AOC activity (IU/mL)	1.67^a	0.62^c	0.66^c	0.84^b,c	0.86^b,c	1.03^b,c	1.18^b	1.15^b	1.13^b	0.93^b,c	0.139	<.0001	.0637	0.0020
SOD activity (U/mL)	20.13^a	3.36^f	5.89^e	9.71^d	9.82^d	12.38^c	17.89^b	17.68^b	16.54^b	16.22^b	0.573	<.0001	<.0001	<.0001
CAT activity (U/mL)	2.19^a	0.53^f	0.99^e	1.24^d,e	1.36^d	1.50^c,d	1.90^a,b	1.78^b,c	1.78^b,c	1.57^b,c,d	0.111	<.0001	.0002	<.0001
MDA content (nmol/mgPr)	2.66^d	4.57^a	3.83^a,b,c	3.72^a,b,c,d	3.67^a,b,c,d	3.34^b,c,d	2.69^d	2.83^c,d	3.27^b,c,d	3.93^a,b	0.302	.0037	.5344	.0002
ROS activity (fluorescence intensity/mL)	283^f	450^a	423^a,b	315^a,b,c	409^b,c,d	378^c,d	309^e,f	316^e,f	326^e	374^d	12.600	<.0001	.0010	<.0001
iNOS activity (IU/mL)	16.89^g	39.47^a	38.10^a,b	37.34^b	35.30^c	31.80^d	21.93^f	22.22^f	25.57^e	26.66^e	0.526	<.0001	<.0001	<.0001
NO content (μmol/L)	132.10^d	175.30^a	173.60^a,b	164.60^a,b,c	160.60^b,c	157.90^c	134.30^d	159.00^c	161.00^b,c	164.40^a,b,c	3.429	<.0001	.8885	.0139
IL-1 content (ng/L)	36.00^b	72.50^a	58.70^a,b	56.20^a,b	48.80^a,b	45.60^a,b	27.90^b	29.30^b	39.90^a,b	44.40^a,b	8.221	.0549	.0556	.0016
IL-6 content (ng/L)	15.90^a	30.70^a	29.10^a	21.60^a	16.00^a	14.80^a	12.10^a	14.40^a	17.90^a	18.10^a	3.753	.2128	.2454	.0479
TNF-α content (ng/L)	126^a	148^a	1308^a	124^a	123^a	119^a	102^a	122^a	123^a	124^a	11.500	.8108	.4247	.4567

^a VA: vitamin A; LPS: lipopolysaccharide; BMECs: bovine mammary epithelial cells.

^b GPx: glutathione peroxidase; T-AOC: total antioxidant capacity; SOD: superoxide dismutase; CAT: catalase; MDA: malondialdehyde; ROS: reactive oxygen species; iNOS: inducible nitric oxide synthase; NO: nitric oxide; IL-1: interleukin-1; IL-6: interleukin-6; TNF-α: tumour necrosis factor. The cells were randomly divided into 10 groups with six replicates.

^c SEM: standard error of the mean.

^a–e Means in the same row not followed by the same letter differ significantly (p < .05), whereas the differences were considered to be a statistical trend when .05 < p < .10. The number of observations for each mean value was six (n = 6).

Table 3. Effect of VA on the gene expression of selenoprotein, inflammatory cytokines, Nrf2, and NF-κB pathway of BMECs upon LPS damage^a.

Index^b	Treatment										SEM^c	p-Value
	Control	LPS	0.05VA + LPS	0.1VA + LPS	0.2VA + LPS	0.5VA + LPS	1VA + LPS	2VA + LPS	3VA + LPS	4VA + LPS		Treatment	Linear	Quadratic
GPx1	1.36^a,b,c	0.45^d	0.69^c,d	0.83^b,c,d	1.27^a,b,c	1.45^a,b	1.61^a	1.45^a,b	1.11^a,b,c,d	0.88^b,c,d	0.200	.0090	.4743	.0004
GPx4	0.88^a	0.22^d	0.37^c,d	0.48^b,c,d	0.63^a,b,c	0.72^a,b	0.78^a	0.65^a,b,c	0.38^c,d	0.25^d	0.084	<.0001	.2175	.0004
IL-1β	1.20^e	24.90^a	20.60^a,b	16.10^b,c	15.90^b,c	15.00^c,d	11.10^d	17.10^b,c	17.90^b,c	22.70^a	1.248	<.0001	.1670	.0015
IL-6	0.79^c	1.45^a	1.40^a,b	1.21^a,b	1.05^a,b,c	0.95^a,b,c	0.69^c	1.28^b,c	1.43^a,b,c	1.58^a,b	0.104	.0002	.1199	.0046
TNF-α	1.03^d	4.94^a	4.33^a,b	4.11^a,b	3.63^a,b,c	2.89^b,c	2.26^c,d	3.05^b,c	4.03^a,b	4.22^a,b	0.385	<.0001	.9238	.0069
iNOS	1.10^d	39.40^a	34.90^a,b	33.20^a,b	28.50^a,b,c	24.10^b,c	19.60^c	23.50^b,c	27.60^b,c	29.30^a,b,c	3.057	<.0001	.2750	.0037
NF-κBp50	1.01^c	1.85^a	1.83^a	1.74^a	1.69^a	1.45^a,b,c	1.17^b,c	1.50^a,b	1.72^a	1.83^a	0.142	.0021	.9046	.0126
NF-κBp65	1.04^f	2.76^a	2.08^b	1.74^b,c,d	1.45^d,e	1.34^e,f	0.97^f	1.49^c,d,e	1.68^c,d,e	1.86^b,c	0.104	<.0001	.3169	.0020
Nrf2	1.12^d	0.48^e	1.50^c,d	1.72^b,c	1.88^a,b,c	1.97^a,b	2.31^a	2.07^a,b	1.83^a,b,c	1.37^b,c	0.138	<.0001	.4345	.0013

^a VA: vitamin A; LPS: lipopolysaccharide; BMEC: bovine mammary epithelial cells.

^b GPx1: glutathione peroxidase1; GPx4: glutathione peroxidase4; IL-1β: interleukin-1β; IL-6: interleukin-6; TNF-α: tumour necrosis factor; iNOS: inducible nitric oxide synthase; NF-κBp50: nuclear factor kappa-Bp50; NF-κBp65: nuclear factor kappa-Bp65; Nrf2: nuclear factor (erythroid-derived 2)-like 2. The cells were randomly divided into 10 groups with six replicates.

^c SEM: standard error of the mean.

^a–e Means in the same row not followed by the same letter differ significantly (p <.05), whereas the differences were considered to be a statistical trend when .05 < p < .10. The number of observations for each mean value was six (n = 6).

Figure 2. Effect of VA on LPS-induced selenoproteins, RARα, IL-1β protein expression, and phosphorylation of Nrf2 and NF-κB pathways in BMECs. The cells were randomly divided into 10 groups with six replicates. The first group was used as control: without vitamin A (VA) and lipopolysaccharide (LPS) (Sigma-Aldrich, St. Louis, MO, USA) for 30 h. Group 2 was the LPS-treated group: without VA for 24 h before treated with LPS (1 μg/mL) for an additional 6 h. Groups 3–10 were eight doses of VA plus LPS treated groups: pre-treated bovine mammary epithelial cells (BMECs) with 0.05, 0.1, 0.2, 0.5, 1, 2, 3, or 4 μg/mL of VA for 24 h and then incubated in the presence of 1 μg/mL of LPS and VA for a further 6 h. Expressions of retinoic acid receptor α (RARα) (A), nuclear factor E2-related factor 2 (Nrf2) (B), glutathione peroxidase1 (GPx1) (C), Kelch-like ECH2 associated protein 1 (Keap1) (D), interleukin-1β (IL-1β) (H), inducible nitric oxide synthase (iNOS) (I), and phosphorylated IκB kinase β (P-IKKβ) (E), inhibition kappa-B α (P-IκBα) (F), nuclear factor kappa-Bp65 (P-NF-κBp65) (G) protein levels were detected by Western blotting and normalised to glyceraldehyde phosphate dehydrogenase (GAPDH) levels.

Table 4. Effect of VA on LPS-induced selenoproteins, RARα, IL-1β protein expression, and phosphorylation of Nrf2 and NF-κB pathways in BMECs^a.

Index^b	Treatment										SEM^c	p-Value
	Control	LPS	0.05VA + LPS	0.1VA + LPS	0.2VA + LPS	0.5VA + LPS	1VA + LPS	2VA + LPS	3VA + LPS	4VA + LPS		Treatment	Linear	Quadratic
RARα	1.00^b,c	0.67^d	0.73^d	0.81^c,d	0.82^c,d	1.08^a,b	1.32^a	1.08^a,b	0.92^b,c,d	0.78^c,d	0.079	.0003	.5986	.0003
Nrf2	1.00^c,d,e	0.47^f	0.66^ef	0.73^d,e,f	0.99^c,d,e	1.03^c,d	1.58^a	1.40^a,b	1.39^a,b	1.14^b,c	0.109	<.0001	.0039	<.0001
GPx1	1.00^b	0.52^e	0.70^d	0.94^b,c	0.89^c	1.01^b	1.17^b	0.56^e	0.67^d	0.66^d	0.024	<.0001	.1587	.1644
Keap1	1.00^b	1.32^a	0.84^c	0.78^c,d	0.66^d	0.46^e	0.31^f	0.86^b,c	0.86^b,c	0.93^b,c	0.049	<.0001	.6425	.0562
IKKβ	1.00^b	1.24^a	1.04^b	0.77^c	0.65^c,d	0.52^d	0.06^e	0.09^e	0.19^e	0.99^b	0.050	<.0001	.1714	<.0001
IκBα	1.00^d,e	2.16^a	1.15^c	1.07^c,d	0.89^f	0.88^f	0.58^g	0.94^e,f	1.07^c,d	1.26^b	0.034	<.0001	.6096	.0123
NF-κBp65	1.00^b,c	1.52^a	1.09^b,c	1.04^b,c	0.97^b,c	0.83^b,c,d	0.40^e	0.65^d,e	0.80^c,d	1.15^b	0.097	<.0001	.3905	<.0001
IL-1β	1.00^g	9.56^a	8.18^b	3.88^d,e	3.24^e	2.51^f	1.65^g	2.50^f	4.27^d	5.00^c	0.229	<.0001	.4044	.0025
iNOS	1.00^f,g	3.53^a	3.35^a	2.54^b,c	2.06^c,d	1.84^d,e	0.66^g	1.49^e,f	2.74^b	3.01^a,b	0.170	<.0001	.9778	<.0001

^a VA: vitamin A; LPS: lipopolysaccharide; BMEC: bovine mammary epithelial cells.

^b RARα: retinoic acid receptor α; Nrf2: nuclear factor (erythroid-derived 2)-like 2; GPx1: glutathione peroxidase1; Keap1: Kelch-like ECH2-associated protein 1; IKKβ: IκB kinase β; IκBα: inhibition kappa-B α; NF-κBp65: nuclear factor kappa-Bp65; IL-1β: interleukin-1β; iNOS: inducible nitric oxide synthase.

^c SEM: standard error of the mean. The number of observations for each mean value was six (n = 6).

Robinson TL, Sutherland IA, Sutherland J. 2007. Validation of candidate bovine reference genes for use with real-time PCR. Vet Immunol Immunopathol. 115:160–165.

 PubMed Web of Science ®Google Scholar