Protective effect of chitosan oligosaccharide against oxidative damage of peripheral blood mononuclear cells in dairy cows induced by diethylenetriamine/nitric oxide via NF-κB signalling pathway

Figures & data

Table 1. Primers sequences used in real-time PCR.

Genes^a	GenBank no.	Primer sequences (5′−3′)^b	Length, bp	Annealing temperature, °C
GAPDH	NM_001034034	F: ATCAAGTGGGGTGATGCTGG	167	60
		R: TACTTCTCGTGGTTCACGCC
β-actin	NM_001252479	F: GGGTCATCCTCTCTGCACCT	177	60
		R: GGTCATGAGTCCCTCCACGA
18S rRNA	(Robinson et al., 2007)	F: GTAACCCGTTGAACCCCATT	152	60
		R: CCATCCAATCGGTAGTAGCG
iNOS	NM_001076799.1	F: TGTCAGCGGCAAGCACCACATT	289	60
		R: CGGCTGGTTGCATGGGAAAACT
IL-1β	NM_174093.1	F: GCCTTGGGTATCAAGGACAA	90	60
		R: TTTGGGGTCTACTTCCTCCA
IL-6	NM_173923.2	F: ACAAGCGCCTTCACTCCATTCG	242	60
		R: GCCAGTGTCTCCTTGCTGCTTT
TNF-α	NM_173966.2	F: TGCTTGTGCCTCAGCCTCTTCT	254	60
		R: ACGAGGGCATTGGCATACGAGT
NF-κBp50	NM_001076409.1	F: CAGATGGGCTACACTGAGGC	179	60
		R: AAGGAGGTCTCTACACCGCT
NF-κBp65	NM_001080242	F: TCACAGACCTGGCATCTGTC	123	60
		R: CCAGGCGAGTTATAGCCTCA

^aGAPDH: glyceraldehyde-3-phosphate dehydrogenase; iNOS: inducible nitric oxide synthase; IL-1β: interleukin-1β; IL-6: interleukin-6; TNF-a: tumour necrosis factor-alpha; NF-κBp50: nuclear factor kappa-Bp50; NF-κBp65: nuclear factor kappa-Bp65.

^bF: forward primer; R: reverse primer.

Figure 1. Relative cell viability for peripheral blood mononuclear cells incubated with 40, 80, 160, or 320 μg/mL of chitosan oligosaccharides (COS40, COS80, COS160, and COS320). Values are expressed as an optical density ratio between treated and control (CTR) wells. The different letter superscripts mean significant difference (p < .05).

Table 2. Effect of chitosan oligosaccharides on the antioxidant enzyme activity and concentration in peripheral blood mononuclear cells.

	Treatment^B						p Value
Item^A	CTR	COS40	COS80	COS160	COS320	SEM	Treatment	Linear	Quadratic
CAT (U/mL)	1.89^b	2.30^ab	2.32^ab	2.44^ab	2.75^a	0.17	.07	.01	.25
SOD (U/mL)	0.37^c	0.39^bc	0.40^b	0.44^a	0.37^c	0.01	<.01	.93	<.01
GPx (U/mL)	19.55^d	28.33^cd	39.71^cb	66.66^a	47.47^b	5.70	<.01	.02	<.01
TrxR (pg/mL)	5.67^c	6.03^b	6.48^a	6.74^a	6.45^a	0.21	<.01	<.01	<.01

^ACAT: Catalase; SOD: superoxide dismutase; GPx: glutathione peroxidase; TrxR: thioredoxin reductase.

^BCTR = control treatment, without chitosan oligosaccharides addition; COS40, COS80, COS160, and COS320= treated with 40, 80, 160, and 320 μg/mL chitosan oligosaccharides, respectively.

^a–d Values within a row with different superscripts differ significantly at p < .05.

Table 3. Effect of chitosan oligosaccharides on the inflammatory cytokines content and in peripheral blood mononuclear cells.

	Treatment^B						p Value
Item^A	CTR	COS40	COS80	COS160	COS320	SEM	Treatment	Linear	Quadratic
IL-1β, ng/L	203.62^a	190.41^b	178.90^c	145.61^d	174.73^c	2.36	<.01	.04	<.01
IL-6, ng/L	237.04^a	222.85^ab	210.82^cb	188.49^cd	196.49^d	6.00	<.01	<.01	<.01
TNF-α, ng/L	290.34^a	232.77^b	211.40^c	205.14^d	210.56^c	1.61	<.01	.01	<.01
ROS, IU/mL	695.33^a	589.30^b	575.19^c	500.65^e	567.43^d	2.37	<.01	.03	<.01
NO, μmol/L	172.55^a	169.09^ab	162.24^c	160.27^c	167.79^b	1.15	<.01	.32	<.01
iNOS, U/mL	17.10^a	16.80^a	16.53^a	15.74^b	14.25^c	0.24	<.01	<.01	<.01

^A IL-1β: interleukin-1β; IL-6: Interleukin-6; ROS: reactive oxygen species; TNF-α: tumour necrosis factor-α; NO: nitric oxide; iNOS: inducible nitric oxide synthase.

^BCTR = control treatment, without chitosan oligosaccharides addition; COS40, COS80, COS160, and COS320 = treated with 40, 80, 160, and 320 μg/mL chitosan oligosaccharides respectively.

^a–eValues within a row with different superscripts differ significantly at p < 0.05.

Table 4. Effect of chitosan oligosaccharides on genes expression of cytokines, inducible nitric oxide synthase and NF-κB pathway.

	Treatment^B						p Value
Item^A	CTR	COS40	COS80	COS160	COS320	SEM	Treatment	Linear	Quadratic
IL-1β	1.00^a	0.83^b	0.77^b	0.74^b	0.57^c	0.04	<.01	<.01	<.01
IL-6	1.00^a	0.75^b	0.49^c	0.37^d	0.35^d	0.03	<.01	<.01	<.01
TNF-α	1.00^a	0.89^a	0.87^a	0.47^b	0.36^b	0.04	<.01	<.01	<.01
NF-κBp50	1.00^a	0.96^a	0.70^b	0.68^b	0.54^c	0.05	<.01	<.01	<.01
NF-κBp65	1.00^a	0.89^b	0.87^b	0.78^c	0.74^c	0.02	<.01	<.01	<.01
iNOS	1.00^a	0.97^a	0.89^a^b	0.87^a^b	0.75^b	0.05	.01	<.01	<.01

^AIL-1β: interleukin-1β; IL-6: interleukin-6; NF-κBp65: nuclear factor kappa-Bp65; NF-κBp50: nuclear factor kappa-Bp50; TNF-α: tumour necrosis factor-α; iNOS: inducible nitric oxide synthase.

B CTR = control treatment, without chitosan oligosaccharides addition; COS40, COS80, COS160, and COS320= treated with 40, 80, 160, and 320 μg/mL chitosan oligosaccharides respectively.

^a–dValues within a row with different superscripts differ significantly at p < .05.

Figure 2. Effects of chitosan oligosaccharideon on the phosphorylation level of NF-kB pathway, the protein expression level of nitric oxide synthase (iNOS) and interleukin-1β (IL-1β). Expressions of IL-1β(A); phosphorylated IκB kinase β (P-IKKβ) (B); phosphorylated inhibitor of nuclear factor kappa-Bα (P-IκBα) (C); phosphorylated nuclear factor kappa-Bp65(P-NF-κBp65) (D); iNOS (E), protein levels were detected by western blotting and normalised to beta-actin (β-Actin) levels. CTR = control treatment, without chitosan oligosaccharides addition; COS40, COS80, COS160, and COS320= treated with 40, 80, 160, and 320 μg/mL chitosan oligosaccharides respectively.

Table 5. Effect of chitosan oligosaccharides on inducible nitric oxide synthase, interleukin-1β protein expression, and phosphorylation of NF-κB pathways in PBMC.

	Treatment^b						p Value
Item^A	CTR	COS40	COS80	COS160	COS320	SEM	Treatment	Linear	Quadratic
IL-1β	1.21^a	1.21^a	0.96^b	0.83^c	0.89^b^c	0.03	<.01	<.01	<.01
IKKβ	1.05^a	0.78^b	0.60^c	0.50^c	0.49^c	0.04	<.01	<.01	<.01
IkBα	0.43^a	0.40^a^b	0.37^b	0.35^b^c	0.33^c	0.01	.01	<.01	<.01
NF-κBp65	1.62^a	1.21^b	0.93^c	0.61^d	0.59^d	0.04	<.01	<.01	<.01
iNOS	1.86^a	1.75^a^b	1.63^b^c	1.49^c	1.51^c	0.06	<.01	<.01	<.01

^AIL-1β: expressions of interleukin-1β (A); iNOS: inducible nitric oxide synthase (B); P-IKKβ: phosphorylated inhibitor of nuclear factor kappa-Bα kinase β (C); IκBα: inhibitor of nuclear factor kappa-Bα (D); NF-κBp65: nuclear factor kappa-Bp65 (E) protein levels were detected by western blotting.

^bCTR = control treatment, without chitosan oligosaccharides addition; COS40, COS80, COS160, and COS320= treated with 40, 80, 160, and 320 μg/mL chitosan oligosaccharides respectively.

^a–dValues within a row with different superscripts differ significantly at p < .05.

Robinson TL, Sutherland IA, Sutherland J. 2007. Validation of candidate bovine reference genes for use with real-time PCR. Veterinary Immunology and Immunopathology. 115(1-2):160–165. doi:10.1016/j.vetimm.2006.09.012.

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