Dietary intake of the citrus flavonoid hesperidin affects stress-resilience and brain kynurenine levels in a subchronic and mild social defeat stress model in mice

Figures & data

Table 1. Dietary ingredients and food composition list of semi-purified diet (AIN-93G).

Dietary ingredients1		Food composition1
Casein (%)	20.0	Water (%)	9.0
L-Cystine (%)	0.30	Crude protein (%)	17.9
Corn starch (%)	52.9	Crude fat (%)	7.0
Sucrose (%)	10.0	Crude ash (%)	2.5
Soy oil (%)	7.0	Crude fiber (%)	3.0
Cellulose powder (%)	5.0	Nitrogen free extract (%)	60.6
AIN-93 mineral mix (%)	3.5
AIN-93 vitamin mix (%)	1.0
Choline bitartrate (%)	0.25
Tertiary butyl hydroquinone (%)	0.001
Total (%)	100.0	Calorie (kcal/100 g)	377.0

Data from Oriental Yeast, Tokyo, Japan.

Figure 1. Experimental design of the subchronic and mild social defeat stress (sCSDS) paradigm, behavioral tests, and sampling.

Table 2. Summary of number. of susceptible and resilient mice and resilient rate in each group.

	sCSDS	sCSDS + CT	Fisher’s exact test
total no. of mice	27	27
no. of susceptible	21	16
no. of resilient	6	11
resilient rate (%)	22.2	40.7
sCSDS vs. sCSDS + CT			p = 0.2409

A 2 × 2 contingency table with Fisher’s exact test was generated for each condition.

The mice that showed susceptible-like and resilient-like phenotypes were counted as susceptible-mice and resilient-mice, respectively.

Table 3. Social interaction test.

	sCSDS	sCSDS + CT	T-test
Time spent in interaction zone time (s)with ICR mouse	41.9 ± 5.3	56.4 ± 7.9	p = 0.1460
Time spent in interaction zone time (s) without ICR mouse	57.4 ± 3.8	71.1 ± 3.5	p = 0.4832
Time spent in corner zone time (s) with ICR mouse	39.0 ± 8.4	34.3 ± 7.1	p = 0.6775
Time spent in corner zone time (s) without ICR mouse	23.2 ± 2.1	20.0 ± 1.6	p = 0.2387
Total distance traveled (cm) with ICR mouse	419.1 ± 34.0	406.8 ± 28.0	p = 0.7860
Total distance traveled (cm) without ICR mouse	974.5 ± 53.1	871.5 ± 42.6	p = 0.1441

Data are expressed mean ± SEM.

Figure 2. Social interaction scores in the sCSDS (n = 27) and sCSDS + CT (n = 27) groups. The bar represents the mean ± SEM. Individual values are represented by circles. p values are for the unpaired two-tailed Student’s t-test. †p < 0.1. sCSDS, subchronic and mild social defeat stress.

Figure 3. Social interaction scores in the control (n = 8), sCSDS (n = 15), and sCSDS + Hes (n = 17) groups. The bar represents the mean ± SEM. Individual values are represented by circles. p values are for Bonferroni’s post-hoc test. †p < 0.1, ***p < 0.001. sCSDS, subchronic and mild social defeat stress.

Table 4. Summary of number. of susceptible and resilient mice and resilient rate in each group.

	Control	sCSDS	sCSDS + Hes	Fisher’s exact test
total no. of mice	8	15	17
no. of susceptible	2	10	3
no. of resilient	6	5	14
resilient rate (%)	75.0	33.3	82.4
Control vs. sCSDS				p = 0.0894
Control vs. sCSDS + Hes				p = 1.0000
sCSDS vs. sCSDS + Hes				p = 0.0145

A 2 × 2 contingency table with Fisher’s exact test was generated for each condition.

The mice that showed susceptible-like and resilient-like phenotypes were counted as susceptible-mice and resilient-mice, respectively.

Table 5. Social interaction test.

	Control	sCSDS	sCSDS + Hes
Time spent in interaction zone time (s) with ICR mouse	71.8 ±9.1	52.0 ± 6.7	92.5 ± 6.4^bbb
Time spent in interaction zone time (s) without ICR mouse	57.0 ± 4.2	63.3 ± 5.1	67.4 ± 4.8
Time spent in corner zone time (s) with ICR mouse	19.7 ± 5.1	34.0 ± 8.9	17.8 ± 5.0
Time spent in corner zone time (s) without ICR mouse	15.1 ± 1.8	21.5 ± 2.5	18.7 ± 1.4
Total distance traveld (cm) with ICR mouse	662.7 ± 40.2	502.7 ± 61.1	616.2 ± 43.2
Total distance traveled (cm) without ICR mouse	1006.8 ± 47.8	1021.5 ± 51.5	1005.8 ± 63.1

Data are expressed mean ± SEM.

bbb, p < 0.001 comparison sCSDS group.

Table 6. Interleukin-6, interleukin-1β, and corticosterone levels in plasma.

	IL-6 (pg/ml)	IL-1β (pg/ml)	Corticosterone (ng/ml)
Control	5.3 ± 2.0	8.0 ± 0.2	82.2 ± 8.1
sCSDS	13.4 ± 2.9	8.2 ± 0.4	76.8 ± 9.2
sCSDS + Hes	13.5 ± 2.6^#	8.1 ± 0.1	55.0 ± 8.9
R	−0.09, p = 0.3394	−0.12, p = 0.2883	−0.25, p = 0.1128

Mice were behaviorally phenotyped 10 day after sCSDS by measuring social interaction (Figure 1). Pearson product-moment correlation coefficient was used to the correlation between SI score and kynurenine was analyzed. Correlations are listed in row R.

Data are expressed mean ± SEM.

# p < 0.1, comparison Control group.

Table 7. Kynurenine and tryptophan levels in brain.

		KYN (nmol/mg)	Trp (nmol/mg)	KYN/Trp (%)
Hippocampus	Control	1.99 ± 0.14	370.4 ± 17.6	0.54 ± 0.03
	sCSDS	2.81 ± 0.16^aaa	438.8 ± 16.9^a	0.65 ± 0.04^#
	sCSDS + Hes	2.04 ± 0.08^bbbb	374.5 ± 13.4^b	0.55 ± 0.02^$
	R	−0.29, p = 0.0346	−0.22, p = 0.0851	−0.18, p = 0.1311
Prefrontal cortex	Control	2.14 ± 0.13	466.5 ± 39.8	0.47 ± 0.03
	sCSDS	2.87 ± 0.18^aa	440.7 ± 12.2	0.66 ± 0.04^aa
	sCSDS + Hes	2.07 ± 0.08^bbb	463.1 ± 10.5	0.45 ± 0.02^bbbb
	R	−0.33, p = 0.0178	0.04, p = 0.3989	−0.30, p = 0.0283

Mice were behaviorally phenotyped 10 day after sCSDS by measuring social interaction (Figure 1). Pearson product-moment correlation coefficient was used to the correlation between SI score and kynurenine was analyzed. Correlations are listed in row R.

Data are expressed mean ± SEM.

^a p < 0.05, ^aa p < 0.01, ^aaa p < 0.001, # p < 0.1, comparison Control group.

^b p < 0.05, ^bbb p < 0.001, ^bbbb p < 0.0001, $ p < 0.1, comparison sCSDS group.

Table 8. Kynurenine and tryptophan levels in blood plasma.

		KYN (μM)	Trp (μM)	KYN/Trp (%)
Plasma	Control	0.70 ± 0.04	76.8 ± 4.3	0.93 ± 0.08
	sCSDS	0.73 ± 0.04	74.2 ± 3.3	0.99 ± 0.06
	sCSDS + Hes	0.63 ± 0.03	75.4 ± 2.2	0.84 ± 0.05
	R	−0.14, p = 0.1967	0.26, p = 0.0555	−0.30, p = 0.0302

Mice were behaviorally phenotyped 10 day after sCSDS by measuring social interaction (Figure 1). Pearson product-moment correlation coefficient was used to the correlation between SI score and kynurenine was analyzed. Correlations are listed in row R.

Data are expressed mean ± SEM.