Oral administration of docosahexaenoic acid activates the GDNF-MAPK-CERB pathway in hippocampus of natural aged rat

Figures & data

Table 1. The comparison of fatty acids in diet and total fatty acids is about 6.8 % in diet.

Fatty acids	Diet (%)
SFAs	11.49
C16:00	7.39
C18:00	2.67
C20:00	1.43
MUFAs	64.58
C18:1n-9	61.76
C18:1n-7	1.27
C20:1n-9	1.55
Total n − 3 PUFAs	4.07
C18:3	3.81
C20:5	0.17
C22:6	0.09
Total n − 6 PUFAs	18.41
C18:2	17.84
C20:4	0.57
Unknown	1.45

MUFAs, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; SFAs, saturated fatty acids; Unknown; the substance is unknown.

Table 2. The effects of DHA on fatty acid composition in hippocampus tissues (% of total).

Fatty acids	Adult group	Aged group	Low-dose group	High dose group
SFAs	48.24 ± 3.47	53.57 ± 4.11^aa	48.56 ± 4.03^bb	45.82 ± 3.67^bb
MUFAs	18.29 ± 2.15	22.82 ± 2.06^aa	20.86 ± 1.43	20.24 ± 1.56
Total n − 7	4.35 ± 0.33	6.87 ± 0.35^aa	5.79 ± 0.87^aa,b	5.76 ± 0.23^aa,b
Total n − 9	13.94 ± 0.45	15.95 ± 0.76	15.07 ± 0.93	14.48 ± 0.76
PUFAs	31.19 ± 2.88	21.72 ± 3.41^aa	29.02 ± 3.89^bb	32.89 ± 3.65^bb
Total n − 3	16.40 ± 2.67	9.88 ± 1.78^aa	14.74 ± 1.83^a,bb	17.23 ± 2.05^bb
C20:5	1.45 ± 0.12	1.09 ± 0.14^aa	1.59 ± 0.25^aa,bb	1.63 ± 0.12^aa,bb
C22:6	14.95 ± 1.34	8.79 ± 1.46^aa	13.15 ± 1.48^bb	15.60 ± 2.25^bb
Total n − 6	14.79 ± 1.78	11.84 ± 1.65^aa	14.28 ± 1.36^bb	15.66 ± 1.78^bb
C18:2	0.61 ± 0.23	0.42 ± 0.16^aa	0.58 ± 0.22^bb	0.59 ± 0.24^bb
C22:4	2.51 ± 0.32	1.44 ± 0.22	3.43 ± 0.14^aa,bb	3.85 ± 0.25^aa,bb
C22:5	2.62 ± 0.21	3.12 ± 0.23^aa	2.32 ± 0.26^bb	2.23 ± 0.11^bb
C20:4	9.05 ± 1.23	6.86 ± 1.54^aa	7.95 ± 1.65^aa	8.99 ± 1.76^bb
Unknown	2.28 ± 0.13	1.89 ± 0.21	1.56 ± 0.18	1.05 ± 0.35
n − 3/n − 6	1.11 ± 0.13	0.83 ± 0.12^aa	1.03 ± 0.14^b	1.10 ± 0.11^bb
DHA/AA	1.65 ± 0.21	1.28 ± 0.15^aa	1.65 ± 0.24^bb	1.74 ± 0.22^bb

MUFAs, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; SEM, standard error of the mean; SFAs, saturated fatty acids. Data are expressed as means ± SEM. n = 10. Adult group, 5-month aged female rat; Aged group, 24-month aged female rat; low-dose group, 24-month rat treated with DHA 80 mg/kg body weight/day; high dose group, 24-month aged rat treated with DHA 160 mg/kg body weight/day.

^ap < 0.05, ^aap < 0.01 versus adult group; ^bp < 0.05, ^bbp < 0.01 versus aged group.

Table 3. The effects of DHA on superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and thiobarbituric acid reactive substances (TBARS) in hippocampus tissues.

	Adult group	Aged group	Low-dose group	High dose group
SOD (U/mg protein)	53.79 ± 5.29	28.97 ± 4.63^aa	35.54 ± 4.50^aa,bb	41.45 ± 4.36^aa,bb
GSH-Px (U/mg protein)	10.35 ± 1.86	7.06 ± 1.30^aa	9.01 ± 1.67^aa,bb	9.36 ± 2.37^a,bb
CAT (U/mg protein)	5.27 ± 0.33	2.14 ± 0.29^aa	2.29 ± 0.18^aa	2.32 ± 0.13^aa
TBARS(nmol/mgrotein)	2.13 ± 0.23	4.95 ± 0.82^aa	3.54 ± 0.31^aa,^bb	3.31 ± 0.51^aa,bb

CAT, catalase; GSH-Px, glutathione peroxidase; SEM, standard error of the mean; SOD, superoxide dismutase; TBARS, thiobarbituric acid reactive substances. Data are expressed as means ± SEM. n = 10. Data are expressed as means ± SEM. n = 8. Adult group, 5-month aged female rat; Aged group, 24-month aged female rat; Low-dose group, 24-month rat treated with DHA 80 mg/kg body weight/day; high dose group, 24-month aged rat treated with DHA 160 mg/kg body weight/day.

^ap < 0.05, ^aap < 0.01 versus adult group; ^bbp < 0.01 versus aged group.

Table 4. Effect of DHA on GDNF levels in hippocampus tissues.

Groups	Hippocampus tissue GDNF (pg/mg)
Adult group	13.58 ± 1.28
Aged group	6.74 ± 0.64^aa
Low-dose group	8.35 ± 1.59^aa,bb
High-dose group	9.81 ± 1.37^aa,bb

DHA, docosahexaenoic acid; GDNF, glial derived neurotrophic factor; SEM, standard error of the mean. Data are expressed as means ± SEM. n = 10. Data are expressed as means ± SEM. n = 8. Adult group, 5-month aged female rat; Aged group, 24-month aged female rat; Low-dose group, 24-month rat treated with DHA 80 mg/kg body weight/day; large dose group, 24-month aged rat treated with DHA 160 mg/kg body weight/day.

^aap < 0.01 versus adult group; ^bbp < 0.01 versus aged group.

Figure 1. GFRa1 mRNA and protein level in the hippocampus tissue. Rat treatment with 80 mg/kg/day and 160 mg/kg/day DHA 50 days significantly influence the expression of GFRa1 in hippocampus tissue. Summary of the results derived from real-time RT-PCR. GFRa1 mRNA levels were first normalized to GAPDH, calculated by the 2^–ΔΔCT method. Relative expression units were determined by dividing the apparent levels of by the expression level of adult group. Compared with the aged rats, the treatment with 80 mg/kg/day and 160 mg/kg/day DHA 50 days significantly increased the protein expression of GFRα1. The quantity of the applied protein was normalized by Western blotting analysis with anti-actin polyclonal (n = 5). ^aap < 0.05 versus aged group. ^bbp < 0.01, DHA treatment group versus aged group. n = 5 in each group.

Figure 2. Western blot analysis of phosphorylation of receptor tyrosine kinase RET expression in the hippocampus tissue. Compared with aged rats treatment with 80 mg/kg/day and 160 mg/kg/day DHA 50 days significantly increased the protein expression of phosphorylation of receptor tyrosine kinase RET. The quantity of the applied protein was normalized by Western analysis with anti-RET (n = 5). ^aap < 0.05 versus aged group. ^bbp < 0.01, DHA treatment group versus aged group. n = 5 in each group.

Figure 3. Western blot analysis of phosphorylation Raf expression in hippocampus tissue. Compared with aged rats, treatment with 80 mg/kg/day and 160 mg/kg/day DHA 50 days significantly increased the levels protein expression of phosphorylation of Raf. The quantity of the applied protein was normalized by Western analysis with anti-Raf (n = 5). ^aap < 0.05 versus aged group. ^bbp < 0.01, DHA treatment group versus aged group. n = 5 in each group.

Figure 4. Western blot analysis of phosphorylation MEK expression in hippocampus tissue. Compared with aged rats, treatment with 80 mg/kg/day and 160 mg/kg/day DHA 50 days significantly increased the levels protein expression of phosphorylation of MEK. The quantity of the applied protein was normalized by Western analysis with anti-total MEK (n = 5). ^aap < 0.05 versus aged group. ^bbp < 0.01, DHA treatment group versus aged group. n = 5 in each group.

Figure 5. Western blot analysis of phosphorylation ERK expression in hippocampus tissue. Compared with aged rats, treatment with 80 mg/kg/day and 160 mg/kg/day DHA 50 days significantly increased the levels protein expression of phosphorylation of EEK. The quantity of the applied protein was normalized by Western analysis with anti-total ERK (n = 5). ^aap < 0.05 versus aged group. ^bbp < 0.01, DHA treatment group versus aged group. n = 5 in each group.

Figure 6. Western blot analysis of phosphorylation CERB expression in hippocampus tissue. Compared with aged rats, treatment with 80 mg/kg/day and 160 mg/kg/day DHA 50 days significantly increased the levels protein expression of phosphorylation of CERB. The quantity of the applied protein was normalized by Western analysis with actin. ^aap < 0.05 versus aged group. ^bb p < 0.01, DHA treatment group versus aged group. n = 5 in each group.