Polyunsaturated fatty acids ameliorate aging via redox-telomere-antioncogene axis

Polyunsaturated fatty acids (PUFA), a group of nourishing and health-promoting nutrients, ameliorate age-related chronic diseases. However, how PUFA especially n-3 PUFA exert anti-aging functions remains poorly understood. Here we link fish oil, docosahexaenoic acid (DHA) and arachidonic acid (AA) to the aging etiology via a redox-telomere-antioncogene axis based on D-galactose-induced aging mice. Both fish oil and PUFA enhanced hepatic superoxide dismutase (SOD) and catalase activities and cardiac SOD activities within the range of 18%-46%, 26%-65% and 19%-58%, respectively, whereas reduced cerebral monoamine oxidase activity, plasma F2-isoprostane level and cerebral lipid peroxidation level by 56%-90%, 20%-79% and 16%-54%, respectively. Thus, PUFA improve the in vivo redox and oxidative stress induced aging process, which however does not exhibit a dose-dependent manner. Notably, both PUFA and fish oil effectively inactivated testicular telomerase and inhibited c-Myc-mediated telomerase reverse transcriptase expression, whereas n-3 PUFA rather than n-6 PUFA protected liver and testes against telomere shortening within the range of 13%-25% and 25%-27%, respectively. Therefore, n-3 PUFA may be better at inhibiting the DNA damage induced aging process. Surprisingly, only DHA significantly suppressed cellular senescence pathway evidenced by testicular antioncogene p16 and p53 expression. This work provides evident support for the crosstalk between PUFA especially n-3 PUFA and the aging process via maintaining the in vivo redox homeostasis, rescuing age-related telomere attrition and down-regulating the antioncogene expression.

and vitamin E, respectively. FO1 and FO2 were analyzed via gas chromatography (GC) using the 7890A gas chromatograph equipped with a flame ionization detector (Agilent, Santa Clara, CA, USA) according to a previously published work [1]. A close constituent of monounsaturated fat (20%), total carbohydrate (20%), protein (less than 20%) and antioxidants (negligible) was present in FO1 and FO2, which was inferred from the supplement facts. Therefore, the major difference between FO1 and

Animals and experimental design
One hundred twenty Institute of Cancer Research (ICR) male mice (8 weeks old,

Preparation of blood and tissues
The femoral arterial blood of 8-h fasted mice was collected, and the mice were subsequently sacrificed to obtain their livers, hearts and brains. These tissues were shock-frozen in liquid nitrogen after collection and stored at -80 °C until analysis.
The serum and plasma isolated from the collected blood samples were used for the determination of antioxidase activities and F 2 -isoprostane levels, respectively. The tissue preparation for the determination of antioxidase activity was performed as follows. A piece (approx. 100 mg) was cut from the frozen tissue sample and transferred to a sterile micro-centrifuge tube. Following the addition of pre-cooled saline (1 mL per 100 mg of tissue), the tissue sample was subsequently homogenized (IKA-T10 homogenizer; IKA, Staufen, Germany) on ice until a uniform consistency was achieved. The mixture was then centrifuged (500 ×g) at 4 °C for 10 min to collect the supernatant.

Determination of antioxidase activities
Superoxide dismutase (SOD) activities in the brain, liver and heart were assessed using a SOD detection kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Briefly, the superoxide radicals generated from the xanthine-xanthine oxidase (XOD) system reduce the formation of a water-soluble tetrazolium salt (WST-1) formazan, which may be inhibited by SOD from the supernatants of the samples and spectrophotometrically measured at 550 nm. The SOD activities were calculated as follows, in which 50% represents the inhibition rate.
One unit of SOD activity is defined as the amount of protein that inhibits half of the WST-1 formazan formation. The tissue SOD activities were expressed as U/mg protein.
The principle is that GSH-Px catalyzes the oxidation of GSH in the presence of H 2 O 2 , and the remaining GSH may be quantified using a reaction with 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) at 422 nm. The GSH-Px activities were calculated as follows, in which 20 represents the standard concentration (μmol/L).
The hepatic and serum GSH-Px activities were expressed as U/mg protein and U/mL, respectively.

Determination of monoamine oxidase (MAO) activities
Brain MAO activities were evaluated with a kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Briefly, benzaldehyde formation using benzylamine as the substrate is catalyzed by MAO, and benzaldehyde extracted by cyclohexane exhibits a maximum absorbance at 242 nm. The MAO activities were calculated as follows, and one unit of MAO activity is defined as per milligram of protein generating 0.01 OD value. The brain MAO activities were expressed as U/mg protein.
Brain MAO activity = sample OD 0.01 × reaction time × sample amount × sample protein conc.

Determination of thiobarbituric acid reactive substances (TBARS) levels
The TBARS contents in the liver, heart and brain were determined using a well-established thiobarbituric acid (TBA) test with a kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Malonaldehyde (MDA) and related compounds in the supernatants of the samples may react with TBA in acidic media at 100 °C, and the reaction mixture is spectrophotometrically measured at 532 nm. Therefore, the TBARS level is typically adapted to assess the MDA level. The MDA levels were calculated as follows, in which 10 represents the standard concentration (μmol/L). The results were expressed as nmol/mg protein.

Determination of F 2 -isoprostane levels
The plasma Fisher Scientific Inc., Waltham, MA, USA) [2]. The plasma F 2 -isoprostane levels were expressed as pg/mL following standard calibration.

Telomere length (TL) analysis
The relative TL in the liver and testes, which was expressed as a ratio of the telomere repeat (T) copy number to the single-copy gene (S) copy number (T/S), was measured via quantitative real-time polymerase chain reaction (PCR) as previously described. The 36B4 (acidic ribosomal phosphoprotein PO) gene is well accepted as the single-copy gene [3]. Briefly, genomic DNA was extracted from the liver and For PCR reactions, two separate plates were used to specifically amplify T or S.

Each sample assayed for both T and S amplifications was analyzed in quadruple.
Therefore, four identical 2-μL aliquots of the DNA sample (20 ng) were added into one plate, and an additional four aliquots were added into the same well position in another plate. For each standard curve, a reference DNA sample was serially diluted in TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 7.5) by two fold per dilution to produce eight concentrations of DNA that ranged 0.395-50 ng/μL. Three identical 2 μL of each concentration were distributed to the standard curve wells on each plate.
The reference DNA sample to which all experimental samples were compared was a pooled sample from isolated DNA samples from all groups. In addition, the 20 mL-PCR reaction system also contained 10 μL SYBR Premix Ex Taq II (Takara Bio seconds. The samples were normalized to single-copy genes as indicated, and the fold change was calculated using the ΔΔCt method (T/S = 2 −ΔΔCt ).

Telomerase activity assay
The testicular telomerase activities in all groups were measured using a TeloTAGGG telomerase PCR ELISA kit (Roche applied science, Indianapolis, IN, USA) according to the manufacturer's instructions. Following determination of the protein concentration with a Bradford Protein Assay (Bio-rad Laboratories, Hercules, CA), 9 μg protein were added to the reaction mixture to participate in the telomeric repeat amplification protocol (TRAP) assay [4]. The amplified PCR product was subsequently subjected to treatment for the enzyme-linked immunosorbent assay, and the color change was detected using a microplate reader (Varioskan Flash, Thermo Fisher Scientific Inc., Waltham, MA, USA). The relative activity was measured compared with the lysis buffer control.

Whole-mount and in situ SA-β-gal staining
Whole-mount testes SA-β-gal staining and in situ SA-β-gal staining in testicular cryosections were processed with a Senescence β-Galactosidase Staining Kit (Cell Signaling Technology, Beverly, USA) with modifications. Thawed mouse testes were fixed in 1 × Fixation Buffer supplied from the kit for 90 min. After washing with PBS three times, the testes were incubated at 37 °C for 16 h with a staining mixture that contains pH 6.0 buffer and X-gal substrate. The images were captured using a NIKON SMZ18 microscope (Nikon, Tokyo, Japan).

Western blotting analysis
Sections from the frozen testicle samples were prepared in RIPA Lysis Buffer (Millipore, Temecula, CA) with Protease inhibitors (Fdbio Science, Hangzhou, China) and subsequently homogenized (IKA-T10 homogenizer; IKA, Staufen, Germany) on ice until there was a uniform consistency. Following a 30-minute incubation on ice, the lysate was centrifuged at 13,500 × g for 20 minutes at 4 °C. The protein concentration in the removed supernatant was determined as previously described. A standard western blotting procedure was subsequently conducted as previously described [5]

Immunofluorescence
Frozen testes sections (~8 μm) were fixed with formaldehyde for 10 min at room temperature and permeabilized with 0.1% triton X-100 (v/v) in PBS for 4 min at room temperature, followed by a block with 1% BSA (w/v) in PBS for 1 hour at room temperature. The sections were subsequently incubated with rabbit polyclonal anti-TERT antibody (sc-7212; Santa Cruz Bio., Santa Cruz, CA, USA; 1:50) overnight at 4 °C, followed by incubation with anti-goat Alexa Fluor 488-conjugated secondary antibody (Invitrogen Life Technologies, Carlsbad, CA; 1:1000) for 1 h at room temperature. After the last wash with PBS-Triton (0.1%), the nuclei were stained with DAPI (Beyotime, Haimen, China; 1:10000) for 5 min prior to mounting with glycerol jelly mounting medium (Beyotime, Haimen, China). Images were captured on an N-SIM super-resolution microscope system (Nikon, Tokyo, Japan) and cropped in NIS-Elements C software.