2.1. Recruitment of healthy volunteers and genotyping of ApoE polymorphism
The healthy subjects (forty-two age- and sex-matched, Table 1) were engaged from the Sports Medicine Unit (Department of Clinical and Experimental Medicine, University of Pisa). This study was approved by the Ethics Committee of the Great North West Area of Tuscany (271/2014 to F.F.), and it was carried out in accordance with the Declaration of Helsinki. All subjects gave informed consent to participate in the study. Fully informed consent was obtained from each subject entering the study [21].
The blood was collected from each subject and, subsequently, genomic DNA was extracted from the whole blood. The restriction fragment length polymorphism (RFLP) technique has been employed to classify the subjects in ApoE ε4 carriers and non-ε4 carriers. Briefly, the polymerase chain reaction (PCR) was made with 1.5 pmol of each primer (forward 5′-TCG-GCCGCA-GGG-CGC-TGA-TGG-3′ and reverse 5′-CTCGCG-GGC-CCC-GGC-CTG-GTA-3′), 250 µmol/L dNTPs, GC-rich (10% of the final volume), 2 units of Taq DNA polymerase (Applied Biosystems Inc., Branchburg, NJ), 10 ng/µL of genomic DNA, 25 mM MgCl2, and buffer 10X. A thermal cycler (PerkinElmer) was employed for reactions: one cycle at 94 °C for 6 min, 30 cycles at 94 °C for 40 s, 67 °C for 30 s, 72 °C for 45 s, and a final extension at 72 °C for 5 min. The amplified fragments, resulting from digestion with 3 U of HhaI restriction enzyme, were divided through agarose (5%) gel electrophoresis. The restriction patterns were displayed using ethidium bromide staining and UV light.
The subjects’ genotypes were established by ABI PRISM310 Automated Sequencer (Applied Biosystems, Forster City, CA, USA). Thus, the subjects have been classified in ApoE ε4 carriers (sixteen, age- and sex-matched, Table 1), who included heterozygotes subjects ε4/ε3, and ApoE non-ε4 carriers (twenty-six, age- and sex-matched, Table 1), who included heterozygotes subjects (ε2/ε3) or homozygotes ones (ε3/ε3). The lowered number of ApoE ε4 carriers is due to a reduced extent of this genotype in the human race compared to other polymorphisms of the same protein (ApoE ε2 or ApoE ε3) [5, 18]. When the DNA concentration was too low to allow a correct discrimination of ApoE alleles by RFLP, subjects’ genotypes were established by ABI PRISM310 Automated Sequencer.
Table 1
Cohort oh healthy volunteers engaged in the study.
Groups | Number of subjects (N) | Age (years) | Sex (M/F) | Physical activity level (Borg scale) |
ApoE ε4 carriers | 16 | 39±14 | 7/9 | 9±3 |
ApoE non-ε4 carriers | 26 | 40±13 | 12/14 | 10±4 |
NA ApoE ε4 carriers | 8 | 38±11 | 3/5 | 7±1 |
A ApoE ε4 carriers | 8 | 41±21 | 4/4 | 13±2 |
NA ApoE non-ε4 carriers | 13 | 41±14 | 5/8 | 7±1 |
A ApoE non-ε4 carriers | 13 | 39±12 | 7/6 | 13±2 |
The subjects were grouped in ApoE ε4 carriers and ApoE non-ε4 carriers, on the base of ApoE polymorphism (Sect. 2.1.). In each group, the subjects are further classified in non-active (NA) and active (A), on the base of physical activity level (Sect. 2.3.). The number of recruited subjects (N), the age (years), and sex (M/F) are indicated. Values are expressed as mean ± SD.
2.2. Clinical parameters of the enrolled subjects
Italian healthy subjects with an upper-middle socio-economic status have been recruited for the current study. Each participant shows neither cardiovascular disease nor other major medical disorders, thus established by clinical history, physical examination, blood pressure, blood chemistry, haematology, urine analysis, basal and stress electrocardiography, with a maximal graded cycle ergometry test executed by a cardiologist blinded to the other data [18, 19, 24]. Familiar AD cases were excluded from subjects’ sampling.
Generally, the major inclusion criteria were as follows: diastolic arterial blood pressure lower than 90 mmHg, systolic arterial blood pressure lower than 140 mmHg, body mass index lower than 30 kg/m2, plasma triglycerides from 30 to 150 mg/ml, total plasma cholesterol ranging from 120 to 220 mg/ml, and HDL cholesterol from 26 to 75 mg/ml. Smokers and subjects in treatment with drug/nutraceutical were excluded from the study [18, 19].
2.3. Levels of physical activity of the enrolled subjects
The participants were grouped into non-active (NA) and active (A) based on the habit’s questionnaire (Table 1). According to the World Health Organization (WHO) [25], a non-active subject performs less than 150 minutes per week of physical activity. Moreover, the Borg Rating and Perceived Exertion (RPE) Scale has been employed to evaluate the intensity level of physical activity [26]. The scale ranges from 6 to 20: 6 corresponds to no exertion at all, 7.5 to extremely light, 9 to very light, 11 to light, 13 to somewhat hard, 15 to hard, 17 to very hard, 19 to extremely hard, and 20 to maximal exertion [18].
2.4. Blood specimen collection
The whole blood was collected from each volunteer at least 48 h later the last exercise bout and it was conserved into an anticoagulant EDTA tube. The blood was centrifuged at 200 x g at 4 °C for 10 min to separate erythrocytes from plasma.
The plasma supernatant was isolated and conserved at -20 °C until use. The erythrocyte pellet was suspended in 3 mL of PBS, centrifuged at 1000 x g for 10 min, and washed with PBS. Following further centrifugation at 1500 x g for 10 min, the isolated erythrocytes were conserved at -20 °C until use [18].
2.5. Assessment of the total antioxidant capability (AOC) in plasma
The total antioxidant capability (AOC) in plasma was assessed using the total oxyradical scavenging capacity (TOSC) assay, a gas chromatographic assay able to define oxyradical scavenging capacity of biological fluids [18, 19, 27]. Hydroxyl radicals were generated at 35 °C by the iron plus ascorbate-driven Fenton reaction (1.8 mM Fe3+, 3.6 mM EDTA, and 180 mM ascorbic acid in 100 mM PBS, pH 7.4). Reactions with 0.2 mM KMBA (alpha-keto gamma-methylthiobutyric acid) were performed in 10 mL vials sealed with gas-tight Mininert valves (Supelco, Bellefonte, PA) in a final volume of 1 mL. Ethylene production was quantified by gas chromatographic analysis of 200 µL aliquots taken from the headspace of vials at timed intervals during the reaction (Hewlett-Packard gas chromatograph, HP 7820A Series, Andoven, M, equipped with a Supelco DB-1 capillary column and a flame ionization detector, FID). Total ethylene formation was measured from the area under the curves that best define the experimental points obtained for control reactions and after the addition of plasma during the reaction [18, 27, 28]. The equation TOSC = 100 − (SA / CA x 100) was used to determine the TOSC values: SA is the area under the curve (AUC) for the sample and CA is the control reaction. A TOSC value of 100 is correlated with a sample able to suppresses the ethylene formation, while a negative TOSC value is attributed to a pro-oxidant sample. A TOSC value of 0 corresponds to a sample without scavenging capacity [29]. Each experiment was performed twice to consider the intrinsic variability of the method. The results were indicated in TOSC units/ml [18, 27, 30].
2.6. Quantification of Amyloid Beta (Aβ) in Erythrocytes
The concentration of Aβ in erythrocytes was measured by an enzyme-linked immunosorbent assay (ELISA), as described [18, 19]. The plate was pre-coated with a specific antibody to Aβ (sc-9129, Santa Cruz Biotechnology), diluted in poli-L-ornithine, and maintained overnight at 4 °C. Following washing with PBS-T (PBS, containing 0.01% Tween 20), to block non-specific sites, BSA 1% was added and incubated for 2 h at 37 °C. After washes with PBS-T, erythrocytes (0.05 mg/100 µL) were added to each well (100 µL/well) and incubated for 1 h at 25 °C. Then, a polyclonal antibody to Aβ (sc-5399, Santa Cruz Biotechnology) was employed and incubated for 1.5 h at 25 °C. Consequently, a HRP antibody (Santa Cruz Biotechnology) was added to each well and incubated for 1 h at 37 °C. The 3,3′,5,5′-tetramethylbenzidine (TMB) (Thermo Scientific) and, consequently, the Stop Solution (H2SO4), were added and the absorbance was read at 450 nm (EnSight Multimode Plate Reade, PerkinElmer). All measurements were performed in duplicate. The standard curve for ELISA assay was constructed using recombinant human Aβ solution at different concentrations [18, 19, 21].
2.7. Evaluation of erythrocyte amyloid precursor protein (APP)
The amyloid precursor protein (APP) levels in erythrocytes were evaluated through a sandwich ELISA kit (Human Amyloid Precursor Protein, ELISA kit, MyBioSource, #MBS731247).
Erythrocytes (50 µL), isolated from whole blood as already described (Sect. 2.4.), were diluted 1:10 in PBS pH = 7.0-7.2 and incubated in the wells of the pre-coated plate, together with balance solution (5 µL) and conjugate (100 µL), for 1 h at 37 °C. Then, each well was thoroughly washed to remove all unbound components. Substrate solutions were added to each well. After a short incubation period necessary to the substrate to react with the enzyme (HRP), and following the addition of sulphuric acid to terminate the enzyme-substrate reaction, the absorbance was read at 450 nm (EnSight Multimode Plate Reade, PerkinElmer). A standard curve was designed relating the intensity of the colour (O.D.) to the concentration of standards. The APP concentration (ng/mg of total proteins) was interpolated from the standard curve.
2.8. Evaluation of erythrocyte expression of β-secretase 1 (BACE1)
The β-secretase 1 (BACE1) amount in erythrocytes was evaluated through a sandwich ELISA kit (Human Beta-secretase 1, ELISA kit, Thermo Scientific Pierce, #EHBACE1).
Erythrocytes (100 µL), isolated from whole blood as already described (Sect. 2.4.), were diluted 1:25 in 1X assay diluent and incubated for 2.5 h at room temperature with gentle shaking. Following washing, a 1X biotinylated antibody (100 µL) was added to each well and incubated for 1 h at room temperature with gentle shaking. After washing, Streptavidin-HRP solution (100 µL) was added to each well and incubated for 45 min at room temperature with gentle shaking. Then, the wells were washed and TMB substrate (100 µL) was added to each well. The colorimetric reaction was stopped by the adding of the stop solution (50 µL) to each well. The absorbance was read at 450 nm (EnSight Multimode Plate Reade, PerkinElmer). The standard curve was generated by plotting the absorbance obtained from each standard. The BACE1 concentration (ng/mL) was quantified according to the standard curve.
2.9. Quantification of the total amount of erythrocyte nuclear factor erythroid 2-related factor 2 (Nrf2)
The nuclear factor erythroid 2-related factor 2 (Nrf2) was quantified in erythrocytes by a high throughput assay, that combines a quick ELISA assay with a sensitive and specific non-radioactive one for transcription factor activation (Nrf2 Transcription Factor Assay Kit, Colorimetric, abcam, #ab207223). Through this assay, only active Nrf2 that is present in the sample is detected by a primary antibody that recognizes an epitope of Nrf2 accessible only when the protein is activated.
Erythrocytes (10 µL, i.e. 5–20 µg), separated from the whole blood as above described (Sect. 2.4.), were diluted in the completed binding buffer and incubated for 1 h at room temperature with mild agitation (100 rpm). After extensive washes, a primary antibody (100 µL) was added and incubated for 1 h at room temperature without shaking. Following washing, a secondary antibody (100 µL) was added and incubated for 1 h at room temperature without shaking. Then, the wells were washed and the developing solution was added and incubated. After the addition of the stop solution, the absorbance was read at 450 nm (EnSight Multimode Plate Reade, PerkinElmer). The Nrf2 amount was calculated from Nrf2 activation absorbance and normalised to the absorbance of the total proteins in the sample (µg/µL).
2.10. Measurement of erythrocyte histone deacetylase 6 (HDAC6)
The histone deacetylase 6 (HDAC6) was detected in erythrocytes with a competitive ELISA kit (Human Histone Deacetylase 6, HDAC6, Elisa Kit, Competitive ELISA, MyBioSource, #MBS7254230).
Erythrocytes (100 µL), isolated from whole blood as already described (Sect. 2.4.), were diluted 1:10 in PBS pH = 7.0-7.2 and incubated in the wells of the pre-coated plate, together with balance solution (10 µL) and conjugate (50 µL), for 1 h at 37 °C. Afterward, the wells were carefully washed to remove all unbound components. Substrate solutions were added to each well and incubated for a few minutes. Following the addition of sulphuric acid to terminate the enzyme-substrate reaction, the absorbance was read at 450 nm (EnSight Multimode Plate Reade, PerkinElmer). A standard curve was designed relating the intensity of the colour (O.D.) to the concentration of standards. The HDAC6 concentration (pg/mg of total proteins) was interpolated from the standard curve.
2.11. Expression of plasma Kelch-like ECH-associated protein 1 (Keap1)
The expression of plasma Kelch-like ECH-associated protein 1 (Keap1) was detected by western-blot analysis.
Briefly, plasma (10 µg of total proteins, quantified through Lowry assay), opportunely isolated from the whole blood as above described (Sect. 2.4.), with additional Laemmli solution, was resolved by electrophoresis using a 4–20% Criterion TGX stain-free precast gel (Bio-Rad, #5678094). Afterward, the samples were transferred by the Trans-Blot Turbo transfer system (Bio-Rad) to Trans-Blot Turbo Midi 0.2 µM PVDF membrane (Bio-Rad, #1704157). Then, the membrane was incubated for at least 1 h with a buffer able to block non-specific sites (5% Milk,). Primary antibody against Keap1 (rabbit, #AV38981, Sigma-Aldrich) was used and incubated overnight at 4 °C, under continuous agitation. Following the incubation with a secondary antibody HRP-conjugated, protein bands were detected with a chemiluminescent substrate (Clarity Western ECL Substrate, Bio-Rad, #1705061). Densitometry was performed by ImageJ Software. Images were obtained in different western blots using a reference standard for each running gel, due to the impossibility to show all the samples at the same time.
2.12. Analysis of the expression of circulating miRNAs
Plasma, isolated from the whole blood as previously described (Sect. 2.4.), were processed by miRNeasy Serum/Plasma Mini Kit (Qiagen, Hilden, Germany) to isolate total RNA, including microRNAs (miRNAs). Retro-transcription was carried out using miRCURY LNA miRNA RT Kit (Qiagen, Hilden, Germany) and the obtained cDNA was diluted 1:30, immediately before use. Real-time PCR was run on the MiniOpticon CFX 48 real-time PCR Detection System (Bio-Rad, Hercules, CA, USA) using miRCURY LNA miRNA SYBR Green PCR and specific miRCURY LNA miRNA PCR Assay (Qiagen, Hilden, Germany), as previously reported [31]. MiRCURY Primer Assay specific for hsa-miR-195-5p (MIMAT0000461), hsa-miR-153-3p (MIMAT0000439) and hsa-miR-93-5p (MIMAT0000093) were purchased by Qiagen (Hilden, Germany).
The relative miRNA expression was calculated using the Ct method and normalized on miR-93-5p. Several pieces of evidence reported high stability of miR-93-5p in biofluids [32–35] thus miR-93-5p was suggested as plasmatic reference gene in manufacture’s handbook. According to this, the expression levels of miR-93-5p in plasma samples of our cohort showed comparable expression levels without significant difference among groups (data unshown).
2.13. Statistical analysis
The data are shown as the mean value ± S.D. (Standard Deviation). The data relative to Aβ, APP, BACE1 were depicted as median values. Sample size calculator was performed to estimate the accuracy of the results. Accordingly, the study group required 40 patients to obtain the same difference with α = 0.05 and a statistical power of 85%.
Kolmogorov–Smirnov tests were used to data meeting the assumption of a normality distribution. The variance between groups was statistically significant using Bartlett’s test. One-way analysis of variance (ANOVA) test was used to evaluate differences among groups for data meeting the assumption of homogeneity of variance. Pearson correlation analysis and t-tests were applied when only two groups were present for data with distributions that met parametric assumptions. Chi-square tests (Pearson’s, Yates-adjusted or Fisher’s exact test according to sample size), Mann–Whitney U tests, and Spearman correlation analysis were employed in situations where parametric assumptions were not met. Tukey’s multiple comparison was applied for the densitometry analysis. Statistical analysis for miRNA expression was performed using the Kruskal-Wallis (non-parametric) followed by Dunn’s multiple comparisons test.
Correlation between variables was determined by linear regression analysis, while interactions between variables were analysed by correlation and multiple regression analyses. P values < 0.05 were considered significantly different. All statistical analysis were carried out by commercial software (GraphPad Prism, version 7.0; GraphPad Software Inc., San Diego, CA) [18, 21, 28].