Design and population
Among 285 subjects who attended to Sina hospital neurology clinic in Tehran, 80 men and women were recruited to this double-blind (neither the participants nor the experimenters) randomized controlled trial on July 2015. Participants were adults and had a current diagnosis of episodic migraine in accordance with the IHS criteria (≥ 15 headache days per month for more than 3 months or ≥ 1 attack per week) (15). All recruiting criteria are shown in Table 1.
Omega-3 and nanocurcumin supplementation
The stratified randomization method based on sex and BMI was used to assign participants in 4 groups to receive 2 months supplementation of Group 1) 2 capsules containing 600 mg EPA + 300 mg DHA + 100 mg other W-3 FAs + 1 placebo capsule of nanocurcumin, Group 1) 1 capsule containing 80 mg nanocurcumin + 2 placebo capsule of W-3, 3) 2 capsules containing 600 mg EPA + 300 mg DHA + 100 mg other W-3 FAs + 1 capsule containing 80 mg nanocurcumin (group 3) and 4) 2 placebo capsule of W-3 + 1 placebo capsule of nanocurcumin (group 4 or control group). Placebo capsules were composed of edible paraffin with the same appearance as W-3 and nanocurcumin capsules. Random allocation and enrolling participants to the study were done by trained people who were outside the investigation. Administration of 25–50 mg from three types of cyclic antidepressants (amitriptyline or nortriptyline) and 20–40 mg of β-blockers (eg propranolol) (as a prophylaxis treatment) along with study supplements was given to all groups. In comparison to W-3 FAs and nanocurcumin, β-blockers and cyclic antidepressants have a different mechanism of action in reducing inflammatory state (16). Patients were asked to abstain from altering their dietary habits and physical activity during the intervention. Moreover, we instructed patients to consume NSAIDS in times of severe headache attacks.
Questionnaires
A predesigned questionnaire was used to consider general characteristics of migraine patients including gender, age, financial perception, migraine duration, educational status, history of other diseases and use of medications and dietary supplements. Severity, numbers and duration of headaches were examined by a trained clinician. Scales for headache severity was within 1–10 in which score 1 and 10 were indicative of minimum and maximum pain of headaches. We also sought dietary information via 3-day dietary recall method to obtain an estimate of energy intake.
Anthropometric indices
Measured anthropometric indices included: i) weight (through 803, Seca Clara, Germany with 0.1 kg accuracy with light clothing and no shoes); ii) height (by a stadiometer (Seca) with 0.1 cm accuracy); and iii) WC (with a common tape after normal exhalation).
Statistical analysis
Sample size was computed based on the VCAM-1 variable considering the difference of 1.817 ng / dl between the intervention and control groups via the following formula (If α = 0.05, 1- β = 1.28 (Power = 90%)) (17)
N = 17 was obtained for each study group. By considering 20% missing in study sample size during intervention, 20 subjects were selected for each study group. Data normality was checked through Kolmogorov–Smirnov distribution. For comparison of normal quantitative data means among groups and in each group, ANOVA paired t-tests were conducted, respectively. We used kruskal-wallis and Wilcoxon to compare abnormal quantitative variables between groups and in each single group, respectively. We also conducted ANCOVA test to compare study outcomes between groups with adjustment of confounding factors (BMI, energy intake and age). The data were finally analyzed by SPSS version 22, in which P-value ≤ 0.05 was considered as significant.
Measurement of serum concentration and gene expression of VCAM
Laboratory measurements were done at the laboratory of School of Public health, Tehran University of Medical Sciences. At baseline and after 2-month intervention, two blood samples from subjects were collected including 10 ml in tubes containing heparin for PBMCs separation and 5 ml that was centrifuged quickly (10min) at 3500 RPM for serum separation. Heparin-containing tubes were diluted in a 1:1 ratio in PBS. Afterwards, lymphocytes and monocytes were separated in Ficoll gradient-gel by centrifugation at 800 RPM for 40 min at 4°C. Obtained cells were washed in PBS for two times and then centrifuged at 600 RPM for 10 min at 4°C for the second time. Total RNA from cells were obtained using RNeasy Plus Mini Kit (Qiagen, Valencia, CA, USA). For RNA quality and quantity assessment, a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) was applied, in which RNAs were considered as pure, if had absorbance ratios 260/ 280 nm between 1.9–2.1. The purified RNA was reverse transcribed to result in the synthesis of DNA using QuantiTect Reverse Transcription Kit (Qiagen, Germany). In the PCR step, appropriate primer for VCAM was designed using Primer Express 3 software (Applied Biosystems). Whole genome sequences of VCAM primer were forward: 5'-TAGCGTGTACCCCCTTGACC-3' and reverse: 3'-AACTTAGCCTGACAAACAAGAGC-5'. PCR Analysis were performed through the StepOne system (Applied Biosystems, Foster City, CA, USA) using 7 µL of Enzymes (Taq polymerase, SYBR Green, etc.) (Applied Biosystems, Foster City, CA, USA), 2 µL of cDNA, 0.5 µL of each forward and reverse primers in an ultimate volume of 20 µL (18). Finally, the calculation of VCAM gene expression was conducted by Ct (2-ΔΔCt) equation. Blood serums were stored in -80 ° C until serum VCAM analysis via ELISA method (Bioassay Technology Laboratory, Chain).