The method of this study was based on ethical standards from the Helsinki Declaration, approved by the ethics committee of Ahwaz Jundishapur University of Medical Sciences ( IR.AJUMS.REC.1398.865) and registered on the Iranian Registry of Clinical Trials (IRCT) ( http://www.irct.ir: IRCT20181210041918N2). Informed consent was obtained from all patients.
2.1. Study design and participants
The present study was a 24-week randomized, double-blind clinical trial which conducted between July 2019 and April 2020. The participants enrolling in this trial were patients referred to the MS association of Khuzestan, Iran, who were included in the study if they met the following criteria: age range between 18 to 50, clinical definite MS according to McDonald criteria and an Expanded Disability Status Scale (EDSS) score £4.5. Exclusion criteria included the presence of any concomitant disease such as rheumatoid arthritis, Inflammatory bowel disease (IBD), rheumatoid arthritis, systemic lupus, type 1 diabetes and other autoimmune diseases, use of anti-inflammatory drugs, omega-3 and probiotic supplements or other antioxidant and anti-inflammatory supplements, corticosteroid therapy, current smoker, disease duration of less than 1 year, malnourishment (BMI under 18.5) or morbid obesity (BMI>35), impaired Th1/Th2 balance, (such as asthma, rheumatoid arthritis and type 1 diabetes mellitus), pregnancy and lactation. All patients were under the guidance of two neurologists and no restrictions were set on concomitant immunomodulatory treatment (i.e. Interferon beta-1a (IFN β-1a) (Avonex), glatiramer acetate, or natalizumab).
Finally, 70 patients with MS were randomly allocated in the intervention and control groups, so that 35 patients were divided into the intervention group and 35 patients into the control group. Randomization lists were computer-generated by a statistician and given to the interviewer. The randomization process took place in a way that researchers, neurologist, staff and patients were blinded.
Patients which participated in the study were randomly divided into two groups receiving probiotic supplement and placebo. Patients in the intervention group received 2 multi-strain probiotic capsules/day for six months, each containing minimum 2 billion live microor-ganisms (2 × 109 CFU/capsule), equivalent to 10 billion live microorganisms per gram (1 × 1010 CFU/gram) of14 strains (Bacillus subtilis PXN 21, Bifidobacterium bifidum PXN 23, Bifidobacterium breve PXN 25, Bifidobacterium infantis PXN 27, Bifidobacterium longum PXN 30, Lactobacillus acidophilus PXN 35, Lactobacillus delbrueckii ssp. bulgaricus PXN 39, Lactobacillus caseiPXN 37, Lactobacillus plantarum PXN 47, Lactobacillusrhamnosus PXN 54, Lactobacillus helveticus PXN 45,Lactobacillus salivarius PXN 57, Lactococcus lactis ssp.lactis PXN 63, Streptococcus thermophilus PXN 66), cellulose (bulking agent) and vegetable capsule (Hydroxy-propylmethyl Cellulose). Participants in the control group received the same amount of placebo for 6 months, and the placebo capsules contained microcellules, so that there was no difference in appearance or smell between probiotic and placebo capsules.
At the beginning of the study, after fully explaining the study protocol and receiving informed consent from patients, patients' demographic information was recorded and then probiotic and placebo boxes were given to patients for 6 weeks. Subsequent probiotic and placebo boxes were presented to patients in the sixth, twelfth and eighteenth weeks. During the 24-week intervention period, patients were reminded of the use of probiotics and placebo by texting and calling. Patients who did not consumed more than 10% of the given capsules in each period were excluded from the study.
Anthropometric measures, dietary intake and physical activity
To evaluate anthropometric variables, at the beginning and after week 24, patients' height and weight were measured by a trained researcher. Patients 'weight was measured using an Inbody device (Inbody BDM370, Korea) without shoes, light clothing with the precision of 0.1 kg and patients' height was measured using a Seca scale without shoes to the nearest 0.5 cm. The standard formula was also used to calculate body mass index (BMI).
To evaluate the dietary intake of the study participants, 3 days 24 hours' dietary recall on one holiday and two working days at the beginning and end of the study was recorded from all participants. The analysis of 24-h food recall questionnaires was done using Nutritionist IV (N4) (First Databank, Hearst Corp, San Bruno, CA, USA).
Physical activity was assessed by the metabolic equivalent of task (MET) questionnaire at
the beginning and the end of the study.
Serum biochemical measurement
To evaluate biochemical variables, at the beginning and end of the study, 10 cc of blood was taken from patients after 12 hours of fasting. Blood samples were allowed to clot at room temperature (20-25°C) for20 min in a vertical position. Then, the samples were centrifuged at 3000 rpm for 10 min and serum samples were frozen at −70°C, until biochemical marker measurement. ELISA kits (Diaclone Research, Besançon, France) were used to assess the concentration of Interferon gamma (IFNγ), Interleukin 17 (IL-17) and Interleukin 35 (IL-35). Also, for evaluation serum levels of TGF-β and FOXP3, we used from the Crystal Day Elisa kits (Shanghai Crystal Day Biotech, China).
All statistical analyses were performed with SPSS 19 software. Kolmogorov–Smirnov test was used to assess the normality of the data. Numerical data were presented as mean ± SD. Mann- Student’st-test was used to compare the continuous data and alternative non-parametric tests were used if the data not normally distributed. Categorical data were compared using the Chi-square test. Paired t-test or McNemar test was applied for intra-group comparison. Also, analysis of covariance was used to remove confounding variables. All of the analysis were adjusted for the age, sex, disease duration and calorie intake. P< 0.05 was considered significant.