Investigating the Effect of Nanomicellar Silymarin on Immunological Indicators in Patients with Rheumatoid Arthritis.

Evaluating effect of nanomicellar silymarin on clinical and immunological indicators in rheumatoid arthritis

A randomized, triple-blind, superiority, parallel group trial with three arms (intervention, placebo, control). Sample size: 45 patients (15 per group). Randomization: block randomization (block size 4) using computer-generated random sequence from www.randomization.com with allocation concealment by sequentially numbered, opaque, sealed envelopes. Single center (Ghaem Hospital, Mashhad, Iran). Phase 3.

Single-center trial in Mashhad. 45 RA patients randomized to 3 groups: silymarin+DMARDs, placebo+DMARDs, DMARDs alone. 8 weeks. Triple-blind (patient, assessor, analyst). Independent person seals allocation.

Inclusion: Active RA (ACR/EULAR 2010, score≥6); age 18-60y; disease duration 1-2y; on standard DMARDs (MTX≤10mg/wk + prednisolone≤5mg/d); no biologics. Exclusion: Malignancy; diabetes/CKD/heart failure; pregnancy/breastfeeding; BMI≥35; K/L grade≥4; unwillingness.

Group1 (Intervention): Standard DMARDs + nanomicellar silymarin (SinaLiv) 70mg, 1 cap/day, 8 weeks. Group 2 (Placebo): Standard DMARDs + identical placebo (gelatin capsule with corn starch), 1 cap/day, 8 weeks. Group 3 (Control): Standard DMARDs only.

Primary outcomes (measured at baseline and week 8): 1. Gene expression changes of 22 immune-related genes including IL-1, IL-4, IL-10, IL-17, IL-18, IL-22, IL-23, TGF-β, IFN-γ, CCL2, CCL5, CXCL8, NLRP3, P2XR, BCMA, APRIL, TACI, BAFF, T-bet, GATA3, ROR-γt, and FOXP3 measured by real-time PCR. 2. Serum levels of CRP, RF, and Anti-CCP. Secondary outcomes (measured at baseline and week 8): 1. DAS28-ESR score (clinical disease activity index). 2. VAS score (pain assessment).

Randomization method: Block randomization (stratified block randomization). Unit of randomization: Individual patient. Randomization strata: Patients will be stratified based on age (18-40 years and 41-60 years) and disease duration (1-1.5 years and 1.5-2 years) to ensure balance between groups. Tools used: The random sequence will be generated using www.randomization.com website with a table of random numbers. Block size of 4 will be used with 8 blocks total. How the random sequence will be built: A computer-generated random allocation sequence will be created by an independent statistician who will not be involved in patient enrollment or outcome assessment. Block sizes of 4 will be used to ensure equal allocation to three groups (intervention, placebo, control) with 15 patients per group. Allocation concealment: Yes, allocation concealment will be performed using sequentially numbered, opaque, sealed envelopes. The envelopes will be prepared by a person not involved in clinical assessments or data analysis. The randomization code will remain sealed and will be kept by this independent person until final data analysis is completed. The key to the code will not be accessible to patients, clinicians, or outcome assessors until the study is unblinded after final analysis

This is a triple-blind trial. Blinding will be implemented as follows: - Participants: Patients will not know their group allocation. Intervention and placebo capsules are identical in color, size, and packaging. - Principal investigator: The PI will be blinded and will not be involved in preparation or distribution of the study medications. - Healthcare providers: Routine care physicians (other than the outcome assessor) are not blinded because the intervention is an add-on to standard care. - Data collector: The person collecting blood samples and laboratory data will not know group allocation. - Outcome assessor: The rheumatologist performing clinical evaluations (DAS28-ESR, VAS) will be blinded to group assignment. - Data analyst: The statistician will receive a de-identified dataset with coded groups (e.g., A, B, C) and will remain blinded until all analyses are completed. - DSMB: Not applicable for this pilot trial. Blinding method: An independent person not involved in patient care, assessment, or analysis will prepare sequentially numbered, opaque, sealed envelopes containing the allocation code. This person will package and code the identical capsules. The allocation key will remain sealed until final data analysis. Emergency unblinding is possible according to protocol and will be documented. Blinding will be checked by asking blinded personnel about their guesses at the end of the study.

The current study protocol does not include a plan for public sharing of individual participant data (IPD). Summary results will be published in a peer-reviewed journal."