Proteomic analysis of Moringa oleifera's anti-arthritic effects on human fibroblasts-like synoviocytes

Abstract

Rheumatoid arthritis (RA) affects more than 1.3 million Americans, making it the most common auto-immune arthritic disorder in the U.S. Current treatments are largely based on non-steroidal anti-inflammatory drugs (NSAIDs) and corticosteroids. However, these drugs are either ineffective, costly or have a plethora of side effects, notably immune suppression and increased risks of heart attacks. Therefore, here, we sought to examine the anti-arthritic effects of a tropical medicinal plant, Moringa oleifera (MO), and its underlying mechanism using proteomics analysis and primary human fibroblast-like synoviocytes (HFLS) harvested from healthy people and a patient with arthritis (HFLS-RA). Initial experiments optimized MO's optimal dosage by examining MO's effect on cell viability using MTT assay. Cells were divided into the following groups: (1) Negative control groups [(a) HFLS and (b) HFLS-RA, 0.1 M phosphate-buffered saline (PBS) only] and (2) MO treatment group (HFLS and HFLS-RA), dose-dependent treatment (500, 750, 1000, 50, 75, 100, 150 and 200 mg/mL). For proteomics studies, cells were divided into the following groups: (1) Negative control group (HFLS-RA), vehicle only (as above) and (2) MO Treatment group (HFLS-RA), 75 mg/mL (optimal concentration). After 24 h of treatment, cells were harvested and analyzed for cell viability (MTT assay) or proteome-wide expression (Proteomics). Data generated from proteomics were verified by confocal immunofluorescence. The MTT assay data shows that below 75 mg/mL, MO is not cytotoxic and therefore does not affect cell viability. Proteomics data revealed that MO alters expression of all the 40 proteins that are aberrantly expressed in HFLS-RA by either mitigating their expression (35 proteins) or enhancing (5 proteins) them. Specifically, the 35 proteins were mostly associated with pathological processes, such as inflammation, aberrant proliferation and cell adhesion, whereas the 5 proteins (Heat shock 70 kDa protein 1A/1B, Kallistatin, programmed cell death 6-interacting protein, Hemoglobin subunit alpha, and Aldo-keto reductase family 1 member C1) were associated overall with normal protective processes, such as anti-inflammatory and apoptotic activities, and were downregulated in HFLS-RA. We conclude that MO is potentially a good candidate for developing alternative therapy for managing arthritis.