Litsea salicifolia Roxb. bark ethanolic extract attenuates rheumatoid arthritis by inhibiting TLR4/NF-κB and activating SIRT1-Nrf2/HO-1 signaling axis: In-vitro and in-vivo evaluations

Abstract

BackgroundLitsea salicifolia Roxb. (LS) is a pioneer medicinal plant that can be found in tropical and subtropical countries like India, China and Japan. LS is traditionally used for treating chronic pulmonary inflammatory diseases like asthma and wound healing. Also, LS contains an essential oil with powerful anti-oxidant and excite-repellency properties. However, no research findings have been reported about its protective activity on chronic inflammatory disease like rheumatoid arthritis (RA) till yet. PurposeThis study aims to investigate the anti-rheumatic potential of ethanolic extract of LS (LSE) in-vitro on RAW 264.7 cells for its anti-oxidant and anti-inflammatory activities and in-vivo CFA-induced adjuvant arthritis in the rat model. MethodsThe chemical characterization of LSE was identified by using LC-MS/MS. The in-vitro effects of LSE on RAW 264.7 cells stimulated with LPS 1 µg/ml were investigated to its potential therapeutic effects. Freund's complete adjuvant (CFA) arthritis was induced by 100 µl of CFA intradermally administered to rats. LSE 100 and 200 mg/kg were administered orally from day1 to day 28. Parameters such as paw volumes measured, X-ray radiography analysis, anti-oxidant status, Gene expression studies, western blot analysis and histological analysis were performed to check the anti-oxidant and anti-inflammatory effects of LSE. ResultsOur in-vitro results revealed that LSE at 5 μg/ml and 10 μg/ml reduced the LPS-induced oxidative stress burden evidenced by reduced ROS levels and improved endogenous anti-oxidants Nrf2 and SIRT1 in RAW 264.7 cells. Also, LSE inhibited the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α, PGE2, COX-2 and NO levels induced by LPS in murine macrophage cells at 5 μg/ml and 10 μg/ml in a dose dependent manner. Our in-vivo results revealed that LSE significantly attenuated the CFA-induced inflammation by restoring body weight, arthritis index, nitrosative and lipid peroxidation assays. LSE treatment significantly reduced the pannus formation and bone erosion evidenced by histopathology of joint tissues. Additionally treatment with LSE significantly reduced inflammation-mediated protein expression levels like TLR4, NF-κB, COX-2 and iNOS levels and improved the Nrf2/HO-1/SIRT1 levels in paw tissues evidenced by immunoblotting. ConclusionLSE alleviated RA through its anti-oxidant and anti-inflammatory properties by inhibiting TLR4-mediated NF-κB nuclear translocation and by activating the Nrf2/SIRT1 signaling pathways, according to our in-vitro and in-vivo findings.