Screening assessment of trimethoxy flavonoid and - (-)-epigallocatechin-3-gallate against formalin-induced arthritis in Swiss albino rats and binding properties on NF-κB-MMP9 proteins

Abstract

The isolated trimethoxy flavonoid 4a,5,8,8a-tetrahydro-5-hydroxy-3,7,8-trimethoxy-2-(3,4-dimethoxyphenyl) chromen-4-one (TMF) from methanolic stem extract of T chrysantha (METC) and - (-)-epigallocatechin-3-gallate (EGCG) can be used to suppress acute inflammation and arthritis as an ethical medicine in Ayurveda. The nuclear factor kappa beta (NF-κB) signaling is involved in the expression of inflammatory mediators such as TNF-α and IL-1β. A successive investigation of NF-κB–MMP9 signaling during the production of inflammatory mediators needs to be developed. The docking studies of compounds TMF and EGCG were carried out using Autodock 4.0 and Discovery studio Biovia 2017 software to find out the interaction between ligand and the target proteins. The anti-arthritic potential of TMF, EGCG, and indomethacin was evaluated against formalin-induced arthritis in Swiss albino rats. Arthritis was assessed by checking the mean increase in paw diameter for 6 days via digital vernier caliper. The blood cell counter and diagnostic kits measured the different blood parameters and Rheumatoid factor (RF, IU/mL). The interleukin-1β (IL-1β) and tumor necrosis factor (TNFα) in serum were determined by ELISA, and the pERK, MMP9, and NF-κB expressions in the inflamed tissue were determined by Western blotting, respectively. The mRNA expression for inflammatory marker enzymes such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) was determined by qRT-PCR. Based on grid score, interactions, and IC50 values in molecular docking studies, the TMF and EGCG can be effectively combined with proteins NF-kB and MMP9. The TMF-HD and EGCG-HD better suppressed the acute inflammation and arthritis with marked low-density pERK, MMP9, NF-κB, iNOS, COX-2 levels. The endogenous antioxidant levels were increased in TMF and EGCG treated rats. The TMF and EGCG effectively unraveled acute inflammation and arthritis by suppressing NF-κB mediated MMP9 and cytokines.