Computational Deciphering of the Role of S100A8 and S100A9 Proteins and Their Changes in the Structure Assembly Influences Their Interaction with TLR4, RAGE, and CD36.

S100A8 and S100A9 belong to the calcium-binding, damage associated molecular pattern (DAMP) proteins shown to aggravate the pathogenesis of rheumatoid arthritis (RA) through their interaction with the TLR4, RAGE and CD36 receptors. S100A8 and S100A9 proteins tend to exist in monomeric, homo and heterodimeric forms, which have been implicated in the pathogenesis of RA, via interacting with Pattern Recognition receptors (PRRs). The study aims to assess the influence of changes in the structure and biological assembly of S100A8 and S100A9 proteins as well as their interaction with significant receptors in RA through computational methods and surface plasmon resonance (SPR) analysis. Molecular docking analysis revealed that the S100A9 homodimer and S100A8/A9 heterodimer showed higher binding affinity towards the target receptors. Most S100 proteins showed good binding affinity towards TLR4 compared to other receptors. Based on the 50 ns MD simulations, TLR4, RAGE, and CD36 formed stable complexes with the monomeric and dimeric forms of S100A8 and S100A9 proteins. However, SPR analysis showed that the S100A8/A9 heterodimers formed stable complexes and exhibited high binding affinity towards the receptors. SPR data also indicated that TLR4 and its interactions with S100A8/A9 proteins may play a primary role in the pathogenesis of RA, with additional contributions from CD36 and RAGE interactions. Subsequent in vitro and in vivo investigations are warranted to corroborate the involvement of S100A8/A9 and the expression of TLR4, RAGE, and CD36 in the pathophysiology of RA.

Methanolic extract of O.umbellata L. exhibits anti-osteoporotic effect via promoting osteoblast proliferation in MG-63 cells and inhibiting osteoclastogenesis in RANKL-stimulated RAW 264.7 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Oldenlandia umbellata L., belonging to the Rubiaceae family, is an annual plant possessing anti-inflammatory and antipyretic, anti-nociceptive, anti-bacterial, anti-helminthic, antioxidant and hepatoprotective activities and used in traditional medicine to treat inflammation and respiratory diseases. AIM OF THE STUDY: The present study aims to evaluate the anti-osteoporotic effect of Methanolic extract of O.umbellata in MG-63 cells and RANKL-stimulated RAW 264.7 cells. MATERIALS AND METHODS: The methanolic extract from the aerial parts of O.umbellata was subjected to metabolite profiling. The anti-osteoporotic effect of MOU was assessed in MG-63 cells and RANKL-stimulated RAW 264.7 cells. In MG-63 cells, the proliferative effect of MOU was evaluated using MTT assay, ALP assay, Alizarin red staining, ELISA and western blot. Similarly, the anti-osteoclastogenic effect of MOU was assessed in RANKL-stimulated RAW 264.7 cells via MTT, TRAP staining and western blot. RESULTS: LC-MS metabolite profiling showed the presence of 59 phytoconstituents including scandoside, scandoside methyl ester, deacetylasperuloside, asperulosidic acid, and cedrelopsin in MOU. In MG-63 cells, MOU has increased the proliferation of osteoblast cells and ALP activity, thereby increasing bone mineralization. ELISA results showed increased levels of osteogenic markers such as osteocalcin and osteopontin in the culture media. Western blot analysis showed inhibition of GSK3ß protein expression and increased the expression levels of ß-catenin, Runx-2, col 1 and osterix, promoting osteoblast differentiation. In RANKL-stimulated RAW 264.7 cells, MOU did not elicit any significant cytotoxicity; instead, it suppressed the osteoclastogenesis reducing the osteoclast number. MOU has reduced TRAP activity in a dose-dependent manner. MOU inhibited the TRAF6, NFATc1, c-Jun, C-fos and cathepsin K expression, thereby inhibiting osteoclast formation. CONCLUSION: In conclusion, MOU promoted osteoblast differentiation via inhibiting GSK3ß and activating Wnt/ß catenin signalling and its transcription factors, including ß catenin, Runx2 and Osterix. Similarly, MOU inhibited osteoclast formation by inhibiting the expression of TRAF6, NFATc1, c-Jun, C-fos and cathepsin K in RANK-RANKL signalling. Finally, it can be emphasised that O.umbellata is a potential source of therapeutic leads for the treatment of osteoporosis.

Underpinning Endogeneous Damp EDA-Fibronectin in the Activation of Molecular Targets of Rheumatoid Arthritis and Identifcation of its Effective Inhibitors by Computational Methods.

Rheumatoid arthritis (RA) is one of the most severe inflammatory diseases that cause swelling, stiffness and pain in the joints, which pose a significant threat worldwide. Damage-associated molecular patterns (DAMPs) are danger molecules of endogenous origin, released during cell injury or cell death, interacts with various Pattern recognition receptors (PRRs) and activates various inflammatory diseases. One of the DAMP molecules, so-called EDA-fibronectin (Fn) is also responsible for causing RA. EDA-Fn triggers RA through its interaction with TLR4. Apart from TLR4, it is divulged that certain other PRR's are also responsible for RA, but the identity and mechanism of those PRRs remain unknown until now. Hence, for the first time, we tried to reveal those PRR's interaction with EDA-Fn in RA through computational methods. Protein-protein interaction (PPI) was checked using ClusPro between EDA-Fn and certain Pattern recognition receptors (PRRs) to explore the binding affinities of the potential PRRs. Protein-protein docking unveiled that TLR5, TLR2 and RAGE has good interaction with EDA-Fn than the well-reported TLR4. Macromolecular simulation was performed for TLR5, TLR2 and RAGE complexes along with the control group TLR4 for 50 ns to further investigate the stability, leading to the identification of TLR2, TLR5 and RAGE as the stable complexes. Hence, TLR2, TLR5 and RAGE on interaction with EDA-Fn may lead to the progression of RA that may need additional validations through in vitro and in vivo animal models. Molecular docking was used to analyse the binding force of the top 33 active anti-arthritic compounds with the target protein EDA-Fn. Molecular docking study showed that withaferin A has a good binding activity with EDA-fibronectin target. Hence, it is emphasized that guggulsterone and berberine could modulate the EDA-Fn-mediated TLR5/TLR2/RAGE pathways, thereby it could inhibit the deteriorating effects of RA which needs further in vitro and in vivo experimental validations.

Identification of galangin as the bioactive compound from Alpinia calcarata (Haw.) Roscoe rhizomes to inhibit IRAK-1/ MAPK/ NF-κB p65 and JAK-1 signaling in LPS stimulated RAW 264.7 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Alpinia calcarata (Haw.) Roscoe rhizomes are used to treat diabetes, rheumatism, gastrointestinal problems, inflammatory diseases, cough and respiratory problems in traditional practices. The primary objective of the study is to identify and isolate anti-inflammatory bioactive compounds from A.calcarata rhizomes and to assess its molecular mechanism. MATERIALS AND METHODS: The bioassay-guided fractionation of methanolic extract of A. calcarata rhizomes yielded chloroform fraction as the effective fraction and galangin as the bioactive compound identified by NMR studies. The anti-inflammatory action of galangin was evaluated by determining NO and cytokine production in LPS stimulated RAW264.7 cells. Further, its mechanism was studied on the expression levels of mRNA and protein targets by qPCR and Western blot analysis. RESULTS: Based on the MTT assay, the concentration of 3.1-25 µM of galangin was selected for further studies. Galangin reduced the levels of NO and proinflammatory cytokines (TNF-α, IL-1ß and IL-6) production in LPS induced RAW 264.7 cells in a dose-dependent manner. In addition, the qPCR analysis revealed a reduction in the mRNA expression levels of COX-2, IRAK 1 and JAK 1 in galangin treated LPS stimulated RAW 264.7 cells in a dose-dependent manner. Western blot analysis implicated that galangin has markedly reduced the protein expression levels of cell signaling regulators (JAK-1, IRAK-1, MyD88, MAPK (p38 and ERK) and NF-κB p65). CONCLUSION: From the results, it is evident that the inhibition of these cell signaling regulators has contributed to the anti-inflammatory effects of galangin. To our knowledge, we are the first to report IRAK-1 and JAK-1 as therapeutic targets of galangin for its anti-inflammatory effect.

Nepafenac loaded silica nanoparticles dispersed in-situ gel systems: Development and characterization.

This research was aimed to develop and evaluate nepafenac loaded silica nanoparticles dispersed in-situ gel system for the improved treatment of ocular diseases. The blank silica nanoparticles prepared by stober's process showed the particle size of 151 nm to 285 nm with the zeta potential of -19.6 to -31.9 mV. The nepafenac loaded silica nanoparticles were spherical in shape with smooth outer surface. The nepafenac loaded silica nanoparticles dispersed in poloxamer - chitosan in-situ gelling system showed gelling temperature of 32 °C with sustained release of nepafenac and higher permeation (58.79 µg) across the goat cornea than poloxamer - poloxamer (21.18 µg) in-situ gelling system. Hence the developed in-situ gelling system containing nepafenac loaded silica nanoparticle could be a promising tool for the topical delivery of drugs to the eye.