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This study elucidated the molecular mechanism of the notch pathway in vascular health and the role of NjRBO as a nutraceutical for the modulation of notch-mediated CD4+ Tcell activation in atherosclerotic rats. In this study, male Sprague-Dawley rats weighing 150-200g given standard diet formula were used. After the study duration of 60 days, in order to determine the nutraceutical effects of NjRBO, we sought to study the effects of treatment with NjRBO on notch pathway components in isolated splenic CD4+ T lymphocytes. In the present study, Western blot analysis revealed that upon high-fat diet supplementation resulted in T cell activation evidenced by increased CD28 co-receptor and CD25 marker expressions. In consistent with the above findings, we analyzed the mRNA expression pattern of Notch1, cleaved notch fragment, Notch-11C and Hes1, which showed a consistent up-regulation upon T-cell activation. Immunofluorescence assay also revealed an increase in Notch 1 receptor expression. Up-regulation in the expression of TCR-activated signalosome complexes or CBM complex in the diseased showed an increase indicating that Carma1-Bcl10-Malt1 (CBM) is a crucial event for T- cell receptor-induced NF-κB activation. Additionally, NF-қB translocation was enhanced in causing a concomitant alteration in Th1, Th2 transcription factors, T-bet, GATA-3 and its respective cytokines, IFN-γ and IL-4. Accordingly, we present evidence that Notch-regulated TCR-mediated activation of CD4+T-cell components was altered by NjRBO treatment, thereby revealing a novel role for the same in controlling TCR-mediated activation and inflammatory milieu.

Background: Progression of chronic inflammatory disease, atherosclerosis is a multifactorial process. Cluster of differentiation 36 (CD36) mediated downstream activation of Toll like receptor 2 (TLR2) and NLRP3 (Nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3) inflammasome signaling pathway actively participates during chronic inflammation. Nowadays, synergistic combinations of bioactive compounds attained priority in the field of drug discovery and development as therapeutic agents. An investigation regarding the anti-inflammatory potential of a novel drug formulation, BASk which is a combination of three bioactive compounds Betulinic acid (B):Apigenin (A):Skimmianine (Sk) remains the focus area of this research study. We also elucidate the molecular mechanism behind the therapeutic potential of BASk through CD36 mediated activation TLR2-NLRP3 signaling pathway., Methods: OxLDL induced hPBMCs used to screen out a suitable combination of BASk via MTT, COX, LOX, NOS and MPO assays. Hypercholesterolemia is induced in rabbits by supplementing with 1% cholesterol + 0.5% cholic acid and treated with BASk (2:2:1) (5 mg/Kg) and atorvastatin (10 mg/Kg) for 60 days. CD36, TLR2, NLRP3, NFκB, cytokines, endothelial damage were quantified by reverse transcription, real time PCR, ELISA, flow cytometry and histopathology., Results: hPBMCs pretreated with BASk at 2:2:1 ratio significantly decreased the activities of COX, 15-LOX, NOS and MPO on OxLDL induction than quercetin. Down regulation of CD36, TLR2, MyD88, TRAF6 by BASk further buttressed NLRP3 inflammasome activation mediated by the transcription factor NFκB. This is in correlation with the effect of BASk by balancing pro (IL-1β, IL-18) and anti-inflammatory (TGF-β) mediators in the aortic endothelial cells., Conclusion: BASk exerted its anti-inflammatory potential by reducing pro-inflammatory mediators during cholesterol supplementation via down regulating CD36 mediated TLR2 - NLRP3 inflammasome cascade. This deciphers a synergistic combination named BASk (2:2:1) as a novel drug formulation against chronic inflammatory disease, atherosclerosis., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Toll-like receptor-4 (TLR-4) mediates activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) resulting in induction of proinflammatory genes such as that encoding tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) which played a significant role in cartilage destruction of rheumatoid arthritis (RA). Low risk and better efficacy made herbal drugs more reliable than nonsteroid anti-inflammatory drugs (NSAIDS) in RA treatment. Gugguluthiktam Kashayam (GuK), Punarnavadi Kashayam (PuK) and Balaguluchiadi Kashayam (BgK) are ayurvedic polyherbal formulations prescribed in classical ayurvedic texts Sahasrayogam and Ashtangahridayam as medicines for the treatment of RA., Objective: The objective of the present study was to elucidate the molecular mechanism of anti-arthritic effect of these Kashayams on TLR-4 signal transduction pathway in collagen induced arthritic rats., Material and Methods: The wistar rats grouped into group I - Normal, group II- Collagen induced arthritis (CIA), group III- CIA + BgK, group IV- CIA + PuK, group V- CIA + GuK, group VI - CIA + Indomethacin (3 mg/kg b.wt.). Treatment with Kashayam (2 ml/kg b.wt) started after 14 days of primary immunization with type II collagen and continued for a period of 45 days., Results: Arthritis index, C-reactive protein (CRP), rheumatoid factor (RF) and myeloperoxidase (MPO) in serum and protein level of TLR-4, myeloid differentiation factor 88 (MYD88), NF-κB, TNF-α, IL-1β, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 COX-2) and prostaglandin E-2 (PGE-2) in cartilage were significantly elevated in CIA rats. Further, treatment with Kashayams downregulated all these inflammatory mediators hitherto TLR-4-NF-kB signal transduction pathway except IL-10, an anti-inflammatory cytokine which showed a reverse effect., Conclusion: This molecular mechanism of the investigation confirmed the clinical efficacy of Kashayams in preventing the progression of RA and gave an intuition of the scientific validation of Kashayams, an Ayurvedic classical medicine., (Copyright © 2018 Transdisciplinary University, Bangalore and World Ayurveda Foundation. Published by Elsevier B.V. All rights reserved.)

Imbalance between Th1/Th2 and Th17/Treg is crucial in RA progression. Various dietary factors can modulate the disease severity by restoring the balance in differentiation of CD4+ T cell subsets. Dietary amaranths hold an important part of diet as vegetables, where commonly consumed species includes Amaranthus cruentus (Ac), Amaranthus viridis (Av), and Amaranthus hybridus (Ah). The present study focuses on to evaluate whether these dietary amaranths can modulate the immune activation in collagen-induced arthritis. For in vivo study, Female Wistar rats were immunized with type II collagen and after immunization period, rats were separately supplemented with cooked Ac, Av, and Ah at 500 mg/100 g bwt concentration mixed with standard rat feed for 60 days. HPTLC fingerprint analysis identified peaks for compounds in these three amaranths. The results showed a protective role of immunomodulation in Th1/Th2 response of the three dietary amaranths, by significantly augmenting lymphocyte activation with increased IL-4 secretion, but decreased IFN-γ by cultured spleen lymphocytes subjected to collagen-induced inflammation. Moreover, Th17/Treg imbalance created by increase in IL-17 and decrease in IL-10 was significantly balanced by the three dietary supplemented groups. Furthermore, Th1/Th2 status reflected from Tbet/GATA3 ratio and Th17/Treg status reflected from RORγt/FOXP3 ratio was significantly decreased in the three dietary amaranth supplemented groups. Thus, dietary amaranths provide an immune-modulating role by keeping the balance between Th1/Th2 and Th17/Treg response in collagen-induced inflammation.

Non-steroidal anti-inflammatory drugs (NSAIDs) are a group of molecules which have been found to be active against cancer cells with chemopreventive properties by targeting cyclooxygenase (COX-1 and COX-2) and lipoxygenase (LOX), commonly upregulated (particularly COX-2) in malignant tumors. Arene ruthenium(ii) complexes with a pseudo-octahedral coordination environment containing different ancillary ligands have shown remarkable activity against primary and metastatic tumors as reported earlier. This work describes the synthesis of four novel ruthenium(ii)-arene complexes viz. [Ru(η 6 -p-cymene)(nap)Cl] 1 [Hnap = naproxen or (S)-2-(6-methoxy-2-naphthyl)propionic acid], [Ru(η 6 -p-cymene)(diclo)Cl] 2 [Hdiclo = diclofenac or 2-[(2,6-dichlorophenyl)amino] benzeneacetic acid, [Ru(η 6 -p-cymene)(ibu)Cl] 3 [Hibu = ibuprofen or 2-(4-isobutylphenyl)propanoic acid] and [Ru(η 6 -p-cymene)(asp)Cl] 4 [Hasp = aspirin or 2-acetoxy benzoic acid] using different NSAIDs as chelating ligands. Complexes 1-3 have shown promising antiproliferative activity against three different cell lines with GI 50 (concentration of drug causing 50% inhibition of cell growth) values comparable to adriamycin. At the concentration of 50 μM, complex 3 is more effective in the inhibition of cyclooxygenase and lipooxygenase enzymes, followed by complex 2 and complex 1 in comparison to their respective free NSAID ligands indicating a possible correlation between the inhibition of COX and/or LOX and anticancer properties. Molecular docking studies with COX-2 reveal that complexes 1 and 2 having naproxen and diclofenac ligands exhibit stronger interactions with COX-2 than their respective free NSAIDs and these results are in good agreement with their relative experimentally observed COX inhibition as well as anti-proliferative activities.