RESUMO
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-1ß, IL-18) and anti-inflammatory (TGF-ß) 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.
Assuntos
Apigenina/farmacologia , Antígenos CD36/metabolismo , Colesterol na Dieta/efeitos adversos , Dieta Hiperlipídica , Triterpenos Pentacíclicos/farmacologia , Quinolinas/farmacologia , Transdução de Sinais , Receptor 2 Toll-Like/metabolismo , Animais , Aorta/efeitos dos fármacos , Aorta/patologia , Araquidonato 15-Lipoxigenase/metabolismo , Aterosclerose/sangue , Biomarcadores/sangue , Sobrevivência Celular/efeitos dos fármacos , Humanos , Mediadores da Inflamação/metabolismo , Interleucina-18/genética , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipídeos/sangue , Masculino , Fator 88 de Diferenciação Mieloide/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Óxido Nítrico Sintase/metabolismo , Peroxidase/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Coelhos , Superóxido Dismutase/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Fator de Transcrição RelA/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Ácido BetulínicoRESUMO
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-1ß (IL-1ß) 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-1ß, 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.