Synergistic effect of Betulinic acid, Apigenin and Skimmianine (BASk) in high cholesterol diet rabbit: Involvement of CD36-TLR2 signaling pathway.

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.

Dietary amaranths modulate the immune response via balancing Th1/Th2 and Th17/Treg response in collagen-induced arthritis.

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.