Scientific validation of anti-arthritic effect of Kashayams - A polyherbal formulation in collagen induced arthritic rats

Abstract

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.

Keywords: Anti-Arthritic; Kashayams; Nuclear factor kappa-light-chain-enhancer of activated B cells; Rheumatoid arthritis; Toll-like receptor -4; Type II collagen.

Fig. 1

Changes in Arthritis index and paw volume. The effect of Kashayams BgK, PuK, and GuK studied on collagen induced arthritic rats for a period of 45 days and from arthritic scores determined arthritic index. a) Paw volume varies during the treatment period. b) 14 days after primary immunization in all groups except the normal both paw volume and arthritis index rises. Treatment with BgK, PuK, GuK and positive control IND started from 14th day and continued for 45 days. # represents statistical significance at p < 0.05 when compared to normal (N) and * represents significance at p < 0.05 when compared to collagen induced arthritic (CIA) groups. Data were expressed as the mean ± SD from six separate experiments.

Fig. 2

Effect of BgK, PuK, GuK on TLR4 receptor activation, MyD 88 recruitment and NFκB translocation. a) The quantity of TLR-4 and MyD-88 in the cartilage of joints were determined by ELISA. b) The quantity of cytosolic and nuclear NF-κB p65 transcription factor in cartilage determined by ELISA. The six groups were N-normal groups, CIA-collagen induced arthritic groups, Balaguluchiadi, Punarnavadi, and Gugguluthiktam Kashayam treated groups and indomethacin treated positive control groups. # represents statistical significance at p < 0.05 when compared to normal (N) and * represents significance at p < 0.05 when compared to collagen induced arthritic (CIA) groups. Data were expressed as the mean ± SD from six separate experiments.

Fig. 3

Effect of kashayams on proinflammatory and anti-inflammatory cytokines. a) The quantity of proinflammatory cytokines TNFα and IL-1β in the cartilage of joints were determined by ELISA. b) The quantity of anti-inflammatory cytokine IL-10 in the cartilage of joints determined by ELISA. c) and d) correspondingly represents fold change in the case of IL-1β gene and Il-10 gene respectively as determined by qRT-PCR method. The six groups were N-normal groups, CIA-collagen induced arthritic groups, Balaguluchiadi, Punarnavadi, and Gugguluthiktam Kashayam treated groups and indomethacin treated positive control groups. # represents statistical significance at p < 0.05 when compared to normal (N) and * represents significance at p < 0.05 when compared to collagen induced arthritic (CIA) groups. Data were expressed as the mean ± SD from six separate experiments.

Fig. 4

Effect on COX- 2, 5-LOX, iNOS and PGE-2 synthesis. a) The quantity of COX-2, PGE-2 and iNOS the cartilage of joints were determined by ELISA. b) The activity of anti-inflammatory enzyme 5-LOX in monocytes measured in U/mg protein. c) mRNA expression of TLR-4, TNFα, COX-2, iNOS and their intensity as compared to GAPDH. The six groups were I- normal groups, II- (CIA) collagen induced arthritic groups, III- Balaguluchiadi, IV- Punarnavadi, and V- Gugguluthiktam Kashayam treated groups and VI- indomethacin treated positive control groups. #- statistical significance at p < 0.05 when compared to normal (N) and *- significance at p < 0.05 when compared to collagen induced arthritic (CIA) groups. Data were expressed as the mean ± SD from six separate experiments.