Anti-inflammatory effect of Kaba Sura Kudineer (AYUSH approved COVID-19 drug)-A Siddha poly-herbal formulation against lipopolysaccharide induced inflammatory response in RAW-264.7 macrophages cells

Abstract

Ethnopharmacological relevance: Medicinal importance and potential activity of Siddha herbal formulations have proved over several centuries against a wide range of causative agents as Influenza, Dengue, Chikungunya, and Tuberculosis. The traditional medicine system of Siddha is a valuable therapeutic approach for treating viral respiratory infections like Coronavirus disease 2019 (COVID-19) and can be effectively employed to target the host response and preventive care to boost the immune system. Kaba Sura Kudineer (KSK), an official polyherbal formulation has been used in Siddha traditional medicine for centuries. However, the role of KSK in regulating inflammation and the underlying molecular mechanisms has remained elusive.

Aim of the study: The goal of this study was to evaluate the anti-inflammatory effect of KSK using lipopolysaccharide (LPS) stimulated RAW 264.7 murine macrophage cells.

Materials and methods: Raw 264.7 murine macrophage cells were used for this study. The Inflammatory mediators and cytokines were measured by enzyme-linked immunosorbent assay (ELISA). The NF-κB nulcear translocation and protein expression of iNOS, COX-2 was analyzed with westernblot.

Results: KSK supplementation decreased LPS mediated TLR-4 production and secretion of pro-inflammatory mediators and cytokines including IL-6, TNF-α, COX-2 and PGE-2. Moreover, it inhibited the production of nitric oxide (NO) and thereby inhibited the expression of iNOS in the cell. The Western blot analysis further confirmed that KSK strongly prevented the LPS-induced degradation of IκB which is normally required for the activation of NF-κB and hereby suppressed nuclear translocation of NF-κB. The protein expression of iNOS, COX-2 was significantly decreased with the presence of KSK treatment. Results suggested that KSK manipulates its anti-inflammatory effects mainly through blocking the TLR mediated NF-κB signal transduction pathways.

Conclusions: Together, this study has proven that KSK could be a potential therapeutic drug for alleviating excessive inflammation in many inflammation-associated diseases like COVID-19.

Keywords: Cycloxygenase-2; Inflammation; Interleukins-6; Kaba sura kudineer; NF-ӄB; Nitric oxide.

Graphical abstract

Fig. 1

Cell viability assay was done by MTT assay. RAW 264.7 cells were incubated with different concentrations of KSK (3.125 μg/mL, 6.25 μg/mL, 12.5 μg/mL, 25 μg/mL, 50 μg/mL and 100 μg/mL) for 24 h* Compared with LPS treated group, P ≤ 0.05. # denotes LPS treated group compared with control group, P ≤ 0.05. Values are expressed as mean ± SD (n = 3).

Fig. 2

Morphological analysis of KSK at 20x by using the phase-contrast inverted microscope.

Fig. 3

Effect of KSK on NO production in LPS-induced RAW 264.7 macrophages. # -Statistical difference with the control group at P ≤ 0.05. * -Statistical difference with LPS at P ≤ 0.05.

Fig. 4

Effect of KSK on ROS production in LPS-induced RAW 264.7 macrophages. # -Statistical difference with the control group at P ≤ 0.05. * -Statistical difference with LPS at P ≤ 0.05.

Fig. 5

Effect of KSK on Pro-inflammatory cytokines in LPS-induced RAW 264.7 macrophages. *-Statistical difference with control group, **-Statistical difference with LPS treated group. ***-Statistical difference with LPS and other KSK doses.

Fig. 6

Western blot analysis for the inhibitory effects of KSK on the protein expression level of iNOS and COX-2 level. GAPDH was used as an internal control.

Fig. 7

Western blot analysis for the inhibitory effects of KSK on the degradation of IκB, protein expression of NF-ӄB p65 and TLR-4. GAPDH was used as an internal control.