Effect of Crude Extract of Ethno Botanical Mixture Used in Indigenous Treatment of Tuberculosis on the Release of TNF α, IFN Ƴ and IL 10 by LPS-Stimulated THP-1 Macrophage Cell Line

Introduction: In-vitro study of pro inflammatory cytokine IFN Ƴ, TNF α and anti-inflammatory cytokines IL-10, production by LPS sensitized THP-1 cell line with response to different concentrations of hawan samagri (HS) extract, has not been documented in literature impeding its possible use in TB treatment. This study attempts to fill the above knowledge gap in an effort to explore possibility of effective ethno botanical alternative therapy. IFN Ƴ response is well established as an immune correlate of M. tuberculosis infection. Here in this study, we studied the immune response profile of three cytokines believed to influence immunity to TB- IFN Ƴ, TNF α and IL-10. Materials and Methods: Hawan samagri: Two different hawan samagri HS1 & HS2 were used as test samples. Cell culture: THP-1, a promonocytic cell line was obtained from the ATCC. Treatment for Cytokine gene expression: In RPMI complete medium 1x105 cells/well was seeded to each well of the 96 well micro-titer plate. After 24 hrs. incubation, PMA (10ng/ml) added to 96 well plates to differentiate THP1 cells. Sample Preparation and RNA Isolation: Total RNA from THP-1cells was extracted using TRizol Reagent. RT-PCR: A semi quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was carried out to determine the levels of TNF-α, IL-10 and IFN and GAPDH mRNA expressions. Gel Electrophoresis: Ten microliters of the final amplification product were run on a 2% ethidium-stained agarose gel and photographed. Results: Test samples showed dose dependent increase in, IFN-gamma, anti-inflammatory cytokine mRNA levels. Highest expression of IFN-gamma was observed in HS2 at 62.5μg/mL with mRNA expression of 2.94-fold, followed by HS1 with IFN mRNA levels of 1.98-fold compared to control cells. Conclusion: All test samples show dose dependent anti-inflammatory activity as demonstrated with suppression of proinflammatory cytokines (TNF-alpha and IFN gamma) and stimulation of anti-inflammatory cytokine (IL-10), LPS induced inflammatory THP-1 cell based model