Berberine Chloride Mediates Its Anti-Leishmanial Activity via Differential Regulation of the Mitogen Activated Protein Kinase Pathway in Macrophages

BACKGROUND: A complex interplay between Leishmania and macrophages influences parasite survival and necessitates disruption of signaling molecules, eventually resulting in impairment of macrophage function. In this study, we demonstrate the immunomodulatory activity of Berberine chloride in Leishmania infected macrophages. PRINCIPAL FINDINGS: The IC(50) of Berberine chloride, a quaternary isoquinoline alkaloid was tested in an amastigote macrophage model and its safety index measured by a cell viability assay. It eliminated intracellular amastigotes, the IC(50) being 2.8 fold lower than its IC(50) in promastigotes (7.10 µM vs. 2.54 µM) and showed a safety index >16. Levels of intracellular and extracellular nitric oxide (NO) as measured by flow cytometry and Griess assay respectively showed that Berberine chloride in Leishmania infected macrophages increased production of NO. Measurement of the mRNA expression of iNOS, IL-12 and IL-10 by RT-PCR along with levels of IL-12p40 and IL-10 by ELISA showed that in infected macrophages, Berberine chloride enhanced expression of iNOS and IL-12p40, concomitant with a downregulation of IL-10. The phosphorylation status of extracellular signal related kinase (ERK1/2) and p38 mitogen activated protein kinase (p38 MAPK) was studied by western blotting. In infected macrophages, Berberine chloride caused a time dependent activation of p38 MAPK along with deactivation of ERK1/2; addition of a p38 MAPK inhibitor SB203580 inhibited the increased generation of NO and IL-12p40 by Berberine chloride as also prevented its decrease of IL-10. CONCLUSIONS: Berberine chloride modulated macrophage effector responses via the mitogen activated protein kinase (MAPK) pathway, highlighting the importance of MAPKs as an antiparasite target

Cytotoxicity of Senecio in macrophages is mediated via its induction of oxidative stress

In Arunachal Pradesh and other sub-Himalayan areas of India, accidental consumption of Senecio plants by yaks is often fatal as the plant contains toxic alkaloids like Seneciophylline. The present investigation was undertaken to demonstrate the pro-oxidant effects of an ethanolic extract of Seneciochrysanthemoides (S-EtOH). S-EtOH impaired viability in macrophages, the IC50 being 13.8 ± 1.11 μg/mL. The effect of S-EtOH (1 μg/mL) on generation of reactive oxygen species (ROS) in macrophages was measured by flow cytometry using 2′,7′-dichlorofluorescein diacetate (H2DCFDA) where it caused a significant increase in the mean fluorescence channel (MFC) from 8.55 ± 0.03 to 47.32 ± 2.25 (p < 0.001). S-EtOH also effected a 3.8-fold increase in extracellular nitric oxide (NO) generation from 4.90 ± 0.72 μM to 18.79 ± 0.32 μM (p < 0.001), a 2.2-fold increase in intracellular NO production, the MFC increasing from 14.95 ± 0.48 to 33.34 ± 1.66 (p < 0.001), and concomitantly depleted non protein thiols as analyzed by flow cytometry using mercury orange, with a reduction in MFC from 632.5 ± 49.44 to 407.4 ± 12.61 (p < 0.01). Additionally, S-EtOH (14 μg/mL, 24 h) caused apoptosis as evident by increased Annexin V binding and terminal deoxynucleotidyl transferase mediated dUTP DNA nick end labeling. Taken together, the cytotoxicity of S-EtOH can be partly attributed to its capacity to inflict oxidative damage via generation of both reactive oxygen and nitrogen species culminating in apoptosis