Betulinic acid inhibits endotoxin-stimulated phosphorylation cascade and pro-inflammatory prostaglandin E(2) production in human peripheral blood mononuclear cells
Authors: Vijayan Viji
Varma R Luxmi
Antony Helen
Journal: British Journal of Pharmacology (2011)
Discipline: PHARMACOLOGY & TOXICOLOGY
Keywords: Betulinic acid; LPS; hPBMCs; COX-2; ERK
Affiliations: Department of Biochemistry, University of Kerala, Kariavattom Campus, Thiruvananthapuram, Kerala, India.

Abstract:  Background and purpose: Betulinic acid (BA) is a naturally occurring triterpenoid widely distributed throughout the plant kingdom. We previously reported that BA inhibits lipopolysaccharide (LPS)-induced interleukin-6 production through modulation of nuclear factor κB (NF-κB) in human peripheral blood mononuclear cells (hPBMCs). This study attempted to identify other mechanisms through which BA modulates LPS signalling in mononuclear cells. The effects of BA on signalling pathways downstream were focused on in this study. Experimental approach: We determined the ability of BA to interfere with p38 and extracellular regulated kinase (ERK) phosphorylation as well as Akt phosphorylation and nuclear factor-κB activation using LPS-activated hPBMCs as an in vitro model. LPS-induced endotoxin shock in mice was the in vivo model employed. Key results: BA inhibited LPS-induced COX-2 protein expression and prostaglandin E(2) production and also attenuated LPS-induced ERK and Akt phosphorylation, but not p38 in hPBMCs. BA abolished LPS-induced IκBα phosphorylation and thus normalized the levels of IκBα in cytosol. BA also inhibited LPS-induced reactive oxygen species formation and lactate dehydrogenase release. Interestingly, BA improved the life span of mice in endotoxin shock and also inhibited PGE(2) production and myeloperoxidase activity in vivo. Conclusions and implications: BA modulates LPS-induced COX-2 expression in hPBMCs by inhibiting ERK and Akt pathways as well as by modulating IκBα phosphorylation. At the same time, no cell toxicity was observed. The effect of the drug was confirmed through in vivo experiments. The study gives an insight into the molecular mechanisms of BA. 

Retracted: No

PubPeer comments:
 The b-actin band in the Figure 4A, 4B and 4C were duplicated in the same figure (shown in the red box). The b-actin used in Figure 4A, 4B and 4C was again duplicated in Figure 5 (shown in the red box), where a different experiment and treatment condition was mentioned. As the same b-actin for Figure 4 is shown; Are the proteins probed in a single membrane? Many of the lanes are not matching with the b-actin control. Could the authors show their raw data of a single membrane probing? If separate membranes were used, individual b-actin bands are missing. Figure 4A: The total ERK 44 42 seems to be a doubtful image by cropping two separate images and joining them together. Could the authors produce the raw data, the real gel image uncropped and not in grayscale, with respective protein ladder? Figure 4B: The lanes are not at all matching with the b-actin control or total p38. Are the images probed from a single membrane? Figure 5: Again the ERK44 42 seems to be not convincing. In the arrows shown, a clear gap can be found. Could the authors produce the raw data, the real gel image uncropped and not in grayscale, with respective protein ladder? ![file] , 
 Erratum for the paper is now published, available at https: bpspubs.onlinelibrary.wiley.com doi 10.1111 bph.15519 , 
 Concerns in the published erratum. - The immunoblot images have some unusual backgrounds (blue arrows). - Both total p38 and p-p38 has the same molecular weight, the images in the lanes were not matching. Additionally, the band for the t-p38 of LPS-BA has some unusual black color (red block), but not the same was observed in the p-p38 bands. Whether the authors used a single blot for their results? - Some part of the band for the p-p38 (Basal condition) is missing (Red arrow). - The actins were also not equal (green arrow). The band intensity for the basal condition is comparably less to that of the others. Could the authors clarify with their uncropped original images? ![file] ,