Abstract
BackgroundAn extract of Phyllanthus muellerianus and its constituent geraniin have been reported to exert anti-inflammatory activity in vivo. However, orally consumed geraniin, an ellagitannin, shows low bioavailability and undergoes metabolization to urolithins by gut microbiota. This study aimed at comparing geraniin and urolithin A with respect to inhibition of M1 (LPS) polarization of murine J774.1 macrophages and shedding more light on possible underlying mechanisms. MethodsPhotometric, fluorimetric as well as luminescence-based assays monitored production of reactive oxygen species (ROS) and nitric oxide (NO), cell viability or reporter gene expression. Western blot analyses and confocal microscopy showed abundance and localization of target proteins, respectively. ResultsUrolithin A is a stronger inhibitor of M1 (LPS) macrophage polarization (production of NO, ROS and pro-inflammatory proteins) than geraniin. Urolithin A leads to an elevated autophagic flux in macrophages. Inhibition of autophagy in M1 (LPS) macrophages overcomes the suppressed nuclear translocation of p65 (NF-kB; nuclear factor kB), the reduced expression of pro-inflammatory genes as well as the diminished NO production brought about by urolithin A. The increased autophagic flux is furthermore associated with impaired Akt/mTOR (mammalian target of rapamycin) signaling in urolithin A-treated macrophages. Conclusions and general significanceIntestinal metabolization may boost the potential health benefit of widely consumed dietary ellagitannins, as suggested by side by side comparison of geraniin and urolithin A in M1(LPS) macrophages. Increased activity of the autophagic cellular recycling machinery aids the anti-inflammatory bioactivity of urolithin A.
Highlights
Phyllanthus muellerianus belongs to the family Euphorbiaceae, and leaves, bark and fruits are used in Traditional African Medicine for the treatment of dysentry, wounds and inflammatory disorders, among others [1]
Cell viability was not markedly affected by urolithin A and geraniin in LPS-stimulated macrophages compared to control cells, as assessed by an ATP–based luminescent viability assays (Fig. 1c), and complementary MTT (3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyltetrazoliumbromid), resazurin-(both metabolic activity) and crystal violet assays
LPS stimulation led to a pro-inflammatory M1 polarization in J774.1 macrophages ( on referred to M1(LPS)) which is characterized by elevated pro-IL-1β, iNOS- and Cox2 expression, massive nitric oxide (NO) release and increased reactive oxygen species (ROS) production
Summary
Phyllanthus muellerianus belongs to the family Euphorbiaceae, and leaves, bark and fruits are used in Traditional African Medicine for the treatment of dysentry, wounds and inflammatory disorders, among others [1]. Given the low bioavailability and metabolization of geraniin it is questionable whether mere in vitro data may become relevant in an in vivo setting or whether the active principle beyond the observation made upon oral administration of geraniin is mainly the parent ellagitannin. In this line, it is of note that the reported bioactivities of urolithins partly overlap with those of geraniin, such as growth inhibition and anti-inflammation [18,19,20,21,22]. Increased activity of the autophagic cellular recycling machinery aids the anti-inflammatory bioactivity of urolithin A
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