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Hyperforin is a modulator of inducible nitric oxide synthase and phagocytosis in microglia and macrophages

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Abstract

Upon activation, microglia, the immunocompetent cells in the brain, get highly phagocytic and release pro-inflammatory mediators like nitric oxide (NO). Excessive NO production is pivotal in neurodegenerative disorders, and there is evidence that abnormalities in NO production and inflammatory responses may at least support a range of neuropsychiatric disorders, including depression. Although extracts of St. John’s wort (Hypericum perforatum L.) have been used for centuries in traditional medicine, notably for the treatment of depression, there is still considerable lack in scientific knowledge about the impact on microglia. We used N11 and BV2 mouse microglia, as well as RAW 264.7 macrophages to investigate the effects of St. John’s wort extract and constituents thereof on NO production Moreover, flow cytometry and fluorescence microscopy were employed to analyze the influence on phagocytosis, transcription factor activation states, and cell motility. We found that extracts of St. John’s wort efficiently suppress lipopolysaccharide-induced NO release and identified hyperforin as the responsible compound, being effective at concentrations between 0.25 and 0.75 µM. The reduced NO production was mediated by diminished inducible nitric oxide synthase expression on the mRNA and protein level. In addition, at similar concentrations, hyperforin reduced zymosan phygocytosis to 20–40% and putatively acted by downregulating the CD206 macrophage mannose receptor and modulation of cell motility. We found that the observed effects correlate with a suppression of the activated state of Nf-kappaB and phospho-CREB, while c-JUN, STAT1, and HIF-1alpha activity and cyclooxygenase-2 expression remained unaffected by hyperforin. These results reveal that hyperforin influences pro-inflammatory and immunological responses of microglia that are involved in the progression of neuropathologic disorders.

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Acknowledgements

We thank Dr. S. Kern (Chair of Phytopathology, Technical University Munich) for help with HPLC measurements, Dr. E. Mann (Chair of Phytopathology, Technical University Munich) for providing the H. perforatum extracts and Magdalena Motyl (Pharmaceutical Biology, University of Regensburg) for performing experiments on hyperforin recovery in cultured cells. We are especially grateful to Dr. D. Weiser (Steigerwald Arzneimittelwerk GmbH, Darmstadt, Germany) for granted support. B. Kraus was funded by Steigerwald Arzneimittel GmbH (Darmstadt).

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Authors and Affiliations

  1. Institute of Pharmaceutical Biology, University of Regensburg, Universitätsstrasse 31, Regensburg, Germany

    Birgit Kraus & Jörg Heilmann

  2. Institute of Virology, Helmholtz Zentrum München – German Research Center for Environmental Health, Ingolstädter Landstrasse 1, Neuherberg, Germany

    Horst Wolff

  3. Institute of Phytopathology, Technical University of Munich, Center of Life and Food Sciences Weihenstephan, Am Hochanger 2, Freising, Germany

    Birgit Kraus & Erich F. Elstner

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  1. Birgit Kraus
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Correspondence to Birgit Kraus.

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Supplementary Figure 1

Inhibition of NO production by HPE but not by single major constituents of HPE. A N11 and B RAW 264.7 cells were exposed for 24 h to HPE and concentrations of hypericin (Hc), pseudohypericin (Ps), rutin (Ru), hyperoside (Hd), quercitrin (Qui), quercetin (Que) corresponding to amounts present in HPE. Subsequently, a 24-h LPS treatment followed. In both cell lines, a decrease of nitrite in the culture medium was observed for HPE but not for the individual compounds. Bars show means and standard deviations. Experiments were carried out with eight parallels and repeated independently three times. Asterisks indicate significance for HPE relative to the LPS-only control. (GIF 71 kb)

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Supplementary Figure 2

Influence of Hf on cell viability as determined by MTT assay. A BV2, B N11, and C RAW 264.7 cells were treated for 24 h with solvent control or increasing concentrations of Hf. Data were normalized to the solvent control (100%). Bars show means and standard deviations. Experiments were carried out with eight parallels and repeated independently three times. Asterisks indicate significance relative to the control. (GIF 102 kb)

High resolution image (TIFF 404 kb)

Supplementary Figure 3

Stainings of cellular factors. The panels depict small regions of interest taken from representative images of transcription factor stainings used to determine activation state or expression levels in Fig. 3. Left image: unstimulated cells. Center image: cells stimulated with LPS and IFN-γ. Right image: stimulated cells pre-treated with Hf. Exemplary images from BV2 and N11 cells are shown. Merge images display the cellular factors pseudo-colored in green and nuclei in blue. Scale bar: 20 μm. (GIF 290 kb)

High resolution image (TIFF 13341 kb)

Supplementary Figure 4

Influence of Hf on phagocytosis (FACS histograms). FACS histograms of BV2 phagocytosis of fluorescently labeled zymosan. Cells were either pretreated with EtOH only (control, A) or with increasing concentrations of Hf B 0.25 μM, C 0.5 μM, D 0.625 μM, and E 0.75 μM. In histograms, green fluorescence (FL-1) is shown on a log scale and the percentage of cells that are green positive is indicated. (GIF 48 kb)

High resolution image (TIFF 166 kb)

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Kraus, B., Wolff, H., Elstner, E.F. et al. Hyperforin is a modulator of inducible nitric oxide synthase and phagocytosis in microglia and macrophages. Naunyn-Schmied Arch Pharmacol 381, 541–553 (2010). https://doi.org/10.1007/s00210-010-0512-y

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