Direct interaction of garcinol and related polyisoprenylated benzophenones of Garcinia cambogia fruits with the transcription factor STAT-1 as a likely mechanism of their inhibitory effect on cytokine signaling pathways.

Garcinol (1), a polyisoprenylated benzophenone occurring in Garcinia species, has been reported to exert anti-inflammatory activity in LPS-stimulated macrophages, through inhibition of NF-κB and/or JAK/STAT-1 activation. In order to provide deeper insight into its effects on the cytokine signaling pathway and to clarify the underlying molecular mechanisms, 1 was isolated from the fruits of Garcinia cambogia along with two other polyisoprenylated benzophenones, guttiferones K (2) and guttiferone M (3), differing from each other in their isoprenyl moieties and their positions on the benzophenone core. The affinities of 1-3 for the STAT-1 protein have been evaluated by surface plasmon resonance and molecular docking studies and resulted in KD values in the micromolar range. Consistent with the observed high affinity toward the STAT-1 protein, garcinol and guttiferones K and M were able to modulate cytokine signaling in different cultured cell lines, mainly by inhibiting STAT-1 nuclear transfer and DNA binding, as assessed by an electrophorectic mobility shift assay.

Direct interaction of natural and synthetic catechins with signal transducer activator of transcription 1 affects both its phosphorylation and activity.

Our previous studies showed that (-)-epigallocatechin-3-gallate (EGCG) inhibits signal transducer activator of transcription 1 (STAT1) activation. Since EGCG may be a promising lead compound for new anti-STAT1 drug design, 15 synthetic catechins, characterized by the (-)-gallocatechin-3-gallate stereochemistry, were studied in the human mammary MDA-MB-231 cell line to identify the minimal structural features that preserve the anti-STAT1 activity. We demonstrate that the presence of three hydroxyl groups of B ring and one hydroxyl group in D ring is essential to preserve their inhibitory action. Moreover, a possible molecular target of these compounds in the STAT1 pathway was investigated. Our results demonstrate a direct interaction between STAT1 protein and catechins displaying anti-STAT1 activity. In particular, surface plasmon resonance (SPR) analysis and molecular modeling indicate the presence of two putative binding sites (a and b) with different affinity. Based on docking data, site-directed mutagenesis was performed, and interaction of the most active catechins with STAT1 was studied with SPR to test whether Gln518 on site a and His568 on site b could be important for the catechin-STAT1 interaction. Data indicate that site b has higher affinity for catechins than site a as the highest affinity constant disappears in the H568A-STAT1 mutant. Furthermore, Janus kinase 2 (JAK2) kinase assay data suggest that the contemporary presence in vitro of STAT1 and catechins inhibits JAK2-elicited STAT1 phosphorylation. The very tight catechin-STAT1 interaction prevents STAT1 phosphorylation and represents a novel, specific and efficient molecular mechanism for the inhibition of STAT1 activation.

Phenylpropanoid glycosides from plant cell cultures induce heme oxygenase 1 gene expression in a human keratinocyte cell line by affecting the balance of NRF2 and BACH1 transcription factors.

Phenylpropanoids have several highly significant biological properties in both plants and animals. Four phenylpropanoid glycosides (PPGs), verbascoside (VB), forsythoside B (FB), echinacoside (EC) and campneoside I (CP), were purified and tested for their capability to activate NRF2 and induce phase II cytoprotective enzymes in a human keratinocyte cell line (HaCaT). All four substances showed similar strong antioxidant and radical-scavenging activities as determined by diphenylpicrylhydrazyl assay. Furthermore, in HaCaT cells, FB and EC are strong activators of NRF2, the nuclear transcription factor regulating many phase II detoxifying and cytoprotective enzymes, such as heme oxygenase 1 (HMOX1). In HaCaT cells, FB and EC (200 µM) induced nuclear translocation of NRF2 protein after 24 h and reduced nuclear protein levels of BACH1, a repressor of the antioxidant response element. FB and EC greatly HMOX1 mRNA levels by more than 40-fold in 72 h. Cytoplasmic HMOX1 protein levels were also increased at 48 h after treatment. VB was less active compared to FB and EC, and CP was slightly active only at later times of treatment. We suggest that hydroxytyrosol (HYD) could be a potential bioactive metabolite of PPGs since HYD, in equimolar amounts to PGGs, is able to both activate HO-1 transcription and modify Nrf2/Bach1 nuclear protein levels. This is in agreement with the poor activity of CP, which contains a HYD moiety modified by an O-methyl group. In conclusion, FB and EC from plant cell cultures may provide long-lasting skin protection by induction of phase II cytoprotective capabilities.

Protective effects of glycyrrhizin in a gut hypoxia (ischemia)-reoxygenation (reperfusion) model.

PURPOSE: This study investigated the effects of glycyrrhizin, a potent antioxidant, on tissue injury caused by ischemia/reperfusion (I/R) of the gut. METHODS: I/R injury of the intestine was caused by clamping both the superior mesenteric artery and the celiac trunk for 45 min followed by release of the clamp allowing reperfusion for 1 or 6 h. RESULTS: Administration of glycyrrhizin, significantly reduced the (a) fall of mean arterial blood pressure, (b) mortality rate, (c) myeloperoxidase (MPO) activity, (d) production of pro-inflammatory cytokines [tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta)], (e) histological evidence of gut injury, (f) immunoreactivity of nitrotyrosine, (g) poly ADP-ribose (PAR) formation, (h) the expression of ICAM-1 and P-selectin, (i) activation of nuclear factor-kappaB (NF-kappaB) and (j) signal transducer and activator transcription-3 (STAT-3) induced by splanchnic artery occlusion-reperfusion shock. CONCLUSIONS: This study demonstrates that glycyrrhizin exerts multiple protective effects in splanchnic artery occlusion-reperfusion shock.

Glycyrrhizin reduces secondary inflammatory process after spinal cord compression injury in mice.

Glycyrrhizin, a major active constituent of liquorice root (Glycyrrhiza glabra), has a free radical scavenging property, and its effects were evaluated on an animal model of spinal cord injury (SCI) induced by the application of vascular clips (force of 24 g) to the dura via a four-level T5-T8 laminectomy. Spinal cord injury in mice resulted in severe trauma characterized by edema, tissue damage, and apoptosis (measured by terminal deoxynucleotidyltransferase-mediated dUTP-biotin end labeling staining, Bax, and Bcl-2 expression). Immunohistochemical examination demonstrated a marked increase in immunoreactivity for nitrotyrosine, iNOS, and poly(adenosine diphosphate-ribose) in the spinal cord tissue. Additionally, we demonstrate that these inflammatory events were associated with the activation of nuclear factor-kappaB. In contrast, the degree of (1) spinal cord inflammation and tissue injury (histological score), (2) nitrotyrosine and poly(adenosine diphosphate [ADP] ribose) formation, (3) iNOS expression, (4) nuclear factor-kappaB activation, and (5) apoptosis (terminal deoxynucleotidyltransferase-mediated dUTP-biotin end labeling, Bax, and Bcl-2) was markedly reduced in spinal cord tissue obtained from mice treated with glycyrrhizin extract (10 mg/kg, i.p., 30 min before and 1 and 6 h after SCI). In a separate set of experiments, we have clearly demonstrated that glycyrrhizin extract treatment significantly ameliorated the recovery of limb function (evaluated by motor recovery score). Taken together, our results clearly demonstrate that treatment with glycyrrhizin extract reduces the development of inflammation and tissue injury events associated with spinal cord trauma.

Glycyrrhizin attenuates the development of carrageenan-induced lung injury in mice.

Glycyrrhizin is a triterpene glycoside, a major active constituent of licorice (Glycyrrhiza glabra) root and numerous pharmacological effects like anti-inflammatory, anti-viral, anti-tumour and hepatoprotective activities has been attributed to it. In this study we evaluated the anti-inflammatory activities of glycyrrhizin in mice model of acute inflammation, carrageenan-induced pleurisy. We report here that glycyrrhizin (given at 10 mg/kg i.p. 5 min prior to carrageenan) exerts potent anti-inflammatory effects in this model. Injection of carrageenan into the pleural cavity of mice elicited an acute inflammatory response characterized by fluid accumulation in the pleural cavity which contained a large number of neutrophils (PMNs) as well as an infiltration of PMNs in lung tissues and subsequent lipid peroxidation (as determinated by thiobarbituric acid-reactant substances measurement) and increased production of tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta). All these parameters were attenuated by glycyrrhizin. Furthermore, carrageenan induced an upregulation of the expression of intercellular cell adhesion molecule (ICAM-1), P-selectin, as well as an increase in the amounts of nitrotyrosine and poly(ADP-ribose) (PAR), as determined by immunohistochemical analysis of lung tissues. The degree of staining for the ICAM-1, P-selectin, nitrotyrosine and PAR was significantly reduced by glycyrrhizin. Additionally, we demonstrate that these inflammatory events were associated with the activation of nuclear factor-kappaB (NF-kappaB) and signal transducer and activator transcription-3 (STAT-3) activation in the lung. NF-kappaB and STAT-3 activation were significantly reduced by glycyrrhizin treatment. Taken together, our results indicate that prevention of the activation of NF-kappaB and STAT-3 by glycyrrhizin reduces the development of acute inflammation.