Rahul Checker

Potent anti-inflammatory activity of ursolic acid, a triterpenoid antioxidant, is mediated through suppression of NF-κB, AP-1 and NF-AT.

Background Ursolic acid (UA), a pentacyclic triterpenoid carboxylic acid, is the major component of many plants including apples, basil, cranberries, peppermint, rosemary, oregano and prunes and has been reported to possess antioxidant and anti-tumor properties. These properties of UA have been attributed to its ability to suppress NF-κB (nuclear factor kappa B) activation. Since NF-κB, in co-ordination with NF-AT (nuclear factor of activated T cells) and AP-1(activator protein-1), is known to regulate inflammatory genes, we hypothesized that UA might exhibit potent anti-inflammatory effects. Methodology/Principal Findings The anti-inflammatory effects of UA were assessed in activated T cells, B cells and macrophages. Effects of UA on ERK, JNK, NF-κB, AP-1 and NF-AT were studied to elucidate its mechanism of action. In vivo efficacy of UA was studied using mouse model of graft-versus-host disease. UA inhibited activation, proliferation and cytokine secretion in T cells, B cells and macrophages. UA inhibited mitogen-induced up-regulation of activation markers and co-stimulatory molecules in T and B cells. It inhibited mitogen-induced phosphorylation of ERK and JNK and suppressed the activation of immunoregulatory transcription factors NF-κB, NF-AT and AP-1 in lymphocytes. Treatment of cells with UA prior to allogenic transplantation significantly delayed induction of acute graft-versus-host disease in mice and also significantly reduced the serum levels of pro-inflammatory cytokines IL-6 and IFN-γ. UA treatment inhibited T cell activation even when added post-mitogenic stimulation demonstrating its therapeutic utility as an anti-inflammatory agent. Conclusions/Significance The present study describes the detailed mechanism of anti-inflammatory activity of UA. Further, UA may find application in the treatment of inflammatory disorders.

Anti-inflammatory effects of plumbagin are mediated by inhibition of NF-kappaB activation in lymphocytes.

Plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone), a quinone isolated from the roots of Plumbago zeylanica was recently reported to suppress the activation of NF-kappaB in tumor cells. NF-kappaB, a ubiquitous transcription factor, plays a central role in regulating diverse processes in leukocytes like cellular proliferation, expression of immunoregulatory genes and apoptosis during innate and adaptive immune responses. Consequently, plumbagin might affect the biological functions of leukocytes participating in various immune responses. The present report describes novel immunomodulatory effects of plumbagin. Plumbagin inhibited T cell proliferation in response to polyclonal mitogen Concanavalin A (Con A) by blocking cell cycle progression. It also suppressed expression of early and late activation markers CD69 and CD25 respectively, in activated T cells. At these immunosuppressive doses (up to 5 microM), plumbagin did not reduce the viability of lymphocytes. Further, the inhibition of T cell proliferation by plumbagin was accompanied by a decrease in the levels of Con A induced IL-2, IL-4, IL-6 and IFN-gamma cytokines. Similar immunosuppressive effects of plumbagin on cytokine levels were seen in vivo. To characterize the mechanism of inhibitory action of plumbagin, the mitogen induced IkappaB-alpha degradation and nuclear translocation of NF-kappaB was studied in lymphocytes. Plumbagin completely inhibited Con A induced IkappaB-alpha degradation and NF-kappaB activation. Further, plumbagin prevented Graft Versus Host Disease-induced mortality in mice. To our knowledge this is the first report showing the immunomodulatory effects of plumbagin in lymphocytes via modulation of NF-kappaB activation.

Plumbagin Inhibits Proliferative and Inflammatory Responses of T Cells Independent of ROS Generation But by Modulating Intracellular Thiols

Plumbagin inhibited activation, proliferation, cytokine production, and graft‐versus‐host disease in lymphocytes and inhibited growth of tumor cells by suppressing nuclear factor‐κB (NF‐κB). Plumbagin was also shown to induce reactive oxygen species (ROS) generation in tumor cells via an unknown mechanism. Present report describes a novel role of cellular redox in modulation of immune responses in normal lymphocytes by plumbagin. Plumbagin depleted glutathione (GSH) levels that led to increase in ROS generation. The decrease in GSH levels was due to direct reaction of plumbagin with GSH as evinced by mass spectrometric and HPLC analysis. Further, addition of plumbagin to cells resulted in decrease in free thiol groups on proteins and increase in glutathionylation of proteins. The suppression of mitogen‐induced T‐cell proliferation and cytokine (IL‐2/IL‐4/IL‐6/IFN‐γ) production by plumbagin was abrogated by thiol antioxidants but not by non‐thiol antioxidants confirming that thiols but not ROS play an important role in biological activity of plumbagin. Plumbagin also abrogated mitogen‐induced phosphorylation of ERK, IKK, and degradation of IκB‐α. However, it did not affect phosphorylation of P38, JNK, and AKT. Our results for the first time show that antiproliferative effects of plumbagin are mediated by modulation of cellular redox. These results provide a rationale for application of thiol‐depleting agents as anti‐inflammatory drugs. J. Cell. Biochem. 110: 1082–1093, 2010. Published 2010 Wiley‐Liss, Inc.

Schisandrin B exhibits anti-inflammatory activity through modulation of the redox-sensitive transcription factors Nrf2 and NF-κB

Schisandrin B (SB), a dibenzocyclooctadiene derivative isolated from Schisandra chinensis and used commonly in traditional Chinese medicine for the treatment of hepatitis and myocardial disorders, has been recently shown to modulate cellular redox balance. Since we have shown that cellular redox plays an important role in the modulation of immune responses, the present studies were undertaken to study the effects of SB on activation and effector functions of lymphocytes. SB altered the redox status of lymphocytes by enhancing the basal reactive oxygen species levels and altering the GSH/GSSG ratio in lymphocytes. It also induced nuclear translocation of redox sensitive transcription factor Nrf2 and increased the transcription of its dependent genes. SB inhibited mitogen-induced proliferation and cytokine secretion by lymphocytes. SB also significantly inhibited mitogen-induced upregulation of T cell costimulatory molecules and activation markers. It was observed that SB inhibited mitogen-induced phosphorylation of c-Raf, MEK, ERK, JNK, and p38. It suppressed IκBα degradation and nuclear translocation of NF-κB in activated lymphocytes. Anti-inflammatory effects of SB were significantly abrogated by the inhibitors of Nrf2 and HO-1, suggesting the involvement of this pathway. Similar anti-inflammatory effects of SB on lymphocyte proliferation and cytokine secretion were also observed in vivo. To our knowledge, this is the first report showing that the anti-inflammatory effects of SB are mediated via modulation of Nrf2 and NF-κB in lymphocytes.

Pro-oxidants ameliorate radiation-induced apoptosis through activation of the calcium–ERK1/2–Nrf2 pathway

There are no reports describing the ability of pro-oxidants to protect against radiation-induced apoptosis. Activation of the redox-sensitive transcription factor Nrf2 by low levels of ROS is known to protect against oxidative stress-induced cell death. In this study, hydrogen peroxide, diethylmaleate, and 1,4-naphthoquinone (NQ) exhibited complete protection against radiation-induced cell death in lymphocytes as estimated by propidium iodide staining. Radioprotection by NQ was demonstrated by inhibition of caspase activation, decrease in cell size, DNA fragmentation, nuclear blebbing, and clonogenic assay. Interestingly, NQ offered protection to lymphocytes even when added to cells postirradiation. NQ increased intracellular ROS levels and decreased GSH levels. NQ activated Nrf2 and increased the expression of the cytoprotective gene heme oxygenase-1 in lymphocytes. NQ increased ERK phosphorylation, which is upstream of Nrf2, and this ERK activation was through increased intracellular calcium levels. Administration of NQ to mice offered protection against whole-body irradiation (WBI)-induced apoptosis in splenic lymphocytes and loss of viability of spleen and bone marrow cells. It restored WBI-mediated changes in hematological parameters and functional responses of lymphocytes. Importantly, NQ rescued mice against WBI-induced mortality. These results demonstrated that a pro-oxidant such as NQ can protect against radiation-induced apoptosis by activation of multiple prosurvival mechanisms including activation of the calcium-ERK1/2-Nrf2 pathway.

Role of immunoregulatory transcription factors in differential immunomodulatory effects of tocotrienols

Tocotrienols have been shown to possess antioxidant, antitumor, cardioprotective, and antiproliferative effects. This report describes novel immunomodulatory effects of tocotrienols in murine lymphocytes. γ-Tocotrienol (GT) was more effective in suppressing concanavalin A (Con A)-induced T cell proliferation and cytokine production compared to α-tocotrienol (AT) when present continuously in the culture. GT inhibited T cell activation markers and costimulatory molecule. GT modulated intracellular glutathione in lymphocytes, and the suppressive effects of GT could not be abrogated by thiol or nonthiol antioxidants, indicating a poor link between anti-inflammatory properties of tocotrienols and cellular redox status. It was also observed that GT suppressed Con A-induced activation of NF-κB, AP-1, and NF-κB-dependent gene expression. Cellular uptake studies with tocotrienols showed higher accumulation of GT compared to AT. Similar immunosuppressive effects of GT were also observed when administered to mice. In contrast, transient exposure of lymphocytes to GT (4 h) resulted in higher survival and proliferation of lymphocytes in vitro and in vivo in syngeneic and allogeneic hosts. This was attributed to the ability of GT to induce NF-κB, AP-1, and mTOR activation in lymphocytes upon transient exposure. Our results demonstrated that antioxidants such as tocotrienols may exhibit pleiotropic effects by activating multiple mechanisms in cells.

Immunomodulatory and radioprotective effects of lignans derived from fresh nutmeg mace (Myristica fragrans) in mammalian splenocytes

Recently, the lignans present in the aqueous extract of fresh nutmeg mace (aril of the fruit of Myristica fragrans) were shown to possess antioxidant properties in cell free systems and protected PUC18 plasmid against radiation-induced DNA damage. The present report describes the immunomodulatory and radiomodifying properties of lignans present in the aqueous extract of fresh nutmeg mace in mammalian splenocytes. These macelignans (ML) inhibited the proliferation of splenocytes in response to polyclonal T cell mitogen concanavalin A (Con A). This inhibition of proliferation was due to cell cycle arrest in G1 phase and augmentation of apoptosis as shown by increase in pre G1 cells. The increase in activation induced cell death by ML was dose dependent. It was found to inhibit the transcription of IL-2 and IL-4 genes in response to Con A. The production of IL-2, IL-4 and IFN-gamma cytokines was significantly inhibited by ML in Con A-stimulated lymphocytes in a dose dependent manner. ML protected splenocytes against radiation-induced intracellular ROS production in a dose dependent manner. ML was not cytotoxic towards lymphocytes. On the contrary, it significantly inhibited the radiation-induced DNA damage in splenocytes as indicated by decrease in DNA fragmentation. To our knowledge, this is the first report showing the antioxidant, radioprotective and immunomodulatory effects of lignans in mammalian cells.

Dimethoxycurcumin, a metabolically stable analogue of curcumin, exhibits anti-inflammatory activities in murine and human lymphocytes

The aim of this study was to investigate whether dimethoxycurcumin (DiMC), a synthetic curcumin analogue having higher metabolic stability over curcumin, could exhibit anti-inflammatory activity in murine and human lymphocytes. Both curcumin and DiMC suppressed mitogen as well as antigen driven proliferation of murine splenic lymphocytes. Further, mitogen and antigen-stimulated cytokine (IL-2, IL-4, IL-6 and IFN-γ) secretion by T cells was also abrogated by curcumin and DiMC. Interestingly, curcumin and DiMC suppressed B cell proliferation induced by lipopolysaccharide. Curcumin and DiMC also inhibited Con A-induced activation of early and late T cell activation markers. They scavenged basal reactive oxygen species and depleted GSH levels in lymphocytes. The suppression of mitogen-induced T cell proliferation and cytokine secretion by curcumin and DiMC was significantly abrogated by thiol containing antioxidants suggesting a role for redox in their anti-inflammatory activity. Further, the possibility of curcumin and DiMC directly interacting with thiol-containing antioxidant GSH was monitored by changes in absorbance. Both curcumin and DiMC inhibited Con A induced activation of NF-κB and MAPK. More importantly, curcumin and DiMC inhibited phytohaemagglutinin induced proliferation and cytokine secretion by human peripheral blood mononuclear cells. To explore their therapeutic efficacy, they were added to lymphocytes post-Con A stimulation and we observed a significant suppression of IL-2, IL-6 and IFN-γ. The present study for the first time demonstrates the potent anti-inflammatory activity of DiMC. Further, DiMC could find application as an alternative to curcumin, which is currently used in several clinical studies, due to its superior bioavailability and comparable efficacy.

Plumbagin induces apoptosis in lymphoma cells via oxidative stress mediated glutathionylation and inhibition of mitogen-activated protein kinase phosphatases (MKP1/2).

Maintaining cellular redox homeostasis is imperative for the survival and normal functioning of cells. This study describes the role and regulation of MAPKinases in oxidative stress mediated apoptosis. Plumbagin, a vitamin K3 analog and a pro-oxidant, was employed and it induced apoptosis in both mouse and human T-cell lymphoma cell lines via increased oxidative stress, caspase activity and loss of mitochondrial membrane potential. The pro-oxidant and cytotoxic effects of plumbagin were sensitive to antioxidants indicating a decisive role of cellular redox balance. Plumbagin induced persistent activation of JNK and pharmacological inhibition as well as shRNA-mediated JNK knock-down rescued cells from plumbagin-induced apoptosis. Further, plumbagin induced cytochrome c release, FasL expression and Bax levels via activation of JNK pathway. Exposure of lymphoma cells to plumbagin led to inhibition of total and specific phosphatase activity, increased total protein S-glutathionylation and induced glutathionylation of dual specific phosphatase- 1 and 4 (MKP-1 and MKP-2). The in vivo anti-tumor efficacy of plumbagin was demonstrated using a mouse model. In conclusion, oxidative stress mediated tumor cytotoxicity operates through sustained JNK activation via a novel redox-mediated regulation of MKP-1 and MKP-2.

Plumbagin, a Vitamin K3 Analogue, abrogates Lipopolysaccharide-Induced Oxidative Stress, Inflammation and Endotoxic Shock via NF-κB Suppression

Plumbagin has been reported to modulate cellular redox status and suppress NF-κB. In the present study, we investigated the effect of plumbagin on lipopolysaccharide (LPS)-induced endotoxic shock, oxidative stress and inflammatory parameters in vitro and in vivo. Plumbagin inhibited LPS-induced nitric oxide, TNF-α, IL-6 and prostaglandin-E2 production in a concentration-dependent manner in RAW 264.7 cells without inducing any cell death. Plumbagin modulated cellular redox status in RAW cells. Plumbagin treatment significantly reduced MAPkinase and NF-κB activation in macrophages. Plumbagin prevented mice from endotoxic shock-associated mortality and decreased serum levels of pro-inflammatory markers. Plumbagin administration ameliorated LPS-induced oxidative stress in peritoneal macrophages and splenocytes. Plumbagin also attenuated endotoxic shock-associated changes in liver and lung histopathology and decreased the activation of ERK and NF-κB in liver. These findings demonstrate the efficacy of plumbagin in preventing LPS-induced endotoxemia and also provide mechanistic insights into the anti-inflammatory effects of plumbagin.