Colon cancer is the leading cause of cancer death in both men and women worldwide. The deregulated cell cycle control or decreased apoptosis of normal epithelial cells leading to uncontrolled proliferation is one of the major features of tumor progression. We have previously shown that aldose reductase (AR), a NADPH dependent- aldo-keto reductase, has been shown to be involved in growth factors–induced proliferation of colon cancer cells. Herein, we report that inhibition of AR prevents epidermal growth factor (EGF) - and basic fibroblast growth factor (bFGF)–induced HT29 cell proliferation by accumulating cells at G1 phase of cell cycle. Similar results were observed in SW480 and HCT-116 colon cancer cells. Treatment of HT29 cells with AR inhibitor, sorbinil or zopolrestat prevented EGF– and bFGF-induced DNA binding activity of E2F-1 and phosphorylation of retinoblastoma protein. Inhibition of AR also prevented EGF– and bFGF-induced phosphorylation of cyclin-dependent kinase (cdk)-2 and expression of G1/S transition regulatory proteins such as cyclin D1, cdk-4, PCNA, cyclin E and c-myc. More importantly, inhibition of AR prevented the EGF– and bFGF-induced activation of PI3K/AKT and reactive oxygen species generation in colon cancer cells. Further, inhibition of AR also prevented the tumor growth of human colon cancer cells in nude mice xenografts. Collectively, these results show that AR mediates EGF– and bFGF–induced colon cancer cell proliferation by activating/expressing G1/S phase proteins such as E2F-1, cdks and cyclins through ROS/PI3K/AKT indicating the use of AR inhibitors in the prevention of colon carcinogenesis.
Aldose reductase; cell cycle; E2F-1; colon cancer; cyclins
Metastable aldehydes produced by lipid peroxidation act as 'toxic second messengers' that extend the injurious potential of free radicals. 4-hydroxy 2-nonenal (HNE), a highly toxic and most abundant stable end product of lipid peroxidation, has been implicated in the tissue damage, dysfunction, injury associated with aging and other pathological states such as cancer, Alzheimer, diabetes, cardiovascular and inflammatory complications. Further, HNE has been considered as a oxidative stress marker and it act as a secondary signaling molecule to regulates a number of cell signaling pathways. Biological activity of HNE depends on its intracellular concentration, which can differentially modulate cell death, growth and differentiation. Therefore, the mechanisms responsible for maintaining the intracellular levels of HNE are most important, not only in the defense against oxidative stress but also in the pathophysiology of a number of disease processes. In this review, we discusse the significance of HNE in mediating various disease processes and how regulation of its metabolism could be therapeutically effective.
4-hydroxy 2-nonenal; oxidative stress; cancer; cataract; Alzheimer
Anti-inflammatory effect of piceatannol, a naturally occurring polyphenol and a potent free radical scavenger, on ocular inflammation is not known. We examined the anti-inflammatory role of piceatannol in ocular inflammatory response due to endotoxin-induced uveitis (EIU) in rats. EIU was induced in Lewis rats by subcutaneous injection of lipopolysaccharide (LPS; 150 ug/rat). Piceatannol (30 mg/kg body wt, i.p) was injected either 2 h prior to or 1 h post LPS induction. A significant increase in the number of infiltrating cells, total protein, and various cytokines and chemokines in AqH were observed in the EIU rat eyes as compared to control groups. However, pre- or post- treatment of piceatannol significantly blocked the LPS-induced changes. Further, piceatannol also suppressed the expression of Cox-2, iNOS and activation of NF-κB in the ciliary bodies as well as retina. Further, piceatannol also inhibited the expression of Cox-2, iNOS, and phosphorylation of NF-κB in primary human non-pigmented ciliary epithelial cells (HNPECs) treated with LPS. Similarly, piceatannol also diminished LPS-induced level of NO and PGE2 in HNPECs. Thus our results demonstrate an anti-inflammatory role of piceatannol in suppressing ocular inflammation induced by endotoxin in rats.
Uveitis; piceatannol; inflammation; NF-κB; rats
Oxidative stress plays a critical role in the pathophysiology of a wide range of diseases including cancer. This view has broadened significantly with the recent discoveries that reactive oxygen species initiated lipid peroxidation leads to the formation of potentially toxic lipid aldehyde species such as 4-hydroxy-trans-2-nonenal (HNE), acrolein, and malondialdehyde which activate various signaling intermediates that regulate cellular activity and dysfunction via a process called redox signaling. The lipid aldehyde species formed during synchronized enzymatic pathways result in the posttranslational modification of proteins and DNA leading to cytotoxicity and genotoxicty. Among the lipid aldehyde species, HNE has been widely accepted as a most toxic and abundant lipid aldehyde generated during lipid peroxidation. HNE and its glutathione conjugates have been shown to regulate redox-sensitive transcription factors such as NF-κB and AP-1 via signaling through various protein kinase cascades. Activation of redox-sensitive transcription factors and their nuclear localization leads to transcriptional induction of several genes responsible for cell survival, differentiation, and death. In this review, we describe the mechanisms by which the lipid aldehydes transduce activation of NF-κB signaling pathways that may help to develop therapeutic strategies for the prevention of a number of inflammatory diseases.
Benfotiamine, a lipid-soluble analogue of vitamin B1, is a potent anti-oxidant that is used as a food supplement for the treatment of diabetic complications. Our recent study indicates a novel role of benfotiamine in the prevention of bacterial endotoxin, lipopolysaccharide (LPS)-induced cytotoxicity and inflammatory response in murine macrophages. Nevertheless, it remains unclear how benfotiamine mediates anti-inflammatory effects. In this study, we investigated the anti-inflammatory role of benfotiamine in regulating the arachidonic acid (AA) pathway generated inflammatory lipid mediators in RAW 264.7 macrophages. Benfotiamine prevented the LPS-induced activation of cPLA2 and release of AA metabolites such as leukotrienes (LTB4), prostaglandin E2 (PGE2), thromboxanes 2 (TXB2) and prostacyclin (PGI2) in macrophages. Further, LPS-induced expressions of AA metabolizing enzymes such as COX-2, LOX-5, TXB synthase and PGI2 synthase were significantly blocked by benfotiamine. Furthermore, benfotiamine prevented the LPS-induced phosphorylation of ERK1/2 and expression of transcription factors NF-kB, and Egr-1. Benfotiamine also prevented the LPS-induced oxidative stress and protein-HNE adducts formation. Most importantly, as compared to specific COX-2 and LOX-5 inhibitors, benfotiamine significantly prevented the LPS-induced macrophage death and monocytes adhesion to endothelial cells. Thus, our studies indicate that the dual regulation of COX and LOX pathways in AA metabolism could be a novel mechanism by which benfotiamine exhibits its potential anti-inflammatory response.
Vitamin B1; inflammation; arachidonic acid; Cox and Lox
To investigate the therapeutic effects of metformin, a commonly used antidiabetic drug, in preventing endotoxin-induced uveitis (EIU) in rats.
EIU in Lewis rats was developed by subcutaneous injection of lipopolysaccharide (LPS; 150 μg). Metformin (300 mg/kg body weight, intraperitoneally) or its carrier was injected either 12 hours before or 2 hours after LPS induction. Three and 24 hours after EIU, eyes were enucleated and aqueous humor (AqH) was collected. The MILLIPLEX-MAG Rat cytokine-chemokine magnetic bead array was used to determine inflammatory cytokines. The expression of Cox-2, phosphorylation of AMPK, and NF-κB (p65) were determined immunohistochemically. Primary human nonpigmented ciliary epithelial cells (HNPECs) were used to determine the in vitro efficacy of metformin.
Compared with controls, the EIU rat AqH had significantly increased number of infiltrating cells and increased levels of various cytokines and chemokines (TNF-α, MCP-1, IL-1β, MIP-1α, IL-6, Leptin, and IL-18) and metformin significantly prevented the increase. Metformin also prevented the expression of Cox-2 and phosphorylation of p65, and increased the activation of AMPK in the ciliary bodies and retinal tissues. Moreover, metformin prevented the expression of Cox-2, iNOS, and activation of NF-kB in the HNPECs and decreased the levels of NO and PGE2 in cell culture media.
Our results for the first time demonstrate a novel role of the antidiabetic drug, metformin, in suppressing uveitis in rats and suggest that this drug could be developed to prevent uveitis complications.
Our studies found that metformin, a commonly used drug for diabetes, prevents endotoxin-induced uveitis in rats via activating AMPK and suppressing NF-kB-mediated ocular inflammatory response. Thus, our studies indicate that metformin could be developed as a potential agent to prevent uveitis.
Bacterial endotoxin, lipopolysaccharide (LPS) is known to induce release of arachidonic acid (AA) and its metabolic products which play important role in inflammatory process. We have shown earlier that LPS-induced signals in macrophages are mediated by aldose reductase (AR). Here we have investigated the role of AR in LPS-induced release of AA metabolites and their modulation using a potent pharmacological inhibitor fidarestat and AR-siRNA ablation in RAW 264.7 macrophages, and AR-knockout mice peritoneal macrophages and heart tissue. Inhibition or genetic ablation of AR prevented the LPS-induced synthesis and release of AA metabolites such as PGE2, TXB, PGI2 and LTBs in macrophages. LPS-induced activation of cPLA2 was also prevented by AR inhibition. Similarly, AR inhibition also prevented the calcium ionophore A23187 –induced cPLA2 and LTB4 in macrophages. Further, AR inhibition with fidarestat prevented the expression of AA metabolizing enzymes such as COX-2 and LOX-5 in RAW 264.7 cells and AR-knockout mice derived peritoneal macrophages. LPS-induced expression of AA metabolizing enzymes and their catalyzed metabolic products were significantly lower in peritoneal macrophages and heart tissue from AR-knockout mice. LPS-induced activation of redox-sensitive signaling intermediates such as MAPKs, transcription factor NF-kB as well as Egr-1, a transcription regulator of mPGES-1, which in collaboration with COX-2 leads to the production of PGE2, were also significantly prevented by AR inhibition. Taken together, our results indicate that AR mediates LPS-induced inflammation by regulating AA metabolic pathway and thus provide novel role of AR inhibition in preventing inflammatory complications such as sepsis.
LPS; Inflammation; Aldose reductase; Arachidonic acid; COX-2 and LOX-5
It is strongly established by numerous studies that oxidative stress-induced inflammation is one of the major causative agents in a variety of cancers. Various factors such as bacterial, viral, parasitic infections, chemical irritants, carcinogens are involved in the initiation of oxidative stress-mediated inflammation. Chronic and persistent inflammation promotes the formation of cancerous tumors. Recent investigations strongly suggest that aldose reductase [AR; AKR1B1], a member of aldo-keto reductase superfamily of proteins, is the mediator of inflammatory signals induced by growth factors, cytokines, chemokines, carcinogens etc. Further, AR reduced product(s) of lipid derived aldehydes and their metabolites such as glutathionyl 1,4-dihydroxynonanol (GS-DHN) have been shown to be involved in the activation of transcription factors such as NF-κB and AP-1 which transcribe the genes of inflammatory cytokines. The increased inflammatory cytokines and growth factors promote cell proliferation, a main feature involved in the tumorigenesis process. Inhibition of AR has been shown to prevent cancer cell growth in vitro and in vivo models. In this review, we have described the possible association between AR with oxidative stress- and inflammation- initiated carcinogenesis. A thorough understanding of the role of AR in the inflammation – associated cancers could lead to the use of AR inhibitors as novel chemotherapeutic agents against cancer.
Aldose reductase; Oxidative stress; Inflammation; Cancer and NF-kB
Aldose reductase (AKR1B1), which catalyzes the reduction of glucose to sorbitol and lipid aldehydes to lipid alcohols, has been shown to be involved in secondary diabetic complications including cataractogenesis. Rats have high levels of AKR1B1 in lenses and readily develop diabetic cataracts, whereas mice have very low levels of AKR1B1 in their lenses and are not susceptible to hyperglycemic cataracts. Studies with transgenic mice that over-express AKR1B1 indicate that it is the key protein for the development of diabetic complications including diabetic cataract. However, no such studies were performed in genetically altered AKR1B1 rats. Hence, we developed siRNA-based AKR1B1 knockdown rats (ARKO) using the AKR1B1-siRNA-pSuper vector construct. Genotyping analysis suggested that more than 90% of AKR1B1 was knocked down in the littermates. Interestingly, all the male animals were born dead and only 3 female rats survived. Furthermore, all 3 female animals were not able to give birth to F1 generation. Hence, we could not establish an AKR1B1 rat knockdown colony. However, we examined the effect of AKR1B1 knockdown on sugar-induced lens opacification in ex vivo. Our results indicate that rat lenses obtained from AKR1B1 knockdown rats were resistant to high glucose–induced lens opacification as compared to wild-type (WT) rat lenses. Biochemical analysis of lens homogenates showed that the AKR1B1 activity and sorbitol levels were significantly lower in sugar-treated AKR1B1 knockdown rat lenses as compared to WT rat lenses treated with 50 mM glucose. Our results thus confirmed the significance of AKR1B1 in the mediation of sugar-induced lens opacification and indicate the use of AKR1B1 inhibitors in the prevention of cataractogenesis.
Aldose Reductase; SiRNA knock-down Rats; Lens Opacification; Cataract; Oxidative Stress
We have recently shown that aldose reductase (AR, EC 188.8.131.52) a nicotinamide adenine dinucleotide phosphate-dependent aldo-keto reductase, known to be involved in oxidative stress-signaling, prevents human colon cancer cell growth in culture as well as in nude mice xenografts. Inhibition of AR also prevents azoxymethane-induced aberrant crypt foci formation in mice. In order to understand the chemopreventive mechanism(s) of AR inhibition in colon cancer, we have investigated the role of AR in the mediation of angiogenic signals in vitro and in vivo models. Our results show that inhibition of AR significantly prevented the VEGF- and FGF -induced proliferation and expression of proliferative marker Ki67 in the human umbilical vein endothelial cells (HUVEC). Further, AR inhibition or ablation with siRNA prevented the VEGF-and FGF –induced invasion and migration in HUVEC. AR inhibition also prevented the VEGF-and FGF- induced secretion/expression of IL-6, MMP2, MMP9, ICAM, and VCAM. The anti-angiogenic feature of AR inhibition in HUVEC was associated with inactivation of PI3K/AKT and NF-κB (p65) and suppression of VEGF receptor 2 protein levels. Most importantly, matrigel plug model of angiogenesis in rats showed that inhibition of AR prevented infiltration of blood cells, invasion, migration and formation of capillary like structures, and expression of blood vessels markers CD31 and vWF. Thus, our results demonstrate that AR inhibitors could be novel agents to prevent angiogenesis.
Aldose reductase; angiogenesis; endothelial cells; cancer; inflammation
In the past years aldose reductase (AKR1B1; AR) is thought to be involved in the pathogenesis of secondary diabetic complications such as retinopathy, neuropathy, nephropathy and cataractogenesis. Subsequently, a number of AR inhibitors have been developed and tested for diabetic complications. Although, these inhibitors have found to be safe for human use, they have not been successful at the clinical studies because of limited efficacy. Recently, the potential physiological role of AR has been reassessed from a different point of view. Diverse groups suggested that AR besides reducing glucose, also efficiently reduces oxidative stress-generated lipid peroxidation-derived aldehydes and their glutathione conjugates. Since lipid aldehydes alter cellular signals by regulating the activation of transcription factors such as NF-kB and AP1, inhibition of AR could inhibit such events. Indeed, a wide array of recent experimental evidence indicates that the inhibition of AR prevents oxidative stress-induced activation of NF-kB and AP1 signals that lead to cell death or growth. Further, AR inhibitors have been shown to prevent inflammatory complications such as sepsis, asthma, colon cancer and uveitis in rodent animal models. The new experimental in-vitro and in-vivo data has provided a basis for investigating the clinical efficacy of AR inhibitors in preventing other inflammatory complications than diabetes. This review describes how the recent studies have identified novel plethoric physiological and pathophysiological significance of AR in mediating inflammatory complications, and how the discovery of such new insights for this old enzyme could have considerable importance in envisioning potential new therapeutic strategies for the prevention or treatment of inflammatory diseases.
Aldose reductase; inflammation; oxidative stress; sepsis; cancer; uveitis; diabetes
The results of this study suggest that AR deficiency protects against autoimmune- and endotoxin-induced uveitis in mice. Thus, inhibition of this enzyme could be a novel approach for the therapeutic interventions of ocular inflammatory complications such as uveitis. Since AR inhibitors have already undergone phase III clinical trials for the treatment of diabetic complications and were found to be safe without any major toxicity, they could be developed as novel anti-inflammatory drugs for the prevention of uveitis complications in a relatively shorter time.
To investigate the effect of aldose reductase (AR) deficiency in protecting the chronic experimental autoimmune (EAU) and acute endotoxin-induced uveitis (EIU) in c57BL/6 mice.
The WT and AR-null (ARKO) mice were immunized with human interphotoreceptor retinoid-binding peptide (hIRPB-1–20), to induce EAU, or were injected subcutaneously with lipopolysaccharide (LPS; 100 μg) to induce EIU. The mice were killed on day 21 for EAU and at 24 hours for EIU, when the disease was at its peak, and the eyes were immediately enucleated for histologic and biochemical studies. Spleen-derived T-lymphocytes were used to study the antigen-specific immune response in vitro and in vivo.
In WT-EAU mice, severe damage to the retinal wall, especially to the photoreceptor layer was observed, corresponding to a pathologic score of ∼2, which was significantly prevented in the ARKO or AR inhibitor–treated mice. The levels of cytokines and chemokines increased markedly in the whole-eye homogenates of WT-EAU mice, but not in ARKO-EAU mice. Further, expression of inflammatory marker proteins such as inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-α, and vascular cell adhesion molecule (VCAM)-1 was increased in the WT-EIU mouse eyes but not in the ARKO-EIU eyes. The T cells proliferated vigorously when exposed to the hIRPB antigen in vitro and secreted various cytokines and chemokines, which were significantly inhibited in the T cells isolated from the ARKO mice.
These findings suggest that AR-deficiency/inhibition protects against acute as well as chronic forms of ocular inflammatory complications such as uveitis.
The authors' studies indicate that the treatment of rats with an aldose reductase (AR) inhibitor significantly prevented leukocyte infiltration and increased inflammatory cytokines and chemokines in rat aqueous humor and in eye tissues in response to experimental autoimmune-induced uveitis. These results provide a novel therapeutic approach to preventing uveitis complications by using AR inhibitors.
Recently, the authors showed that the inhibition of aldose reductase (AR) prevents bacterial endotoxin-induced uveitis in rats. They have now investigated the efficacy of AR inhibitors in the prevention of experimental autoimmune-induced uveitis (EAU) in rats.
Lewis rats were immunized with bovine interphotoreceptor retinoid-binding peptide (IRBP) to develop EAU. Two or 8 days after immunization, the rats started receiving the AR inhibitor fidarestat (7 mg/kg/d; intraperitoneally). They were killed when the disease was at its peak; aqueous humor (AqH) was collected from one eye, and the other eye of each rat was used for histologic studies. The protein concentration and the levels of inflammatory markers were determined in AqH. Immunohistochemical analysis of eye sections was performed to determine the expression of inflammatory markers. The effect of AR inhibition on immune response was investigated in isolated T lymphocytes.
Immunization of rats by IRBP peptide resulted in a significant infiltration of leukocytes in the posterior and the anterior chambers of the eye. Further, EAU caused an increase in the concentration of proteins, inflammatory cytokines, and chemokines in AqH, and the expression of inflammatory markers such as inducible-nitric oxide synthase and cycloxygenase-2 in the rat eye ciliary bodies and retina. Treatment with fidarestat significantly prevented the EAU-induced ocular inflammatory changes. AR inhibition also prevented the proliferation of spleen-derived T cells isolated from EAU rats in response to the IRBP antigen.
These results suggest that AR could be a novel mediator of bovine IRBP-induced uveitis in rats.
Current understanding of the role of oxidative stress in ocular inflammatory diseases indicates that antioxidant therapy may be important to optimize the treatment. Recently investigated antioxidant therapies for ocular inflammatory diseases include various vitamins, plant products and reactive oxygen species scavengers. Oxidative stress plays a causative role in both non-infectious and infectious uveitis complications, and novel strategies to diminish tissue damage and dysfunction with antioxidant therapy may ameliorate visual complications. Preclinical studies with experimental animals and cell culture demonstrate significance of anti-inflammatory effects of a number of promising antioxidant agents. Many of these antioxidants are under clinical trial for various inflammatory diseases other than uveitis such as cardiovascular, rheumatoid arthritis and cancer. Well planned interventional clinical studies of the ocular inflammation will be necessary to sufficiently investigate the potential medical benefits of antioxidant therapies for uveitis. This review summarizes the recent investigation of novel antioxidant agents for ocular inflammation, with selected studies focused on uveitis.
Antioxidants; eye; ocular inflammation; oxidative stress and uveitis
Our studies indicate that ethyl pyruvate prevents lipopolysaccharide-induced nuclear factor kappa B–dependent ocular inflammatory signals in cultured cells and in rat eyes. Therefore, supplementation of ethyl pyruvate could be a novel approach to control ocular inflammation, especially uveitis.
Recent studies indicate that ethyl pyruvate (EP) exerts anti-inflammatory properties; however, the effect of EP on ocular inflammation is not known. The efficacy of EP in endotoxin-induced uveitis (EIU) in rats was investigated.
EIU in Lewis rats was developed by the subcutaneous injection of lipopolysaccharide (LPS; 150 μg). EP (30 mg/kg body weight) or its carrier was injected intraperitoneally 1 hour before or 2 hours after lipopolysaccharide injection. Animals were killed after 3 and 24 hours followed by enucleation of eyes and collection of the aqueous humor (AqH). The number of infiltrating cells and levels of proteins in the AqH were determined. The rat cytokine/chemokine multiplex method was used to determine level of cytokines and chemokines in the AqH. TNF-α and phospho-nuclear factor kappa B (NF-κB) expression in ocular tissues were determined immunohistochemically. Human primary nonpigmented ciliary epithelial cells (HNPECs) were used to determine the in vitro efficacy of EP on lipopolysaccharide-induced inflammatory response.
Compared to controls, AqH from the EIU rat eyes had a significantly higher number of infiltrating cells, total protein, and inflammatory cytokines/chemokines, and the treatment of EP prevented EIU-induced increases. In addition, EP also prevented the expression of TNF-α and activation of NF-κB in the ciliary bodies and retina of the eye. Moreover, in HNPECs, EP inhibited lipopolysaccharide-induced activation of NF-κB and expression of Cox-2, inducible nitric oxide synthase, and TNF-α.
Our results indicate that EP prevents ocular inflammation in EIU, suggesting that the supplementation of EP could be a novel approach for the treatment of ocular inflammation, specifically uveitis.
To investigate the anti-inflammatory effects of guggulsterone, an antioxidant and anti-tumor agent, in endotoxin-induced uveitis (EIU) in rats and to elucidate the underlying molecular mechanism(s) related to ocular inflammation.
EIU was induced by subcutaneous injection of lipopolysaccharide (LPS) (150 ug) in Lewis rats treated with guggulsterone (30 mg/kg body wt, i.p) or its carrier. After 24 h the rats were sacrificed, eyes were enucleated and aqueous humors (AqH) were collected. The number of infiltrating cells, levels of metrix metalloproteinase-2 (MMP-2), nitric oxide (NO), and prostaglandin E2 (PGE2) were determined in AqH by specific ELISAs. An antibody array was used to measure the expression of various inflammatory cytokines in AqH. The expression of MMP-2, iNOS, Cox-2, phospho-IκB and phospho-NF-κB were determined immunohistochemically. Human primary non-pigment ciliary epithelial cells (HNPECs) were used to determine the in vitro efficacy of guggulsterone on LPS-induced inflammatory response.
Compared to control, the EIU rat eye AqH had significantly higher number of infiltrating cells, total protein, and inflammatory markers such as MMP-2, NO, and PGE2 and treatment of guggulsterone prevented EIU-induced increases. Further, guggulsterone also prevented the expression of MMP-2, iNOS, and Cox-2 proteins as well as IκB and NF-κB in various eye tissues. Moreover, in cultured HNPECs guggulsterone inhibited LPS-induced expression of inflammatory proteins.
Our results for the first time demonstrate that the plant sterol guggulsterone suppresses the ocular inflammation in EIU suggesting that supplementation of guggulsterone could be a novel approach for the treatment of ocular inflammation.
Uveitis; guggulsterone; Inflammation; NF-kB; LPS; cytokines
Aldose reductase, although identified initially as a glucose-reducing enzyme via polyol pathway, is believed to be an important component of antioxidant defense system as well as a key mediator of oxidative stress-induced molecular signaling. The dual role played by AR has made it a very important enzyme for the regulation of not only the cellular redox state by detoxifying the reactive lipid-aldehydes generated by lipid peroxidation which is crucial in the cellular homeostasis, but also in the regulation of molecular signaling cascade that may regulate oxidative stress-induced cytotoxic events. Search for the new molecular targets to restrain the oxidative stress-induced inflammation has resulted in the identification of AR as an unanticipated mediator of oxidative stress-induced signaling. Although, in last one decade or so AR has been implicated in various inflammation-related disease conditions ranging from diabetes, sepsis, cancer, cardiovascular and airway inflammation, however, a critical evaluation of the clinical efficacy of AR inhibitors awaits a better understanding of the role of AR in regulating inflammation, especially in ocular inflammation.
Aldose reductase; inflammation; uveitis; NF-κB; GS-DHN; infection; autoimmunity
Tumor necrosis factor-alpha (TNF-α) –converting enzyme (TACE), a member of the family of metalloproteinase disintegrin proteins, is responsible for the conversion of inactive TNF-α precursor from to active mature form. TNF-α is a pleiotropic cytokine that contributes to cellular immunity and inflammatory response in wide range of inflammatory pathologies. Although a large number of studies indicate the use of TACE inhibitors, which prevents processing of TNF-α as potential therapeutic drugs for the treatment of inflammatory diseases including rheumatoid arthritis, Crohn’s disease and cancer, very few studies indicate its use in ocular pathologies. It is still not clearly understood how the TACE-mediated shedding of cytokines and growth factors in various ocular tissues plays a critical role in the cytotoxic signals causing tissue dysfunction and damage leading to blindness. Regulation of TACE activity is likely to have wide implications for ocular immunology and inflammatory diseases. Specifically, since anti-TNF-α therapies have been used to prevent ocular inflammatory complications, the use of TACE inhibitors could be a novel therapeutic approach for ocular inflammatory diseases especially uveitis.
TNF-alpha; TACE; Uveitis; inflammation; Eye
Macrophages which play a central role in the injury, infection and sepsis, use glucose as their primary source of metabolic energy. Increased glucose uptake in inflammatory cells is well known to be one of the responsible processes that cause inflammatory response and cytotoxicity. We have shown recently that the inhibition of aldose reductase (AR) prevents bacterial endotoxin, lipopolysaccharide (LPS)-induced cytotoxicity and inflammatory response in macrophages. However, it is not known how AR inhibition prevents LPS-induced inflammation. Here in, we examined the effect of AR inhibition on LPS-induced glucose uptake and the expression of glucose transporter 3 (GLUT-3) in RAW264.7 murine macrophages. Stimulation of macrophages with LPS-increased glucose uptake as measured by using C14 labeled methyl-D-glucose and inhibition of AR prevented it. Similarly, ablation of AR by using AR–siRNA also prevented the LPS-induced glucose uptake in macrophages. Further, AR inhibition also prevented the LPS-induced upregulation of GLUT-3 expression, cyclic adenosine monophosphate (cAMP) accumulation and protein kinase A (PKA) activation in RAW264.7 cells. Moreover, LPS-induced down-regulation of cAMP response element modulator (CREM), phosphorylation of cAMP response element-binding protein (CREB) and DNA binding of CREB were also prevented by AR inhibition. Further, inhibition of AR or PKA also prevented the LPS-induced levels of GLUT-3 protein as well as mRNA in macrophages. These results indicate that AR mediates LPS-induced glucose uptake and expression of glucose transporter-3 via cAMP/PKA/CREB pathway and thus represents a novel mechanism by which AR regulates LPS-induced inflammation.
cAMP; PKA; Aldose Reductase; CREB; LPS; Glucose transporters
The study has been designed to investigate the molecular mechanisms by which benfotiamine, a lipid-soluble analogue of Vitamin B1 effects lipopolysaccharide (LPS) – induced inflammatory signals leading to cytotoxicity in mouse macrophage cell line RAW264.7. Benfotiamine prevented LPS-induced apoptosis, expression of Bcl-2 family of pro-apoptotic proteins, caspase-3 activation and PARP cleavage, altered mitochondrial membrane potential and release of cytochrome-c and apoptosis inducing factor (AIF), phosphorylation and subsequent activation of p38-MAPK, stress activated kinases (SAPK/JNK), Protein kinase C, and cytoplasmic-phospholipase A2 in RAW cells. Further, phosphorylation and degradation of inhibitory kappa B (IκB) and consequent activation and nuclear translocation of redox-sensitive transcription factor NF-κB was significantly prevented by benfotiamine. The LPS-induced increased expression of cytokines and chemokines and other inflammatory marker proteins iNOS and COX-2 and their metabolic products NO and PGE2 were also blocked significantly. Thus, our results elucidate the molecular mechanism of anti-inflammatory action of benfotiamine in LPS-induced inflammation in murine macrophage. Benfotiamine suppresses oxidative stress-induced NF-κB activation and prevents the bacterial endotoxin-induced inflammation indicating that vitamin B1 supplementation could be beneficial in the treatment of inflammatory diseases.
Benfotiamine; vitamin B1; Oxidative stress; NF-κB; Macrophages; Inflammation
Chronic inflammatory diseases such as autoimmune and bacterial infections are associated with an elevated risk of ocular inflammation. Ciliary epithelial cells that play an important role in maintaining aqueous humor dynamics and homeostasis of anterior segment of eye are continuously exposed to inflammatory markers during infections and injury. Lipopolysachharide (LPS), a gram negative bacterial endotoxin, dysregulates aqueous humor (AqH) homeostasis by inducing inflammatory changes. We have investigated how inhibition of a polyol pathway enzyme, aldose reductase (AR), alters LPS-induced inflammatory changes in human non-pigmented ciliary epithelial cells (hNPECs). The stimulation of hNPECs with LPS (1 μg/ml) caused increased secretion of inflammatory markers such as PGE2 and NO in the culture medium as well as increased expression of COX-2 and iNOS proteins in cell extracts. LPS also increased phosphorylation of MAPKs (ERK1/2) and SAPK/JNK and activation of redox-sensitive transcription factors NF-κB and AP-1 in hNPECs and inhibition of AR by zopolrestat and sorbinil ameliorated these changes. Further, LPS-induced decrease in the expression of Na/K-ATPase in hNPECs was restored by AR inhibitors. Similar results were observed in ciliary bodies of LPS-injected rats. Taken together, our results suggest that AR plays an important role in the LPS-induced inflammatory changes in hNPECs and that inhibition of AR could be a novel therapeutic approach for ocular inflammation.
hNPECs; oxidative stress; LPS; Aldose reductase; inflammation
The authors suggest that the plant sterol guggulsterone suppresses bacterial endotoxin-induced inflammatory signaling through the PI3K/AKT/NF-κB pathway, leading to uveitis, and that it could therefore be used as a novel therapeutic agent for the treatment of ocular inflammation, especially uveitis.
To investigate the anti-inflammatory effects of guggulsterone, an antioxidant and antitumor agent, in endotoxin-induced uveitis (EIU) in rats and to elucidate the underlying molecular mechanism or mechanisms related to ocular inflammation.
EIU was induced by subcutaneous injection of lipopolysaccharide (LPS; 150 μg) into Lewis rats treated with guggulsterone (30 mg/kg body weight, intraperitoneally) or its carrier. After 24 hours the rats were killed, eyes were enucleated, and aqueous humor (AqH) was collected. Numbers of infiltrating cells and levels of matrix metalloproteinase-2 (MMP-2), nitric oxide (NO), and prostaglandin E2 (PGE2) were determined in AqH by specific ELISAs. An antibody array was used to measure the expression of various inflammatory cytokines in AqH. The expression of MMP-2, iNOS, Cox-2, phospho-IκB, and phospho-NF-κB was determined immunohistochemically. Human primary nonpigment ciliary epithelial cells (HNPECs) were used to determine the in vitro efficacy of guggulsterone on the LPS-induced inflammatory response.
Compared with control, the EIU rat eye AqH had a significantly higher number of infiltrating cells, total protein, and inflammatory markers, such as MMP-2, NO, and PGE2, and the treatment of guggulsterone prevented EIU-induced increases. Guggulsterone also prevented the expression of MMP-2, iNOS, and Cox-2 proteins and of IκB and NF-κB in various eye tissues. Moreover, in cultured HNPECs, guggulsterone inhibited LPS-induced expression of inflammatory proteins.
These results for the first time demonstrate that the plant sterol guggulsterone suppresses ocular inflammation in EIU, suggesting that the supplementation of guggulsterone could be a novel approach for the treatment of ocular inflammation.
Aldose reductase (AR), that catalyzes the rate limiting step of the polyol pathway of glucose metabolism, besides reducing glucose to sorbitol, reduces a number of lipid peroxidation –derived aldehydes and their glutathione conjugates. Recent studies suggest that apart from its involvement in diabetic complications, AR’s catalytic activity plays a key role in a number of inflammatory diseases such as atherosclerosis, sepsis, asthma, uveitis, and colon cancer. Furthermore, AR is overexpressed in human cancers such as liver, colon, breast, cervical and ovarian. Since AR inhibitors have already undergone up to phase-iii clinical trials for diabetic complications, they could be safe anti-inflammatory drugs. Therefore the future use of AR inhibitors in down-regulating major inflammatory pathologies such as cancer and cardiovascular diseases could relieve some of the major health concerns of worldwide.
Aldose reductase; inflammation; oxidative stress; NF-kB; aldehydes