withaferin-a and Inflammation

The discovery of inflammasomes has enriched our knowledge in the pathogenesis of multiple inflammatory diseases. The NLR pyrin domain-containing protein 3 (NLRP3) has emerged as the most versatile and well-characterized inflammasome, consisting of an intracellular multi-protein complex that acts as a central driver of inflammation. Its activation depends on a tightly regulated two-step process, which includes a wide variety of unrelated stimuli. It is therefore not surprising that the specific regulatory mechanisms of NLRP3 inflammasome activation remain unclear. Inflammasome-mediated inflammation has become increasingly important in acute pancreatitis, an inflammatory disorder of the pancreas that is one of the fatal diseases of the gastrointestinal tract. This review presents an update on the progress of research into the contribution of the NLRP3 inflammasome to acute pancreatic injury, examining the mechanisms of NLRP3 activation by multiple signaling events, the downstream interleukin 1 family of cytokines involved and the current state of the literature on NLRP3 inflammasome-specific inhibitors.

Topics: Animals; Anti-Inflammatory Agents; Gene Expression Regulation; Glyburide; Humans; Indomethacin; Inflammasomes; Inflammation; Interleukin-18; Interleukin-1beta; Myeloid Differentiation Factor 88; NLR Family, Pyrin Domain-Containing 3 Protein; Pancreas; Pancreatitis; Signal Transduction; Toll-Like Receptor 3; Withanolides

Withania somnifera, also called 'Indian ginseng', is an important medicinal plant of the Indian subcontinent. It is widely used, singly or in combination, with other herbs against many ailments in Indian Systems of Medicine since time immemorial. Withania somnifera contains a spectrum of diverse phytochemicals enabling it to have a broad range of biological implications. In preclinical studies, it has shown anti-microbial, anti-inflammatory, anti-tumor, anti-stress, neuroprotective, cardioprotective, and anti-diabetic properties. Additionally, it has demonstrated the ability to reduce reactive oxygen species, modulate mitochondrial function, regulate apoptosis, and reduce inflammation and enhance endothelial function. In view of these pharmacologic properties, W. somnifera is a potential drug candidate to treat various clinical conditions, particularly related to the nervous system. In this review, we summarize the pharmacologic characteristics and discuss the mechanisms of action and potential therapeutic applications of the plant and its active constituents.

Topics: Alzheimer Disease; Animals; Anti-Infective Agents; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents, Phytogenic; Cardiotonic Agents; Humans; India; Inflammation; Medicine, Ayurvedic; Neuroprotective Agents; Parkinson Disease; Plants, Medicinal; Withania; Withanolides

Infections due to multidrug-resistant Pseudomonas aeruginosa are prevalent among patients with cystic fibrosis. The emergence of antibiotic-resistant pathogens necessitated the development of novel low-risk natural antibacterial compounds. Herbal medicines are used from dates of the origin of mankind and still serve their purpose as therapeutic agents. We demonstrated the antibacterial activity of Withaferin A extracted from the traditional herb, ashwagandha or winter cherry (Withania somnifera). Withaferin A exhibits strong antibacterial activity against P. aeruginosa with a minimum inhibitory concentration of 60 μM and minimum bactericidal concentration of 80 μM. Results obtained from membrane stabilization assay and electron microscopic analysis showed that Withaferin A acts by damaging the cell membrane of P. aeruginosa. Additionally, we investigated oxidative stress and inflammatory response after Withaferin A treatment in P. aeruginosa infected zebrafish larvae model. The results indicate that the level of ROS, and its related lipid peroxidation and apoptosis were significantly reduced after treated with Withaferin A. Consequently, an increment in antioxidant enzymes level such as superoxide dismutase (SOD) and catalase (CAT) was observed. Macrophage localization experiment showed a smaller number of localized macrophages in zebrafish, which indicates the reduction in inflammatory response. In conclusion, Withaferin A could serve as an alternative natural product in the treatment of infections caused by P. aeruginosa.

Topics: Animals; Anti-Bacterial Agents; Antioxidants; Biological Products; Catalase; Inflammation; Larva; Pseudomonas aeruginosa; Reactive Oxygen Species; Superoxide Dismutase; Withania; Zebrafish

The current aim is to evaluate the effect of ashwagandha root extract (AE) on the neurochemical changes induced in the cortex and hippocampus as a consequence of thyroid dysfunction induced by propylthiouracil (PTU).

Topics: Animals; Cerebral Cortex; Disease Models, Animal; Hypothyroidism; Inflammation; Male; Neuroprotective Agents; Oxidative Stress; Plant Extracts; Plant Roots; Rats; Rats, Wistar

Systemic inflammation driven neuroinflammation is an event which correlates with pathogenesis of several neurodegenerative diseases. Therefore, targeting peripheral and central inflammation simultaneously could be a promising approach for the management of these diseases. Nowadays, herbal medicines are emerging as potent therapeutics against various brain pathologies. Therefore, in this contemporary study, the neuroprotective activity of Ashwagandha (Withania somnifera) was elucidated against the inflammation associated neurodegeneration and cognitive impairments induced by systemic LPS administration using in vivo rat model system.. To achieve this aim, young adult wistar strain male albino rats were randomized into four groups: (i) Control, (ii) LPS alone, (iii) LPS + ASH-WEX, (iv) ASH-WEX alone. Post regimen, the animals were subjected to Rotarod, Narrow Beam Walking and Novel Object Recognition test to analyze their neuromuscular coordination, working memory and learning functions. The rats were then sacrificed to isolate the brain regions and expression of proteins associated with synaptic plasticity and cell survival was studied using Western blotting and Quantitative real time PCR. Further, neuroprotective potential of ASH-WEX and its active fraction (FIV) against inflammatory neurodegeneration was studied and validated using in vitro model system of microglial conditioned medium-treated neuronal cultures and microglial-neuronal co-cultures.. Orally administered ASH-WEX significantly suppressed the cognitive and motor-coordination impairments in rats. On the molecular basis, ASH-WEX supplementation also regulated the expression of various proteins involved in synaptic plasticity and neuronal cell survival. Since microglial-neuronal crosstalk is crucial for maintaining CNS homeostasis, the current study was further extended to ascertain whether LPS-mediated microglial activation caused damage to neurons via direct cell to cell contact or through secretion of inflammatory mediators. ASH-WEX and FIV pretreatment was found to restore neurite outgrowth and protect neurons from apoptotic cell death caused by LPS-induced neuroinflammation in both activated microglial conditioned medium-treated neuronal cultures as well as microglial-neuronal co-cultures.. This extensive study using in vivo and in vitro model systems provides first ever pre-clinical evidence that ASH-WEX can be used as a promising natural therapeutic remedial for the prevention of neurodegeneration and cognitive impairments associated with peripheral inflammation and neuroinflammation.

Topics: Animals; Apoptosis; Cells, Cultured; Cognition; Cognitive Dysfunction; Inflammation; Male; Microglia; Neurons; Neuroprotective Agents; Plant Extracts; Rats; Rats, Wistar

Withaferin A (WA), a bioactive constituent derived from Withania somnifera plant, has been shown to exhibit many qualifying properties in attenuating several metabolic diseases. The current investigation sought to elucidate the protective mechanisms of WA (1.25 mg/kg/day) on pre-existing obese mice mediated by high-fat diet (HFD) for 12 weeks. Following dietary administration of WA, significant metabolic improvements in hepatic insulin sensitivity, adipocytokines with enhanced glucose tolerance were observed. The hepatic oxidative functions of obese mice treated with WA were improved via augmented antioxidant enzyme activities. The levels of serum pro-inflammatory cytokines and hepatic mRNA expressions of toll-like receptor (TLR4), nuclear factor κB (NF-κB), tumor necrosis factor-α (TNF-α), chemokine (C-C motif) ligand-receptor, and cyclooxygenase 2 (COX2) in HFD-induced obese mice were reduced. Mechanistically, WA increased hepatic mRNA expression of peroxisome proliferator-activated receptors (PPARs), cluster of differentiation 36 (CD36), fatty acid synthase (FAS), carnitine palmitoyltransferase 1 (CPT1), glucokinase (GCK), phosphofructokinase (PFK), and phosphoenolpyruvate carboxykinase (PCK1) that were associated with enhanced lipid and glucose metabolism. Taken together, these results indicate that WA exhibits protective effects against HFD-induced obesity through attenuation of hepatic inflammation, oxidative stress, and insulin resistance in mice.

Topics: Animals; Blood Glucose; Body Weight; Cytokines; Diet, High-Fat; Enzyme-Linked Immunosorbent Assay; Fatty Liver; Feeding Behavior; Inflammation; Inflammation Mediators; Insulin Resistance; Lipid Metabolism; Male; Mice; Mice, Inbred C57BL; Obesity; Oxidative Stress; RNA, Messenger; Withanolides

Topics: Animals; Cell Line; Collagen; Cytokines; Gene Expression; Humans; Inflammasomes; Inflammation; Male; Mice, Inbred C57BL; NLR Family, Pyrin Domain-Containing 3 Protein; Ovalbumin; Pulmonary Fibrosis; Respiratory System; Withanolides

Chronic kidney disease (CKD) is one of the major global health concerns and is responsible for end-stage renal disease (ESRD) complications. Inflammation plays a pivotal role in the progression of CKD. In the present study, we evaluated the renoprotective effects of a potent immunomodulator steroidal lactone, Withaferin A (WfA), in an animal model of renal injury (unilateral ureteral obstruction, UUO) and further investigated if the inhibition of inflammatory signaling can be a useful approach to reduce renal injury. Animals were randomly divided into five groups: Sham control, UUO control, WfA control, WfA low dose (1 mg/kg), and WfA high dose (3 mg/kg). Oxidative stress was measured by the estimation of reduced glutathione and lipid peroxidation levels. H&E and Picrosirius Red staining were performed to assess the extent of histological damage and collagen deposition. Furthermore, the molecular mechanism of the WfA effects was explored by immunohistochemistry, enzyme-linked immunosorbent assay, multiplex analysis of transforming growth factor β (TGF-β) pathway, and an array of inflammatory cytokines/chemokines. Interestingly, our pharmacological intervention significantly attenuated tissue collagen, inflammatory signaling, and macrophage signaling. WfA intervention abrogated the inflammatory signaling as evident from the modulated levels of chemokines and cytokines. The levels of TGF-β along with downstream signaling molecules were also attenuated by WfA treatment as revealed by inhibition in the expression of TGF-β1, TGF-β2, p-Smad2, p-Smad3, total Smad4, p-Akt, and p-ERK. We, to the best of our knowledge, prove for the first time that WfA has potential renoprotective activity against UUO-induced nephropathy due to its outstanding anti-inflammatory properties.

Topics: Animals; Anti-Inflammatory Agents; Antioxidants; Disease Models, Animal; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation; Glutathione; Inflammation; Kidney; Lipid Peroxidation; Male; Mice; Oxidative Stress; Proto-Oncogene Proteins c-akt; Signal Transduction; Smad2 Protein; Smad3 Protein; Smad4 Protein; Transforming Growth Factor beta1; Transforming Growth Factor beta2; Treatment Outcome; Ureteral Obstruction; Withanolides

Despite the usefulness of glucocorticoids, they may cause hazardous side effects that limit their use. Searching for compounds that are as equally efficient as glucocorticoids, but with less side effects, the current study compared plant steroids, namely, glycyrrhetinic acid, guggulsterone, boswellic acid, withaferin A, and diosgenin with the classical glucocorticoid, fluticasone. This was approached both

Topics: Animals; Anti-Inflammatory Agents; Croton Oil; Diosgenin; Ear Diseases; Edema; Enzyme-Linked Immunosorbent Assay; Glycyrrhetinic Acid; Inflammation; Interleukin-6; Mice; Molecular Docking Simulation; Phytosterols; Pregnenediones; Rats; Receptors, Glucocorticoid; Software; Thymus Gland; Triterpenes; Tumor Necrosis Factor-alpha; Withanolides

Topics: Adenosine Triphosphate; Chemokines; Cytokines; Gene Expression Profiling; Gene Expression Regulation; Humans; Inflammasomes; Inflammation; NF-kappa B; Nigericin; Oligonucleotide Array Sequence Analysis; Plant Extracts; Promoter Regions, Genetic; Signal Transduction; THP-1 Cells; Withanolides

A paste composed of the boiled leaves and roots of the Ashwagandha plant is used to cure ulcer and swelling in Ayurvedic medicine. However, the effects of the hot water extract of Ashwagandha roots (ASH‑WEX), which is also used in Ayurveda, on skin have not been fully elucidated. Therefore, the present study investigated the anti‑inflammatory activity of ASH‑WEX on skin, by using the human keratinocyte cell line HaCaT. The results indicated that ASH‑WEX significantly inhibited mRNA expression of inflammatory cytokines, including interleukin (IL)‑8, IL‑6, tumor necrosis factor (TNF‑α), IL‑1β and IL‑12, and promoted the mRNA expression of the anti‑inflammatory cytokine transforming growth factor (TGF)‑β1 in HaCaT cells. In addition, ASH‑WEX inhibited the lipopolysaccharide‑induced phosphorylation of p38 and c‑Jun N‑terminal kinase, as well as the nuclear translocation of nuclear factor (NF)‑κB p65. Downregulation of TNF‑α mRNA and upregulation of TGF‑β1 mRNA were also observed in vivo following ASH‑WEX treatment of mouse skin. In conclusion, the present study demonstrated that the anti‑inflammatory effect of ASH‑WEX may be due to its ability to suppress the NF‑κB and mitogen‑activated protein kinase pathways, and to modulate cytokine expression. These results suggest that ASH‑WEX can potentially protect against skin inflammation.

Topics: Animals; Anti-Inflammatory Agents; Cell Line; Cytokines; Down-Regulation; Humans; Inflammation; Inflammation Mediators; Keratinocytes; Lipopolysaccharides; Male; MAP Kinase Signaling System; Mice, Inbred C57BL; NF-kappa B; Plant Extracts; Plant Roots; RNA, Messenger; Up-Regulation; Water; Wound Healing

Type 1 diabetes mellitus (T1DM) is one of the major metabolic disorders with life-long dependence on insulin. The present study was designed to evaluate the antioxidant and anti-diabetic potential of Withaferin A (WA), the active constituent of Withania somnifera in multiple low doses of Streptozotocin (MLD-STZ) induced T1DM. STZ (40 mg/Kg) was administered intraperitoneally (i.p.) for 5 consecutive days to male Swiss albino mice to induce T1DM. Mice were concurrently treated with WA (2 & 10 mg/Kg). Blood glucose levels, intraperitoneal glucose tolerance test, oxidative stress parameters were estimated biochemically (MDA, GSH) and immunohistochemically (Nrf2, NFκB). In addition, inflammatory cytokines, and insulin levels were quantified by ELISA method. Apoptosis was assessed by immunohistochemical staining for cleaved-caspase-3 and TUNEL assay. WA treatment significantly reduced the blood glucose levels and improved glucose clearance. Strikingly, we observed a significant reduction in the incidence of diabetes upon WA treatment and only 2 out of 8 (2/8 = 25%) animals were diabetic. WA ameliorated the MLD-STZ induced oxidative and nitrosative stress. Furthermore, WA exhibited promising anti-inflammatory effect as evident from reduction in the levels of IL-6 (p < 0.05) and TNF-α (p < 0.05) compared to diabetic mice. In addition, insulitis scoring and IHC for Nrf2 and NFκB indicated promising anti-diabetic effect. WA reduced MLD-STZ induced DNA fragmentation and apoptosis, further supporting the observed protective effect. We, to the best of our knowledge, report for the first time that WA can effectively combat MLD-STZ induced T1DM via modulation of Nrf2/NFκB signaling and holds substantial potential for therapy of T1DM.

Topics: Animals; Anti-Inflammatory Agents; Antioxidants; Apoptosis; Biomarkers; Blood Glucose; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Dose-Response Relationship, Drug; Hypoglycemic Agents; Inflammation; Inflammation Mediators; Insulin; Interleukin-6; Male; Mice; NF-E2-Related Factor 2; NF-kappa B; Nitrosative Stress; Oxidative Stress; Pancreas; Streptozocin; Time Factors; Tumor Necrosis Factor-alpha; Withanolides

Chemopreventive effects and associated mechanisms of withaferin A (WA) against intestinal and colon carcinogenesis remain unknown. We investigated the chemopreventive effect of WA on transgenic adenomatous polyposis coli (APCMin/+) mouse and chemically induced azoxymethane/dextran sodium sulfate (AOM/DSS) models of intestinal and colon carcinogenesis. Oral WA administration (4 and 3 mg/kg) inhibited tumor initiation and progression of intestinal polyps formation in APCMin/+ mice and colon carcinogenesis in the AOM/DSS mouse model. WA-administered mice showed a significant reduction in both number [duodenum, 33% (P > 0.05); jejunum, 32% (P < 0.025); ileum, 43% ( P < 0.001); and colon 59% (P < 0.01] and size of polyps in APCMin/+ mice compared with the respective controls. Similarly, tumor multiplicity was significantly reduced (P < 0.05) in the colon of WA-administered AOM/DSS mice. Pathological analysis showed reduced adenomas and tissue inflammation in WA-administered mouse models. Molecular studies suggested that WA inhibited the expression of inflammatory (interluekin-6, tumor necrosis factor-alpha and cyclooxygenase-2), pro-survival (pAKT, Notch1 and NF-κB) markers in APCMin/+ and AOM/DSS models. The results suggest that WA is a potent agent for preventing colon carcinogenesis and further investigation is required to show clinical utility of the agent.

Topics: Animals; Anticarcinogenic Agents; Carcinogenesis; Chemoprevention; Colon; Colonic Neoplasms; Disease Models, Animal; Female; Inflammation; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Withanolides

Acute pancreatitis is an inflammatory disorder of the pancreas that may precipitate due to various reasons such as chronic alcoholism, gall stone obstruction, and life style. Current treatment options offer limited efficacy, as they provide only symptomatic relief. This study is an attempt to study the effects of Withaferin A (WFA) against Cerulein-induced acute pancreatitis in mice. Animals were pretreated with WFA via intraperitoneal route, for 7 days. Plasma amylase and lipase, tissue malondialdehyde (MDA), and glutathione were evaluated for all groups. Western blot analysis; haematoxylin and eosin staining of the liver, lung, and pancreas; immunohistochemistry for nitrotyrosine; and myeloperoxidase activity were performed. Haematoxylin and eosin stained sections significantly revealed the altered architecture and thereby damage in the pancreas, lungs, and liver that has been low in treatment groups. Increased myeloperoxidase and nitrotyrosine have also been reduced upon treatment with WFA. Increased levels of MDA, NO, and expression of myeloperoxidase and nitrotyrosine in the parameters estimated add evidence to the role of oxidative stress and inflammation in acute pancreatitis. WFA evidently altered these conditions upon pretreatment. Our study shows that this novel steroidal compound has potent anti-inflammatory property. Natural compounds can therefore be good remedies against many diseases if incorporated in routine diet as dietary supplement.

Topics: Acute Disease; Animals; Ceruletide; Inflammation; Male; Mice; Oxidative Stress; Panax; Pancreatitis; Withania; Withanolides

The demand for the chemopreventive drug from the plant source is increasing in recent times, owing to its various biological activities without any adverse effect. The intention of this current study was to examine the anti-glioma effect of Withaferin A (WFA) on C6 glioma cell line model.. C6 glioma cells were administrated with different concentration of WFA (50, 100, 200 and 500 μg/mL) and DMSO (control) group to examine its anti-proliferative, anti-inflammatory and pro-apoptotic activities.. Treatment with WFA showed a significant decline in the glioma cell count in a dose-dependent manner and thus proving its anti-proliferative effect. Similarly, inflammatory markers were also substantially lowered upon treatment with different concentration of WFA. However, DNA fragmentation and apoptotic markers like Caspase-3 and 9 were concomitantly enhanced after co-cultured with different concentration of WFA and thus exhibiting its cytotoxicity efficacy. Furthermore, the protein expression of Bcl2 and Bax were markedly downregulated and upregulated respectively; upon treatment with WFA on C6 glioma cells.. The outcome of this study evidently demonstrates that C6 glioma cells co-cultured with increased concentration of WFA, showed an anti-proliferative, anti-inflammatory and pro-apoptotic effect in a dose-dependent fashion.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Biological Transport; Caspases; Cell Line, Tumor; Cell Proliferation; DNA Fragmentation; Enzyme Activation; Glioma; Inflammation; NF-kappa B; Phytotherapy; Plant Extracts; Proto-Oncogene Proteins c-bcl-2; Rats; Withania; Withanolides

Withaferin A (WFA) exhibits diverse pharmaceutical applications on human diseases, including rheumatoid arthritis, cancers and microbial infection.. We evaluated the neuroprotective role of WFA using a mouse model of spinal cord injury (SCI).. BALB/c mice were administrated 10 mg/kg of WFA. Gene expression was measured by real-time PCR, western blot and immunohistochemistry. Cell morphology and apoptosis were determined by H&E staining and TUNEL assay. Motor function was evaluated by the BBB functional scale for continuous 7 weeks.. WFA significantly improved neurobehavioural function and alleviated histological alteration of spinal cord tissues in traumatized mice. Brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) significantly increased in WFA-treated mice. Meanwhile, the expression of Nogo-A and RhoA remarkably decreased in the presence of WFA. Furthermore, the apoptotic cell death was attenuated in mice treated with WFA (31.48 ± 2.50% vs. 50.08 ± 2.08%) accompanied by decreased bax and increased bcl-2. In addition, WFA decreased the expression of pro-inflammatory mediators such as IL-1β (11.20 ± 1.96 ng/mL vs. 17.59 ± 1.42 ng/mL) and TNF-α (57.38 ± 3.57 pg/mL vs. 95.06 ± 9.13 pg/mL). The anti-inflammatory cytokines including TGF-β1 (14.32 ± 1.04 pg/mL vs. 9.37 ± 1.17 pg/mL) and IL-10 (116.80 ± 6.91 pg/mL vs. 72.33 ± 9.35 pg/mL) were elevated after WFA administration.. This study demonstrated that WFA has a neuroprotective role by inhibition of apoptosis and inflammation after SCI in mice.

Topics: Animals; Anti-Inflammatory Agents; Apoptosis; Apoptosis Regulatory Proteins; Behavior, Animal; Brain-Derived Neurotrophic Factor; Disease Models, Animal; Glial Cell Line-Derived Neurotrophic Factor; Inflammation; Inflammation Mediators; Mice, Inbred BALB C; Motor Activity; Neuroprotective Agents; Nogo Proteins; rho GTP-Binding Proteins; rhoA GTP-Binding Protein; Signal Transduction; Spinal Cord; Spinal Cord Injuries; Time Factors; Withanolides

Withaferin A (WA), a withanolide purified from Withania somnifera, has been known to exert anti-inflammatory effects. The present study sought to determine the effects of WA on Helicobacter (H.) pylori-mediated inflammation in the AGS gastric epithelial cell line. Cellular production of interleukin (IL)-8 and vascular endothelial growth factor (VEGF) was measured by ELISA. Western blot analysis was performed to determine the activation of nuclear factor (NF)-κB and mitogen-activated protein kinases (MAPKs) as well as hypoxia-inducible factor 1α stabilization. Bacterial growth was also examined by measuring the optical density. Pre-treatment or co-treatment with WA efficiently reduced IL-8 production by AGS cells in response to H. pylori infection. H. pylori-induced activation of NF-κB, but not MAPKs, was also inhibited by pre-treatment of WA in the cells. However, WA did not affect VEGF production and HIF-1α stabilization induced by H. pylori in AGS cells. In addition, WA did not influence the growth of H. pylori, suggesting that the anti-inflammatory effect of WA was not due to any bactericidal effect. These findings indicate that WA is a potential preventive or therapeutic agent for H. pylori-mediated gastric inflammation.

Topics: Epithelial Cells; Gastric Mucosa; Gene Expression Regulation; Helicobacter Infections; Helicobacter pylori; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Inflammation; Interleukin-8; Mitogen-Activated Protein Kinases; NF-kappa B; Vascular Endothelial Growth Factor A; Withanolides

Bromobenzene is a well-known environmental toxin which causes liver and kidney damage through CYP450-mediated bio-activation to generate reactive metabolites and, consequently, oxidative stress. The present study aimed to evaluate the possible protective role of withaferin A against bromobenzene-induced liver and kidney damage in mice. Withaferin A (10 mg/kg) was administered orally to the mice for 8 days before intragastric intubation of bromobenzene (10 mmol/kg). As results of this experiment, the levels of liver and kidney functional markers, lipid peroxidation, and cytokines (TNF-α and IL-1β) presented an increase and there was a decrease in anti-oxidant activity in the bromobenzene-treated group of mice. Pre-treatment with withaferin A not only significantly decreased the levels of liver and kidney functional markers and cytokines but also reduced oxidative stress, as evidenced by improved anti-oxidant status. In addition, the mitochondrial dysfunction shown through the decrease in the activities of mitochondrial enzymes and imbalance in the Bax/Bcl-2 expression in the livers and kidneys of bromobenzene-treated mice was effectively prevented by pre-administration of withaferin A. These results validated our conviction that bromobenzene caused liver and kidney damage via mitochondrial pathway and withaferin A provided significant protection against it. Thus, withaferin A may have possible usage in clinical liver and kidney diseases in which oxidative stress and mitochondrial dysfunction may be existent.

Topics: Animals; Antioxidants; Bromobenzenes; Chemical and Drug Induced Liver Injury; Female; Inflammation; Interleukin-1beta; Kidney; Kidney Diseases; Lipid Peroxidation; Liver; Male; Mice; Mitochondria; Oxidants; Oxidative Stress; Random Allocation; Tumor Necrosis Factor-alpha; Withanolides

Rheumatoid arthritis is an inflammatory autoimmune disorder. Withania somnifera Dunal (Solanaceae) (WS), is a common medicinal plant used in traditional systems of medicine for the treatment of arthritis, and is an ingredient of anti-arthritic polyherbal formulations such as Habb-e-Asgand® and Arthritin™. In the present study, we evaluated the antioxidant and anti-arthritic activity of aqueous extract of WS root (WSAq) in collagen-induced arthritic (CIA) rats.. CIA rats were treated by using three doses of WSAq (100, 200, 300 mg/kg b. wt., orally) and methotrexate (MTX, 0.25 mg/kg b. wt. i.p.) as a standard reference drug for 20 days. The anti-arthritic effect was assayed by measuring the arthritic index, autoantibodies such as rheumatoid factor (RF), anti-cyclic citrullinated peptide antibody (a-CCP), anti-nuclear antibody (ANA), anti-collagen type II antibody (a-CII) and inflammatory marker like C-reactive protein (CRP). The oxidative stress parameters were also measured.. Treatment with WSAq resulted in a dose-dependent reduction in arthritic index, autoantibodies and CRP (p < 0.05) with maximum effect at dose of 300 mg/kg b. wt. and the results were comparable to that of MTX-treated rats. Similarly, oxidative stress in CIA rats was ameliorated by treatment with different doses of WSAq, as evidenced by a decrease in lipid peroxidation and glutathione-S-transferase activity and an increase in the glutathione content and ferric-reducing ability of plasma (p < 0.05).. The results showed that WSAq exhibited antioxidant and anti-arthritic activity and reduced inflammation in CIA rats and suggests the potential use of this plant in the treatment of arthritis.

Topics: Animals; Antioxidants; Arthritis, Rheumatoid; Autoantibodies; C-Reactive Protein; Collagen; Cytokines; Inflammation; Lipid Peroxidation; Male; Methotrexate; Oxidative Stress; Phytotherapy; Plant Extracts; Plant Roots; Rats, Wistar; Withania

The instant blood-mediated inflammatory response (IBMIR) has been shown as a major factor that causes damage to transplanted islets. Withaferin A (WA), an inhibitor of nuclear factor (NF) κB, was shown to suppress the inflammatory response in islets and improve syngeneic islet graft survival in mice. We investigated how treating islets with NF-κB inhibitors affected IBMIR using an in vitro human autologous blood islet model.. Human islets were pretreated with or without NF-κB inhibitors WA or CAY10512 before mixing autologous blood in a miniaturized in vitro tube model. Plasma samples were collected at multiple time points and used for the measurement of C-peptide, proinsulin, thrombin-antithrombin (TAT) complex, and a panel of proinflammatory cytokines. Infiltration of neutrophils into islets was analyzed using immunohistochemistry.. Rapid release of C-peptide and proinsulin was observed 3 hr after mixing islets and blood in the control group, but not in the NF-κB inhibitor-treated groups, whereas TAT levels were elevated in all three groups with a peak at 6 hr. Significant elevation of proinflammatory cytokines was observed in the control group after 3 hr, but not in the treatment groups. Significant inhibition of neutrophil infiltration was also observed in the WA group compared with the control (P<0.001) and CAY10512 (P<0.001) groups.. A miniaturized in vitro tube model can be useful in investigating IBMIR. The presence of NF-κB inhibitor could alleviate IBMIR, thus improving the survival of transplanted islets. Protection of islets in the peritransplant phase may improve long-term graft outcomes.

Topics: Anti-Inflammatory Agents; Biomarkers; Cytokines; Humans; Immunohistochemistry; In Vitro Techniques; Inflammation; Islets of Langerhans; Islets of Langerhans Transplantation; Neutrophil Infiltration; NF-kappa B; Postoperative Complications; Transplantation, Autologous; Withanolides

Withaferin A (WFA) is a major chemical constituent of Withania somnifera, also known as Indian ginseng. Many recent reports have provided evidence of its anti-tumor, anti-inflammation, anti-oxidant, and immune modulatory activities. Although the compound appears to have a large number of effects, its defined mechanisms of action have not yet been determined. We investigated the effects of WFA on loss of type collagen expression and inflammation in rabbit articular chondrocytes. WFA increased the production of reactive oxygen species, suggesting the induction of oxidative stress, in a dose-dependent manner. Also, we confirmed that WFA causes loss of type collagen expression and inflammation as determined by a decrease of type II collagen expression and an increase of cyclooxygenase-2 (COX-2) expression via western blot analysis in a dose- and time- dependent manner. WFA also reduced the synthesis of sulfated proteoglycan via Alcian blue staining and caused the synthesis of prostaglandin E2 (PGE2) via assay kit in dose- and time-dependent manners. Treatment with N-acetyl-L-cysteine (NAC), an antioxidant, inhibited WFA-induced loss of type II collagen expression and increase in COX-2 expression, accompanied by inhibition of reactive oxygen species production. WFA increased phosphorylation of both Akt and p38. Inhibition of PI3K/Akt, p38, and JNK with LY294002 (LY), SB203580 (SB), or SP600125 (SP) in WFA-treated cells rescued the expression of type II collagen and suppressed the expression of COX-2. These results demonstrate that WFA induces loss of type collagen expression and inflammation via PI3K/Akt, p38, and JNK by generating reactive oxygen species in rabbit articular chondrocytes.

Topics: Animals; Blotting, Western; Cells, Cultured; Chondrocytes; Collagen; Cyclooxygenase 2; Enzymes; Gene Expression Regulation; Immunohistochemistry; Inflammation; MAP Kinase Signaling System; Phosphatidylinositol 3-Kinases; Plant Extracts; Rabbits; Reactive Oxygen Species; Real-Time Polymerase Chain Reaction; Withanolides

Beta cell death triggered by pro-inflammatory cytokines plays a central role in the pathogenesis of type 1 diabetes and loss of transplanted islets. The nuclear factor κB (NF-κB) signalling pathway is a key regulator of beta cell stress response, survival and apoptosis. Withaferin A (WA), a steroidal lactone derived from Withania somnifera, has been demonstrated to be a potent, safe, anti-inflammatory molecule that can inhibit NF-κB signalling. Therefore, we evaluated the ability of WA to protect mouse and human islets from the damaging effects of pro-inflammatory cytokines in vitro and following intraportal transplantation.. Mouse and human islets were treated with a cytokine cocktail, and NF-κB activation was measured by immunoblots, p65 nuclear translocation and chromatin immunoprecipitation of p65-bound DNA. Intraportal transplantation of a marginal mass of syngeneic mouse islets was performed to evaluate the in vivo protective effect of WA.. Treatment with WA substantially improved islet engraftment of syngeneic islets (83% for infusion with 200 islets + WA; 0% for 200 islets + vehicle) in a mouse model of diabetes, compared with marginal graft controls with superior islet function in WA-treated mice confirmed by glucose tolerance test. Treatment of human and mouse islets with WA prevented cytokine-induced cell death, inhibited inflammatory cytokine secretion and protected islet potency.. WA was shown to be a strong inhibitor of the inflammatory response in islets, protecting against cytokine-induced cell damage while improving survival of transplanted islets. These results suggest that WA could be incorporated as an adjunctive treatment to improve islet transplant outcome.

Topics: Active Transport, Cell Nucleus; Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Cell Culture Techniques; Chromatin Immunoprecipitation; Cytokines; Glucose Tolerance Test; Humans; Inflammation; Islets of Langerhans Transplantation; Mice; NF-kappa B; Withanolides

Withaferin A (WFA), an active compound from Withania somnifera, is widely researched for its anti-inflammatory, cardioactive and central nervous system effects. In this study, we first investigated the possible barrier protective effects of WFA against pro-inflammatory responses in human umbilical vein endothelial cells (HUVECs) and in mice induced by high mobility group box 1 protein (HMGB1) and the associated signaling pathways. The barrier protective activities of WFA were determined by measuring permeability, leukocytes adhesion and migration, and activation of pro-inflammatory proteins in HMGB1-activated HUVECs. We found that WFA inhibited lipopolysaccharide (LPS)-induced HMGB1 release and HMGB1-mediated barrier disruption, expression of cell adhesion molecules (CAMs) and adhesion/transendothelial migration of leukocytes to human endothelial cells. WFA also suppressed acetic acid-induced hyperpermeability and carboxymethylcellulose-induced leukocytes migration in vivo. Further studies revealed that WFA suppressed the production of interleukin 6, tumor necrosis factor-α (TNF-α) and activation of nuclear factor-κB (NF-κB) by HMGB1. Collectively, these results suggest that WFA protects vascular barrier integrity by inhibiting hyperpermeability, expression of CAMs, adhesion and migration of leukocytes, thereby endorsing its usefulness as a therapy for vascular inflammatory diseases.

Topics: Animals; Anti-Inflammatory Agents; Capillary Permeability; Cell Adhesion; Cell Adhesion Molecules; Cell Movement; Disease Models, Animal; Female; HMGB1 Protein; Human Umbilical Vein Endothelial Cells; Humans; Inflammation; Leukocytes; Lipopolysaccharides; Mice; Mice, Inbred ICR; Withanolides

Microglia are the major immune effector cells in the brain, and microglia activated by injury and infection can produce inflammatory mediators. A number of studies have reported that withaferin A has anti-inflammatory functions. However, the effects of withaferin A on the microglial inflammatory response have not been investigated. Our results show that withaferin A inhibited lipopolysaccharide (LPS)-induced cyclooxygenase (COX)-2 mRNA and protein expression and prostaglandin E2 (PGE(2)) production in BV2 murine microglial cells. Withaferin A had no effect on LPS-induced Akt and ERK phosphorylation, but phosphorylation of p38 and JNK was slightly decreased by withaferin A. Withaferin A significantly inhibited LPS-induced STAT1 and STAT3 phosphorylation in a dose-dependent manner. Furthermore, withaferin A inhibited nuclear translocation of STAT1 and interferon-gamma activated sequence (GAS)-promoter activity. Taken together, these results suggest that withaferin A inhibits LPS-induced PGE(2) production and COX-2 expression, at least in part, by blocking STAT1 and STAT3 activation.

Topics: Animals; Anti-Inflammatory Agents; Cells, Cultured; Cyclooxygenase 2; Dinoprostone; Down-Regulation; Extracellular Signal-Regulated MAP Kinases; Inflammation; Interferon-gamma; Lipopolysaccharides; MAP Kinase Kinase 4; Microglia; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Promoter Regions, Genetic; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; STAT1 Transcription Factor; STAT3 Transcription Factor; Withanolides

Induction of inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production is thought to have beneficial immunomodulatory effects in acute and chronic inflammatory disorders. In Raw 264.7 cells stimulated with lipopolysaccharide (LPS) to mimic inflammation, withaferin A inhibited LPS-induced expression of both iNOS protein and mRNA in a dose-dependent manner. To investigate the mechanism by which withaferin A inhibits iNOS gene expression, we examined activation of mitogen-activated protein kinases (MAPKs) and Akt in Raw 264.7 cells. We did not observe any significant changes in the phosphorylation of p38 MAPK in cells treated with LPS alone or LPS plus withaferin A. However, LPS-induced Akt phosphorylation was markedly inhibited by withaferin A, while the phosphorylation of p42/p44 extracellular signal-regulated kinases (ERKs) was slightly inhibited by withaferin A treatment. Withaferin A prevented IkappaB phosphorylation, blocking the subsequent nuclear translocation of nuclear factor-kappaB (NF-kappaB) and inhibiting its DNA binding activity. LPS-induced p65 phosphorylation, which is mediated by extracellular signal-regulated kinase (ERK) and Akt pathways, was attenuated by withaferin A treatment. Moreover, LPS-induced NO production and NF-kappaB activation were inhibited by SH-6, a specific inhibitor of Akt. Taken together, these results suggest that withaferin A inhibits inflammation through inhibition of NO production and iNOS expression, at least in part, by blocking Akt and subsequently down-regulating NF-kappaB activity.

Topics: Animals; Cell Line; Dose-Response Relationship, Drug; Down-Regulation; Ergosterol; Extracellular Signal-Regulated MAP Kinases; Inflammation; Lipopolysaccharides; Macrophages; Mice; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase Type II; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; RNA, Messenger; Transcription Factor RelA; Withanolides

Gouty arthritis is a characteristically intense acute inflammatory reaction resulting from the formation of sodium urate crystals in the joint cavity. In the present study, the effect of withaferin A, a steroidal lactone was investigated on monosodium urate crystal-induced inflammation in mice; an experimental model for gouty arthritis and compared it with that of the non-steroidal anti-inflammatory drug, indomethacin.. Paw volume and levels/activities of lysosomal enzymes, lipid peroxidation, anti-oxidant status and inflammatory mediator TNF-alpha were determined in control and monosodium urate crystal-induced mice. The levels of beta-glucuronidase and lactate dehydrogenase were also measured in monosodium urate crystal-incubated polymorphonuclear leucocytes (PMNL).. Paw volume, the levels of lysosomal enzymes, lipid peroxidation, and inflammatory mediator tumour necrosis factor-alpha were found to be increased significantly and the activities of antioxidant status were in turn decreased in monosodium urate crystal-induced mice; however these changes were reverted back to near normal levels in withaferin A (30 mg/kg/b.wt, i.p.) treated monosodium urate crystal-induced mice. In addition, beta-glucuronidase and lactate dehydrogenase level were reduced in withaferin A (100microg/ml) treated monosodium urate crystal-incubated polymorphonuclear leucocytes.. The present findings clearly indicated that withaferin A exerted a strong anti-inflammatory effect against gouty arthritis.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antioxidants; Disease Models, Animal; Ergosterol; Female; Indomethacin; Inflammation; Interferon-alpha; Lipid Peroxidation; Lysosomes; Male; Mice; Neutrophils; Uric Acid; Withanolides

The transcription factor NFkappaB plays a critical role in normal and pathophysiological immune responses. Therefore, NFkappaB and the signaling pathways that regulate its activation have become a major focus of drug development programs. Withania somnifera (WS) is a medicinal plant that is widely used in Palestine for the treatment of various inflammatory disorders. In this study we show that the leave extract of WS, as well as its major constituent withaferin A (WA), potently inhibits NFkappaB activation by preventing the tumor necrosis factor-induced activation of IkappaB kinase beta via a thioalkylation-sensitive redox mechanism, whereas other WS-derived steroidal lactones, such as withanolide A and 12-deoxywithastramonolide, are far less effective. To our knowledge, this is the first communication of IkappaB kinase beta inhibition by a plant-derived inhibitor, coinciding with MEK1/ERK-dependent Ser-181 hyperphosphorylation. This prevents IkappaB phosphorylation and degradation, which subsequently blocks NFkappaB translocation, NFkappaB/DNA binding, and gene transcription. Taken together, our results indicate that pure WA or WA-enriched WS extracts can be considered as a novel class of NFkappaB inhibitors, which hold promise as novel anti-inflammatory agents for treatment of various inflammatory disorders and/or cancer.

Topics: Animals; Anti-Inflammatory Agents; Cell Line, Tumor; Ergosterol; Humans; I-kappa B Kinase; Inflammation; MAP Kinase Kinase 1; Mice; NF-kappa B; Oxidation-Reduction; Phosphorylation; Plant Extracts; Plant Leaves; Protein Binding; Protein Kinase Inhibitors; Protein Processing, Post-Translational; Protein Transport; Transcription, Genetic; Tumor Necrosis Factor-alpha; Withania; Withanolides

The immunomodulatory activities of an Indian Ayurvedic medicinal preparation, i.e. extracts from Ashwagandha, Withania somnifera (L.) Dunal (Solanaceae), namely WST and WS2, were studied in mice for immune inflammation: active paw anaphylaxis and delayed type hypersensitivity (DTH). Immunomodulatory effect was assessed in If IgE-mediated anaphylaxis as reduction of ovalbumin-induced paw edema, in animals treated with WS2 at doses of 150 and 300 mg/kg, and the results were compared with the standard drug disodium chromoglycate. In the DTH model, the modulatory effect was assessed as potentiation or suppression of the reaction, revealing an increase or decrease in mean foot pad thickness, respectively. Potentiation of the DTH reaction was observed in animals treated with cyclophosphamide at a dose of 20 mg/kg, WST at a dose of 1000 mg/kg and WS2 at a dose of 300 mg/kg. On the other hand, cyclophosphamide-induced potentiation of DTH reaction was suppressed in animals treated with WST and WS2. A significant increase in white blood cell counts and platelet counts was observed in animals treated with WST. A protective effect in cyclophosphamide-induced myelosuppression was observed in animals treated with WST and WS2, revealing a significant increase in white blood cell counts and platelet counts. Cyclophosphamide-induced immunosuppression was counteracted by treatment with WS2, revealing significant increase in hemagglutinating antibody responses and hemolytic antibody responses towards sheep red blood cells.

Topics: Adjuvants, Immunologic; Animals; Anti-Inflammatory Agents, Non-Steroidal; Cyclophosphamide; Disease Models, Animal; Dose-Response Relationship, Drug; Hindlimb; Hypersensitivity, Delayed; Immunosuppressive Agents; India; Inflammation; Medicine, Ayurvedic; Mice; Mice, Inbred Strains; Passive Cutaneous Anaphylaxis; Plant Extracts; Withania