Isoproterenol-induced cardiac ischemia and fibrosis: Plant-based approaches for intervention
Allawadhi P, Khurana A, Sayed N, Kumari P and Godugu C
Heart is the most active and incumbent organ of the body, which maintains blood flow, but due to various pathological reasons, several acute and chronic cardiac complications arise out of which myocardial infarction is one of the teething problems. Isoproterenol (ISP)-induced myocardial ischemia is a classical model to screen the cardioprotective effects of various pharmacological interventions. Phytochemicals present a novel option for treating various human maladies including those of the heart. A large number of plant products and their active ingredients have been screened for efficacy in ameliorating ISP-induced myocardial ischemia including coriander, curcumin, Momordica, quercetin, and Withania somnifera. These phytochemicals constituents may play key role in preventing disease and help in cardiac remodeling. Reactive oxygen species scavenging, antiinflammatory, and modulation of various molecular pathways such as Nrf2, NFкB, p-21 activated kinase 1 (PAK1), and p-smad2/3 signaling modulation have been implicated behind the claimed protection. In this review, we have provided a focused overview on the utility of ISP-induced cardiotoxicity, myocardial ischemia, and cardiac fibrosis for preclinical research. In addition, we have also surveyed molecular mechanism of various plant-based interventions screened for cardioprotective effect in ISP-induced cardiotoxicity, and their probable mechanistic profile is summarized.
Withaferin-A attenuates multiple low doses of Streptozotocin (MLD-STZ) induced type 1 diabetes
Tekula S, Khurana A, Anchi P and Godugu C
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.
Withania somnifera Extract Protects Model Neurons from In Vitro Traumatic Injury
Saykally JN, Hatic H, Keeley KL, Jain SC, Ravindranath V and Citron BA
Withania somnifera has been used in traditional medicine for a variety of neural disorders. Recently, chronic neurodegenerative conditions have been shown to benefit from treatment with this extract. To evaluate the action of this extract on traumatically injured neurons, the efficacy of W. somnifera root extract as a neuroprotective agent was examined in cultured model neurons exposed to an in vitro injury system designed to mimic mild traumatic brain injury (TBI). Neuronal health was evaluated by staining with annexin V (an early, apoptotic feature) and monitoring released lactate dehydrogenase activity (a terminal cell loss parameter). Potential mechanisms underlying the observed neuroprotection were examined. Additionally, morphological changes were monitored following injury and treatment. Although no differences were found in the expression of the antioxidant transcription factor nuclear factor erythroid 2-like 2 (Nrf2) or other Nrf2-related downstream components, significant changes were seen in apoptotic signaling. Treatment with the extract resulted in an increased length of neurites projecting from the neuronal cell body after injury. W. somnifera extract treatment also resulted in reduced cell death in the model neuron TBI system. The cell death factor Bax was involved (its expression was reduced 2-fold by the treatment) and injury-induced reduction in neurite lengths and numbers was reversed by the treatment. This all indicates that W. somnifera root extract was neuroprotective and could have therapeutic potential to target factors involved in secondary injury and long-term sequelae of mild TBI.
Withaferin A induces Nrf2-dependent protection against liver injury: Role of Keap1-independent mechanisms
Palliyaguru DL, Chartoumpekis DV, Wakabayashi N, Skoko JJ, Yagishita Y, Singh SV and Kensler TW
Small molecules of plant origin offer presumptively safe opportunities to prevent carcinogenesis, mutagenesis and other forms of toxicity in humans. However, the mechanisms of action of such plant-based agents remain largely unknown. In recent years the stress responsive transcription factor Nrf2 has been validated as a target for disease chemoprevention. Withania somnifera (WS) is a herb used in Ayurveda (an ancient form of medicine in South Asia). In the recent past, withanolides isolated from WS, such as Withaferin A (WA) have been demonstrated to be preventive and therapeutic against multiple diseases in experimental models. The goals of this study are to evaluate withanolides such as WA as well as Withania somnifera root extract as inducers of Nrf2 signaling, to probe the underlying signaling mechanism of WA and to determine whether prevention of acetaminophen (APAP)-induced hepatic toxicity in mice by WA occurs in an Nrf2-dependent manner. We observed that WA profoundly protects wild-type mice but not Nrf2-disrupted mice against APAP hepatotoxicity. WA is a potent inducer of Nrf2-dependent cytoprotective enzyme expression both in vivo and in vitro. Unexpectedly, WA induces Nrf2 signaling at least in part, in a Keap1-independent, Pten/Pi3k/Akt-dependent manner in comparison to prototypical Nrf2 inducers, sulforaphane and CDDO-Im. The identification of WA as an Nrf2 inducer that can signal through a non-canonical, Keap1-independent pathway provides an opportunity to evaluate the role of other regulatory partners of Nrf2 in the dietary and pharmacological induction of Nrf2-mediated cytoprotection.
Withania somnifera and Its Withanolides Attenuate Oxidative and Inflammatory Responses and Up-Regulate Antioxidant Responses in BV-2 Microglial Cells
Sun GY, Li R, Cui J, Hannink M, Gu Z, Fritsche KL, Lubahn DB and Simonyi A
Withania somnifera (L.) Dunal, commonly known as Ashwagandha, has been used in Ayurvedic medicine for promoting health and quality of life. Recent clinical trials together with experimental studies indicated significant neuroprotective effects of Ashwagandha and its constituents. This study is aimed to investigate anti-inflammatory and anti-oxidative properties of this botanical and its two withanolide constituents, namely, Withaferin A and Withanolide A, using the murine immortalized BV-2 microglial cells. Ashwagandha extracts not only effectively inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) and reactive oxygen species (ROS) production in BV-2 cells, but also stimulates the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway, leading to induction of heme oxygenase-1 (HO-1), both in the presence and absence of LPS. Although the withanolides were also capable of inhibiting LPS-induced NO production and stimulating Nrf2/HO-1 pathway, Withaferin A was tenfold more effective than Withanolide A. In serum-free culture, LPS can also induce production of long thin processes (filopodia) between 4 and 8 h in BV-2 cells. This morphological change was significantly suppressed by Ashwagandha and both withanolides at concentrations for suppressing LPS-induced NO production. Taken together, these results suggest an immunomodulatory role for Ashwagandha and its withanolides, and their ability to suppress oxidative and inflammatory responses in microglial cells by simultaneously down-regulating the NF-kB and upregulating the Nrf2 pathways.
Comparative evaluation of the sexual functions and NF-κB and Nrf2 pathways of some aphrodisiac herbal extracts in male rats
Sahin K, Orhan C, Akdemir F, Tuzcu M, Gencoglu H, Sahin N, Turk G, Yilmaz I, Ozercan IH and Juturu V
Mucuna pruriens, Tribulus terrestris and Ashwagandha (Withania somnifera) are widely known as antioxidant effective herbals and have been reported to possess aphrodisiac activities in traditional usages. In this study, we determined the effects of these herbals on sexual functions, serum biochemical parameters, oxidative stress and levels of NF-κB, Nrf2, and HO-1 in reproductive tissues.
Phytochemicals and botanical extracts regulate NF-κB and Nrf2/ARE reporter activities in DI TNC1 astrocytes
Ajit D, Simonyi A, Li R, Chen Z, Hannink M, Fritsche KL, Mossine VV, Smith RE, Dobbs TK, Luo R, Folk WR, Gu Z, Lubahn DB, Weisman GA and Sun GY
The increase in oxidative stress and inflammatory responses associated with neurodegenerative diseases has drawn considerable attention towards understanding the transcriptional signaling pathways involving NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) and Nrf2 (Nuclear Factor Erythroid 2-like 2). Our recent studies with immortalized murine microglial cells (BV-2) demonstrated effects of botanical polyphenols to inhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) and enhance Nrf2-mediated antioxidant responses (Sun et al., 2015). In this study, an immortalized rat astrocyte (DI TNC1) cell line expressing a luciferase reporter driven by the NF-κB or the Nrf2/Antioxidant Response Element (ARE) promoter was used to assess regulation of these two pathways by phytochemicals such as quercetin, rutin, cyanidin, cyanidin-3-O-glucoside, as well as botanical extracts from Withania somnifera (Ashwagandha), Sutherlandia frutescens (Sutherlandia) and Euterpe oleracea (Açaí). Quercetin effectively inhibited LPS-induced NF-κB reporter activity and stimulated Nrf2/ARE reporter activity in DI TNC1 astrocytes. Cyanidin and the glycosides showed similar effects but only at much higher concentrations. All three botanical extracts effectively inhibited LPS-induced NF-κB reporter activity. These extracts were capable of enhancing ARE activity by themselves and further enhanced ARE activity in the presence of LPS. Quercetin and botanical extracts induced Nrf2 and HO-1 protein expression. Interestingly, Ashwagandha extract was more active in inducing Nrf2 and HO-1 expression in DI TNC1 astrocytes as compared to Sutherlandia and Açaí extracts. In summary, this study demonstrated NF-kB and Nrf2/ARE promoter activities in DI TNC1 astrocytes, and further showed differences in ability for specific botanical polyphenols and extracts to down-regulate LPS-induced NF-kB and up-regulate the NRF2/ARE activities in these cells.
Withaferin A induces heme oxygenase (HO-1) expression in endothelial cells via activation of the Keap1/Nrf2 pathway
Heyninck K, Sabbe L, Chirumamilla CS, Szarc Vel Szic K, Vander Veken P, Lemmens KJA, Lahtela-Kakkonen M, Naulaerts S, Op de Beeck K, Laukens K, Van Camp G, Weseler AR, Bast A, Haenen GRMM, Haegeman G and Vanden Berghe W
Withaferin A (WA), a natural phytochemical derived from the plant Withania somnifera, is a well-studied bioactive compound exerting a broad spectrum of health promoting effects. To gain better insight in the potential therapeutic capacity of WA, we evaluated the transcriptional effects of WA on primary human umbilical vein endothelial cells (HUVECs) and an endothelial cell line (EA.hy926). RNA microarray analysis of WA treated HUVEC cells demonstrated increased expression of the antioxidant gene heme oxygenase (HO-1). Transcriptional regulation of this gene is strongly dependent on the transcription factor NF-E2-related factor 2 (Nrf2), which senses chemical changes in the cell and coordinates transcriptional responses to maintain chemical homeostasis via expression of antioxidant genes and cytoprotective Phase II detoxifying enzymes. Under normal conditions, Nrf2 is kept in the cytoplasm by Kelch-like ECH-associated protein 1 (Keap1), an adaptor protein controlling the half-life of Nrf2 via constant proteasomal degradation. In this study we demonstrate that WA time- and concentration-dependently induces HO-1 expression in endothelial cells via upregulation and increased nuclear translocation of Nrf2. According to the crucial negative regulatory role of Keap1 in Nrf2 expression levels, a direct interaction of WA with Keap1 could be demonstrated. In vitro and in silico evaluations suggest that specific cysteine residues in Keap1 might be involved in the interaction with WA.
Upregulation of phase II enzymes through phytochemical activation of Nrf2 protects cardiomyocytes against oxidant stress
Reuland DJ, Khademi S, Castle CJ, Irwin DC, McCord JM, Miller BF and Hamilton KL
Increased production of reactive oxygen species has been implicated in the pathogenesis of cardiovascular disease (CVD), and enhanced endogenous antioxidants have been proposed as a mechanism for regulating redox balance. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a transcriptional regulator of phase II antioxidant enzymes, and activation of Nrf2 has been suggested to be an important step in attenuating oxidative stress associated with CVD. A well-defined combination of five widely studied medicinal plants derived from botanical sources (Bacopa monniera, Silybum marianum (milk thistle), Withania somnifera (Ashwagandha), Camellia sinensis (green tea), and Curcuma longa (turmeric)) has been shown to activate Nrf2 and induce phase II enzymes through the antioxidant response element. The purpose of these experiments was to determine if treatment of cardiomyocytes with this phytochemical composition, marketed as Protandim, activates Nrf2, induces phase II detoxification enzymes, and protects cardiomyocytes from oxidant-induced apoptosis in a Nrf2-dependent manner. In cultured HL-1 cardiomyocytes, phytochemical treatment was associated with nuclear accumulation of Nrf2, significant induction of phase II enzymes, and concomitant protection against hydrogen peroxide-induced apoptosis. The protection against oxidant stress was abolished when Nrf2 was silenced by shRNA, suggesting that our phytochemical treatment worked through the Nrf2 pathway. Interestingly, phytochemical treatment was found to be a more robust activator of Nrf2 than oxidant treatment, supporting the use of the phytochemicals as a potential treatment to increase antioxidant defenses and protect heart cells against an oxidative challenge.
Ashwagandha leaf derived withanone protects normal human cells against the toxicity of methoxyacetic acid, a major industrial metabolite
Priyandoko D, Ishii T, Kaul SC and Wadhwa R
The present day lifestyle heavily depends on industrial chemicals in the form of agriculture, cosmetics, textiles and medical products. Since the toxicity of the industrial chemicals has been a concern to human health, the need for alternative non-toxic natural products or adjuvants that serve as antidotes are in high demand. We have investigated the effects of Ayurvedic herb Ashwagandha (Withania somnifera) leaf extract on methoxyacetic acid (MAA) induced toxicity. MAA is a major metabolite of ester phthalates that are commonly used in industry as gelling, viscosity and stabilizer reagents. We report that the MAA cause premature senescence of normal human cells by mechanisms that involve ROS generation, DNA and mitochondrial damage. Withanone protects cells from MAA-induced toxicity by suppressing the ROS levels, DNA and mitochondrial damage, and induction of cell defense signaling pathways including Nrf2 and proteasomal degradation. These findings warrant further basic and clinical studies that may promote the use of withanone as a health adjuvant in a variety of consumer products where the toxicity has been a concern because of the use of ester phthalates.
Differential activities of the two closely related withanolides, Withaferin A and Withanone: bioinformatics and experimental evidences
Vaishnavi K, Saxena N, Shah N, Singh R, Manjunath K, Uthayakumar M, Kanaujia SP, Kaul SC, Sekar K and Wadhwa R
Withanolides are naturally occurring chemical compounds. They are secondary metabolites produced via oxidation of steroids and structurally consist of a steroid-backbone bound to a lactone or its derivatives. They are known to protect plants against herbivores and have medicinal value including anti-inflammation, anti-cancer, adaptogenic and anti-oxidant effects. Withaferin A (Wi-A) and Withanone (Wi-N) are two structurally similar withanolides isolated from Withania somnifera, also known as Ashwagandha in Indian Ayurvedic medicine. Ashwagandha alcoholic leaf extract (i-Extract), rich in Wi-N, was shown to kill cancer cells selectively. Furthermore, the two closely related purified phytochemicals, Wi-A and Wi-N, showed differential activity in normal and cancer human cells in vitro and in vivo. We had earlier identified several genes involved in cytotoxicity of i-Extract in human cancer cells by loss-of-function assays using either siRNA or randomized ribozyme library.
Withanolide A prevents neurodegeneration by modulating hippocampal glutathione biosynthesis during hypoxia
Baitharu I, Jain V, Deep SN, Shroff S, Sahu JK, Naik PK and Ilavazhagan G
Withania somnifera root extract has been used traditionally in ayurvedic system of medicine as a memory enhancer. Present study explores the ameliorative effect of withanolide A, a major component of withania root extract and its molecular mechanism against hypoxia induced memory impairment. Withanolide A was administered to male Sprague Dawley rats before a period of 21 days pre-exposure and during 07 days of exposure to a simulated altitude of 25,000 ft. Glutathione level and glutathione dependent free radicals scavenging enzyme system, ATP, NADPH level, γ-glutamylcysteinyl ligase (GCLC) activity and oxidative stress markers were assessed in the hippocampus. Expression of apoptotic marker caspase 3 in hippocampus was investigated by immunohistochemistry. Transcriptional alteration and expression of GCLC and Nuclear factor (erythroid-derived 2)-related factor 2 (Nrf2) were investigated by real time PCR and immunoblotting respectively. Exposure to hypobaric hypoxia decreased reduced glutathione (GSH) level and impaired reduced gluatathione dependent free radical scavenging system in hippocampus resulting in elevated oxidative stress. Supplementation of withanolide A during hypoxic exposure increased GSH level, augmented GSH dependent free radicals scavenging system and decreased the number of caspase and hoescht positive cells in hippocampus. While withanolide A reversed hypoxia mediated neurodegeneration, administration of buthionine sulfoximine along with withanolide A blunted its neuroprotective effects. Exogenous administration of corticosterone suppressed Nrf2 and GCLC expression whereas inhibition of corticosterone synthesis upregulated Nrf2 as well as GCLC. Thus present study infers that withanolide A reduces neurodegeneration by restoring hypoxia induced glutathione depletion in hippocampus. Further, Withanolide A increases glutathione biosynthesis in neuronal cells by upregulating GCLC level through Nrf2 pathway in a corticosterone dependenet manner.
Natural Withanolides in the Treatment of Chronic Diseases
White PT, Subramanian C, Motiwala HF and Cohen MS
Withanolides, and in particular extracts from Withania somnifera, have been used for over 3,000 years in traditional Ayurvedic and Unani Indian medical systems as well as within several other Asian countries. Traditionally, the extracts were ascribed a wide range of pharmacologic properties with corresponding medical uses, including adaptogenic, diuretic, anti-inflammatory, sedative/anxiolytic, cytotoxic, antitussive, and immunomodulatory. Since the discovery of the archetype withaferin A in 1965, approximately 900 of these naturally occurring, polyoxygenated steroidal lactones with 28-carbon ergostane skeletons have been discovered across 24 diverse structural types. Subsequently, extensive pharmacologic research has identified multiple mechanisms of action across key inflammatory pathways. In this chapter we identify and describe the major withanolides with anti-inflammatory properties, illustrate their role within essential and supportive inflammatory pathways (including NF-κB, JAK/STAT, AP-1, PPARγ, Hsp90 Nrf2, and HIF-1), and then discuss the clinical application of these withanolides in inflammation-mediated chronic diseases (including arthritis, autoimmune, cancer, neurodegenerative, and neurobehavioral). These naturally derived compounds exhibit remarkable biologic activity across these complex disease processes, while showing minimal adverse effects. As novel compounds and analogs continue to be discovered, characterized, and clinically evaluated, the interest in withanolides as a novel therapeutic only continues to grow.

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