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Reactive oxygen species (ROS) and oxidative stress increase susceptibility to neurodegeneration and other age-related pathologies. We have previously demonstrated that an infusion prepared from Pulicaria incisa (Pi) has protective,... more
Reactive oxygen species (ROS) and oxidative stress increase susceptibility to neurodegeneration and other age-related pathologies. We have previously demonstrated that an infusion prepared from Pulicaria incisa (Pi) has protective, anti-inflammatory, and antioxidative effects in glial cells. However, the neuroprotective activities of Pi infusion in cultured neurons and aging mice have never been studied. In the following study, the effects of Pi infusion were explored in a hydrogen peroxide (H2O2)-induced oxidative stress model in SH-SY5Y human neuroblastoma cells. Profiling of the infusion by gas chromatography–mass spectrometry identified chlorogenic acid, quercetin, and aucubin as some of its main constituents. H2O2-induced ROS accumulation and caspase 3 activity decreased SH-SY5Y viability and were prevented upon the pretreatment of cells with Pi infusion. Additionally, the Pi infusion upregulated cellular levels and the nuclear translocation of nuclear factor erythroid 2–relate...
We chose to focus our investigations on the effect of the active forms, TTF and AcA, rather than the whole (crude) extract. 1. To establish cultivation program designed to develop lead cultivar/s (which will be selected from the different... more
We chose to focus our investigations on the effect of the active forms, TTF and AcA, rather than the whole (crude) extract. 1. To establish cultivation program designed to develop lead cultivar/s (which will be selected from the different Af accessions) with the highest yield of the active compounds TTF and/or achillolide A (AcA). These cultivar/s will be the source for the purification of large amounts of the active compounds when needed in the future for functional foods/drug development. This task was completed. 2. To determine the effect of the Af extract, TTF and AcA on neuronal vulnerability to oxidative stress in cultured neurons expressing FAD-linked mutants.Compounds were tested in N2a neuroblastoma cell line. In addition, we have tested the effects of TTF and AcA on signaling events promoted by H₂O₂ in astrocytes and by β-amyloid in neuronal N2a cells. 3. To determine the effect of the Af extract, TTF and AcA on neuropathology (amyloidosis and tau phosphorylation) in cultu...
Plant-derived substances have been shown to affect potential targets in inflammatory diseases. We have previously purified from the desert plant Achillea fragrantissima, a sesquiterpene lactone named achillolide A, and demonstrated its... more
Plant-derived substances have been shown to affect potential targets in inflammatory diseases. We have previously purified from the desert plant Achillea fragrantissima, a sesquiterpene lactone named achillolide A, and demonstrated its anti-inflammatory activities in cultured brain macrophages named microglial cells. In the present study, we further investigated achillolide A in alleviating atopic dermatitis, a chronic and recurring inflammatory skin disease. We investigated achillolide A for its in vivo anti-inflammatory activity using the oxazolone model of atopic dermatitis in mice, in which oxazolone induces ear swelling. Our results show that mice treated with achillolide A showed a significant decrease in the oxazolone-induced ear swelling. Since macrophages are inflammatory cells that play a pivotal role in the pathogenesis of atopic dermatitis, the anti-inflammatory effects of achillolide A were also studied in spleen cells. We demonstrated that achillolide A reduced the lev...
Myasthenia gravis (MG) is a T cell-regulated, antibody-mediated autoimmune disease. Two peptides representing sequences of the human acetylcholine receptor α-subunit, p195–212 and p259–271, previously were shown to stimulate the... more
Myasthenia gravis (MG) is a T cell-regulated, antibody-mediated autoimmune disease. Two peptides representing sequences of the human acetylcholine receptor α-subunit, p195–212 and p259–271, previously were shown to stimulate the proliferation of peripheral blood lymphocytes of patients with MG and were found to be immunodominant T cell epitopes in SJL and BALB/c mice, respectively. Single amino acid-substituted analogs of p195–212 and p259–271, as well as a dual analog composed of the tandemly arranged two single analogs, were shown to inhibit, in vitro and in vivo , MG-associated autoimmune responses. Stimulation of T cells through the antigen-specific T cell receptor activates tyrosine kinases and phospholipase C (PLC). Therefore, in attempts to understand the mechanism of action of the analogs, we first examined whether the myasthenogenic peptides trigger tyrosine phosphorylation and activation of phospholipase C. For that purpose, we measured generation of inositol phosphates an...
Cytokines have been suggested to be involved in the cross talk between the immune and the nervous systems, under normal and pathological conditions. For example, the cytokine interleukin-2 was suggested to be involved in response to CNS... more
Cytokines have been suggested to be involved in the cross talk between the immune and the nervous systems, under normal and pathological conditions. For example, the cytokine interleukin-2 was suggested to be involved in response to CNS trauma and spontaneous regeneration. Here, we examined whether mammalian CNS has an intrinsic potential to produce interleukin-2 and, if so, what its cellular origin is. mRNA sequences encoding for interleukin-2 were detected in brains of humans and rodents. Northern blot analysis revealed the presence of several interleukin-2 transcripts of different sizes in the brain, all recognized by lymphocyte-derived interleukin-2 cDNA probes. One of the transcripts, a high molecular weight form of approximately 5 kb, appeared to be unique to the brain. Reverse transcription and amplification by PCR of human fetal brain mRNA revealed one cDNA product that, upon sequence analysis, showed a high degree of homology with the human lymphocyte-derived interleukin-2 coding sequence. To identify the possible cellular source of the interleukin-2 transcripts within the mammalian brain, we similarly analyzed mRNA of rat brain cells in culture. Northern blot analysis revealed that astrocytes contain transcripts that hybridize with interleukin-2 cDNA probe. These findings point to the astrocytes as a possible source of brain interleukin-2.
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the increased production of autoantibodies and by T cell dysfunction associated with general clinical manifestations. A model of induced experimental SLE by the... more
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the increased production of autoantibodies and by T cell dysfunction associated with general clinical manifestations. A model of induced experimental SLE by the immunization with the human monoclonal anti-DNA 16/6Id(+) autoantibody and a model of the SLE-prone mice (NZB x NZW)F1 were used in the present study. Two peptides based on the complementarity determining regions (CDR) 1 and 3 of a murine monoclonal anti-DNA 16/6Id(+) autoantibody were shown to ameliorate spontaneous and induced SLE in mice. We demonstrate here that levels of matrix metalloproteinase (MMP)-3 and MMP-9 were elevated in plasma and kidneys of SLE-afflicted mice. Levels of both MMP-3 and MMP-9 were elevated in kidneys of mice with the 16/6Id induced experimental SLE already in the early phases of disease development. However, increased levels of only MMP-3 were detected in the plasma at the early stages of disease, while MMP-9 activity was elevated later, when clinical manifestations were already observed. Treatment of SLE-afflicted mice, with the CDR1-based peptide that ameliorates disease manifestations in mice, led to a reduction in MMP-9 activity and in MMP-3 protein levels both in plasma and in kidneys. We thus suggest that these enzymes may play a pathogenic role in the disease and may serve as markers for the determination of disease progression or amelioration.
Reactive oxygen species (ROS) and oxidative stress increase susceptibility to neurodegeneration and other age-related pathologies. We have previously demonstrated that an infusion prepared from Pulicaria incisa (Pi) has protective,... more
Reactive oxygen species (ROS) and oxidative stress increase susceptibility to neurodegeneration and other age-related pathologies. We have previously demonstrated that an infusion prepared from Pulicaria incisa (Pi) has protective, anti-inflammatory, and antioxidative effects in glial cells. However, the neuroprotective activities of Pi infusion in cultured neurons and aging mice have never been studied. In the following study, the effects of Pi infusion were explored in a hydrogen peroxide (H 2 O 2)-induced oxidative stress model in SH-SY5Y human neuroblastoma cells. Profiling of the infusion by gas chromatography-mass spectrometry identified chlorogenic acid, quercetin, and aucubin as some of its main constituents. H 2 O 2-induced ROS accumulation and caspase Antioxidants 2022, 11, x. https://doi.org/10.3390/xxxxxwww.mdpi.com/journal/antioxidants 3 activity decreased SH-SY5Y viability and were prevented upon the pretreatment of cells with Pi infusion. Additionally, the Pi infusion upregulated cellular levels and the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) as well as the phosphorylation of cyclic AMP response element-binding protein (CREB). Aging mice treated daily for 18 months with Pi infusion exhibited reduced neuronal cell death in the hippocampus as compared to age-matched controls. We, therefore, propose Pi infusion as a candidate regulator of oxidative stress in the brain.
NADPH oxidase is a superoxide-generating, membrane-bound complex activated in stimulated phagocytes or in a reconstituted system consisting of membranes, cytosolic components and arachidonate or SDS. To delineate mechanism of oxidase... more
NADPH oxidase is a superoxide-generating, membrane-bound complex activated in stimulated phagocytes or in a reconstituted system consisting of membranes, cytosolic components and arachidonate or SDS. To delineate mechanism of oxidase activation in the cell-free system, hydrolysis of phosphoinositides in the combined membrane-cytosol oxidase mixture was investigated. Arachidonate promoted hydrolysis of membrane-[3H]-phosphatidylinositol by cytosolic phospholipase C. PI hydrolysis was similarly supported by other unsaturated fatty acids and by SDS. Unlike activation of the NADPH oxidase, PI hydrolysis required the presence of calcium ions. Implications of these findings to the mechanism of NADPH oxidase activation are discussed.
A soluble extract of neutrophil granules interfered with activation of the NADPH oxidase in a cell-free system. The extract had no effect on superoxide production by preactivated enzyme. The inhibitory activity was retained during... more
A soluble extract of neutrophil granules interfered with activation of the NADPH oxidase in a cell-free system. The extract had no effect on superoxide production by preactivated enzyme. The inhibitory activity was retained during dialysis and was lost upon exposure to proteinase K indicating that the active substance was a protein. The inhibitor exhibited a high stability at elevated temperatures. Chromatography of granules extract on ion exchangers implied that the inhibitor was a positively charged protein eluting from S Sepharose cation exchanger above 0.4M concentration of NaCl.
Summary Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the increased production of autoantibodies and by systemic clinical manifestations and damage to multiple organs. The aim of the present study was to... more
Summary Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the increased production of autoantibodies and by systemic clinical manifestations and damage to multiple organs. The aim of the present study was to analyse matrix metalloproteinase (MMP)-9 activity in sera of patients with active and inactive SLE in order to evaluate its role in the pathogenesis and course of the disease, as well as its diagnostic value. We measured activity levels of MMP-9 and MMP-2, using both gel zymography and activity assay kits, in sera of 40 SLE patients and of 25 healthy controls. We found that MMP-9 activity, but not MMP-2 activity, is significantly elevated in the sera of SLE patients compared with sera samples of healthy controls. High activity levels of MMP-9 were determined in sera of 68% of the SLE patients. Elevated levels of MMP-9 were correlated with the presence of discoid rash, Raynaud phenomenon, pneumonitis, mucosal ulcers and anti-phospholipid antibodies. Cha...
Alzheimer's disease (AD) is the most prevalent cause of dementia in adults. Current available drugs for AD transiently alleviate some of the symptoms, but do not modify the disease mechanism or cure it. Therefore, new drugs are... more
Alzheimer's disease (AD) is the most prevalent cause of dementia in adults. Current available drugs for AD transiently alleviate some of the symptoms, but do not modify the disease mechanism or cure it. Therefore, new drugs are desperately needed. Key contributors to AD are amyloid beta (Aβ)- and reactive oxygen species (ROS)-induced cytotoxicities. Plant-derived substances have been shown to affect various potential targets in various diseases including AD. Therefore, phytochemicals which can protect neuronal cells against these insults might help in preventing and treating this disease. In the following research, we have isolated the sesquiterpene lactone achillolide A from the plant Achillea fragrantissima and, for the first time, characterized its effects on Aβ-treated neuroblastoma cells. Aβ is a peptide derived from the sequential cleavage of amyloid precursor protein, and is part of the pathogenesis of AD. Our current study aimed to determine whether achillolide A can int...
The demand for organic food products has increased over the last decades; however, the health effects of organically grown products are controversial and knowledge about how different fertilization regimes affect nutritionally and health... more
The demand for organic food products has increased over the last decades; however, the health effects of organically grown products are controversial and knowledge about how different fertilization regimes affect nutritionally and health relevant components is still limited. The aim of the present study was to determine the effect of organic and conventional mineral-based fertilizers on the quality of sweet red peppers. The parameters tested were anti-proliferative activity against cancer cells and the concentrations of antioxidants, flavonoids, phenolics, and vitamin C. The decay incidence, percentage of weight loss, and total soluble solids (TSS) content were also evaluated. The different parameters were tested in fresh peppers immediately after harvest and after cold storage. Our results show that the anti-proliferative activity of pepper extracts against colon cancer cells is similar in fresh organically and conventionally fertilized sweet red peppers. While in conventionally fe...
Morphogenesis and tissue repair require appropriate cross-talk between the cells and their surrounding milieu, which includes extracellular components and soluble factors, e.g., cytokines and growth factors. The present work deals with... more
Morphogenesis and tissue repair require appropriate cross-talk between the cells and their surrounding milieu, which includes extracellular components and soluble factors, e.g., cytokines and growth factors. The present work deals with this communication needed for recovery after axotomy in the central nervous system (CNS). The failure of CNS axons to regenerate after axonal injury has been attributed, in part, to astrocyte failure to repopulate the injury site. The goal of this work was to provide an in vitro model to mimic the in vivo response of astrocytes to nerve injury and to find ways to modulate this response and create a milieu that favors astrocyte migration and repopulation of the injury site. In an astrocyte scratch wound model, we blocked astrocyte migration by tumor necrosis factor alpha (TNF-alpha). This effect could not be reversed by astrocyte migration-inducing factors such as transforming growth factor beta 1 (TGF-beta 1) or by any of the tested extracellular matr...
Oxidative stress is involved in the pathogenesis of neurodegenerative diseases such as Parkinson's and Alzheimer's diseases. Astrocytes, the most abundant glial cells in the brain, protect neurons from reactive oxygen species... more
Oxidative stress is involved in the pathogenesis of neurodegenerative diseases such as Parkinson's and Alzheimer's diseases. Astrocytes, the most abundant glial cells in the brain, protect neurons from reactive oxygen species (ROS) and provide them with trophic support, such as glial-derived neurotrophic factor (GDNF). Thus, any damage to astrocytes will affect neuronal survival. In the present study, an infusion prepared from the desert plant Pulicaria incisa (Pi) was tested for its protective and antioxidant effects on astrocytes subjected to oxidative stress. The Pi infusion attenuated the intracellular accumulation of ROS following treatment with hydrogen peroxide and zinc and prevented the H(2)O(2)-induced death of astrocytes. The Pi infusion also exhibited an antioxidant effect in vitro and induced GDNF transcription in astrocytes. It is proposed that this Pi infusion be further evaluated for use as a functional beverage for the prevention and/or treatment of brain inj...
Alzheimer's disease (AD) is the most prevalent cause of dementia in adults. Current available drugs for AD transiently alleviate some of the symptoms, but do not modify the disease mechanism or cure it. Therefore, new drugs are... more
Alzheimer's disease (AD) is the most prevalent cause of dementia in adults. Current available drugs for AD transiently alleviate some of the symptoms, but do not modify the disease mechanism or cure it. Therefore, new drugs are desperately needed. Key contributors to AD are amyloid beta (Aβ)- and reactive oxygen species (ROS)-induced cytotoxicities. Plant-derived substances have been shown to affect various potential targets in various diseases including AD. Therefore, phytochemicals which can protect neuronal cells against these insults might help in preventing and treating this disease. In the following research, we have isolated the sesquiterpene lactone achillolide A from the plant Achillea fragrantissima and, for the first time, characterized its effects on Aβ-treated neuroblastoma cells. Aβ is a peptide derived from the sequential cleavage of amyloid precursor protein, and is part of the pathogenesis of AD. Our current study aimed to determine whether achillolide A can int...
Glutamate toxicity is a major contributor to the path-ophysiology of numerous neurodegenerative diseases including amyotrophic lateral sclerosis and Alzheimer's disease. Therefore, protecting neuronal cells against glutamate-induced... more
Glutamate toxicity is a major contributor to the path-ophysiology of numerous neurodegenerative diseases including amyotrophic lateral sclerosis and Alzheimer's disease. Therefore, protecting neuronal cells against glutamate-induced cytotoxicity might be an effective approach for the treatment of these diseases. We have previously purified from the medicinal plant Achillea fragrantissima two bioactive compounds which were not studied before: the sesquiterpene lactone achillolide A and the flavonoid 3,5,4′-trihydroxy-6,7,3′-trimethoxyflavone (TTF). We have shown that these compounds protect astrocytes from oxidative stress-induced cell death and inhibit microglial activation. The current study examined for the first time their effects on differentiated mouse neuroblastoma N2a cells and on glutamate toxicity. We have found that, although these compounds belong to different chemical families, they protect neu-ronal cells from glutamate toxicity. We further demonstrate that this protective effect might be, at least partially, due to inhibi-tory effects of these compounds on the levels of reactive oxygen species produced following treatment with glutamate.
Background: Alzheimer's disease is a neurodegenerative disease, characterized by progressive decline in memory and cognitive functions, that results from loss of neurons in the brain. Amyloid beta (Aβ) protein and oxidative stress are... more
Background: Alzheimer's disease is a neurodegenerative disease, characterized by progressive decline in memory and cognitive functions, that results from loss of neurons in the brain. Amyloid beta (Aβ) protein and oxidative stress are major contributors to Alzheimer's disease, therefore, protecting neuronal cells against Aβ-induced toxicity and oxidative stress might form an effective approach for treatment of this disease. 3,5,4′-trihydroxy-6,7,3′-trimethoxyflavone (TTF) is a flavonoid we have purified from the plant Achillea fragrantissima; and the present study examined, for the first time, the effects of this compound on Aβ-toxicity to neuronal cells.
Activated microglial cells release various mediators, which cause neuronal cell death and have been implicated in different neurological disorders. The present study demonstrates that an infusion prepared from the plant Pulicaria incisa... more
Activated microglial cells release various mediators, which cause neuronal cell death and have been implicated in different neurological disorders. The present study demonstrates that an infusion prepared from the plant Pulicaria incisa (Pi) inhibits microglial activation and down-regulates levels of the inflammatory cytokines interleukin (IL)-1β, IL-6, and of the toxic mediators nitric oxide and glutamate. The infusion was also shown to have antioxi-dant properties in cell-free assays (e.g., differential pulse voltammetry) and in a cellular assay, in which the infusion attenuated the induced accumulation of intracellular reactive oxygen species (ROS). We found that Pi infusion is rich in polyphenols, especially chlorogenic acids and ferulic acids, which might be responsible for the observed activities. It is proposed that Pi infusion be further evaluated for use as a functional beverage for the prevention and/or treatment of chronic diseases, especially neurodegenerative disorders in which microglial activation and oxidative stress play important roles.
Background: The neuroinflammatory process plays a central role in the initiation and progression of neurodegenerative diseases such as Parkinson's and Alzheimer's diseases, and involves the activation of brain microglial cells. During the... more
Background: The neuroinflammatory process plays a central role in the initiation and progression of neurodegenerative diseases such as Parkinson's and Alzheimer's diseases, and involves the activation of brain microglial cells. During the neuroinflammatory process, microglial cells release proinflammatory mediators such as cytokines, matrix metalloproteinases (MMP), Reactive oxygen species (ROS) and nitric oxide (NO). In the present study, extracts from 66 different desert plants were tested for their effect on lipopolysaccharide (LPS)-induced production of NO by primary microglial cells. The extract of Achillea fragrantissima (Af), which is a desert plant that has been used for many years in traditional medicine for the treatment of various diseases, was the most efficient extract, and was further studied for additional anti-neuroinflammatory effects in these cells.
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Achillolide A is a natural sesquiterpene lactone that we have previously shown can inhibit microglial activation. In this study we present evidence for its beneficial effects on astrocytes under oxidative stress, a situation relevant to... more
Achillolide A is a natural sesquiterpene lactone that we have previously shown can inhibit microglial activation. In this study we present evidence for its beneficial effects on astrocytes under oxidative stress, a situation relevant to neurodegenerative diseases and brain injuries. Viability of brain astrocytes (primary cultures) was determined by lactate dehydrogenase (LDH) activity, intracellular ROS levels were detected using 2',7'-dichlorofluorescein diacetate, in vitro antioxidant activity was measured by differential pulse voltammetry, and protein phosphorylation was determined using specific ELISA kits. We have found that achillolide A prevented the H 2 O 2-induced death of astrocytes, and attenuated the induced intracellular accumulation of reactive oxygen species (ROS). These activities could be attributed to the inhibition of the H 2 O 2-induced phosphorylation of MAP/ERK kinase 1 (MEK1) and p44/42 mitogen-activated protein kinases (MAPK), and to the antioxidant activity of achillolide A, but not to H 2 O 2 scavenging. This is the first study that demonstrates its protective effects on brain astrocytes, and its ability to interfere with MAPK activation. We propose that achillolide A deserves further evaluation for its potential to be developed as a drug for the prevention/treatment of neurodegenerative diseases and brain injuries where oxidative stress is part of the pathophysiology.
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Atopic dermatitis is a chronic and recurring inflammatory disease of the skin. This study aimed to evaluate the anti-inflammatory properties of pulichalconoid B, a chalcone that we have isolated from the plant Pulicaria incisa. The in... more
Atopic dermatitis is a chronic and recurring inflammatory disease of the skin. This study aimed to evaluate the anti-inflammatory properties of pulichalconoid B, a chalcone that we have isolated from the plant Pulicaria incisa. The in vivo activity of pulichalconoid B was evaluated using the oxazolone model of cutaneous dermatitis and its in vitro activity was studied using isolated splenocytes. Changes in ear thickness, myeloperoxidase activity and production of different cytokines were considered to be indicators of dermal inflammation. Mice that were treated with pulichalconoid B showed a significantly decreased response to delayed-type hypersensitivity induced by oxazolone. Pulichalconoid B significantly reduced oxazolone-induced ear edema, inhibited myeloperoxidase activity and downregulated several cytokines. Pulichalconoid B also inhibited the secretion of cytokines from activated splenocytes. The data suggest that pulichalconoid B may be useful in treating skin inflammations.
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