Differential Effects of Halofuginone Enantiomers on Muscle Fibrosis and Histopathology in Duchenne Muscular Dystrophy.

Progressive loss of muscle and muscle function is associated with significant fibrosis in Duchenne muscular dystrophy (DMD) patients. Halofuginone, an analog of febrifugine, prevents fibrosis in various animal models, including those of muscular dystrophies. Effects of (+)/(-)-halofuginone enantiomers on motor coordination and diaphragm histopathology in mdx mice, the mouse model for DMD, were examined. Four-week-old male mice were treated with racemic halofuginone, or its separate enantiomers, for 10 weeks. Controls were treated with saline. Racemic halofuginone-treated mice demonstrated better motor coordination and balance than controls. However, (+)-halofuginone surpassed the racemic form's effect. No effect was observed for (-)-halofuginone, which behaved like the control. A significant reduction in collagen content and degenerative areas, and an increase in utrophin levels were observed in diaphragms of mice treated with racemic halofuginone. Again, (+)-halofuginone was more effective than the racemic form, whereas (-)-halofuginone had no effect. Both racemic and (+)-halofuginone increased diaphragm myofiber diameters, with no effect for (-)-halofuginone. No effects were observed for any of the compounds tested in an in-vitro cell viability assay. These results, demonstrating a differential effect of the halofuginone enantiomers and superiority of (+)-halofuginone, are of great importance for future use of (+)-halofuginone as a DMD antifibrotic therapy.

Early posthatch thermal stress causes long-term adverse effects on pectoralis muscle development in broilers.

Broiler chicks in the immediate posthatch handling period are exposed to thermal stress, with potentially harmful consequences for muscle growth and structure (e.g., less protein and more fat deposition). We addressed the effects of broiler chicks' exposure to various ambient temperatures during the first 13 D posthatch on their performance, as well as on muscle development and structure, up to day 35. Body weight and pectoralis muscle growth were lower throughout the entire period in the high-heat-exposed chicks (39°C, Hot) and to a lesser extent in the mild-heat-exposed chicks (35°C, Hot Mild) than in the Control chicks that were raised under a commercial protocol. In the cold-exposed chicks (29oC, Cold), BW and pectoralis muscle absolute growth were similar to the Control group throughout the entire period. The lower body and muscle growth in the Hot and Hot Mild groups were reflected in a lower number of myonuclei expressing proliferating cell nuclear cell in pectoralis major muscle cross sections sampled on day 8, in the distribution of myofibers as the experiment progressed, and in mean myofiber diameter on day 35, whereas in the Cold group, these numbers exceeded that of the Control group. However, TUNEL assay revealed similar cell survival in all groups. Hematoxylin-eosin and Oil red O staining revealed the highest fat deposition in the pectoralis muscle derived from the Hot group, whereas lower fat deposition was observed in the Control Cold group. These results were corroborated by immunostaining for CCAAT/enhancer binding protein ß in the pectoralis muscle, the levels of which were significantly higher in the Hot and Hot Mild groups on day 35 than in the Control group. Similar results were observed with Sirius red staining for collagen content in the pectoralis muscle. Together, the results imply long-term effects of chronic heat stress vs. cold stress in the early posthatch period on the broiler's body and muscle growth in general and myodegeneration of the pectoralis muscle in particular.

Hydroxy group requirement for halofuginone-dependent inhibition of muscle fibrosis and improvement of histopathology in the mdx mouse model for Duchenne muscular dystrophy.

In Duchenne muscular dystrophy (DMD), the progressive loss of muscle and its ability to function is associated with significant fibrosis, representing the major disease complication in patients. Halofuginone, a halogenated analog of the naturally occurring febrifugine, has been shown to prevent fibrosis in various animal models, including those of muscular dystrophies. Here, two optically active enantiomers of deoxyhalofuginone - a halofuginone analogue in which the hydroxy group in position 3 was removed from the piperidinyl entity - were evaluated with respect to their effect on muscle histopathology in mdx mice. Male mdx mice were treated with either deoxyhalofuginone (as single enantiomers or in racemic form), or halofuginone, for 10 weeks, starting at the age of 4 weeks. Halofuginone caused a significant reduction in total collagen content, degenerative areas, as well as in utrophin and phosphorylated-Smad3 levels in the mdx diaphragms. However, neither the deoxyhalofuginone enantiomers, nor its racemic form had any effect on these parameters. A positive effect of the deoxyhalofuginone (+)-enantiomer was observed on myofiber diameters; however, it was lesser than that of halofuginone. It is concluded that the hydroxy group plays a key role in halofuginone's effects related to fibrosis in DMD, and points towards the transforming growth factor ß/Smad3 signaling pathway being involved in this inhibition. Elucidation of the structure-function relationship of halofuginone, in relation to inhibiting fibrosis in muscular dystrophies, is of the utmost importance for creating the next generation of anti-fibrotic therapies that will be more efficacious and less toxic, hence improving life quality of patients.

Downregulation of microglial activation by achillolide A.

Chronic inflammation has been implicated in the pathogenesis of various neurodegenerative diseases. During the neuroinflammatory process, microglial cells release neurotoxic and proinflammatory mediators. In the present study, using activity-guided fractionation, we have purified an anti-inflammatory compound determined by spectroscopic methods to be a sesquiterpene lactone named achillolide A from Achillea fragrantissima (Forsk.) Sch. Bip. In primary cultures of lipopolysaccharide-activated microglial cells, achillolide A inhibited the lipopolysaccharide-induced levels of proinflammatory and toxic mediators including glutamate, nitric oxide, matrix metalloproteinase-9, cyclooxygenase-2, induced nitric oxide synthase, interleukin-1ß, and tumor necrosis factor-α. Achillolide A also exhibited an antioxidant capacity, as was shown in a cell free system as well as by its ability to reduce intracellular reactive oxygen species levels in microglial cells. Thus, achillolide A might have therapeutic potential for treatment of neurodegenerative diseases and deserves further studies.

3,5,4'-Trihydroxy-6,7,3'-trimethoxyflavone protects astrocytes against oxidative stress via interference with cell signaling and by reducing the levels of intracellular reactive oxygen species.

Oxidative stress is tightly involved in various neurodegenerative diseases such as Parkinson's and Alzheimer's diseases, and conditions such as ischemia. Astrocytes, the most abundant glial cells in the brain, protect neurons from reactive oxygen species (ROS) and provide them with trophic support. Therefore, any damage to astrocytes will affect neuronal survival. In a previous study we have demonstrated that an extract prepared from the plant Achillea fragrantissima (Af) prevented the oxidative stress-induced death of astrocytes and attenuated the intracellular accumulation of ROS in astrocytes under oxidative stress. In the present study, using activity guided fractionation, we have purified from this plant the active compound, determined to be a flavonoid named 3,5,4'-trihydroxy-6,7,3'-trimethoxyflavone (TTF). The effects of TTF in any biological system have not been studied previously, and this is the first study to characterize the anti-oxidant and protective effects of this compound in the context of neurodegenerative diseases. Using primary cultures of astrocytes we have found that TTF prevented the hydrogen peroxide (H2O2)-induced death of astrocytes, and attenuated the intracellular accumulation of ROS following treatment of these cells with H2O2 or the peroxyl radicals generating molecule 2,2'-Azobis(amidinopropane) (ABAP). TTF also interfered with cell signaling events and inhibited the phosphorylation of the signaling proteins stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), extracellular signal regulated kinase (ERK 1/2) and mitogen activated protein kinase kinase (MEK1) and the phosphorylation of the transcription factor cyclic AMP response element-binding protein (CREB). The mechanism of the protective effect of TTF against H2O2-cytotoxicity could not be attributed to a direct H2O2 scavenging but rather to the scavenging of free radicals as was shown in cell free systems. Thus, TTF might be a therapeutic candidate for the prevention/treatment of neurodegenerative diseases where oxidative stress is part of the pathophysiology.

Protective and antioxidant effects of a chalconoid from Pulicaria incisa on brain astrocytes.

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, by activity-guided fractionation, we have purified from the desert plant Pulicaria incisa two protective compounds and determined their structures by spectroscopic methods. The compounds were found to be new chalcones-pulichalconoid B and pulichalconoid C. This is the first study to characterize the antioxidant and protective effects of these compounds in any biological system. Using primary cultures of astrocytes, we have found that pulichalconoid B attenuated the accumulation of ROS following treatment of these cells with hydrogen peroxide by 89% and prevented 89% of the H2O2-induced death of astrocytes. Pulichalconoid B exhibited an antioxidant effect both in vitro and in the cellular antioxidant assay in astrocytes and microglial cells. Pulichalconoid B also caused a fourfold increase in GDNF transcription in these cells. Thus, this chalcone deserves further studies in order to evaluate if beneficial therapeutic effect exists.

Antioxidant and astroprotective effects of a Pulicaria incisa infusion.

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 injuries and neurodegenerative diseases in which oxidative stress plays a role.

Anti-neuroinflammatory effects of the extract of Achillea fragrantissima.

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. METHODS: In the present study, the ethanolic extract prepared from Af was tested for its anti-inflammatory effects on lipopolysaccharide (LPS)-activated primary cultures of brain microglial cells. The levels of the proinflammatory cytokines interleukin1ß (IL-1ß) and tumor necrosis factor-α (TNFα) secreted by the cells were determined by reverse transcriptase-PCR and Enzyme-linked immunosorbent assay (ELISA), respectively. NO levels secreted by the activate cells were measured using Griess reagent, ROS levels were measured by 2'7'-dichlorofluorescein diacetate (DCF-DA), MMP-9 activity was measured using gel zymography, and the protein levels of the proinflammatory enzymes cyclooxygenase-2 (COX-2) and induced nitric oxide synthase (iNOS) were measured by Western blot analysis. Cell viability was assessed using Lactate dehydrogenase (LDH) activity in the media conditioned by the cells or by the crystal violet cell staining. RESULTS: We have found that out of the 66 desert plants tested, the extract of Af was the most efficient extract and inhibited ~70% of the NO produced by the LPS-activated microglial cells, without affecting cell viability. In addition, this extract inhibited the LPS - elicited expression of the proinflammatory mediators IL-1ß, TNFα, MMP-9, COX-2 and iNOS in these cells. CONCLUSIONS: Thus, phytochemicals present in the Af extract could be beneficial in preventing/treating neurodegenerative diseases in which neuroinflammation is part of the pathophysiology.

Protective Effects of the Essential Oil of Salvia fruticosa and Its Constituents on Astrocytic Susceptibility to Hydrogen Peroxide-Induced Cell Death.

Oxidative stress has been implicated in pathologic processes associated with neurodegenerative diseases. Astrocytes, the most abundant glial cell type in the brain, protect neurons from reactive oxygen species (ROS), and any damage to them will affect neuronal survival. This study compares the ability of essential oils prepared from different herbs and spices to protect cultured primary brain astrocytes from H2O2-induced death. The results show that the essential oil of Salvia fruticosa (Sf) among the tested essential oils demonstrated remarkable protective activity. The protective effect of Sf could be attributed to alpha-humulene and alpha-pinene. Following incubation, alpha-humulene and trans-beta-caryophyllene could be found in the cytosol of astrocytes. It is proposed that Sf, by attenuating H2O2-induced cell death, might be used as a functional food or may be offered as a means of therapy in the treatment of neurodegenerative diseases.

Dopamine, through the extracellular signal-regulated kinase pathway, downregulates CD4+CD25+ regulatory T-cell activity: implications for neurodegeneration.

Fighting off neuronal degeneration requires a well controlled T-cell response against self-antigens residing in sites of the CNS damage. The ability to evoke this response is normally suppressed by naturally occurring CD4+CD25+ regulatory T-cells (Treg). No physiological compound that controls Treg activity has yet been identified. Here, we show that dopamine, acting via type 1 dopamine receptors (found here to be preferentially expressed by Treg), reduces the suppressive activity and the adhesive and migratory abilities of Treg. Treg activity was correlated with activation of the ERK1/2 (extracellular signal-regulated kinase 1/2) signaling pathway. Systemic injection of dopamine or an agonist of its type 1 receptors significantly enhanced, via a T-cell-dependent mechanism, protection against neuronal death after CNS mechanical and biochemical injury. These findings shed light on the physiological mechanisms controlling Treg and might open the way to novel therapeutic strategies for downregulating Treg activity (e.g., in neuronal degeneration) or for strengthening it (in autoimmune diseases).