The Protective Effects of Ecdysterone on Cognitive Impairment through Regulating Akt/GSK-3ß/Nrf2 Signaling Pathway and Oxidative Stress in Cognitive Mice Model and Aß-Induced Cell Neurotoxicity.

BACKGROUND: Severe neurological condition like Alzheimer's disease (AD) has a significantly negative impact on families and society, wherein there is no proven cure. As one of the principal active constituents of Achyranthes bidentata Blume, ecdysterone (ECR) has demonstrated antioxidant and cognitive dysfunction improvement effects. Nonetheless, the mechanism underlying the improvement of cognitive dysfunction by ECR remains unclear. This study sought to ascertain whether ECR may allebviate cognitive impairment by reducing oxidative stress via activation of the nuclear factor erythroid-2-related factor-2 (Nrf2) antioxidant system through Akt/GSK3ß pathway. METHODS: In terms of the experimental procedure, we determined the neuroprotective benefits of ECR in vivo via a cognitive impairment model of senescence-accelerated mouse prone 8 (SAMP8), we performed procedures such as behavioral testing, biochemical assaying, Nissl and TUNEL stainings, as well as flow cytometry, immunohistochemistry and western blotting. Furthermore, we investigated the underlying mechanistic action of ECR by activating PC12 cells with ß-amyloid peptide fragment 25-35 (Aß25-35). RESULTS: In vivo studies showed that ECR effectively improved cognitive impairment in SAMP8 via enhancement of learning and memory capabilities, but decreased oxidative stress, apoptosis and neuronal damage in the hippocampus. During the in vitro study, we observed that ECR dose-dependently reduced the oxidative stress and apoptosis that were induced in PC12 cells by Aß25-35. Additionally, the use of Akt inhibitors further established the potential of ECR to control Nrf2 through activation of the Akt/GSK3ß pathway and protect the PC12 cells from Aß25-35 induced damage. CONCLUSIONS: These findings offer proof that ECR reduces cognitive impairment by triggering the Nrf2 antioxidant system via the Akt/GSK3ß pathway and offer fresh information on ECR's potential as a promising therapeutic development candidate for AD.

Effects of the combination of high-intensity interval training and Ecdysterone on learning and memory abilities, antioxidant enzyme activities, and neuronal population in an Amyloid-beta-induced rat model of Alzheimer's disease.

AIMS: Oxidative stress and neuronal death are the primary reasons for the progression of amyloid-beta (Aß) deposition and cognitive deficits in Alzheimer's disease (AD). Ecdysterone (ecdy), a common derivative of ecdysteroids, possesses free radical scavenging and cognitive-improving effects. High-intensity interval training (HIIT) can be a therapeutic strategy for improving cognitive decline and oxidative stress. The present study was aimed to evaluate the effect of HIIT exercise and ecdy consumption synergistically on the changes in learning and memory functions, activities of hippocampal antioxidant enzymes, and neuronal population after AD induced by Aß in male rats. MATERIALS AND METHODS: Following ten days of Aß injection, HIIT exercise and ecdy treatment (10 mg/kg/day; P.O.) were initiated and continued for eight consecutive weeks in rats. At the end of the treatment period, the rat's learning and memory functions were assessed using Morris water maze and passive avoidance tests. The activity of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GRx), and changes in neuronal population were evaluated in rats' brains. RESULTS: The results indicated that Aß injection disrupted spatial/passive avoidance learning and memory in both tests, accompanied by a decrease in the SOD and CAT (as endogenous antioxidants) in rats' hippocampus. Additionally, Aß injection resulted in neuronal loss in the cerebral cortex and hippocampus. Although the consumption of ecdy separately improved spatial/passive avoidance learning and memory impairments, recovered hippocampal activity of SOD, CAT, GRx, and prevented the hippocampal neuronal loss, its combination along with HIIT resulted in a more powerful and effective amelioration in all the above-mentioned Aß-neuropathological changes. CONCLUSION: Our results confirm that a combination of HIIT and ecdy treatment could be a promising potential therapeutic option against AD-associated cognitive decline, owing to their free radical scavenging and neuroprotective properties.

Antiprotozoal effect of steroid hormone 20-hydroxyecdysone in giardiosis patients.

The World Health Organization reports that approximately 200 million people are infected with Giardia (G.) lamblia worldwide. Taking into account the emergence of resistance and the high toxicity of conventional drugs, research into new strategies to fight against G. lamblia is increasing. The aim of the study was to assess the antiprotozoal activity of 20-hydroxyecdysone in water sports athletes with giardiosis. A randomized, double-blinded, placebocontrolled clinical study was conducted. Seventy-six athletes with G. lamblia infection participated in the study and were divided into 20-hydroxyecdysone, metronidazole and placebo groups. Clinical, parasitological, haematological and biochemical analyses were performed. Positive results for antiprotozoal therapy were revealed in the 20-hydroxyecdysone and metronidazole groups. After therapy, elimination of G. lamblia was observed in 100.0% of the athletes included in the 20-hydroxyecdysone group. However, G. lamblia was resistant to metronidazole in 4.0% of athletes included in the metronidazole group. A positive clinical response to the therapy occurred in the 20-hydroxyecdysone and metronidazole groups. Our study reveals high antiprotozoal activity of 20-hydroxyecdysone against G. lamblia. Further clinical studies are necessary to evaluate the antiprotozoal efficacy of 20-hydroxyecdysone.

Testing the efficacy and safety of BIO101, for the prevention of respiratory deterioration, in patients with COVID-19 pneumonia (COVA study): a structured summary of a study protocol for a randomised controlled trial.

OBJECTIVES: As of December, 1st, 2020, coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2, resulted in more than 1 472 917 deaths worldwide and death toll is still increasing exponentially. Many COVID-19 infected people are asymptomatic or experience moderate symptoms and recover without medical intervention. However, older people and those with comorbid hypertension, diabetes, obesity, or heart disease are at higher risk of mortality. Because current therapeutic options for COVID-19 patients are limited specifically for this elderly population at risk, Biophytis is developing BIO101 (20-hydroxyecdysone, a Mas receptor activator) as a new treatment option for managing patients with SARS-CoV-2 infection at the severe stage. The angiotensin converting enzyme 2 (ACE2) serves as a receptor for SARS-CoV-2. Interaction between ACE2 and SARS-CoV2 spike protein seems to alter the function of ACE2, a key player in the renin-angiotensin system (RAS). The clinical picture of COVID-19 includes acute respiratory distress syndrome (ARDS), cardiomyopathy, multiorgan dysfunction and shock, all of which might result from an imbalance of the RAS. We propose that RAS balance could be restored in COVID-19 patients through MasR activation downstream of ACE2 activity, with 20-hydroxyecdysone (BIO101) a non-peptidic Mas receptor (MasR) activator. Indeed, MasR activation by 20-hydroxyecdysone harbours anti-inflammatory, anti-thrombotic, and anti-fibrotic properties. BIO101, a 97% pharmaceutical grade 20-hydroxyecdysone could then offer a new therapeutic option by improving the respiratory function and ultimately promoting survival in COVID-19 patients that develop severe forms of this devastating disease. Therefore, the objective of this COVA study is to evaluate the safety and efficacy of BIO101, whose active principle is 20-hydroxyecdysone, in COVID-19 patients with severe pneumonia. TRIAL DESIGN: Randomized, double-blind, placebo-controlled, multi-centre, group sequential and adaptive which will be conducted in 2 parts. Part 1: Ascertain the safety and tolerability of BIO101 and obtain preliminary indication of the activity of BIO101, in preventing respiratory deterioration in the target population Part 2: Re-assessment of the sample size needed for the confirmatory part 2 and confirmation of the effect of BIO101 observed in part 1 in the target population. The study is designed as group sequential to allow an efficient run-through, from obtaining an early indication of activity to a final confirmation. And adaptive - to allow accumulation of early data and adapt sample size in part 2 in order to inform the final design of the confirmatory part of the trial. PARTICIPANTS: Inclusion criteria 1. Age: 45 and above 2. A confirmed diagnosis of COVID-19 infection, within the last 14 days, prior to randomization, as determined by PCR or other approved commercial or public health assay, in a specimen as specified by the test used. 3. Hospitalized, in observation or planned to be hospitalized due to COVID-19 infection symptoms with anticipated hospitalization duration ≥3 days 4. With evidence of pneumonia based on all of the following: a. Clinical findings on a physical examination b. Respiratory symptoms developed within the past 7 days 5. With evidence of respiratory decompensation that started not more than 4 days before start of study medication and present at screening, meeting one of the following criteria, as assessed by healthcare staff: a. Tachypnea: ≥25 breaths per minute b. Arterial oxygen saturation ≤92% c. A special note should be made if there is suspicion of COVID-19-related myocarditis or pericarditis, as the presence of these is a stratification criterion 6. Without a significant deterioration in liver function tests: a. ALT and AST ≤ 5x upper limit of normal (ULN) b. Gamma-glutamyl transferase (GGT) ≤ 5x ULN c. Total bilirubin ≤ 5×ULN 7. Willing to participate and able to sign an informed consent form (ICF). Or, when relevant, a legally authorized representative (LAR) might sign the ICF on behalf of the study participant 8. Female participants should be: at least 5 years post-menopausal (i.e., persistent amenorrhea 5 years in the absence of an alternative medical cause) or surgically sterile; OR a. Have a negative urine pregnancy test at screening b. Be willing to use a contraceptive method as outlined in inclusion criterion 9 from screening to 30 days after last dose. 9. Male participants who are sexually active with a female partner must agree to the use of an effective method of birth control throughout the study and until 3 months after the last administration of the investigational product. (Note: medically acceptable methods of contraception that may be used by the participant and/or partner include combined oral contraceptive, contraceptive vaginal ring, contraceptive injection, intrauterine device, etonogestrel implant, each supplemented with a condom, as well as sterilization and vasectomy). 10. Female participants who are lactating must agree not to breastfeed during the study and up to 14 days after the intervention. 11. Male participants must agree not to donate sperm for the purpose of reproduction throughout the study and until 3 months after the last administration of the investigational product. 12. For France only: Being affiliated with a European Social Security. Exclusion criteria 1. Not needing or not willing to remain in a healthcare facility during the study 2. Moribund condition (death likely in days) or not expected to survive for >7 days - due to other and non-COVID-19 related conditions 3. Participant on invasive mechanical ventilation via an endotracheal tube, or extracorporeal membrane oxygenation (ECMO), or high-flow Oxygen (delivery of oxygen at a flow of ≥16 L/min.). 4. Participant is not able to take medications by mouth (as capsules or as a powder, mixed in water). 5. Disallowed concomitant medication: Consumption of any herbal products containing 20-hydroxyecdysone and derived from Leuzea carthamoides; Cyanotis vaga or Cyanotis arachnoidea is not allowed (e.g. performance enhancing agents). 6. Any known hypersensitivity to any of the ingredients, or excipients of the study medication, BIO101. 7. Renal disease requiring dialysis, or known renal insufficiency (eGFR≤30 mL/min/1.73 m2, based on Cockcroft & Gault formula). 8. In France only: a. Non-affiliation to compulsory French social security scheme (beneficiary or right-holder). b. Being under tutelage or legal guardianship. Participants will be recruited from approximately 30 clinical centres in Belgium, France, the UK, USA and Brazil. Maximum patients' participation in the study will last 28 days. Follow-up of participants discharged from hospital will be performed through post-intervention phone calls at 14 (± 2) and 60 (± 4) days. INTERVENTION AND COMPARATOR: Two treatment arms will be tested in this study: interventional arm 350 mg b.i.d. of BIO101 (AP 20-hydroxyecdysone) and placebo comparator arm 350 mg b.i.d of placebo. Administration of daily dose is the same throughout the whole treatment period. Participants will receive the study medication while hospitalized for up to 28 days or until a clinical endpoint is reached (i.e., 'negative' or 'positive' event). Participants who are officially discharged from hospital care will no longer receive study medication. MAIN OUTCOMES: Primary study endpoint: The proportion of participants with 'negative' events up to 28 days. 'Negative' events are defined as respiratory deterioration and all-cause mortality. For the purpose of this study, respiratory deterioration will be defined as any of the following: Requiring mechanical ventilation (including cases that will not be intubated due to resource restrictions and triage). Requiring extracorporeal membrane oxygenation (ECMO). Requiring high-flow oxygen defined as delivery of oxygen at a flow of ≥16 L/min. Only if the primary endpoint is significant at the primary final analysis the following Key secondary endpoints will be tested in that order: Proportion of participants with events of respiratory failure at Day 28 Proportion of participants with 'positive' events at Day 28. Proportion of participants with events of all-cause mortality at Day 28 A 'positive' event is defined as the official discharge from hospital care by the department due to improvement in participant condition. Secondary and exploratory endpoints: In addition, a variety of functional measures and biomarkers (including the SpO2 / FiO2 ratio, viral load and markers related to inflammation, muscles, tissue and the RAS / MAS pathways) will also be collected. RANDOMIZATION: Randomization is performed using an IBM clinical development IWRS system during the baseline visit. Block-permuted randomization will be used to assign eligible participants in a 1:1 ratio. In part 1, randomization will be stratified by RAS pathway modulator use (yes/no) and co-morbidities (none vs. 1 and above). In Part 2, randomization will be stratified by centre, gender, RAS pathway modulator use (yes/no), co-morbidities (none vs. 1 and above), receiving Continuous Positive Airway Pressure/Bi-level Positive Airway Pressure (CPAP/BiPAP) at study entry (Yes/No) and suspicion of COVID-19 related myocarditis or pericarditis (present or not). BLINDING (MASKING): Participants, caregivers, and the study team assessing the outcomes are blinded to group assignment. All therapeutic units (TU), BIO101 b.i.d. or placebo b.i.d., cannot be distinguished in compliance with the double-blind process. An independent data-monitoring committee (DMC) will conduct 2 interim analyses. A first one based on the data from part 1 and a second from the data from parts 1 and 2. The first will inform about BIO101 safety, to allow the start of recruitment into part 2 followed by an analysis of the efficacydata, to obtain an indication of activity. The second interim analysis will inform about the sample size that will be required for part 2, in order to achieve adequate statistical power. Numbers to be randomised (sample size) Number of participants randomized: up to 465, in total Part 1: 50 (to obtain the proof of concept in COVID-19 patients). Part 2: 310, potentially increased by 50% (up to 465, based on interim analysis 2) (to confirm the effects of BIO101 observed in part 1). TRIAL STATUS: The current protocol Version is V 10.0, dated on 24.09.2020. The recruitment that started on September 1st 2020 is ongoing and is anticipated to finish for the whole study by March2021. TRIAL REGISTRATION: The trial was registered before trial start in trial registries: EudraCT , No. 2020-001498-63, registered May 18, 2020; and Clinicaltrials.gov, identifier NCT04472728 , registered July 15, 2020. FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.

Experimental diabetes induced by streptozotocin in the desert gerbil, Gerbillus gerbillus, and the effects of short-term 20-hydroxyecdysone administration.

The present work was aimed at studying the effects of streptozotocin (STZ; 130 mg/kg) in the desert gerbil, Gerbillus gerbillus, and at evaluating the impact of the short-term administration of 20-hydroxyecdysone (20E; 5 mg/kg). We observed that administration of streptozotocin caused a significant increase in plasmatic glucose and a decrease in insulin levels. The plasma lipid profile and liver glycogen content were also altered. The activities of antioxidant enzymes and malondialdehyde (MDA) levels were increased in the pancreatic tissue of STZ-treated gerbils. Moreover, histopathological and immunohistochemical analysis showed degenerative damage in the pancreas with a decline in the percentage area of ß-cells. Treatment of STZ-treated gerbils with 20E counteracted metabolic disorders and reduced lipid peroxidation. Histological and immunohistochemical studies showed moderate amelioration in the pancreatic structure. These findings indicate that streptozotocin administration induced experimental diabetes in gerbils and that short-term administration of 20E has beneficial effects in glucose homeostasis in STZ-treated gerbils suggesting that 20E may stimulate surviving ß-cells to release more insulin.

Dual inhibition of BACE1 and Aß aggregation by ß-ecdysone: Application of a phytoecdysteroid scaffold in Alzheimer's disease therapeutics.

Current medications for the complex neurological disorder, Alzheimer's disease (AD), can neither stop disease progression nor revert back disease pathogenesis. The present study demonstrates the applicability of a phytoecdysteroid, ß-ecdysone, as a multi-potent agent in AD therapeutics. ß-ecdysone strongly binds to the active site cavity of BACE1 with calculated dissociation constant of 1.75±0.1µM. Steady-state and time-resolved fluorescence spectroscopy reveal that binding of ß-ecdysone induces conformational transition of the protein from open to closed form thereby blocking substrate binding. Even 500nM of the compound completely blocks the enzyme activity. Furthermore, ß-ecdysone strongly inhibits Aß aggregation, evident from ANS and ThT binding assay. Co-incubation of equimolar peptide and ß-ecdysone completely inhibits Aß fibril formation which is further complemented by the AFM study. Low systemic toxicity of ß-ecdysone further extends the applicability of the compound as functional food and dietary supplement for disease management.

Effects of 20-hydroxyecdysone on improving memory deficits in streptozotocin-induced type 1 diabetes mellitus in rat.

We investigate the effects of 20-hydroxyecdysone (20E) on improving memory deficits in the current study by using an animal model of type 1 diabetes mellitus in rats. Animals in control group went on a normal diet. Rats that developed diabetes were divided into 4 groups, including STZ-induced diabetic group which was treated with saline and three 20E groups received different 20E concentrations for 12 weeks. Spatial memory performance was measured in rats by the Morris water maze. The level of nuclear factor-кB (NF-кB) in the brain was determined by real-time quantitative PCR. The mRNA levels and enzyme activities of superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px) and glutathione reductase (GR) were analyzed by real-time quantitative PCR and spectrophotometry. The concentrations of brain-derived neurotrophic factor (BDNF) in the brain were detected by ELISA. Compared with the control group, rats in the STZ-induced diabetic group that developed type 1 diabetes exhibited significant memory loss. In addition to the hippocampus CA1 area that displayed severe damage, significantly higher expression levels of NF-кB were observed in these rats. Furthermore, the expression levels of SOD, catalase, GSH-Px GR and BDNF were significantly decreased in rats with diabetes. By contrast, the treatment with 20E, especially at higher concentrations, reversed the above-mentioned conditions caused by diabetes. The results suggest that the 20E has a protective role in counteracting memory deficits in rats with diabetes of rat, possibly through enhancing the antioxidative ability in the brain.

Ecdysterone protects gerbil brain from temporal global cerebral ischemia/reperfusion injury via preventing neuron apoptosis and deactivating astrocytes and microglia cells.

Ecdysterone (EDS), a common derivative of ecdysteroid, has shown its effects on alleviating cognitive impairment and improving the cognition and memory. However, the mechanisms remain unknown. Using temporal global forebrain ischemia and reperfusion-induced brain injury as a model system, we investigated the roles of EDS in improving cognitive impairment in gerbil. Our results demonstrated that intraperitoneal injection of EDS obviously increased the number of surviving neuron cells by Nissl and neuronal nuclei (NeuN) staining. Indeed, the protecting effects of EDS are because of its ability to prevent the apoptosis of neuron cells as evidenced by TUNEL staining and caspase-3 deactivation in the brain of temporal global forebrain ischemia/reperfusion-treated gerbil. Moreover, EDS administration suppressed the ischemia stimulated activity of astrocytes and microglia cells by inhibiting the production of tumor necrosis alpha (TNF-α) in the brain of gerbil. More importantly, these actions of neurons and astrocytes/microglia cells in response to EDS treatment played pivotal roles in ameliorating the cognitive impairment in the ischemia/reperfusion-injured gerbil. In view of these observations, we not only decipher the mechanisms of EDS in reducing the syndrome of ischemia, but also provide novel perspectives to combat ischemic stroke.

Protective role of 20-OH ecdysone on lipid profile and tissue fatty acid changes in streptozotocin induced diabetic rats.

Hyperlipidemia is an associated complication of diabetes mellitus. The association of hyperglycemia with an alteration of lipid parameters presents a major risk for cardiovascular complications in diabetes. The present study was designed to examine the antihyperlipidemic effect of 20-OH ecdysone on lipid profile and tissue fatty acid changes in streptozotocin induced diabetic rats. The levels of blood glucose, cholesterol, triglycerides, free fatty acids, phospholipids, low density lipoprotein, very low density lipoprotein, high density lipoprotein, lipoprotein lipase, lecithin cholesterol acyl transferase, 3-hydroxy 3-methylglutaryl coenzyme A reductase and fatty acid composition were estimated in plasma, liver and kidneys of control and experimental groups of rats. Oral administration of 20-OH ecdysone at a dose of 5mg/kg bodyweight per day to STZ-induced diabetic rats for a period of 30 days resulted in a significant reduction in fasting blood glucose, cholesterol, triglycerides, free fatty acids, phospholipids, low density lipoprotein, very low density lipoprotein, 3-hydroxy 3-methylglutaryl coenzyme A reductase and elevation of high density lipoprotein, lipoprotein lipase and lecithin cholesterol acyl transferasein comparison with diabetic untreated rats. Moreover, administration of 20-OH ecdysone to diabetic rats also decreased the concentrations of fatty acids, viz., palmitic, stearic (16:1) and oleic acid (18:1), whereas linolenic (18:3) and arachidonic acid (20:4) were elevated. The antihyperlipidemic effect of 20-OH ecdysone was compared with glibenclamide a well-known antihyperglycemic drug. The result of the present study indicates that 20-OH ecdysone showed an antihyperlipidemic effect in addition to its antidiabetic effect in experimental diabetes.

20-Hydroxyecdysone attenuates TGF-ß1-induced renal cellular fibrosis in proximal tubule cells.

UNLABELLED: Renal fibrosis progresses to end stage of diabetes kidney disease, which causes irreversible progressive proximal tubular injury. In a previous study, 20-hydroxyecdysterone (20-HE), a phytoecdysteroid, attenuated renal injury in diabetes models. However, the fibrosis regulatory role remains to be investigated. METHODS: The proximal tubular epithelial cells (designated as HK-2) were treated for 48 h with TGF-ß1 (5 ng/ml) in different concentrations of 20-HE (0 to 500 nM/ml) in the last 24 h of culture. The extracellular fibronectin was measured by ELISA assay. Western blot and immunofluorescence were used to evaluate the expression of TGF-ß1/Smads transducer (including Smad2/3, 4, and 7), epithelial and mesenchymal markers (e.g. E-cadherin and α-smooth muscle actin) and Snail (transcriptional regulators for EMT). RESULTS: 20-HE reverses TGF-ß1-induced increase in fibronectin (both intracellular and extracellular fibronectin). Simultaneously, 20-HE reverses TGF-ß1-induced down-regulation of Smad7. In addition, 20-HE significantly attenuates TGF-ß1-induced upregulation of Smad2/3 and pSmad2/3, and downregulation of E-Cadherin. Moreover, 20-HE dramatically suppresses TGF-ß1-induced increases in the expression of Snail. CONCLUSION: We propose that 20-HE is a potential fibrosis antagonist for renal proximal tubule cells. 20-HE might act through suppressing post-receptor signaling of TGF-ß1 and restoring tubule epithelial character by blocking the expression of Snail.

[Experience in using ecdisten in the treatment of hymenolepiasis].

The paper presents the results of clinical and parasitological studies in 22 patients with hymenolepiasis after treatment with ecdisten. The drug was used in a dose of 5 ml thrice a day during two weeks. It normalized all clinical symptoms in 8 patients and improved most of them in other patients. The results of helminthoovoscopy were interpreted in terms of clinical data. The parasitological efficacy of ecdisten is 36.4%. Its good tolerance, no contraindications, and easiness-to-use permit the drug to be recommended as a supplementary health-improving medication.

Ecdysterone and its activity on some degenerative diseases.

Beside ecdysone (1), ecdysterone (2) is one of the most common 5beta-cholest-7-en-6-one (ecdysteroid) derivatives, which, besides having a hormonal effect on invertebrates, possesses a number of favorable non-hormonal biological effects on mammals. The most interesting of these is that on degenerative diseases, one of which, up to now not clarified in detail, is the so-called adaptogenic effect (protection of the organism against adverse stress factors) associated with anabolic, gastroprotective, and antioxidant effects. A second group of favorable effects is the possibility of suppression of neurodegenerative processes and protection of the cardiovascular system (metabolic syndrome symptom suppression, antidiabetic activity, and protection of heart and blood vessels). Because of these properties, ecdysterone has the potential to be developed as a medicinal agent.

Enhanced angiogenesis and astrocyte activation by ecdysterone treatment in a focal cerebral ischemia rat model.

BACKGROUND AND PURPOSE: We reported previously that ecdysterone (EDS) improves neurologic function after experimental stroke. However, the underlying mechanism remained unclear. The present study was conducted to test whether ecdysterone improves neurologic function by enhancing astrocyte activation and angiogenesis after focal cerebral ischemia in rats. METHODS: Focal cerebral ischemia model was conducted by middle cerebral artery occlusion (MCAO). EDS was intraperitoneally injected at 20 mg kg1 daily for 7 days after MCAO. Neurologic recovery was assessed using the neurologic severity scores. Microvessel density and GFAP expression were detected with immunostaining and analyzed quantitatively with image system. RESULTS: Treatment with EDS significantly improved functional recovery, along with increases in density of cerebral microvessels and astrocyte activation. Microvessel density was significantly higher in EDS treated group than in ischemia control group at all time points, and reached a peak on day 14. EDS treated group had substantial increment in GFAP immunoreactive cells, darker staining color, more and longer nerve processes, higher GFAP expression and area of immunoreactive cells at each time point. CONCLUSION: Our data suggest that EDS treatment enhanced angiogenesis and astrocyte activation which could contribute to functional recovery.

Diverse ecdysterones show different effects on amyloid-ß42 aggregation but all uniformly inhibit amyloid-ß42-induced cytotoxicity.

Amyloid-ß (Aß) plays a pivotal role in Alzheimer's disease (AD) pathogenesis and in toxic mechanisms such as oxidative stress, mitochondrial dysfunction, calcium turbulence, and apoptosis induction. Therefore, interfering with Aß aggregation has long been one of the most promising strategies for AD treatment. Ecdysterones (ECRs) are steroidal hormones in insects and terrestrial plants that have high structural diversity and multiple beneficial pharmacological activities. Here, we studied the effects of six ECRs on Aß aggregation and cytotoxicity. Two ECRs with an acetoxyl group at the 2 or 3 position and saturated chains as side groups showed apparent promotion of Aß42 fibrilization, resulting in less Aß42 oligomers in the samples. Another three with unsaturated side chains clearly inhibited Aß aggregation and disaggregated preformed fibrils, but increased the Aß42 oligomer levels. Nevertheless, our MTT results showed that all ECRs tested inhibited Aß42-induced cytotoxicity. This protective activity may be partly attributable to ECR-mediated amelioration of A&beta42-induced release of reactive oxygen species. Taken together, our findings suggest that ECRs, a series of natural compounds in many plants and insects, have therapeutic potential in AD and that the deduced structure-activity relationships may be beneficial in drug design for the treatment of AD and other amyloidoses.

Beta-ecdysone has bone protective but no estrogenic effects in ovariectomized rats.

Estrogens exert beneficial effects in the bone. Their chronic use however bares several risks. Therefore intensive search for non-estrogenic, bone protective compounds is going on. We observed that an extract of Tinospora cordifolia has antiosteoporotic effects and identified 20-OH-Ecdysone (beta-Ecdysone=Ecd) as a possible candidate for this action. Ovariectomized (ovx) rats were treated orally over 3 months with no Ecd (control) or 18, 57 or 121 mg Ecd/day/animal. Estradiol-17beta benzoate (E2) 159 microg/day/animal) fed animals served as positive controls. Bone mineral density (BMD) of tibia was measured by quantitative computer tomography, serum Osteocalcin and CrossLaps were measured in a ligand binding assay. Utilizing an estrogen receptor (ER) containing cytosolic extract of porcine uteri the capability of Ecd to bind to ER was tested. Ecd did not bind to ER. BMD was reduced by more than 50% in the control. In the Ecd animals BMD was dose dependently higher. Serum CrossLaps was lower in the Ecd and E2 group while serum Osteocalcin levels were decreased in the E2 but increased in the Ecd fed animals. Ecd has an antiosteoporotic effect which does not involve activation of ER.

Beneficial effects of beta-Ecdysone on the joint, epiphyseal cartilage tissue and trabecular bone in ovariectomized rats.

Ecdysteroids are steroids found in invertebrates and plants. In mammals they have protein anabolic effects. We have recently published antiosteoporotic effects of Tinospora cordifolia (TC) extract and the search for the possible active ingredients yielded the presence of beta-Ecdysone (Ecd). Therefore, we investigated the effects of pure Ecd in ovariectomized rats on morphological changes in joint, epiphyseal cartilage and trabecular tissue. Following ovariectomy rats were fed for 1 month with Ecd containing food at a dose of 52.8 mg/day/animal. Positive and negative control animals received 17-beta Estradiol (E(2), 132 microg/day/animal) and soy free (sf) food respectively. At sacrifice, specimens consisting of upper tibiae-lower femurs and knee joint were harvested and processed for histomorphometry. The parameters measured included thickness of the joint cartilage, thickness of the whole epiphyseal growth plate and its three zones. Furthermore, the percentage of trabecular bone in the metaphysis region of tibiae was quantified. Ecd and E(2) induced a significant increase in the thickness of joint cartilage. The whole epiphyseal growth plate and its proliferative and hypertrophic zones were also increased by Ecd whereas E(2) reduced their size. The percentage of trabecular area in the metaphysis of tibia was significantly increased in Ecd and E(2) treated animals. Results provide a plausible explanation for the antiosteoporotic effects of TC. Hence, TC as well as other Ecd producing plants or pure Ecd may be of value in the prevention and treatment of osteoporosis and osteoarthritis which is of increasing importance due to aging and obesity among individuals.

Synergistic effects of the combination of 20-hydroxyecdysone with ampicillin and gentamicin against methicillin-resistant Staphylococcus aureus.

The emergence of methicillin-resistant of Staphylococcus aureus (MRSA) has led to an urgent need for the discovery and development of new antibacterial agents. As part of an ongoing investigation into the antibacterial properties of the natural products, 20-Hydroxyecdysone (20E), isolated from the roots of Achyranthes japonica Nakai, was found to be active methicillin-resistant Staphylococcus aureus (MRSA), either alone or in combination with ampicillin (AM) or gentamicin (GM), vis checkerboard assay. This study investigated the antibacterial activity of 20E, which exhibited poor antibacterial activity (MIC=250-500 microg/mL) against all the bacterial strains tested. The combined activity of ampicillin (AM), gentamicin (GE) plus 20E against MRSA resulted in fractional inhibitory concentrations (FICs) ranging from 4.00 to 0.031 microg/mL, respectively. Meanwhile, the FIC index ranged from 0.16-4.50, indicating a marked synergistic relationship between AM, GE and 20E against MRSA with enterotoxin gene. Time-kill assays also showed a decrease remarkably between the combination and the more active compound. Therefore, this study demonstrated that AM, GE, and 20E can act synergistically in inhibiting MRSA in vitro.

Beta-ecdysterone induces osteogenic differentiation in mouse mesenchymal stem cells and relieves osteoporosis.

The effect on bone tissue of beta-ecdysterone, a type of ecdysteroid found in many plants, has not been previously investigated. In this study, we found that beta-ecdysterone treatment significantly induced alkaline phosphatase (ALP) activity in mesenchymal stem cells in a dose-dependent manner. Real-time polymerase chain reaction (PCR) showed that Runx2, osteocalcin, and type I collagen expression also increased. ICI182780, a specific estrogen receptor antagonist, inhibited the upregulation of ALP activity. Moreover, beta-ecdysterone promoted estrogen receptor (ER) reporter gene activity; however, ICI182780 reversed its effect, suggesting that beta-ecdysterone has stimulatory effects on osteogenic differentiation via the ER. Furthermore, beta-ecdysterone alleviated osteoporosis symptoms in a mouse model without obvious side effects. Therefore beta-ecdysterone may be a promising candidate drug for the treatment of osteoporosis.

[Role of non-haem iron in protecting effect of ecdysterone on development of streptozocin-induced hyperglycaemia in rats].

Chronic hyperglycaemia (60 days) which developed after streptozotocine (STZ) administration (5 mg/100 g) in rats was accompanied with development of severe endothelial dysfunction as well as with disturbed non-haem iron metabolism. It was established by EPR spectroscopy method that STZ administration reduced transferrin levels in the blood as well as pools of iron associated with blood transferrin and with ferritin in the heart and aorta of rats with hyperglycaemia. Chronic ecdysterone administration (100 ng/100 g, 60 days) protects hyperglycaemia development by preventing of non-haem iron metabolism disturbance. These data suppose participation of non-haem iron in mechanisms of ecdysterone protection of streptozotocine-induced hyperglycaemia and ischemia.

The effect of ecdysterone on cerebral vasospasm following experimental subarachnoid hemorrhage in vitro and in vivo.

OBJECTIVES: Cerebral vasospasm has been the dreaded complication of ruptured intracranial aneurysms. Worldwide effort has led to many promising experimental treatments but none was confirmed to be effective in clinical trials. Ecdysterone is an insect steroid hormone. Our previous study showed that ecdysterone might prevent cerebral vasospasm in vitro. Even after all these works, rare attempts have been made to test the effect of ecdysterone on vascular adventitial fibroblast (VAF) proliferation, a process known to play an important role in various pathogenic vascular conditions. Thus, we tested the hypothesis that ecdysterone could affect VAF characteristics and have an effect on SAH induced cerebral vasospasm. METHODS: OxyHb of 100 microM was used in the in vitro study to mimic the clinical situation. The effect of OxyHb on the cell proliferation and migration of cultured aortic smooth muscle cells was investigated. In the in vivo study, 20 rabbits were equally divided into four groups: control group, SAH group, SAH/nimodipine group and SAH/ecdysterone group. Changes in neurological function and cerebral angiograms were observed after SAH. RESULTS: OxyHb increased the proliferation of vascular adventitial fibroblasts at 24 hours. Ecdysterone co-treatment was apparently similar to the suppression of proliferation. Cell cycle analysis indicated that ecdysterone inhibited the progression of vascular adventitial fibroblasts from G1 to S. The results of the migration assay showed that 100 microM OxyHb obviously prompted vascular adventitial fibroblast migration and that ecdysterone would attenuate this effect. In the SAH/nimodipine and SAH/ecdysterone groups, neurological deficit, cerebral vasospasm and structural changes in basilar artery were alleviated with nimodipine or ecdysterone treatment. CONCLUSION: Ecdysterone could affect vascular adventitial fibroblast characteristics and attenuate vasospasm after SAH.