Molecular characterization of the cytochrome P450 enzyme CYP18A1 in Henosepilachna vigintioctopunctata.

In insects, the expression of 20E response genes that initiate metamorphosis is triggered by a pulse of 20-hydroxyecdysone (20E). The 20E pulse is generated through two processes: synthesis, which increases its level, and inactivation, which decreases its titer. CYP18A1 functions as an ecdysteroid 26-hydroxylase and plays a role in 20E removal in several representative insects. However, applying 20E degradation activity of CYP18A1 to other insects remains a significant challenge. In this study, we discovered high levels of Hvcyp18a1 during the larval and late pupal stages, particularly in the larval epidermis and fat body of Henosepilachna vigintioctopunctata, a damaging Coleopteran pest of potatoes. RNA interference (RNAi) targeting Hvcyp18a1 disrupted the pupation. Approximately 75% of the Hvcyp18a1 RNAi larvae experienced developmental arrest and remained as stunted prepupae. Subsequently, they gradually turned black and eventually died. Among the Hvcyp18a1-depleted animals that successfully pupated, around half became malformed pupae with swollen elytra and hindwings. The emerged adults from these deformed pupae appeared misshapen, with shriveled elytra and hindwings, and were wrapped in the pupal exuviae. Furthermore, RNAi of Hvcyp18a1 increased the expression of a 20E receptor gene (HvEcR) and four 20E response transcripts (HvE75, HvHR3, HvBrC, and HvαFTZ-F1), while decreased the transcription of HvßFTZ-F1. Our findings confirm the vital role of CYP18A1 in the pupation, potentially involved in the degradation of 20E in H. vigintioctopunctata.

MicroRNA miR-7-5p targets MARK2 to control metamorphosis in Galeruca daurica.

The MARK2 gene, coding microtubule affinity-regulating kinase or serine/threonine protein kinase, is an important modulator in organism microtubule generation and cell polarity. However, its role in the metamorphosis of insects remains unknown. In this study, we found a conserved miRNA, miR-7-5p, which targets MARK2 to participate in the regulation of the larval-pupal metamorphosis in Galeruca daurica. The dual luciferase reporter assay showed that miR-7-5p interacted with the 3' UTR of MARK2 and repressed its expression. The expression profiling of miR-7-5p and MARK2 displayed an opposite trend during the larval-adult development process. In in-vivo experiments, overexpression of miR-7-5p by injecting miR-7-5p agomir in the final instar larvae down-regulated MARK2 and up-regulated main ecdysone signaling pathway genes including E74, E75, ECR, FTZ-F1 and HR3, which was similar to the results from knockdown of MARK2 by RNAi. In contrast, repression of miR-7-5p by injecting miR-7-5p antagomir obtained opposite effects. Notably, both overexpression and repression of miR-7-5p in the final instar larvae caused abnormal molting and high mortality during the larval-pupal transition, and high mortality during the pupal-adult transition. The 20-hydroxyecdysone (20E) injection experiment showed that 20E up-regulated miR-7-5p whereas down-regulated MARK2. This study reveals that the accurate regulation of miRNAs and their target genes is indispensable for insect metamorphosis.

A comprehensive review of Pfaffia glomerata botany, ethnopharmacology, phytochemistry, biological activities, and biotechnology.

ETHNOPHARMACOLOGICAL RELEVANCE: Pfaffia glomerata (Spreng.) Pedersen, Amaranthaceae, is found in South America, mainly in Brazil, where it is considered a species of great medicinal interest owing to its popular use as a tonic, aphrodisiac, anti-inflammatory, and analgesic. These properties can be attributed to the presence of the phytosteroid, 20-Hydroxyecdysone (ß-ecdysone), the main compound found in its roots. AIM OF THE REVIEW: This review aims to provide information about the botanical characteristics, ethnomedicinal uses, the phytochemistry, the biological activities, and the biotechnology of P. glomerata, an important species to local communities and groups researching medicinal plants of South America. MATERIALS AND METHODS: The information available on P. glomerata was collected from scientific databases (ScienceDirect, PubMed/MEDLINE, SciELO, and Scopus) until June 7, 2023, using the search terms "Pfaffia glomerata", "Pfaffia glomerata (Spreng.) Pedersen", and "Brazilian ginseng". The review includes studies that evaluated the botanical, ethnopharmacological, and phytochemical aspects, biological properties, nutraceutical uses, and the application of biotechnology for improving the biosynthesis of metabolites of interest. RESULTS: A total of 207 studies were identified, with 81 articles read in full. Seventy-six studies were included for qualitative synthesis. Overall, 40 compounds belonging to different classes are presented in this review, including ecdysteroids, triterpenes, saponins, flavonoids, anthraquinones, tannins, coumarins, alkaloids, and polysaccharides. Among them, flavonoids, anthraquinones, tannins, coumarins, and alkaloids were only putatively identified. ß-Ecdysone, triterpenes, saponins, and polysaccharides are the chemical components most frequently identified and isolated from P. glomerata and possibly responsible for ethnopharmacological use and the biological activities of this species, with important in vitro and in vivo activities, such as anti-inflammatory, antidepressant, aphrodisiac, analgesic, gastroprotective, antioxidant, and prebiotic. CONCLUSIONS: This review summarizes discussions about the P. glomerata species, highlighting its ethnopharmacological, chemical, biotechnological, and nutraceutical importance. New scientific studies on this species are encouraged in the search for new therapeutic molecules with pharmaceutical potential and nutraceutical applications.

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.

Indomethacin and 20-hydroxyecdysone influence protein expression in a Spodoptera frugiperda nervous system cell line.

Insecticide mode of action studies provide insights into how new insecticidal actives function and contribute to assessing safety to humans and nontarget organisms. Insect cell lines that express potential target sites can serve as valuable tools in this effort. In this paper, we report on the influence of two signaling molecules on protein expression in a nervous system cell line established from Spodoptera frugiperda (Bayer/BCIRL-SfNS2-0714-TR). We selected this line because we established it in our laboratory and we are experienced in using it. Cells were exposed to the insect developmental hormone (1 µg/mL 20-hydroxyecdysone, 20E) and/or a cyclooxygenase (COX) inhibitor (25 µM indomethacin, INDO; inhibits prostaglandin [PG] biosynthesis) for 24 h (Day 2), 72 h (Day 4), or 120 h (Day 6). We selected a PG biosynthesis inhibitor because PGs act in many aspects of insect biology, such as embryonic development, immunity, and protein phosphorylation. We selected the developmental hormone, 20E, because it also acts in fundamental aspects of insect biology. We identified specific proteins via in silico analysis. Changes in protein expression levels were determined using liquid chromatography-mass spectrometry (MS) + MS-MS. The largest number of changes in protein expression occurred on Day 2. The combination of 20E plus INDO led to 222 differentially expressed proteins, which documents the deep significance of PGs and 20E in insect biology. 20E and, separately, INDO led to changes in 30 proteins each (p value < 0.01; >2X or <0.5X-fold changes). We recorded changes in the expression of 9 or 12 proteins (20E), 10 or 6 proteins (INDO), and 21 or 20 proteins (20E + INDO) on D4 and D6, respectively. While the cell line was established from neuronal tissue, the differentially expressed proteins act in a variety of fundamental cell processes. In this paper, we moved beyond a list of proteins by providing detailed, Gene Ontology term analyses and enrichment, which offers an in-depth understanding of the influence of these treatments on the SfNS2 cells. Because proteins are active components of cell physiology in their roles as enzymes, receptors, elements of signaling transduction pathways, and cellular structures, changes in their expression levels under the influence of signaling molecules provide insights into their function in insect cell physiology.

KAT8 is upregulated and recruited to the promoter of Atg8 by FOXO to induce H4 acetylation for autophagy under 20-hydroxyecdysone regulation.

Selective gene expression in cells in physiological or pathological conditions is important for the growth and development of organisms. Acetylation of histone H4 at K16 (H4K16ac) catalyzed by histone acetyltransferase 8 (KAT8) is known to promote gene transcription; however, the regulation of KAT8 transcription and the mechanism by which KAT8 acetylates H4K16ac to promote specific gene expression are unclear. Using the lepidopteran insect Helicoverpa armigera as a model, we reveal that the transcription factor FOXO promotes KAT8 expression and recruits KAT8 to the promoter region of autophagy-related gene 8 (Atg8) to increase H4 acetylation at that location, enabling Atg8 transcription under the steroid hormone 20-hydroxyecdysone (20E) regulation. H4K16ac levels are increased in the midgut during metamorphosis, which is consistent with the expression profiles of KAT8 and ATG8. Knockdown of Kat8 using RNA interference results in delayed pupation and repression of midgut autophagy and decreases H4K16ac levels. Overexpression of KAT8-GFP promotes autophagy and increases H4K16ac levels. FOXO, KAT8, and H4K16ac colocalized at the FOXO-binding region to promote Atg8 transcription under 20E regulation. Acetylated FOXO at K180 and K183 catalyzed by KAT8 promotes gene transcription for autophagy. 20E via FOXO promotes Kat8 transcription. Knockdown or overexpression of FOXO appeared to give similar results as knockdown or overexpression of KAT8. Therefore, FOXO upregulates KAT8 expression and recruits KAT8 to the promoter region of Atg8, where the KAT8 induces H4 acetylation to promote Atg8 transcription for autophagy under 20E regulation. This study reveals the mechanism that KAT8 promotes transcription of a specific gene.

E93 gene in the swimming crab, Portunus trituberculatus: Responsiveness to 20-hydroxyecdysone and methyl farnesoate and role on regulating ecdysteroid synthesis.

Ecdysone-induced protein 93 (E93) is a metamorphic determinant involved in crosstalk between 20-hydroxyecdysone (20E) and juvenile hormone (JH) during the insect molting process. The present study identified the E93 gene from the swimming crab, P. trituberculatus, and found it was widely distributed in adult tissues. PtE93 mRNA levels in Y-organ and epidermis fluctuated during the molt cycle, suggesting its involvement in juvenile molting. In vitro and in vivo treatments with 20E led to an induction of PtE93 expression in Y-organ and epidermis, while we found the opposite effect for methyl farnesoate (MF) treatments, a crustacean equivalent of insect JH. We also observed that two genes for ecdysteroid biosynthesis, Spook (Spo) and Shadow (Sad), were suppressed by 20E and induced by MF, showing a negative correlation between PtE93 and ecdysteroid biosynthesis. PtE93 RNA interference (RNAi) induced Spo and Sad expression levels, elevated ecdysteroid content in culture medium, and relieved the 20E inhibitory effect on ecdysteroid synthesis, indicating an inhibitory role of PtE93 on ecdysteroid synthesis. Overall, our results suggest that E93 may be involved in the crosstalk between 20E and MF during crustacean molting, and its presence in Y-organ is closely related to ecdysteroid synthesis.

Identification of genes regulated by 20-Hydroxyecdysone in Macrobrachium nipponense using comparative transcriptomic analysis.

BACKGROUND: Macrobrachium nipponense is a freshwater prawn of economic importance in China. Its reproductive molt is crucial for seedling rearing and directly impacts the industry's economic efficiency. 20-hydroxyecdysone (20E) controls various physiological behaviors in crustaceans, among which is the initiation of molt. Previous studies have shown that 20E plays a vital role in regulating molt and oviposition in M. nipponense. However, research on the molecular mechanisms underlying the reproductive molt and role of 20E in M. nipponense is still limited. RESULTS: A total of 240.24 Gb of data was obtained from 18 tissue samples by transcriptome sequencing, with > 6 Gb of clean reads per sample. The efficiency of comparison with the reference transcriptome ranged from 87.05 to 92.48%. A total of 2532 differentially expressed genes (DEGs) were identified. Eighty-seven DEGs associated with molt or 20E were screened in the transcriptomes of the different tissues sampled in both the experimental and control groups. The reliability of the RNA sequencing data was confirmed using Quantitative Real-Time PCR. The expression levels of the eight strong candidate genes showed significant variation at the different stages of molt. CONCLUSION: This study established the first transcriptome library for the different tissues of M. nipponense in response to 20E and demonstrated the dominant role of 20E in the molting process of this species. The discovery of a large number of 20E-regulated strong candidate DEGs further confirms the extensive regulatory role of 20E and provides a foundation for the deeper understanding of its molecular regulatory mechanisms.

Ecdysone and gene expressions for chromatin remodeling, histone modification, and Broad Complex in relation to pupal commitment in Bombyx mori.

In the present study, we tried to clarify when and how pupal commitment (PT) better to use PC occurs and what is involved in the PT of Bombyx mori. To clarify this, we examined the responsiveness of a wing disc to ecdysone, referring to metamorphosis-related BR-C, development-related Myc and Wnt, and chromatin remodeling-related genes at around the predicted PT stage of the Bombyx wing disc. Wing disc responsiveness to juvenile hormone (JH) and ecdysone was examined using Methoprene and 20-hydroxyecdysone (20E) in vitro. The body weight of B. mori increased after the last larval ecdysis, peaked at Day 5 of the fifth larval instar (D5L5), and then decreased. The responsiveness of the wing disc to JH decreased after the last larval ecdysis up to D3L5. Bmbr-c (the Broad Complex of B. mori) showed enhanced expression in D4L5 wing discs with 20E treatment. Some chromatin remodeler and histone modifier genes (Bmsnr1, Bmutx, and Bmtip60) showed upregulation after being cultured with 20E in D4L5 wing discs. A low concentration of 20E is suggested to induce responsiveness to 20E in D4L5 wing discs. Bmbr-c, Bmsnr1, Bmutx, and Bmtip60 were upregulated after being cultured with a low concentration of 20E in D4L5 wing discs. The expression of Bmmyc and Bmwnt1 did not show a change after being cultured with or without 20E in D4L5 wing discs, while enhanced expression was observed with 20E in D5L5 wing discs. From the present results, we concluded that PT of the wing disc of B. mori occurred beginning on D4L5 with the secretion of low concentrations of ecdysteroids. Bmsnr1, Bmutx, Bmtip60, and BR-C are also involved.

Genome-wide CRISPR screening reveals key genes and pathways associated with 20-hydroxyecdysone signal transduction in the silkworm (Bombyx mori).

Metamorphosis is a complex developmental process involving multiple pathways and a large number of genes that are regulated by juvenile hormone (JH) and 20-hydroxyecdysone (20E). Despite important progress in understanding various aspects of silkworm biology, the hormone signaling pathway in the silkworm remains poorly understood. Genome-wide screening using clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein 9 (Cas9)-based libraries has recently emerged as a novel method for analyzing genome function, enabling further research into essential genes, drug targets, and virus-host interaction. Previously, we constructed a genome-wide CRISPR/Cas9-based library of the silkworm (Bombyx mori) and successfully revealed the genes involved in biotic or abiotic stress factor responses. In this study, we used our silkworm CRISPR library and large-scale genome-wide screening to analyze the key genes in the silkworm 20E signaling pathway and their mechanisms of action. Functional annotation showed that 20E regulates key proteins in processes that mainly occur in the cytoplasm and nucleus. Pathway enrichment analysis showed that 20E can activate phosphorylation and may affect innate immunity, interfere with intracellular nutrition and energy metabolism, and eventually cause cell apoptosis. The screening results were experimentally validated by generating cells with knockout alleles of the relevant genes, which had increased tolerance to 20E. Our findings provide a panoramic overview of signaling in response to 20E in the silkworm, underscoring the utility of genome-wide CRISPR mutant libraries in deciphering hormone signaling pathways and the mechanisms that regulate metamorphosis in insects.

Feeding cessation after feeding on 20-hydroxyecdysone in the Formosan subterranean termite.

BACKGROUND: To control subterranean termite pests, chitin synthesis inhibitor (CSI) baits have been widely applied. Despite CSI baits having low impacts on the environment, they require a lengthy time period to eliminate colonies. 20-hydroxyecdysone (20E) was proposed to speed up the baiting process as it showed faster mortality than CSI baits. However, the efficacy of 20E has previously not been tested at the colony level prior to applying in the field. RESULTS: We compared the effect of 20E, 20E + noviflumuron, noviflumuron and untreated control using colonies of Coptotermes formosanus. Our result revealed that both 20E and 20E + noviflumuron did not accelerate colony elimination and termite activity remained relatively stable during the observation periods. To determine the limited effects of 20E, we further investigated feeding duration and consumption amount of 20E with different concentrations (control, 100 and 1000 ppm) for 10 days. Termites ceased feeding after 1 day in 100 and 1000 ppm treatment and 100% mortality was observed within 10 days in 1000 ppm 20E, while mortality in the 100 ppm 20E treated group was much lower than that in the 1000 ppm group. Furthermore, no termites molted in the control and termites died from hyperecdysonism in 1000 ppm 20E treatment, whereas about 20% of termites molted in 100 ppm 20E. CONCLUSION: This study demonstrated that 20E may not be suitable as a sole active ingredient to accelerate elimination of a subterranean termite colony, while CSI baits and lower concentrations of 20E may reduce the lengthy time period in colony elimination. © 2023 Society of Chemical Industry.

Dpp-mediated TGF-ß signaling regulates vitellogenesis through 20-hydroxyecdysone signaling in the cabbage beetle, Colaphellus bowringi.

The Dpp signaling, as one of the branches within the TGF-ß superfamily, plays a crucial role in regulating various biological processes in insects. However, its impact on female reproduction through vitellogenesis remains unclear. In this study, the expression profiles implied that the Dpp signaling genes, including Dpp, Punt, Mad, and Medea, were up-regulated during reproductive development in the ovary of Colaphellus bowringi. Knockdown of these five Dpp signaling genes revealed significant effects of Dpp, Tkv, Mad, and Medea on ovarian development through vitellogenesis in the fat body. Our finding further indicated that Dpp signaling influences the expression of 20-hydroxyecdysone (20E) receptor and responsive genes in the fat body. Additionally, knockdown of 20E receptor EcR resulted in similar phenotypes as observed in the Dpp pathway genes knockdown, implying a regulatory role for Dpp signaling via EcR in vitellogenesis. Furthermore, knocking down Dpp, Tkv, and EcR in female adults led to a reduction in total dry weight and protein content, as well as the expression of mTOR, a factor linked to protein intake. These results suggest that the Dpp signaling pathway modulates vitellogenesis by impacting the AA/TOR-mediated 20E pathway in the fat body, providing novel insights into the network governing insect reproduction and offering potential targets for controlling female pest reproduction.

Ecdiesteroides y su actividad farmacológica sobre el músculo esquelético / Ecdysteroids and their pharmacological activity on skeletal muscle

Introducción: Los ecdiesteroides presentan cualidades únicas dentro del reino vegetal y animal. Su similitud a esteroides endógenos de mamíferos les otorga actividad biológica sobre el tejido muscular esquelético. Sin embargo, su mecanismo de acción está por definirse en su totalidad. Método: Se realizó una revisión narrativa utilizando la evidencia científica más relevante. Se consultaron de las bases de datos Medline, Google Scholar, Scielo y Wiley, incluyéndose y excluyéndose trabajos acordes a los criterios del autor. Resultados: La actividad de los ecdiesteroides, principalmente de la Ecdisterona (Ec), podría deberse a la interacción con Mas, receptor acoplado a proteína-G transmembrana (GPCR), y la posterior activación del receptor de estrógenos β (ER β) no nuclear. Dicho mecanismo de acción induce la activación de la ruta alternativa del Sistema Renina-Angiotensina-Aldosterona (RAA) aboliendo los mecanismos de degradación muscular y, mediante la activación indirecta de Erβ, se suprime la expresión del gen de la miostatina. Esta actividad biológica pudiera conferir a los ecdiesteroides propiedades farmacológicas óptimas para impedir la degradación proteico-muscular, tales como la regeneración y reparación del tejido. Conclusiones: Ec ha demostrado poseer propiedades farmacológicas interesantes para el abordaje alternativo de patologías musculodegenerativas por sus efectos anticatabólicos. Aunque prosigue la investigación para su implementación en la clínica, esta siendo utilizada en la industria deportiva y en ensayos para el tratamiento de diferentes patologías. (AU)

Introduction: Ecdysteroids present unique qualities within the plant and animal kingdoms. Their similarity to en-dogenous mammalian steroids allows them to present biological activity on skeletal muscle tissue. However, this molecule’s action mechanism remains to be fully understood. Method: A narrative review was carried out using the most relevant scientific evidence. Different databases such as Medline, Google Scholar, Scielo and Wiley were consulted. Works were included or excluded according to the author ́s criterium. Results: Ecdysteroids’ activity, mostly that of ecdysterone, might be due to the interaction with Mas receptor, a transmembrane G-Protein Coupled Receptor (GPCR), and the subsequent indirect activation of β-Estrogen Recep-tor ́s (β-ER) non-nuclear form. Said action mechanism induces the alternative pathway activation of the Renin-An-giotensin-Aldosterone System (RAAS), abolishing muscular degradation mechanism. Finally, through β-ER activa-tion, the myostatin gene is supressed. This biological activity could provide ecdysteroids optimal pharmacological properties to prevent muscular protein degradation. These include tissue regeneration and repair. Conclusions: Due to its anticatabolic effects, Ec has shown great pharmacological properties that could make it work as an alternative treatment for degenerative muscle pathologies. Although investigations regarding Ec are still in progress, it has already been used by the sports’ industry and in several clinical trials that focus on the treatment of other diseases. (AU)

Are 20-hydroxyecdysone and related genes potential biomarkers of sublethal exposure to lipid-altering contaminants?

In Daphnia magna, 20-hydroecdysone (20E) is the main molting hormone and its metabolism is of interest to identify new biomarkers of exposure to contaminants. The present study aimed to (i) assess baseline levels of 20E and transcription levels of four related-genes (shade, neverland, ultraspiracle, and ecdysteroid receptor); and (ii) evaluate effects in D. magna after 21 days of exposure to fenarimol (anti-ecdysteroid) and a mixture of gemfibrozil and clofibric acid (lipid-lowering drugs) at sublethal concentrations. Endpoints included transcription of the target genes and quantification of 20E, mortality, and reproduction of daphnids. Baseline results showed that average responses were relatively similar and did not vary more than 2-fold. However, intra-day variation was generally high and could be explained by sampling individuals with slightly different stages of their development. Exposure tests indicated a significant decrease in daphnid reproduction following chronic exposure to a concentration of 565 µg/L of fenarimol. However, no difference was observed between the control and exposed groups for any of the investigated genes, nor for the levels of 20E after 21 days of exposure. Following exposition to gemfibrozil and clofibric acid at 1 µg/L, no changes were observed for the measured parameters. These results suggest that changes in transcription levels of the target genes and concentrations of 20E may not be sensitive endpoints that can be used as biomarkers of sublethal exposure to the target compounds in D. magna. Measuring multiple time points instead of a single measure as well as additional molecular endpoints obtained from transcriptomic and metabolomic studies could afford more insights on the changes occurring in exposed daphnids to lipid-altering compounds and identify efficient biomarkers of sublethal exposure.

Investigations into the human metabolism of ecdysterone.

The possible performance-enhancing effects and medical benefits of ecdysterone (ECDY) have been discussed several times throughout the last decades. In 2020, the World Anti-Doping Agency include ECDY in their monitoring programme and continued this prevalence study until now. Only little is known about the human metabolism of ECDY besides the first study performed on human subjects in the field of sports drug testing that was already conducted in 2001. Aim of this study was the in-depth investigation on human ECDY metabolism to improve its detectability and to support the decision-making processes as to how ECDY can be implemented most effectively into sports drug testing regulations. In a first trial, one male volunteer was administered with threefold deuterated ECDY to enable the detection and potential identification of all urinary metabolites still comprising the deuterium label by employing hydrogen isotope ratio mass spectrometry and high-resolution/high-accuracy mass spectrometry. Samples were collected for up to 14 days, and metabolites excreted unconjugated, glucuronidated, and sulphated were investigated. The detected deuterated metabolites were confirmed in a second administration trial encompassing two male and one female volunteers. After the administration of 50 mg of unlabelled ECDY, urine samples were collected for up to 7 days. Besides the already described urinary metabolites of ECDY, more than 20 new metabolites were detected encompassing all expected metabolic conversions including side chain cleavage at C21. A large interindividual variation in the amounts of excreted metabolites was visible, and considerable differences in abundances of early- and late-excretion phase metabolites were observed.

The Role of 20-Hydroxyecdysone in the Control of Carbohydrate Levels in Drosophila melanogaster under Short-Term Heat Stress.

It is known that 20-hydroxyecdysone is one of the most important hormonal regulators of development, reproduction and adaptation to unfavorable conditions in insects. Here, we show for the first time that exogenous 20-hydroxyecdysone increases the content of two main insect carbohydrates, trehalose and glucose, in Drosophila melanogaster females both in normal conditions and under short-term heat stress. It is found that the levels of both trehalose and glucose increase after 39 min of heat exposure and return to their original levels after 1.5 h. A scheme of hormonal regulation of carbohydrate content under heat stress, involving 20-hydroxyecdysone, juvenile hormone, and dopamine, is suggested.

Identification of quality markers for Cyanotis arachnoidea and analysis of its physiological mechanism based on chemical pattern recognition, network pharmacology, and experimental validation.

Cyanotis arachnoidea C. B. Clarke is a traditional Chinese medicinal herb that has a limited clinical use in the treatment of diabetes mellitus (DM) in minority areas of Guizhou in China. However, few prior reports are available on the quality control of Cyanotis arachnoidea, and its quality markers and hypoglycemic mechanism are still unclear. The purpose of this study is to explore the quality markers (Q-markers) of Cyanotis arachnoidea and predict its hypoglycemic mechanism. In this study, ultra-high-performance liquid chromatography (UHPLC) fingerprint combined with chemical pattern recognition were performed, and four differential components were screened out as quality markers, including 20-Hydroxyecdysone, 3-O-acetyl-20-hydroxyecdysone, Ajugasterone C, and 2-O-acetyl-20-hydroxyecdysone. Network pharmacology analysis revealed 107 therapeutic target genes of Cyanotis arachnoidea in DM treatment, and the key targets were Akt1, TNF, IL-6, MAPK3, and JUN. The hypoglycemic mode of action of Cyanotis arachnoidea may be mediated by tumor necrosis factor (TNF) signaling, cancer, insulin resistance, and JAK-STAT pathways. Molecular docking analysis disclosed that the foregoing quality markers effectively bound their key target genes. An in vitro experiment conducted on pancreatic islet ß-cells indicated that the forenamed active components of Cyanotis arachnoidea had hypoglycemic efficacy by promoting PI3K/Akt and inhibiting MAPK signaling. UHPLC also accurately quantified the quality markers. The identification and analysis of quality markers for Cyanotis arachnoidea is expected to provide references for the establishment of a quality control evaluation system and clarify the material basis and hypoglycemic mechanisms of this traditional Chinese medicine (TCM).

Estrogen-related receptor functions via the 20-hydroxyecdysone and IIS/TOR signaling pathways to regulate the development and morphology changes of ant Polyrhachis vicina Roger (Hymenoptera, Formicidae).

Estrogen-related receptor (ERR) is a key regulator of insect growth, development, and metabolic processes in insects; however, the molecular mechanisms underlying its effects are not fully understood. We investigated roles of 20-hydroxyecdysone (20E) and insulin/insulin-like signaling/target of rapamycin (IIS/TOR) signaling pathways in the effects of PvERR on larval development, metamorphosis, and adult growth in ant Polyrhachis vicina Roger. PvFOXO expression levels depended on caste and developmental stage. PvERR RNAi significantly reduced the expression levels of IIS/TOR signaling pathway genes and 20E signaling pathway genes in fourth-instar larvae, pupae, females, and workers and significantly increased the expression levels of IIS/TOR signaling pathway genes PvFOXO and PvAkt in males. PvFOXO RNAi resulted in developmental defects and increased mortality. After PvFOXO RNAi, the expression of PvERR, 20E signaling pathway genes, and IIS/TOR signaling pathway genes decreased significantly in pupae, females, and workers and increased significantly in fourth-instar larvae. Exogenous 20E attenuated expression changes induced by PvFOXO RNAi in a sex- and stage-specific manner. These results indicate that ERR interacts with 20E and IIS/TOR signaling pathways to regulate caste determination, metamorphosis, and male fertility in P. vicina and that correlations between PvERR and PvFOXO are caste- and stage-specific.

Chemical profiling and simultaneous quantification of major bioactive constituents from Cocculus hirsutus by UPLC-QqQ-MS.

Cocculus hirsutus is a widely used herb in traditional systems of medicine for the treatment of various diseases. In the present study, five alkaloids (1-5), two flavonoids (6-7), one triterpenoid (8), and three steroids (9-10) were isolated from the roots of Cocculus hirsutus and further crude extract was analyzed by LC-Q-Tof-MS/MS in positive ionization mode leading to the identification of ten metabolites through comparison of exact molecular masses from their MS/MS spectra, mass fragmentation studies and with literature data. In addition, a method was developed and validated for the quantification of four bio-active compounds [Sinococuline (1), Magnoflorine (2), (E)-N-feruloyltyramine (3), and 20-Hydroxyecdysone (10)] using UPLC-QqQ-MS in multiple reaction monitoring (MRM) mode for the first time. The method has shown good linearity with correlation coefficients (r2) higher than 0.9916 for all four compounds. The intra- and inter-day precision were in the range of 0.3-6.1% and from 0.7% to 8.8%, respectively. The matrix effects of all the four analytes were found in the range of 94.7 ± 2.8-112.7 ± 3.7%. Overall, our study provides a reliable and rapid approach by hyphenated LC-MS/MS using high-resolution mass spectrometers for identification and quantification of bioactive constituents from the root extracts of Cocculus hirsutus.

The effects of 20-hydroxyecdysone on nuclear shape, heterochromatin quantity and gray-level co-occurrence matrix texture analysis of adrenal zona fasciculata cells in an obese gerbil (Gerbillus tarabuli) model for metabolic syndrome: a correlational study.

The aim of this study was to reveal the effects of obesity and phytotherapy with 20-hydroxyecdysone (20E) on the nuclei of adrenal zona fasciculata (ZF) in the gerbil Gerbillus tarabuli by analyzing nuclear shape and gray-level co-occurrence matrix (GLCM) texture characteristics and by quantifying heterochromatin. Twelve gerbils were divided into three groups: control (C), HC and HC-20E (animals receiving a high-calorie-diet without or with a supplement of 20E, respectively). The adrenals were removed and fixed for histological and statistical analysis. Principal component analysis showed a positive correlation of area, perimeter and textural correlation in C. Nevertheless, a negative correlation was recorded for contrast and entropy. The obesity caused a disorder in nuclear texture; negative correlation was noted with heterochromatin fraction, which may be related to increased ZF activity. However, administration of 20E seems to improve the nuclear state by preserving circularity, uniformity and homogeneity of nuclei as well as the proportion of heterochromatin, which could be a sign of a downregulation of cell activity.Our results suggest that new techniques of image processing could contribute to the understanding of nuclear changes associated with obesity and its possible therapy in this gerbil model for metabolic syndrome.