This study identified 8 members including NjBIN2 of the GSK3 family in Nardostachys jatamansi by bioinformatics analysis. Moreover, the phylogenetic tree revealed that the GKS3 family members of N. jatamansi had a close relationship with those of Arabidopsis. RT-qPCR results showed that NjBIN2 presented a tissue-specific expression pattern with the highest expression in roots, suggesting that NjBIN2 played a role in root growth and development. In addition, the application of epibrassinolide or the brassinosteroid(BR) synthesis inhibitor(brassinazole) altered the expression pattern of NjBIN2 and influenced the photomorphogenesis(cotyledon opening) and root development of N. jatamansi, which provided direct evidence about the functions of NjBIN2. In conclusion, this study highlights the roles of BIN2 in regulating the growth and development of N. jatamansi by analyzing the expression pattern and biological function of NjBIN2. It not only enriches the understanding about the regulatory mechanism of the growth and development of N. jatamansi but also provides a theoretical basis and potential gene targets for molecular breeding of N. jatamansi with improved quality in the future.
Brassinosteroids/metabolism* ; Steroids, Heterocyclic/metabolism* ; Gene Expression Regulation, Plant/drug effects* ; Plant Proteins/metabolism* ; Phylogeny ; Nardostachys/metabolism* ; Plant Growth Regulators/pharmacology* ; Plant Roots/drug effects*

OBJECTIVES: To assess the efficacy and safety of Tiaozhou Ziyin (TZZY) recipe for treatment of premature ovarian insufficiency (POI) and explore the possible mechanisms. METHODS: We used bioinformatics analyses and network pharmacology to identify the main active ingredients in TZZY recipe and their core targets, which were verified by Western blotting. We tested the efficacy and safety of the recipe in 60 POI patients, who were randomized into control group (n=30) with Femoston treatment and TZZY group (n=30) with additional TZZY recipe treatment for 3 menstrual cycles. RESULTS: The core active ingredients of TZZY recipe included kaempferol, β-sitosterol, luteolin, and quercetin. The core targets included SRC, TP53, STAT3, PIK3CA, and MAPK3, which were involved in positive regulation of cell movement and protein phosphorylation, the cancer pathways and the PI3K-Akt signaling pathway. Molecular docking showed that the core active ingredients had good binding ability with the core targets. In female rat models of POI, TZZY recipe treatment significantly up-regulated ovarian expressions of p-PI3K and p-Akt proteins. In the clinical trial, treatment with Femoston and Femoston plus TZZY recipe both significantly increased E₂ levels and reduced FSH and LH levels and Kupperman scores of the patients, and the combined treatment produced significantly stronger effects. Both treatments increased the number of antral follicles of the patients, but the combined treatment also significantly increased the levels of AMH. CONCLUSIONS The therapeutic mechanism of TZZY recipe for POI involves multiple active ingredients, multiple therapeutic targets and multiple pathways, and activating the PI3K /Akt pathway is one of its main mechanisms of action, to improve ovarian reserve function, alleviate clinical symptoms, and enhance clinical efficacy in POI patients.
Female ; Primary Ovarian Insufficiency/drug therapy* ; Humans ; Drugs, Chinese Herbal/therapeutic use* ; Animals ; Rats ; Molecular Docking Simulation ; Signal Transduction ; Sitosterols/therapeutic use* ; Kaempferols/therapeutic use*

OBJECTIVES: To elucidate the anti-aging effect of β-sitosterol (BS), an important component in the fruits of Alpinia oxyphylla Miq., in C. elegans and its regulatory effect on ETS-5 gene to modulate ferroptosis. METHODS: C. elegans treated with 10 µg/mL BS were monitored for survival time and changes in body length, motility, and reproductive function. The effect of ETS-5 gene knockdown on survival time of C. elegans was observed, and the changes in fat accumulation and lipid redox homeostasis in the transfected C. elegans were assessed using Oil Red O staining and by detecting MDA levels and the GSH/GSSG ratio. The mRNA expression levels of ferroptosis-related genes (FTN-1, GPX-1 and AAT-9) were detected using qPCR. The effects of BS treatment and ETS-5 knockdown on AAT-9 enzyme activity in C. elegans were examined. The effect of BS on nuclear localization of FEV (the human homolog of ETS-5) was validated in cultured human umbilical venous endothelial cells (HUVECs). RESULTS: Both BS treatment and ETS-5 knockdown significantly prolonged the lifespan, promoted lipid accumulation and reduced lipid peroxidation in C. elegans. ETS-5 knockdown resulted in upregulated expressions of the ferroptosis repressors GPX-1, AAT-9 and FTN-1 and increased the GSH/GSSG ratio in C. elegans. CONCLUSIONS BS inhibits ferroptosis in C. elegans by suppressing the expression of ETS-5 transcription factor and hence the activity of AAT-9 enzyme, a key gene for ferroptosis, which in turn prolongs the lifespan of C. elegans.
Animals ; Caenorhabditis elegans/physiology* ; Ferroptosis/drug effects* ; Alpinia/chemistry* ; Sitosterols/pharmacology* ; Longevity/drug effects* ; Fruit/chemistry* ; Humans

A comprehensive phytochemical investigation of the leaves and twigs of Physalis angulata. var. villosa resulted in the isolation of 23 withanolide derivatives, including one novel 13,20-γ-lactone withanolide derivative (1) and three new withanolide derivatives (2-4). Architecturally, physalinin A (1) represents the first identified type B withanolide featuring a 13,20-γ-lactone moiety. The molecular structures of all isolates were elucidated using an integrated approach combining nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), infrared (IR) spectroscopy, and quantum chemical calculations to confirm structural assignments. The antiproliferative activities of all isolated withanolides were evaluated against four human cancer cell lines (HEL, HCT-116, Colo320DM, and MDA-MB-231). Among them, eight derivatives (2, 5-8, 14, 15, and 23) exhibited significant inhibitory effects, with half-maximal inhibitory concentration (IC₅₀) values of 0.18 ± 0.03 to 17.02 ± 0.21 μmol·L^-1. Structure-activity relationship (SAR) analysis suggested that the presence of an epoxide ring enhances anticancer activity, potentially through increased reactivity or specific interactions with molecular targets involved in cancer progression. These findings underscore the pharmacological potential of withanolides as promising lead compounds for the development of novel anticancer therapeutics.
Withanolides/isolation & purification* ; Physalis/chemistry* ; Humans ; Molecular Structure ; Cell Line, Tumor ; Antineoplastic Agents, Phytogenic/isolation & purification* ; Cell Proliferation/drug effects* ; Plant Leaves/chemistry* ; Plant Extracts/pharmacology*

Stigmasterol is a plant sterol with anti-apoptotic, anti-oxidative and anti-inflammatory effect through multiple mechanisms. In this study, we further assessed whether it exerts protective effect on human brain microvessel endothelial cells (HBMECs) against ischemia-reperfusion injury and explored the underlying mechanisms. HBMECs were used to establish an in vitro oxygen and glucose deprivation/reperfusion (OGD/R) model, while a middle cerebral artery occlusion (MCAO) model of rats were constructed. The interaction between stigmasterol and EPHA2 was detected by surface plasmon resonance (SPR) and cellular thermal shift assay (CETSA). The results showed that 10 μmol·L^-1 stigmasterol significantly protected cell viability, alleviated the loss of tight junction proteins and attenuated the blood-brain barrier (BBB) damage induced by OGD/R in thein vitro model. Subsequent molecular docking showed that stigmasterol might interact with EPHA2 at multiple sites, including T692, a critical gatekeep residue of this receptor. Exogenous ephrin-A1 (an EPHA2 ligand) exacerbated OGD/R-induced EPHA2 phosphorylation at S897, facilitated ZO-1/claudin-5 loss, and promoted BBB leakage in vitro, which were significantly attenuated after stigmasterol treatment. The rat MCAO model confirmed these protective effects in vivo. In summary, these findings suggest that stigmasterol protects HBMECs against ischemia-reperfusion injury by maintaining cell viability, reducing the loss of tight junction proteins, and attenuating the BBB damage. These protective effects are at least meditated by its interaction with EPHA2 and inhibitory effect on EPHA2 phosphorylation.
Humans ; Animals ; Rats ; Stigmasterol ; Phosphorylation ; Endothelial Cells ; Molecular Docking Simulation ; Reperfusion Injury ; Blood-Brain Barrier ; Glucose ; Microvessels ; Oxygen

Saccharomyces cerevisiae/genetics* ; Ergosterol ; Fermentation

Sterols are a class of cyclopentano-perhydrophenanthrene derivatives widely present in living organisms. Sterols are important components of cell membranes. In addition, they also have important physiological and pharmacological activities. With the development of synthetic biology and metabolic engineering technology, yeast cells are increasingly used for the heterologous synthesis of sterols in recent years. Nevertheless, since sterols are hydrophobic macromolecules, they tend to accumulate in the membrane fraction of yeast cells and consequently trigger cytotoxicity, which hampers the further improvement of sterols yield. Therefore, revealing the mechanism of sterol transport in yeast, especially understanding the working principle of sterol transporters, is vital for designing strategies to relieve the toxicity of sterol accumulation and increasing sterol yield in yeast cell factories. In yeast, sterols are mainly transported through protein-mediated non-vesicular transport mechanisms. This review summarizes five types of sterol transport-related proteins that have been reported in yeast, namely OSBP/ORPs family proteins, LAM family proteins, ABC transport family proteins, CAP superfamily proteins, and NPC-like sterol transport proteins. These transporters play important roles in intracellular sterol gradient distribution and homeostasis maintenance. In addition, we also review the current status of practical applications of sterol transport proteins in yeast cell factories.
Saccharomyces cerevisiae/genetics* ; Sterols ; Phytosterols ; Biological Transport ; ATP-Binding Cassette Transporters/genetics*

Steroids are a class of medicines with important physiological and pharmacological effects. In pharmaceutical industry, steroidal intermediates are mainly prepared through Mycobacteria transformation, and then modified chemically or enzymatically into advanced steroidal compounds. Compared with the "diosgenin-dienolone" route, Mycobacteria transformation has the advantages of abundant raw materials, cost-effective, short reaction route, high yield and environmental friendliness. Based on genomics and metabolomics, the key enzymes in the phytosterol degradation pathway of Mycobacteria and their catalytic mechanisms are further revealed, which makes it possible for Mycobacteria to be used as chassis cells. This review summarizes the progress in the discovery of steroid-converting enzymes from different species, the modification of Mycobacteria genes and the overexpression of heterologous genes, and the optimization and modification of Mycobacteria as chassis cells.
Mycobacterium/metabolism* ; Steroids/metabolism* ; Phytosterols/metabolism* ; Genomics

In this study, the effect of brassinosteroid(BR) on the physiological and biochemical conditions of 2-year-old Panax notoginseng under the cadmium stress was investigated by the pot experiments. The results showed that cadmium treatment at 10 mg·kg~(-1) inhibited the root viability of P. notoginseng, significantly increased the content of H_2O_2 and MDA in the leaves and roots of P. noto-ginseng, caused oxidative damage of P. notoginseng, and reduced the activities of SOD and CAT. Cadmium stress reduced the chlorophyll content of P. notoginseng, increased leaf F_o, reduced F_m, F_v/F_m, and PIABS, and damaged the photosynthesis system of P. notoginseng. Cadmium treatment increased the soluble sugar content of P. notoginseng leaves and roots, inhibited the synthesis of soluble proteins, reduced the fresh weight and dry weight, and inhibited the growth of P. notoginseng. External spray application of 0.1 mg·L~(-1) BR reduced the H_2O_2 and MDA content in P. notoginseng leaves and roots under the cadmium stress, alleviated cadmium-induced oxidative damage to P. notoginseng, improved the antioxidant enzyme activity and root activity of P. notoginseng, increased the content of chlorophyll, reduced the F_o of P. notoginseng leaves, increased F_m, F_v/F_m, and PIABS, alleviated the cadmium-induced damage to the photosynthesis system, and improved the synthesis ability of soluble proteins. In summary, BR can enhance the anti-cadmium stress ability of P. notoginseng by regulating the antioxidant enzyme system and photosynthesis system of P. notoginseng under the cadmium stress. In the context of 0.1 mg·L~(-1) BR, P. notoginseng can better absorb and utilize light energy and synthesize more nutrients, which is more suitable for the growth and development of P. notoginseng.
Cadmium/metabolism* ; Antioxidants/pharmacology* ; Panax notoginseng ; Brassinosteroids/pharmacology* ; Chlorophyll/metabolism* ; Plant Roots/metabolism* ; Stress, Physiological

Plant adaptation to adverse environment depends on transmitting the external stress signals into internal signaling pathways, and thus forming a variety of stress response mechanisms during evolution. Brassinosteroids (BRs) is a steroid hormone and widely involved in plant growth, development and stress response. BR is perceived by cell surface receptors, including the receptor brassinosteroid-insensitive 1 (BRI1) and the co-receptor BRI1-associated-kinase 1 (BAK1), which in turn trigger a signaling cascade that leads to the inhibition of BIN2 and activation of BES1/BZR1 transcription factors. BES1/BZR1 can directly regulate the expression of thousands of downstream responsive genes. Studies in the model plant Arabidopsis thaliana have shown that members of BR biosynthesis and signal transduction pathways, particularly protein kinase BIN2 and its downstream transcription factors BES1/BZR1, can be extensively regulated by a variety of environmental factors. In this paper, we summarize recent progresses on how BR biosynthesis and signal transduction are regulated by complex environmental factors, as well as how BR and environmental factors co-regulate crop agronomic traits, cold and salt stress responses.
Arabidopsis/metabolism* ; Brassinosteroids/pharmacology* ; DNA-Binding Proteins/metabolism* ; Gene Expression Regulation, Plant ; Stress, Physiological