stigmasterol and Hypoxia

To explore the mechanism of Xiaoqinglong decoction (XQLD) in the treatment of infantile asthma (IA) based on network pharmacology and molecular docking. The active ingredients of fdrugs in XQLD were retrieved from Traditional Chinese Medicine Systems Pharmacology database and then the targets of drug ingredients were screened. The disease targets of IA were obtained from OMIM and Gencards databases, and the intersection targets of XQLD in the treatment of IA were obtained by Venny 2.1 mapping of ingredient targets and disease targets. Cytoscape software was used to construct active ingredient-intersection target network. The potential targets of XQLD in the treatment of IA were analyzed by protein-protein interaction network using STRING platform, and the Gene Ontology function and Kyoto Encyclopedia of Genes and Genomes enrichment analysis were obtained by R Studio software. AutoDock was used to perform molecular docking for verification. In this study, 150 active ingredients of XQLD were obtained, including quercetin, kaempferol, β-sitosterol, luteolin, stigmasterol, and so on. And 92 intersection targets of drugs and diseases were obtained, including interleukin 6 (IL6), cystatin 3, estrogen receptor 1, hypoxia inducible factor 1A, HSP90AA1, epidermal growth factor receptor and so on. There were 127 items of Gene Ontology enrichment analysis and 125 Kyoto Encyclopedia of Genes and Genomes enrichment results, showing that apoptosis, IL-17 signaling pathway, tumor necrosis factor signaling pathway, P13K-Akt signaling pathway and other pathways may play a key role in the treatment of IA by XQLD. The results of molecular docking showed that the key active ingredients including quercetin, kaempferol, β-sitosterol, luteolin, stigmasterol, and the core targets including IL6, cystatin 3, estrogen receptor 1, hypoxia inducible factor 1A, HSP90AA1, and epidermal growth factor receptor had good binding activity. Through network pharmacology and molecular docking, the potential targets and modern biological mechanisms of XQLD in the treatment of IA were preliminarily revealed in the study, which will provide reference for subsequent animal experiments and clinical trials.

Topics: Animals; Asthma; Cystatin C; Drugs, Chinese Herbal; ErbB Receptors; Estrogen Receptor alpha; Hypoxia; Interleukin-6; Kaempferols; Luteolin; Medicine, Chinese Traditional; Molecular Docking Simulation; Network Pharmacology; Quercetin; Stigmasterol

Neuronal excitotoxicity induces a plethora of downstream signaling pathways, resulting in the calcium overload-induced excitotoxic cell death, a well-known phenomenon in cerebrovascular and neurodegenerative disorders. The naturally occurring phytosterol, stigmasterol (ST) is known for its potential role in cholesterol homeostasis and neuronal development. However, the ability of ST to protect against the induced excitotoxicity in hippocampal neurons has not been investigated yet.. The present study aimed to investigate whether ST could protect against hypoxia/reoxygenation (H/R)-induced excitotoxicity in hippocampal neurons.. After H/R, neurons were initially subjected to trypan blue exclusion assay for the assessment of cell viability. Live staining using fluorescence dyes namely JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide), DCFDA (2',7'-dichlorofluorescein diacetate) and FM1-43 (N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) were used to measure MMP, ROS and synaptic vesicle pool size. Immunostaining was performed to analyze the expression levels of vesicular glutamate transporter 1 (VGLUT1), N-methyl-D-acetate receptor subunit 2B (GluN2B), LC3BII, p62, and PTEN induced protein kinase 1 (PINK1) in neuron after H/R. Western blotting was carried out to measure the protein expression of GluN2B. The molecular dynamics simulation was employed to elucidate the LXRβ agonistic conformation of ST.. Pre-incubation of neuronal cultures with ST (20 μM) protected against excitotoxicity, and attenuated reactive oxygen species (ROS) generation, double-stranded DNA break, and mitochondrial membrane potential (MMP) loss. ST treatment also resulted in the downregulation of the expressions of VGLUT1 and GluN2B and the reduction of the size of recyclable synaptic vesicle (SV) pool. Like LXRβ agonist GW3695, ST suppressed the expression of GluN2B. Furthermore, ST induced mitophagy through upregulating the expressions of LC3BII, p62, and PINK1. The molecular simulation study showed that ST interacted with the ligand binding domain of liver X receptor β (LXRβ), a known binding receptor of ST, through multiple hydrogen bonding.. Collectively, these findings revealed that ST exhibited a promising neuroprotective effect by regulating both pre- and post-synaptic events following H/R, particularly, attenuation of GluN2B-mediated excitotoxicity and oxidative stress, and induction of mitophagy, and suggested that ST might be a therapeutic promise against ischemic stroke and its associated neurological disorders.

Topics: Animals; Cell Death; Cell Survival; Hippocampus; Hypoxia; Liver X Receptors; Male; Membrane Potential, Mitochondrial; Mitophagy; Molecular Docking Simulation; Neurons; Neuroprotective Agents; Oxidative Stress; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Reperfusion Injury; Stigmasterol