The potential LXRß agonist stigmasterol protects against hypoxia/reoxygenation injury by modulating mitophagy in primary hippocampal neurons.

BACKGROUND: 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. PURPOSE: The present study aimed to investigate whether ST could protect against hypoxia/reoxygenation (H/R)-induced excitotoxicity in hippocampal neurons. METHODS: 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 LXRß agonistic conformation of ST. RESULT: 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 LXRß 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 ß (LXRß), a known binding receptor of ST, through multiple hydrogen bonding. CONCLUSION: 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.

Integrated System Pharmacology and In Silico Analysis Elucidating Neuropharmacological Actions of Withania somnifera in the Treatment of Alzheimer's Disease.

BACKGROUND: Withania somnifera (WS), also referred to as Medhya Rasayana (nootropic or rejuvenating), has traditionally been prescribed for various neurological ailments, including dementia. Despite substantial evidence, pharmacological roles of WS, neither as nootropic nor as an antidementia agent, are well-understood at the cellular and molecular levels. OBJECTIVES: We aimed at elucidating the pharmacological action mechanisms of WS root constituents against Alzheimer's Disease (AD) pathology. METHODS: Various bioinformatics tools and resources, including DAVID, Cytoscape, NetworkAnalyst and KEGG pathway database were employed to analyze the interaction of WS root bioactive molecules with the protein targets of AD-associated cellular processes. We also used a molecular simulation approach to validate the interaction of compounds with selected protein targets. RESULTS: Network analysis revealed that ß-sitosterol, withaferin A, stigmasterol, withanolide A, and withanolide D are the major constituents of WS root that primarily target the cellular pathways such as PI3K/Akt signaling, neurotrophin signaling and toll-like receptor signaling and proteins such as Tropomyosin receptor Kinase B (TrkB), Glycogen Synthase Kinase-3ß (GSK-3ß), Toll-Like Receptor 2/4 (TLR2/4), and ß-secretase (BACE-1). Also, the in silico analysis further validated the interaction patterns and binding affinity of the major WS compounds, particularly stigmasterol, withanolide A, withanolide D and ß-sitosterol with TrkB, GSK-3ß, TLR2/4, and BACE-1. CONCLUSION: The present findings demonstrate that stigmasterol, withanolide A, withanolide D and ß-sitosterol are the major metabolites that are responsible for the neuropharmacological action of WS root against AD-associated pathobiology, and TrkB, GSK-3ß, TLR2/4, and BACE-1 could be the potential druggable targets.

The Biological Safety of Stainless Steel Needles Used in Warm-needling.

Warm-needling (also called thermo-acupuncture) is a combination of acupuncture and moxibustion. Due to the intense heat involved, there have been concerns over the biological safety of the acuneedles used in the treatment. This paper reports two phases of a safety test. For a preliminary test, we compared the temperature change patterns of stainless steel (SS304) needles and traditional gold alloy needles, which have been increasingly replaced by the former. To verify the effects of the presence of coating materials, the main test involved three different kinds of SS304: silicone-coated, salicylic acid-coated and non-coated needles. Each group of needles was tested for pH level, heavy metals and UV absorbance spectrum along with biological tests on the cytotoxicity and hemolysis of the needle. All the tests on the extractants from the needles were negative. In the biological tests, each test result showed a significant difference from the positive control samples, while no significant difference was observed compared with the negative control samples. In the hemolysis tests, all samples satisfied the Korean Government Standards. All the results suggest that SS304 needles are biologically safe to be used in warm-needling, though they can be improved to perform as well as the gold alloy needles in terms of temperature fluctuations.