Zihao Wang

Like phosphorylation, the addition of O-linked beta-N-acetylglucosamine (O-GlcNAcylation) is a ubiquitous, reversible process that modifies serine and threonine residues on nuclear and cytoplasmic proteins. Overexpression of the enzyme that adds O-GlcNAc to target proteins, O-GlcNAc transferase (OGT), perturbs cytokinesis and promotes polyploidy, but the molecular targets of OGT that are important for its cell cycle functions are unknown. Here, we identify 141 previously unknown O-GlcNAc sites on proteins that function in spindle assembly and cytokinesis. Many of these O-GlcNAcylation sites are either identical to known phosphorylation sites or in close proximity to them. Furthermore, we found that O-GlcNAcylation altered the phosphorylation of key proteins associated with the mitotic spindle and midbody. Forced overexpression of OGT increased the inhibitory phosphorylation of cyclin-dependent kinase 1 (CDK1) and reduced the phosphorylation of CDK1 target proteins. The increased phosphorylation of CDK1 is explained by increased activation of its upstream kinase, MYT1, and by a concomitant reduction in the transcript for the CDK1 phosphatase, CDC25C. OGT overexpression also caused a reduction in both messenger RNA expression and protein abundance of Polo-like kinase 1, which is upstream of both MYT1 and CDC25C. The data not only illustrate the crosstalk between O-GlcNAcylation and phosphorylation of proteins that are regulators of crucial signaling pathways but also uncover a mechanism for the role of O-GlcNAcylation in regulation of cell division.

BACKGROUND: Iron homeostasis plays a positive role in articular cartilage health. Excessive iron or iron overload can induce oxidative stress damage in chondrocytes and ferroptosis cell death, advancing knee osteoarthritis (KOA). However, up to date, few effective agents treat iron overload-induced KOA (IOKOA). Chinese herbal medicine (CHM) provides abundant resources for drug selection to manage bone metabolic conditions, including osteoporosis. Biochanin A (BCA) is a novel bioactive multifunctional natural compound isolated from Huangqi, which has protective effects on bone loss. Nevertheless, the function and mechanism of BCA in treating IOKOA are still elusive. PURPOSE: This study seeks to uncover the potential therapeutic targets and mechanisms of BCA in the management of KOA with iron accumulation. METHODS: Iron dextrin (500 mg/kg) was intraperitoneally injected into mice to establish the iron overloaded mice model. OA was induced through surgery, and the progression was evaluated eight weeks following surgery. OA severity was evaluated with micro-CT and Safranin-O/Fast green staining in vivo. Iron deposition in the knee joint and synovium was assessed using Perl's Prussian blue staining. Ferric ammonium citrate (FAC) was then administered to primary chondrocytes to evaluate iron regulators mediated iron homeostasis. Toluidine blue staining was utilized to identify chondrocytes in vitro. The vitality of the cells was assessed using the CCK-8 test. The apoptosis rate of cells was measured using Annexin V-FITC/PI assay. The intracellular iron level was detected utilizing the calcein-AM test. Reactive oxygen species (ROS), lipid-ROS, and mitochondrial membrane potentiality were reflected via fluorescence density. Utilizing RT-qPCR and western blotting, the expression level was determined. RESULTS: Micro-CT and histological staining of knee joints showed greater cartilage degradation and higher iron buildup detected in iron-overloaded mice. BCA can reduce iron deposition and the severity of KOA. Toluidine blue staining and the CCK-8 assay indicated that BCA could rescue chondrocytes killed by iron. Cell apoptosis rates were increased due to iron overload but improved by BCA. Further, the intracellular content of iron, ROS, and lipid-ROS was increased with ferric ammonium citrate (FAC) treatment but restored after treatment with different concentrations of BCA. JC-1 staining revealed that BCA could reduce mitochondrial damage induced by iron overload. CONCLUSION: Iron overload was shown to promote chondrocyte ferroptosis in vivo and in vitro. Moreover, iron overload suppressed the expression of collagen II and induced MMP expression by catalyzing ROS generation with mitochondrial dysfunction. Our results showed that BCA could directly reduce intracellular iron concentration by inhibiting TfR1 and promoting FPN but also target the Nrf2/system xc-/GPX4 signaling pathway to scavenge free radicals and prevent lipid peroxidation. The results of this research indicate that BCA regulates iron homeostasis during the progression of osteoarthritis, which can open a new field of treatment for KOA.

Alzheimer’s disease (AD) is an age-related neurodegenerative disorder with cognitive deficits, which is becoming markedly more common in the world. Currently, the exact cause of AD is still unclear, and no curative therapy is available for preventing or mitigating the disease progression. Caffeic acid phenethyl ester (CAPE), a natural phenolic compound derived from honeybee hive propolis, has been reported as a potential therapeutic agent against AD, while its application is limited due to the low water solubility and poor bioavailability. Here, caffeic acid phenethyl ester 4- O -glucoside (FA-97) is synthesized. We validate that FA-97 attenuates H 2 O 2 -induced apoptosis in SH-SY5Y and PC12 cells and suppresses H 2 O 2 -induced oxidative stress by inhibiting the ROS level, malondialdehyde (MDA) level, and protein carbonylation level, as well as induces cellular glutathione (GSH) and superoxide dismutase (SOD). Mechanistically, FA-97 promotes the nuclear translocation and transcriptional activity of Nrf2 associated with the upregulated expression of HO-1 and NQO-1. The prime importance of Nrf2 activation in the neuroprotective and antioxidant effects of FA-97 is verified by Nrf2 siRNA transfection. In addition, FA-97 prevents scopolamine- (SCOP-) induced learning and memory impairments in vivo via reducing neuronal apoptosis and protecting against cholinergic system dysfunction in the hippocampus and cortex. Moreover, the increased MDA level and low total antioxidant capacity in SCOP-treated mouse brains are reversed by FA-97, with the increased expression of HO-1, NQO-1, and nuclear Nrf2. In conclusion, FA-97 protects against oxidative stress-mediated neuronal cell apoptosis and SCOP-induced cognitive impairment by activating Nrf2/HO-1 signaling, which might be developed as a therapeutic drug for AD.

Neutrophil extracellular traps (NETs) have been considered a significant unfavorable factor for wound healing in diabetes, but the mechanisms remain unclear. The therapeutic application of small extracellular vesicles (sEVs) derived from mesenchymal stem cells (MSCs) has received considerable attention for their properties. Hypoxic preconditioning is reported to enhance the therapeutic potential of MSC-derived sEVs in regenerative medicine. Therefore, the aim of this study is to illustrate the detailed mechanism of NETs in impairment of diabetic wound healing and develop a promising NET-targeting treatment based on hypoxic pretreated MSC-derived sEVs (Hypo-sEVs). Excessive NETs were found in diabetic wounds and in high glucose (HG)-induced neutrophils. Further research showed that high concentration of NETs impaired the function of fibroblasts through activating endoplasmic reticulum (ER) stress. Hypo-sEVs efficiently promoted diabetic wound healing and reduced the excessive NET formation by transferring miR-17-5p. Bioinformatic analysis and RNA interference experiment revealed that miR-17-5p in Hypo-sEVs obstructed the NET formation by targeting TLR4/ROS/MAPK pathway. Additionally, miR-17-5p overexpression decreased NET formation and overcame NET-induced impairment in fibroblasts, similar to the effects of Hypo-sEVs. Overall, we identify a previously unrecognized NET-related mechanism in diabetic wounds and provide a promising NET-targeting strategy for wound treatment.