Anti-inflammatory effect of Danhong injection through inhibition of GSDMD-mediated pyroptosis.

BACKGROUND: Pyroptosis is an inflammatory form of cell death that has been implicated in various infectious and non-infectious diseases. Gasdermin family proteins are the key executors of pyroptotic cell death, thus they are considered as novel therapeutic targets for inflammatory diseases. However, only limited gasdermin specific inhibitors have been identified to date. Traditional Chinese medicines have been applied in clinic for centuries and exhibit potential in anti-inflammation and anti-pyroptosis. We attempted to find candidate Chinese botanical drugs which specifically target gasdermin D (GSDMD) and inhibit pyroptosis. METHODS: In this study, we performed high-throughput screening using a botanical drug library to identify pyroptosis specific inhibitors. The assay was based on a cell pyroptosis model induced by lipopolysaccharides (LPS) and nigericin. Cell pyroptosis levels were then evaluated by cell cytotoxicity assay, propidium iodide (PI) staining and immunoblotting. We then overexpressed GSDMD-N in cell lines to investigate the direct inhibitory effect of the drug to GSDMD-N oligomerization. Mass spectrometry studies were applied to identify the active components of the botanical drug. Finally, a mouse model of sepsis and a mouse model of diabetic myocardial infarction were constructed to verify the protective effect of the drug in disease models of inflammation. RESULTS: High-throughput screening identified Danhong injection (DHI) as a pyroptosis inhibitor. DHI remarkably inhibited pyroptotic cell death in a murine macrophage cell line and bone marrow-derived macrophages. Molecular assays demonstrated the direct blockade of GSDMD-N oligomerization and pore formation by DHI. Mass spectrometry studies identified the major active components of DHI, and further activity assays revealed salvianolic acid E (SAE) as the most potent molecule among these components, and SAE has a strong binding affinity to mouse GSDMD Cys192. We further demonstrated the protective effects of DHI in mouse sepsis and mouse myocardial infarction with type 2 diabetes. CONCLUSION: These findings provide new insights for drug development from Chinese herbal medicine like DHI against diabetic myocardial injury and sepsis through blocking GSDMD-mediated macrophage pyroptosis.

Quantification of Cardiomyocyte Contraction In Vitro and Drug Screening by MyocytoBeats.

Cardiomyocyte contractility is the crucial feature of heart function. Quantifying cardiomyocyte contraction in vitro is essential for disease phenotype characterization, mechanism illumination, and drug screening. Although many experimental methods have been employed to determine contraction dynamics in vitro, a time-saving and easy-to-use software is still needed to be developed. We presented a reliable tool, named MyocytoBeats, to measure cardiomyocyte contraction by processing recorded videos. Analysis results by MyocytoBeats of various experimental models have shown a significant linear relationship with another validated software. We also performed pharmacology screen in the platform, and astragaloside IV was identified to stabilize the frequency and amplitude of cardiomyocyte in the arrhythmia model. MyocytoBeats is a high-performance tool for generating cardiomyocyte contraction data of vitro study and shows a great potential in cardiac pharmacology study.

Cardioprotective mechanism of SGLT2 inhibitor against myocardial infarction is through reduction of autosis.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors reduce cardiovascular mortality in patients with diabetes mellitus but the protective mechanism remains elusive. Here we demonstrated that the SGLT2 inhibitor, Empagliflozin (EMPA), suppresses cardiomyocytes autosis (autophagic cell death) to confer cardioprotective effects. Using myocardial infarction (MI) mouse models with and without diabetes mellitus, EMPA treatment significantly reduced infarct size, and myocardial fibrosis, thereby leading to improved cardiac function and survival. In the context of ischemia and nutritional glucose deprivation where autosis is already highly stimulated, EMPA directly inhibits the activity of the Na+/H+ exchanger 1 (NHE1) in the cardiomyocytes to regulate excessive autophagy. Knockdown of NHE1 significantly rescued glucose deprivation-induced autosis. In contrast, overexpression of NHE1 aggravated the cardiomyocytes death in response to starvation, which was effectively rescued by EMPA treatment. Furthermore, in vitro and in vivo analysis of NHE1 and Beclin 1 knockout mice validated that EMPA's cardioprotective effects are at least in part through downregulation of autophagic flux. These findings provide new insights for drug development, specifically targeting NHE1 and autosis for ventricular remodeling and heart failure after MI in both diabetic and non-diabetic patients.

Gasdermin D inhibition confers antineutrophil-mediated cardioprotection in acute myocardial infarction.

Acute myocardial infarction (AMI) induces blood leukocytosis, which correlates inversely with patient survival. The molecular mechanisms leading to leukocytosis in the infarcted heart remain poorly understood. Using an AMI mouse model, we identified gasdermin D (GSDMD) in activated leukocytes early in AMI. We demonstrated that GSDMD is required for enhanced early mobilization of neutrophils to the infarcted heart. Loss of GSDMD resulted in attenuated IL-1ß release from neutrophils and subsequent decreased neutrophils and monocytes in the infarcted heart. Knockout of GSDMD in mice significantly reduced infarct size, improved cardiac function, and increased post-AMI survival. Through a series of bone marrow transplantation studies and leukocyte depletion experiments, we further clarified that excessive bone marrow-derived and GSDMD-dependent early neutrophil production and mobilization (24 hours after AMI) contributed to the detrimental immunopathology after AMI. Pharmacological inhibition of GSDMD also conferred cardioprotection after AMI through a reduction in scar size and enhancement of heart function. Our study provides mechanistic insights into molecular regulation of neutrophil generation and mobilization after AMI, and supports GSDMD as a new target for improved ventricular remodeling and reduced heart failure after AMI.

The prokaryotic Argonaute proteins enhance homology sequence-directed recombination in bacteria.

Argonaute proteins are present and conserved in all domains of life. Recently characterized prokaryotic Argonaute proteins (pAgos) participates in host defense by DNA interference. Here, we report that the Natronobacterium gregoryi Argonaute (NgAgo) enhances gene insertions or deletions in Pasteurella multocida and Escherichia coli at efficiencies of 80-100%. Additionally, the effects are in a homologous arms-dependent but guide DNA- and potential enzyme activity-independent manner. Interestingly, such effects were also observed in other pAgos fragments including Thermus thermophilus Argonaute (TtAgo), Aquifex aeolicus Argonaute (AaAgo) and Pyrococcus furiosus Argonaute (PfAgo). The underlying mechanism of the NgAgo system is a positive selection process mainly through its PIWI-like domain interacting with recombinase A (recA) to enhance recA-mediated DNA strand exchange. Our study reveals a novel system for enhancing homologous sequence-guided gene editing in bacteria.

Diabetes Exacerbates Myocardial Ischemia/Reperfusion Injury by Down-Regulation of MicroRNA and Up-Regulation of O-GlcNAcylation.

Management for patients with diabetes experiencing myocardial infarction remains a challenge. Here the authors show that hyperglycemia- and hyperinsulinemia-induced microRNA-24 (miR-24) reduction and O-GlcNAcylation in the diabetic heart contribute to poor survival and increased infarct size in diabetic myocardial ischemia/reperfusion (I/R). In a mouse model of myocardial I/R, pharmacological or genetic overexpression of miR-24 in hearts significantly reduced myocardial infarct size. Experimental validation revealed that miR-24 targets multiple key proteins, including O-GlcNac transferase, ATG4A, and BIM, to coordinately protect the myocardium from I/R injury. These results establish miR-24 as a promising therapeutic candidate for diabetic I/R injury.