LPS and ATP-induced Death of PMA-differentiated THP-1 Macrophages and its Validation.

Cell death is a fundamental process in all living organisms. The protocol establishes a lipopolysaccharide (LPS) and adenosine triphosphate (ATP)-induced phorbol-12-myristate-13-acetate (PMA)-differentiated lipid deposition in human monocyte (THP-1) macrophage model to observe cell death. LPS combined with ATP is a classic inflammatory induction method, often used to study pyroptosis, but apoptosis and necroptosis also respond to stimulation by LPS/ATP. Under normal circumstances, phosphatidylserine is only localized in the inner leaflet of the plasma membrane. However, in the early stages of pyroptosis, apoptosis, and necroptosis, the cell membrane remains intact and exposed to phosphatidylserine, and in the later stages, the cell membrane loses its integrity. Here, flow cytometry was used to analyze Annexin V and 7-Aminoactinomycin D (AAD) double staining to detect the cell death from the whole cells. The results show that substantial cells died after stimulation with LPS/ATP. Using scanning electron microscopy, we observe the possible forms of cell death in individual cells. The results indicate that cells may undergo pyroptosis, apoptosis, or necroptosis after stimulation with LPS/ATP. This protocol focuses on observing the death of macrophages after stimulation with LPS/ATP. The results showed that cell death after LPS and ATP stimulation is not limited to pyroptosis and that apoptosis and necrotic apoptosis can also occur, helping researchers better understand cell death after LPS and ATP stimulation and choose a better experimental method.

The Scutellaria-Coptis herb couple and its active small-molecule ingredient wogonoside alleviate cytokine storm by regulating the CD39/NLRP3/GSDMD signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: A drug pair is a fundamental aspect of traditional Chinese medicine prescriptions. Scutellaria baicalensis Georgi and Coptis chinensis Franch, commonly used as an herb couple (SBCC), are representative heat-clearing and dampness-drying drugs. They possess functions such as clearing heat, drying dampness, purging fire, and detoxifying. These herbs are used in both traditional and modern medicine for treating inflammation. AIM OF THE STUDY: This study investigated the effects of SBCC on cytokine storm syndrome (CSS) and explored its potential regulatory mechanism. MATERIALS AND METHODS: We assessed the impact of SBCC in a sepsis-induced acute lung injury mouse model by administering an intraperitoneal injection of LPS (15 mg/kg). The cytokine levels in the serum and lungs, the wet-to-dry ratio of the lungs, and lung histopathological changes were evaluated. The macrophages in the lung tissue were examined through transmission electron microscopy. Western blot was used to measure the levels of the CD39/NLRP3/GSDMD pathway-related proteins. Immunofluorescence imaging was used to assess the activation of pro-caspase-1 and ASC and their interaction. AMP-Glo™ assay was used to screen for active ingredients in SBCC targeting CD39. One of the ingredients was selected, and its effect on cell viability was assessed. We induced inflammation in macrophages using LPS + ATP and detected the levels of proinflammatory factors. The images of cell membrane large pores were captured using scanning electron microscopy, the interaction between NLRP3 and ASC was detected using immunofluorescence imaging, and the levels of CD39/NLRP3/GSDMD pathway-related proteins were assessed using Western blot. RESULTS: SBCC administration effectively mitigated LPS-induced cytokine storm, pulmonary edema and lung injury. Furthermore, it repressed the programmed death of lung tissue macrophages by inhibiting the NLRP3/GSDMD pyroptosis pathway and regulating the CD39 purinergic pathway. Based on the results of the AMP-Glo™ assay, we selected wogonoside for further valuation. Wogonoside alleviated LPS + ATP-induced inflammatory damage by regulating the inhibiting the NLRP3/GSDMD pyroptosis pathway and regulating the CD39 purinergic pathway. However, its effect on NLRP3 is not mediated though CD39. CONCLUSION: SBCC and its active small-molecule ingredient, wogonoside, improved CSS by regulating the NLRP3/GSDMD pyroptosis pathway and its upstream CD39 purinergic pathway. It is essential to note that the regulatory effect of wogonoside on NLRP3 is not mediated by CD39.

Sepsis-Induced Acute Lung Injury Is Alleviated by Small Molecules from Dietary Plants via Pyroptosis Modulation.

Sepsis-induced acute respiratory distress syndrome (ARDS) has high morbidity and mortality, and it has three major pathogeneses, namely alveolar-capillary barrier destruction, elevated gut permeability, and reduced neutrophil extracellular traps (NETS), all of which are pyroptosis-involved. Due to limitations of current agents like adverse reaction superposition, inevitable drug resistance, and relatively heavier financial burden, naturally extracted small-molecule compounds have a broad market even though chemically modified drugs have straightforward efficacy. Despite increased understanding of the molecular biology and mechanism underlying sepsis-induced ARDS, there are no specific reviews concerning how small molecules from dietary plants alleviate sepsis-induced acute lung injury (ALI) via regulating pyroptotic cell death. Herein, we traced and reviewed the molecular underpinnings of sepsis-induced ALI with a focus on small-molecule compounds from dietary plants, the top three categories of which are respectively flavonoids and flavone, terpenoids, and polyphenol and phenolic acids, and how they rescued septic ALI by restraining pyroptosis.

Sennoside A is a novel inhibitor targeting caspase-1.

The assembly of inflammasomes drives caspase-1 activation, which further promotes proinflammatory cytokine secretion and downstream pyroptosis. The discovery of novel caspase-1 inhibitors is pivotal to developing new therapeutic means for inflammasome-involved diseases. In our present study, sennoside A (Sen A), a popular ingredient in multiple weight-loss medicines and dietary supplements, is found to potently inhibit the enzymatic activity of caspase-1 in vitro. Sen A considerably decreased IL-1ß production in macrophages stimulated by LPS plus ATP, nigericin or MSU as well as poly(dA:dT) transfection, and remedied ROS-involved pyroptosis via caspase-1 inhibition. Mechanistically, Sen A not only suppressed the assembly of both NLRP3 and AIM2 inflammasome but also affected the priming process of NLRP3 inflammasome by blocking NF-κB signaling. Sen A significantly ameliorated the pathophysiological effect in LPS-, MSU- and carrageenan-challenged rodent models by suppressing inflammasome activation. Furthermore, P2X7 was indispensable for Sen A inhibiting NLRP3 inflammasome since it failed to further decrease IL-1ß and IL-18 production in LPS plus ATP-stimulated BMDMs that were transfected with P2X7 siRNA. Sen A also restrained the large pore-forming functionalities of the P2X7R as verified by the YO-PRO-1 uptake assay. Taken together, Sen A inactivates caspase-1 to inhibit NLRP3 and AIM2 inflammasome-involved inflammation in a P2X7-dependent manner, making it an attractive candidate as a caspase-1 small-molecular inhibitor.

Cytokine storm-calming property of the isoquinoline alkaloids in Coptis chinensis Franch.

Coronavirus disease (COVID-19) has spread worldwide and its effects have been more devastating than any other infectious disease. Importantly, patients with severe COVID-19 show conspicuous increases in cytokines, including interleukin (IL)-6, monocyte chemoattractant protein (MCP)-1, IL-8, tumor necrosis factor (TNF)-α, IL-1, IL-18, and IL-17, with characteristics of the cytokine storm (CS). Although recently studied cytokine inhibitors are considered as potent and targeted approaches, once an immunological complication like CS happens, anti-viral or anti-inflammation based monotherapy alone is not enough. Interestingly, certain isoquinoline alkaloids in Coptis chinensis Franch. (CCFIAs) exerted a multitude of biological activities such as anti-inflammatory, antioxidant, antibacterial, and immunomodulatory etc, revealing a great potential for calming CS. Therefore, in this timeline review, we report and compare the effects of CCFIAs to attenuate the exacerbation of inflammatory responses by modulating signaling pathways like NF-ĸB, mitogen-activated protein kinase, JAK/STAT, and NLRP3. In addition, we also discuss the role of berberine (BBR) in two different triggers of CS, namely sepsis and viral infections, as well as its clinical applications. These evidence provide a rationale for considering CCFIAs as therapeutic agents against inflammatory CS and this suggestion requires further validation with clinical studies.