Potassium Efflux Research Articles

Voltage-gated potassium channels as a potential therapeutic target for the treatment of neurological and psychiatric disorders.

Voltage-gated potassium channels are a widely distributed subgroup of potassium channels responsible for the efflux of potassium in the repolarisation of the cell membrane, and hence contribute to the latency and propagation of action potentials. As they are causal to synaptic transmission, alterations to the structure of these channels can lead to a variety of neurological and psychiatric diseases. The Kv3 subfamily of voltage-gated potassium channels are found on many neurons in the brain, including inhibitory interneurons where they contribute to fast-frequency firing. Changes to the firing ability of these interneurons can lead to an imbalance of inhibitory and excitatory neurotransmission. To date, we have little understanding of the mechanism by which excitatory and inhibitory inputs become imbalanced. This imbalance is associated with cognitive deficits seen across neurological and neuropsychiatric disorders, which are currently difficult to treat. In this review, we collate evidence supporting the hypothesis that voltage-gated potassium channels, specifically the Kv3 subfamily, are central to many neurological and psychiatric disorders, and may thus be considered as an effective drug target. The collective evidence provided by the studies reviewed here demonstrates that Kv3 channels may be amenable to novel treatments that modulate the activity of these channels, with the prospect of improved patient outcome.

Application of a modified multifunctional short peptide in the treatment of periodontitis

Periodontitis is a chronic inflammatory disease involving plaque biofilm as a pathogenic factor. Potassium ion plays an important role in cellular homeostasis; a large outflow of potassium may lead to local inflammation progression. In this work, the multifunctional short peptide molecule BmKTX-33 was designed by modifying the BmKTX, a Kv1.3 potassium channel inhibitor. This was to explore its antibacterial properties, capability of maintaining cell ion homeostasis, and bone-forming capacity. The results showed that BmKTX-33 had inhibitory effects on S. gordonii, F. nucleatum, and P. gingivalis. Moreover, BmKTX-33 also inhibited excessive potassium outflow in inflammatory environments. Finally, BmKTX-33 promoted MC3T3-E1 early osteogenesis while suppressing the NLRP3 inflammasome's production. In conclusion, BmKTX-33 not only has antibacterial properties, but also can inhibit the expression of NLRP3 inflammasome and play an anti-inflammatory role.

G protein-gated inwardly rectifying K+ (GIRK/Kir3) channels: Molecular, cellular, and subcellular diversity.

G protein-gated inwardly rectifying K+ (GIRK/Kir3) channels are mainly expressed in excitable cells such as neurons and atrial myocytes, where they can respond to a wide variety of neurotransmitters. Four GIRK subunits have been found in mammals (GIRK1-4) and act as downstream targets for various Gαi/o-linked G protein-coupled receptors (GPCRs). Activation of GIRK channels produces a postsynaptic efflux of potassium from the cell, responsible for hyperpolarization/inhibition of the neuron. A growing body of evidence suggests that dysregulation of GIRK signalling can lead to excessive or deficient neuronal excitability, which contributes to neurological diseases and disorders. Therefore, GIRK channels are proposed as new pharmacological targets. The function of GIRK channels in neurons is not only determined by their biophysical properties but also by their cellular and subcellular localization patterns and densities on the neuronal surface. GIRK channels can be located within several subcellular compartments, where they have many different functional implications. This subcellular localization changes dynamically along the neuronal surface in response to drug intake. Ongoing research is focusing on determining the proteins that form macromolecular complexes with GIRK channels and are responsible for fast and precise signalling under physiological conditions, and how their alteration is implicated in pathological conditions. In this review, the distinct regional, cellular, and subcellular distribution of GIRK channel subunits in the brain will be discussed in view of their possible functional and pathological implications.

Life-Threatening Hypokalemic Paralysis and Prevention of Severe Rebound Hyperkalemia in a Female with Barium Poisoning: A Rare Case Report

Hypokalemic paralysis is a clinical syndrome characterized by acute flaccid paralysis with concomitant hypokalemia. Complications, such as acute respiratory failure and cardiac arrhythmias, can be fatal. If treated appropriately, the patient can recover without any sequelae. We present a rare case of life-threatening hypokalemic paralysis following the ingestion of an unknown substance. At presentation, her serum potassium concentration was 1.9 mmol/L. A review of the patient’s history confirmed the ingestion of barium chloride. She was diagnosed with acute barium poisoning characterized by high serum and urine barium levels. Aggressive potassium repletion was administered intravenously and orally. Her serum potassium concentration dropped to 1.5 mmol/L and peaked at 5.4 mmol/L following treatment. The patient achieved a complete recovery and was discharged without sequelae. Barium can competitively block the potassium inward rectifier channels and interfere with the efflux of intracellular potassium, leading to severe hypokalemia. Our report illustrates a rare presentation of acute barium intoxication and a differential diagnosis indicating hypokalemic paralysis. We also discuss the pathophysiological features and compare the clinical findings with cases of rebound hyperkalemia.

ML335 inhibits TWIK2 channel-mediated potassium efflux and attenuates mitochondrial damage in MSU crystal-induced inflammation

BackgroundActivation of the NLRP3 inflammasome is critical in the inflammatory response to gout. Potassium ion (K+) efflux mediated by the TWIK2 channel is an important upstream mechanism for NLRP3 inflammasome activation. Therefore, the TWIK2 channel may be a promising therapeutic target for MSU crystal-induced inflammation. In the present study, we investigated the effect of ML335, a known K2P channel modulator, on MSU crystal-induced inflammatory responses and its underlying molecular mechanisms.MethodsBy molecular docking, we calculated the binding energies and inhibition constants of five K2P channel modulators (Hydroxychloroquine, Fluoxetine, DCPIB, ML365 and ML335) with TWIK2. Intracellular potassium ion concentration and mitochondrial function were assessed by flow cytometry. The interaction between MARCH5 and SIRT3 was demonstrated by immunoprecipitation and Western blotting assay. MSU suspensions were injected into mouse paw and peritoneal cavity to induce acute gout model.ResultsML335 has the highest binding energy and the lowest inhibition constant with TWIK2 in the five calculated K2P channel modulators. In comparison, among these five compounds, ML335 efficiently inhibited the release of IL-1β from MSU crystal-treated BMDMs. ML335 decreased MSU crystal-induced K+ efflux mainly dependent on TWIK2 channel. More importantly, ML335 can effectively inhibit the expression of the mitochondrial E3 ubiquitin ligase MARCH5 induced by MSU crystals, and MARCH5 can interact with the SIRT3 protein. ML335 blocked MSU crystal-induced ubiquitination of SIRT3 protein by MARCH5. In addition, ML335 improved mitochondrial dynamics homeostasis and mitochondrial function by inhibiting MARCH5 protein expression. ML335 attenuated the inflammatory response induced by MSU crystals in vivo and in vitro.ConclusionInhibition of TWIK2-mediated K+ efflux by ML335 alleviated mitochondrial injury via suppressing March5 expression, suggesting that ML335 may be an effective candidate for the future treatment of gout.

Antibacterial Mechanism of Garviecin LG34 Against S. Aureus and L. Monocytogenes and its Application in Milk Preservation

The objective of this study was to reveal the antibacterial mode of action of garviecin LG34 against S. aureus CICC 21600 and L. monocytogenes CICC 21633 and measure the inhibitions on these two foodborne pathogenic bacteria in milk. Antibacterial mechanism of garviecin LG34 was ascertained by its effect on the efflux of Potassium (K+) ions, extracellular electrical conductivity, UV-absorbing substances, potential across the membrane (ΔΨ), and cell permeability. The inhibition of garviecin LG34 against S. aureus CICC 21600 and L. monocytogenes CICC 21600 in milk was studied by viable counting method. Supplementation with 160 AU/ml of garviecin LG34 had a bactericidal effect on S. aureus CICC 21600 and L. monocytogenes CICC 21633. A total of 80, 160, and 320 AU/ml of garviecin LG34 resulted in the effusion of potassium ion and UV-absorbing substances, the leakage of cellular electrolytes, and the dissipation of electrical potential across the membrane of these two food-borne bacteria and showed a dose-dependent. Moreover, the increase in cell permeability of both strains was observed by flow cytometer after cells treated with 160 AU/ml of garviecin LG34. Garviecin LG34 significantly inhibited the growth of these two food-borne bacteria in milk, especially in skimmed milk. Garviecin LG34 could cause pore formation, intracellular materials release, and permeability increase of S. aureus CICC 21600 and L. monocytogenes CICC 21633, and could be applied to milk as bio-preservative.

Calcium-activated Potassium Channels as Amplifiers of TRPV4-mediated Pulmonary Edema Formation in Male Mice.

As a mechanosensitive cation channel and key regulator of vascular barrier function, endothelial transient receptor potential vanilloid type 4 (TRPV4) contributes critically to ventilator-induced lung injury and edema formation. Ca2+ influx via TRPV4 can activate Ca2+-activated potassium (KCa) channels, categorized into small (SK1-3), intermediate (IK1), and big (BK) KCa, which may in turn amplify Ca2+ influx by increasing the electrochemical Ca2+ gradient and thus promote lung injury. The authors therefore hypothesized that endothelial KCa channels may contribute to the progression of TRPV4-mediated ventilator-induced lung injury. Male C57Bl/6J mice were ventilated for 2 h with low or high tidal volumes in the presence or absence of the nonselective KCa antagonists apamin and charybdotoxin or the selective IK1 antagonist TRAM34. Lung injury was similarly assessed in overventilated, endothelial-specific TRPV4-deficient mice or TRAM34-treated C57Bl/6J mice challenged with intratracheal acid installation. Changes in intracellular calcium Ca2+ concentration ([Ca2+]i) were monitored by real-time imaging in isolated-perfused lungs in response to airway pressure elevation or in human pulmonary microvascular endothelial cells in response to TRPV4 activation with or without inhibition of KCa channels. Analogously, changes in intracellular potassium concentration ([K+]i) and membrane potential were imaged in vitro. Endothelial TRPV4 deficiency or inhibition of KCa channels, and most prominently inhibition of IK1 by TRAM34, attenuated ventilator-induced lung injury as demonstrated by reduced lung edema, protein leak, and quantitative lung histology. All KCa antagonists reduced the [Ca2+]i response to mechanical stimulation or direct TRPV4 activation in isolated lungs. TRAM34 and charybdotoxin yet not apamin prevented TRPV4-induced potassium efflux and membrane hyperpolarization in human pulmonary microvascular endothelial cells. TRAM34 also attenuated the TRPV4 agonist-induced Ca2+ influx in vitro and reduced acid-induced lung injury in vivo. KCa channels, specifically IK1, act as amplifiers of TRPV4-mediated Ca2+ influx and establish a detrimental feedback that promotes barrier failure and drives the progression of ventilator-induced lung injury.

Mycobacterium tuberculosis Utilizes Serine/Threonine Kinase PknF to Evade NLRP3 Inflammasome-driven Caspase-1 and RIPK3/Caspase-8 Activation in Murine Dendritic Cells.

Dendritic cells (DCs) are crucial for initiating the acquired immune response to infectious diseases such as tuberculosis. Mycobacterium tuberculosis has evolved strategies to inhibit activation of the NLRP3 inflammasome in macrophages via its serine/threonine protein kinase, protein kinase F (PknF). It is not known whether this pathway is conserved in DCs. In this study, we show that the pknF deletion mutant of M. tuberculosis (MtbΔpknF) compared with wild-type M. tuberculosis-infected cells induces increased production of IL-1β and increased pyroptosis in murine bone marrow-derived DCs (BMDCs). As shown for murine macrophages, the enhanced production of IL-1β postinfection of BMDCs with MtbΔpknF is dependent on NLRP3, ASC, and caspase-1/11. In contrast to macrophages, we show that MtbΔpknF mediates RIPK3/caspase-8-dependent IL-1β production in BMDCs. Consistently, infection with MtbΔpknF results in increased activation of caspase-1 and caspase-8 in BMDCs. When compared with M. tuberculosis-infected cells, the IL-6 production by MtbΔpknF-infected cells was unchanged, indicating that the mutant does not affect the priming phase of inflammasome activation. In contrast, the activation phase was impacted because the MtbΔpknF-induced inflammasome activation in BMDCs depended on potassium efflux, chloride efflux, reactive oxygen species generation, and calcium influx. In conclusion, PknF is important for M. tuberculosis to evade NLRP3 inflammasome-mediated activation of caspase-1 and RIPK3/caspase-8 pathways in BMDCs.

Chronic kidney disease leads to microglial potassium efflux and inflammasome activation in the brain

Cognitive impairment is common in extracerebral diseases such as chronic kidney disease (CKD). Kidney transplantation reverses cognitive impairment, indicating that cognitive impairment driven by CKD is therapeutically amendable. However, we lack mechanistic insights allowing development of targeted therapies. Using a combination of mouse models (including mice with neuron-specific IL-1R1 deficiency), single cell analyses (single nuclei RNA sequencing and single cell thallium autometallography), human samples and in vitro experiments we demonstrate that microglia activation impairs neuronal potassium homeostasis and cognition in CKD. CKD disrupts the barrier of brain endothelial cells in vitro and the blood-brain barrier in vivo, establishing that the uremic state modifies vascular permeability in the brain. Exposure to uremic conditions impairs calcium homeostasis in microglia, enhances microglial potassium efflux via the calcium-dependent channel KCa3.1, and induces p38-MAPK associated IL-1β maturation in microglia. Restoring potassium homeostasis in microglia using a KCa3.1-specific inhibitor (TRAM34) improves CKD-triggered cognitive impairment. Likewise, inhibition of the IL-1β receptor 1 (IL-R1) using anakinra or genetically abolishing neuronal IL-1R1 expression in neurons prevent CKD-mediated reduced neuronal potassium turnover and CKD-induced impaired cognition. Accordingly, in CKD mice, impaired cognition can be ameliorated by either preventing microglia activation or inhibiting IL-1R-signaling in neurons. Thus, our data suggest that potassium efflux from microglia triggers their activation, which promotes microglia IL-1β release and IL-1R1-mediated neuronal dysfunction in CKD. Hence, our study provides new mechanistic insight into cognitive impairment in association with CKD and identifies possible new therapeutic approaches.

Loss-of-function/gain-of-function polymorphisms of the ATP sensitive P2X7R influence sepsis, septic shock, pneumonia, and survival outcomes.

Extracellular ATP (eATP) released from damaged cells activates the P2X7 receptor (P2X7R) ion channel on the surface of surrounding cells, resulting in calcium influx, potassium efflux and inflammasome activation. Inherited changes in the P2X7R gene (P2RX7) influence eATP induced responses. Single nucleotide polymorphisms (SNPs) of P2RX7 influence both function and signaling of the receptor, that in addition to ion flux includes pathogen control and immunity. Subjects (n = 105) were admitted to the ICU at the University Hospital Ulm, Germany between June 2018 and August 2019. Of these, subjects with a diagnosis of sepsis (n = 75), were also diagnosed with septic shock (n = 24), and/or pneumonia (n = 42). Subjects with pneumonia (n = 43) included those without sepsis (n = 1), sepsis without shock (n = 29) and pneumonia with septic shock (n = 13). Out of the 75 sepsis/septic shock patients, 33 patients were not diagnosed with pneumonia. Controls (n = 30) were recruited to the study from trauma patients and surgical patients without sepsis, septic shock, or pneumonia. SNP frequencies were determined for 16 P2RX7 SNPs known to affect P2X7R function, and association studies were performed between frequencies of these SNPs in sepsis, septic shock, and pneumonia compared to controls. The loss-of-function (LOF) SNP rs17525809 (T253C) was found more frequently in patients with septic shock, and non-septic trauma patients when compared to sepsis. The LOF SNP rs2230911 (C1096G) was found to be more frequent in patients with sepsis and septic shock than in non-septic trauma patients. The frequencies of these SNPs were even higher in sepsis and septic patients with pneumonia. The current study also confirmed a previous study by our group that showed a five SNP combination that included the GOF SNPs rs208294 (C489T) and rs2230912 (Q460R) that was designated #21211 was associated with increased odds of survival in severe sepsis. The results found an association between expression of LOF P2RX7 SNPs and presentation to the ICU with sepsis, and septic shock compared to control ICU patients. Furthermore, frequencies of LOF SNPs were found to be higher in sepsis patients with pneumonia compared to those without pneumonia. In addition, a five SNP GOF combination was associated with increased odds of survival in severe sepsis. These results suggest that P2RX7 is required to control infection in pneumonia and that inheritance of LOF variants increases the risk of sepsis when associated with pneumonia. This study confirms that P2RX7 genotyping in pneumonia may identify patients at risk of developing sepsis. The study also identifies P2X7R as a target in sepsis associated with an excessive immune response in subjects with GOF SNP combinations.

Pseudomonas aeruginosa senses and responds to epithelial potassium flux via Kdp operon to promote biofilm.

Mucosa-associated biofilms are associated with many human disease states, but the host mechanisms promoting biofilm remain unclear. In chronic respiratory diseases like cystic fibrosis (CF), Pseudomonas aeruginosa establishes chronic infection through biofilm formation. P. aeruginosa can be attracted to interspecies biofilms through potassium currents emanating from the biofilms. We hypothesized that P. aeruginosa could, similarly, sense and respond to the potassium efflux from human airway epithelial cells (AECs) to promote biofilm. Using respiratory epithelial co-culture biofilm imaging assays of P. aeruginosa grown in association with CF bronchial epithelial cells (CFBE41o-), we found that P. aeruginosa biofilm was increased by potassium efflux from AECs, as examined by potentiating large conductance potassium channel, BKCa (NS19504) potassium efflux. This phenotype is driven by increased bacterial attachment and increased coalescence of bacteria into aggregates. Conversely, biofilm formation was reduced when AECs were treated with a BKCa blocker (paxilline). Using an agar-based macroscopic chemotaxis assay, we determined that P. aeruginosa chemotaxes toward potassium and screened transposon mutants to discover that disruption of the high-sensitivity potassium transporter, KdpFABC, and the two-component potassium sensing system, KdpDE, reduces P. aeruginosa potassium chemotaxis. In respiratory epithelial co-culture biofilm imaging assays, a KdpFABCDE deficient P. aeruginosa strain demonstrated reduced biofilm growth in association with AECs while maintaining biofilm formation on abiotic surfaces. Furthermore, we determined that the Kdp operon is expressed in vivo in people with CF and the genes are conserved in CF isolates. Collectively, these data suggest that P. aeruginosa biofilm formation can be increased by attracting bacteria to the mucosal surface and enhancing coalescence into microcolonies through aberrant AEC potassium efflux sensed by the KdpFABCDE system. These findings suggest host electrochemical signaling can enhance biofilm, a novel host-pathogen interaction, and potassium flux could be a therapeutic target to prevent chronic infections in diseases with mucosa-associated biofilms, like CF.

The Silent Threat of Hypokalemia after High Voltage Electrical Injuries: A Case Study and Review of the Literature.

High-voltage electrical injuries, especially from lightning strikes, can cause life-threatening complications due to extreme temperature and voltage exposure. While burns and cardiac complications have been widely described, the documentation of metabolic imbalances, particularly hypokalemia, has not been as prevalent. This report focuses on a patient with profound transient hypokalemia following a lightning strike, alongside a review of three similar cases of transient hypokalemia from the literature. Our patient, a previously healthy young man, was struck by lightning and subsequently suffered transient hypokalemia with lower extremity sensory changes, which resolved after the normalization of serum potassium levels. While the exact underlying mechanisms of transient hypokalemia following high-voltage electrical injuries are unknown, we propose a multifactorial mechanism, which includes massive intracellular shifts of potassium due to elevated epinephrine levels and the prevention of potassium efflux through the electrical disruption of voltage-gated potassium channels. Our report underscores the importance of recognizing hypokalemia in patients with high-voltage electrical injuries and contributes to the understanding of the complex mechanisms involved. Further research is necessary to understand the connection between cellular changes induced by high-voltage exposure and their effects on metabolism, particularly in relation to hypokalemia.

Extracellular histones promote TWIK2-dependent potassium efflux and associated NLRP3 activation in alveolar macrophages during sepsis-induced lung injury.

Sepsis-induced acute lung injury (ALI) is a complex and life-threatening condition lacking specific and efficient clinical treatments. Extracellular histones, identified as a novel type of damage-associated molecular patterns, have been implicated in the inflammatory process of ALI. However, further elucidation is needed regarding the precise mechanism through which extracellular histones induce inflammation. The aim of this study was to investigate whether extracellular histones can activate NLRP3 inflammasome-mediated inflammation in alveolar macrophages (AMs) by affecting TWIK2-dependent potassium efflux. We conducted experiments using cecal ligation and puncture (CLP) C57BL/6 mice and extracellular histone-stimulated LPS-primed MH-Scells. The results demonstrated a significant increase in the levels of extracellular histones in the plasma and bronchoalveolar lavage fluid (BALF) of CLP mice. Furthermore, neutralizing extracellular histone mitigated lung injury and inflammation in CLP-induced ALI mice. In vitro studies confirmed that extracellular histones upregulated the expression of NLRP3 inflammasome activation-related proteins in MH-S cells, and this effect was dependent on increased potassium efflux mediated by the TWIK2 channel on the plasma membrane. Moreover, extracellular histones directly triggered a substantial influx of calcium, leading to increased Rab11 activity and facilitating the trafficking and location of TWIK2 to the plasma membrane. These findings underscore the critical role of extracellular histone-induced upregulation of TWIK2 expression on the plasma membrane of alveolar macrophages (AMs). This upregulation leads to potassium efflux and subsequent activation of the NLRP3 inflammasome, ultimately exacerbating lung inflammation and injury during sepsis.

The complex regulation of Slo1 potassium channels from a structural perspective.

Fast and regulated potassium efflux by Slo1 channels is crucial in many tissues in animals including neurons, the kidney and smooth muscle. During the last decade, structures have revealed many details about the gating mechanism and regulation of these large and complex molecular machines. This review summarizes these findings and the current knowledge about the intricate regulation of these important channels. Slo1 integrates sensing of the membrane potential via a voltage-sensor domain that undergoes subtle but significant structural rearrangements with a calcium-induced expansion of parts of the intracellular gating ring. Together, these two signals synergistically lead to changes in the conformation and chemical nature of the pore domain, allowing potassium ions to be translocated. In many native tissues, Slo1 channels are assembled with at least three classes of auxiliary subunits that change the gating kinetics or allow the channel to open also in absence of one of the two signals. Finally, Slo1 is inhibited, activated or deregulated by natural toxins and synthetic compounds, underlining the importance of the channel for the organism and as a potential target for drugs and other molecules.

C-type inactivation and proton modulation mechanisms of the TASK3 channel

The TWIK-related acid-sensitive K+ channel 3 (TASK3) belongs to the two-pore domain (K2P) potassium channel family, which regulates cell excitability by mediating a constitutive "leak" potassium efflux in the nervous system. Extracellular acidification inhibits TASK3 channel, but the molecular mechanism by which channel inactivation is coupled to pH decrease remains unclear. Here, we report the cryo-electron microscopy structures of human TASK3 at neutral and acidic pH. Structural comparison revealed selectivity filter (SF) rearrangements upon acidification, characteristic of C-type inactivation, but with a unique structural basis. The extracellular mouth of the SF was prominently dilated and simultaneously blocked by a hydrophobic gate. His98 protonation shifted the conformational equilibrium between the conductive and C-type inactivated SF toward the latter by engaging a cation-π interaction with Trp78, consistent with molecular dynamics simulations and electrophysiological experiments. Our work illustrated how TASK3 is gated in response to extracellular pH change and implies how physiological stimuli might directly modulate the C-type gating of K2P channels.

The Impact of Potassium Dynamics on Cardiomyocyte Beating in Hemodialysis Treatment.

Background: Observational studies of intermittent hemodialysis therapy have reported that the excess decrease in K+ concentration in plasma (KP) during treatment is associated with the destabilization of cardiac function. Elucidating the mechanism by which the decrease in KP impairs myocardial excitation is indispensable for a deeper understanding of prescription design. Methods: In this study, by using an electrophysiological mathematical model, we investigated the relationship between KP dynamics and cardiomyocyte excitability for the first time. Results: The excess decrease in KP during treatment destabilized cardiomyocyte excitability through the following events: (1) a decrease in KP led to the prolongation of the depolarization phase of ventricular cells due to the reduced potassium efflux rate of the Kr channel, temporarily enhancing contraction force; (2) an excess decrease in KP activated the transport of K+ and Na+ through the funny channel in sinoatrial nodal cells, disrupting automaticity; (3) the excess decrease in KP also resulted in a significant decrease in the resting membrane potential of ventricular cells, causing contractile dysfunction. Avoiding an excess decrease in KP during treatment contributed to the maintenance of cardiomyocyte excitability. Conclusions: The results of these mathematical analyses showed that it is necessary to implement personal prescription or optimal control of K+ concentration in dialysis fluid based on predialysis KP from the perspective of regulatory science in dialysis treatment.

Anti-inflammatory effect of Trichospira verticillata via suppression of the NLRP3 inflammasome in neutrophilic asthma.

Trichospira verticillata is an annual herb that belongs to the family Asteraceae. Trichospira verticillata extract (TVE) elicits anti-plasmodial activity; however, there has been no detailed report about its anti-inflammatory effects and molecular mechanisms. In addition, herbal plants exhibit anti-inflammatory effects by suppressing the NLRP3 inflammasome. Therefore, the primary goal of this study was to examine the effects of TVE on NLRP3 inflammasome activation by measuring interleukin-1β (IL-1β) secretion. We treated lipopolysaccharides (LPS)-primed J774A.1 and THP-1 cells with TVE, which attenuated NLRP3 inflammasome activation. Notably, TVE did not affect nuclear factor-kappa B (NF-κB) signalling or intracellular reactive oxygen species (ROS) production and potassium efflux, suggesting that it inactivates the NLRP3 inflammasome via other mechanisms. Moreover, TVE suppressed the formation of apoptosis-associated speck-like protein (ASC) speck and oligomerization. Immunoprecipitation data revealed that TVE reduced the binding of NLRP3 to NIMA-related kinase 7 (NEK7), resulting in reduced ASC oligomerization and speck formation. Moreover, TVE alleviated neutrophilic asthma (NA) symptoms in mice. This study demonstrates that TVE modulates the binding of NLPR3 to NEK7, thereby reporting novel insights into the mechanism by which TVE inhibits NLRP3 inflammasome. These findings suggest TVE as a potential therapeutic of NLRP3 inflammasome-mediated diseases, particularly NA.

Aiouea padiformis extract exhibits anti-inflammatory effects by inhibiting the ATPase activity of NLRP3

Inflammation is implicated as a cause in many diseases. Most of the anti-inflammatory agents in use are synthetic and there is an unmet need for natural substance-derived anti-inflammatory agents with minimal side effects. Aiouea padiformis belongs to the Lauraceae family and is primarily found in tropical regions. While some members of the Aiouea genus are known to possess anti-inflammatory properties, the anti-inflammatory properties of Aiouea padiformis extract (AP) have not been investigated. In this study, we aimed to examine the anti-inflammatory function of AP through the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome and elucidate the underlying mechanisms. Treatment with AP inhibited the secretion of interleukin-1 beta (IL-1β) mediated by NLRP3 inflammasome in J774A.1 and THP-1 cells without affecting the viability. In addition, AP treatment did not influence NF-κB signaling, potassium efflux, or intracellular reactive oxygen species (ROS) production—all of which are associated with NLRP3 inflammasome activation. However, intriguingly, AP treatment significantly reduced the ATPase activity of NLRP3, leading to the inhibition of ASC oligomerization and speck formation. Consistent with cellular experiments, the anti-inflammatory property of AP in vivo was also evaluated using an LPS-induced inflammation model in zebrafish, demonstrating that AP hinders NLRP3 inflammasome activation.

Acacetin inhibits inflammation by blocking MAPK/NF-κB pathways and NLRP3 inflammasome activation.

Objective: Our preliminary research indicates that acacetin modulates the nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain containing 3 (NLRP3) inflammasome, providing protection against Alzheimer's Disease (AD) and cerebral ischemic reperfusion injury. The mechanisms of acacetin to inhibit the activation of the NLRP3 inflammasome remain fully elucidated. This study aims to investigate the effects and potential mechanisms of acacetin on various agonists induced NLRP3 inflammasome activation. Methods: A model for the NLRP3 inflammasome activation was established in mouse bone marrow-derived macrophages (BMDMs) using Monosodium Urate (MSU), Nigericin, Adenosine Triphosphate (ATP), and Pam3CSK4, separately. Western blot analysis (WB) was employed to detect Pro-caspase-1, Pro-Interleukin-1β (Pro-IL-1β) in cell lysates, and caspase-1, IL-1β in supernatants. Enzyme-Linked Immunosorbent Assay (ELISA) was used to measured the release of IL-1β, IL-18, and Tumor Necrosis Factor-alpha (TNF-α) in cell supernatants to assess the impact of acacetin on NLRP3 inflammasome activation. The lactate dehydrogenase (LDH) release was also assessed. The Nuclear Factor Kappa B (NF-κB) and Mitogen-Activated Protein Kinase (MAPK) signaling pathways related proteins were evaluated by WB, and NF-κB nuclear translocation was observed via laser scanning confocal microscopy (LSCM). Disuccinimidyl Suberate (DSS) cross-linking was employed to detect oligomerization of Apoptosis-associated Speck-like protein containing a Caspase Recruitment Domain (ASC), and LSCM was also used to observe Reactive Oxygen Species (ROS) production. Inductively Coupled Plasma (ICP) and N-(6-methoxyquinolyl) acetoethyl ester (MQAE) assays were utilized to determined the effects of acacetin on the efflux of potassium (K+) and chloride (Cl-) ions. Results: Acacetin inhibited NLRP3 inflammasome activation induced by various agonists, reducing the release of TNF-α, IL-1β, IL-18, and LDH. It suppressed the expression of Lipopolysaccharides (LPS)-activated Phosphorylated ERK (p-ERK), p-JNK, and p-p38, inhibited NF-κB p65 phosphorylation and nuclear translocation. Acacetin also reduced ROS production and inhibited ASC aggregation, thus suppressing NLRP3 inflammasome activation. Notably, acacetin did not affect K+ and Cl-ions efflux during the activation process. Conclusion: Acacetin shows inhibitory effects on both the priming and assembly processes of the NLRP3 inflammasome, positioning it as a promising new candidate for the treatment of NLRP3 inflammasome-related diseases.

Effect of glutathione-transport-related gene gsiD on desiccation tolerance of Cronobacter sakazakii and its related regulatory mechanism.

The outstanding desiccation tolerance of Cronobacter sakazakii (C. sakazakii) enables long-term persistence in food products with low-water activity to increase the infection risk, especially in low-birth-weight, immuno-compromised neonates, and infants less than 4 weeks of age. In our previous study, the disruption of glutathione transport-related gene gsiD by transposon was found to significantly increase its inactivation rate under drying stress challenges. However, the mechanism underlying the association between glutathione transport and desiccation tolerance of C. sakazakii remains to be clarified. In this study, the mechanism underlying their association was investigated in detail by constructing the gsiD gene deletion mutant. gsiD gene deletion was found to cause the dysfunction of the glutathione transport system GsiABCD and the limitation of glutathione import. The resulting decrease in intracellular glutathione caused the decreased potassium ions uptake and increased potassium ions efflux, inhibited the proline synthesis process, limited extracellular glutathione utilization, increased oxidant stress, reduced biofilm formation, and increased outer membrane permeability, which may be the main reasons for the significant reduction of the desiccation tolerance of C. sakazakii.IMPORTANCEContributing to its superior environmental adaptability, Cronobacter sakazakii can survive under many abiotic stress conditions. The outstanding desiccation tolerance makes this species persist in low-water activity foods, which increases harm to humans. For decades, many studies have focused on the desiccation tolerance of C. sakazakii, but the existing research is still insufficient. Our study found that gsiD gene deletion inhibited glutathione uptake and further decreased intracellular glutathione content, causing a decrease in desiccation tolerance and biofilm formation and an increase in outer membrane permeability. Moreover, the expression level of relative genes verified that gsiD gene deletion made the mutant not conducive to surviving in dry conditions due to restricting potassium ions uptake and efflux, inhibiting the conversion of glutamate to compatible solute proline, and increasing the oxidative stress of C. sakazakii. The above results enrich our knowledge of the desiccation tolerance mechanism of C. sakazakii.