The Angiotensin II Receptor Neprilysin Inhibitor LCZ696 Inhibits the NLRP3 Inflammasome By Reducing Mitochondrial Dysfunction in Macrophages and Alleviates Dextran Sulfate Sodium-induced Colitis in a Mouse Model.

The intracellular sensor protein complex known as the NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome plays a crucial role in regulating inflammatory diseases by overseeing the production of interleukin (IL)-1ß and IL-18. Targeting its abnormal activation with drugs holds significant promise for inflammation treatment. This study highlights LCZ696, an angiotensin receptor-neprilysin inhibitor, as an effective suppressor of NLRP3 inflammasome activation in macrophages stimulated by ATP, nigericin, and monosodium urate. LCZ696 also reduces caspase-11 and GSDMD activation, lactate dehydrogenase release, propidium iodide uptake, and the extracellular release of NLRP3 and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) in ATP-activated macrophages, suggesting a potential mitigation of pyroptosis. Mechanistically, LCZ696 lowers mitochondrial reactive oxygen species and preserves mitochondrial integrity. Importantly, it does not significantly impact NLRP3, proIL-1ß, inducible nitric oxide synthase, cyclooxygenase-2 expression, or NF-κB activation in lipopolysaccharide-activated macrophages. LCZ696 partially inhibits the NLRP3 inflammasome through the induction of autophagy. In an in vivo context, LCZ696 alleviates NLRP3-associated colitis in a mouse model by reducing colonic expression of IL-1ß and tumor necrosis factor-α. Collectively, these findings suggest that LCZ696 holds significant promise as a therapeutic agent for ameliorating NLRP3 inflammasome activation in various inflammatory diseases, extending beyond its established use in hypertension and heart failure treatment.

Leptin Induces MMP-1 Expression Through the RhoA/ERK1/2/NF-κB Axis in Human Intervertebral Disc Cartilage Endplate-Derived Stem Cells.

Purpose: Intervertebral disc (IVD) degeneration, associated with aging, may cause low back pain and disability, with obesity as a significant risk factor. In a prior study, we found a positive correlation between IVD degeneration and levels of matrix metalloproteinase-1 (MMP-1) and leptin. Yet, the interaction between MMP-1 and leptin in IVD degeneration is unclear. Our research seeks to explore leptin's influence on MMP-1 expression and the underlying mechanisms in human intervertebral disc cartilage endplate-derived stem cells, specifically SV40 cells. Methods: The mRNA and protein expression in leptin-stimulated SV40 cells were assessed using RT-real-time PCR and Western blotting or ELISA, respectively. We examined leptin-mediated RhoA activation through a GTP-bound RhoA pull-down assay. Furthermore, the phosphorylation levels of mitogen-activated protein kinases and AKT in leptin-stimulated SV40 cells were analyzed using Western blotting. The activation of NF-κB by leptin was investigated by assessing phosphorylation of IKKα/ß, IκBα, and NF-κB p65, along with the nuclear translocation of NF-κB p65. To understand the underlying mechanism behind leptin-mediated MMP-1 expression, we employed specific inhibitors. Results: Leptin triggered the mRNA and protein expression of MMP-1 in SV40 cells. In-depth mechanistic investigations uncovered that leptin heightened RhoA activity, promoted ERK1/2 phosphorylation, and increased NF-κB activity. However, leptin did not induce phosphorylation of JNK1/2, p38, or AKT. When we inhibited RhoA, ERK1/2, and NF-κB, it resulted in a decrease in MMP-1 expression. Conversely, inhibition of reactive oxygen species and NADPH oxidase did not yield the same outcome. Additionally, inhibiting RhoA or ERK1/2 led to a reduction in leptin-induced NF-κB activation. Moreover, inhibiting RhoA also decreased leptin-mediated ERK1/2 phosphorylation. Conclusion: These results indicated that leptin induced MMP-1 expression in SV40 cells through the RhoA/ERK1/2/NF-κB axis. This study provided the pathogenic role of leptin and suggested the potential therapeutic target for IVD degeneration.

Antrodia cinnamomea May Interfere with the Interaction Between ACE2 and SARS-CoV-2 Spike Protein in vitro and Reduces Lung Inflammation in a Hamster Model of COVID-19.

Purpose: Coronavirus disease 2019 (COVID-19) poses a global health challenge with widespread transmission. Growing concerns about vaccine side effects, diminishing efficacy, and religious-based hesitancy highlight the need for alternative pharmacological approaches. Our study investigates the impact of the ethanol extract of Antrodia cinnamomea (AC), a native medicinal fungus from Taiwan, on COVID-19 in both in vitro and in vivo contexts. Methods: We measured the mRNA and protein levels of angiotensin-converting enzyme-2 (ACE2) in human lung cells using real-time reverse transcriptase-polymerase chain reaction and Western blotting, respectively. Additionally, we determined the enzymatic activity of ACE2 using the fluorogenic peptide substrate Mca-YVADAPK(Dnp)-OH. To assess the impact of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, we used SARS-CoV-2 pseudovirus infections in human embryonic kidney 293T cells expressing ACE2 to measure infection rates. Furthermore, we evaluated the in vivo efficacy of AC in mitigating COVID-19 by conducting experiments on hamsters infected with the Delta variant of SARS-CoV-2. Results: AC effectively decreased ACE2 mRNA and protein levels, a critical host receptor for the SARS-CoV-2 spike protein, in human lung cells. It also prevented the spike protein from binding to human lung cells. Dehydrosulphurenic acid, an isolate from AC, directly inhibited ACE2 protease activity with an inhibitory constant of 1.53 µM. In vitro experiments showed that both AC and dehydrosulphurenic acid significantly reduced the infection rate of SARS-CoV-2 pseudovirus. In hamsters infected with the Delta variant of SARS-CoV-2, oral administration of AC reduced body weight loss and improved lung injury. Notably, AC also inhibited IL-1ß expression in both macrophages and the lung tissues of SARS-CoV-2-infected hamsters. Conclusion: AC shows potential as a nutraceutical for reducing the risk of SARS-CoV-2 infection by disrupting the interaction between ACE2 and the SARS-CoV-2 spike protein, and for preventing COVID-19-associated lung inflammation.

Surfactin Containing Bacillus licheniformis-Fermented Products Alleviate Dextran Sulfate Sodium-Induced Colitis by Inhibiting Colonic Inflammation and the NLRP3 Inflammasome in Mice.

Inflammatory bowel disease (IBD) is a non-infectious disease characterized by chronic inflammation of the gastrointestinal tract. Currently, management of IBD is still a clinical challenge. The purpose of this study was to investigate the therapeutic potential of surfactin containing Bacillus licheniformis-fermented products (SBLF) and commercial surfactin (CS) on the treatment of dextran sulfate sodium (DSS)-induced colitis in a mouse model. We found that mice that received drinking water containing 3% DSS developed significant colitis symptoms, including increased disease activity index, body weight loss, shortening of the colon length, splenomegaly, colonic inflammation and colonic NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome activation. Notably, orally received SBLF, CS or clinical anti-inflammatory drug 5-aminosalicylic acid improved DSS-induced colitis symptoms in mice. These findings show that SBLF can improve IBD in mice by reducing colonic inflammation and inhibiting the NLRP3 inflammasome activation, suggesting that SBLF has the potential to be used as a nutraceutical in humans or a feed additive in economic and companion animals for preventing IBD.

The leaves of the seasoning plant Litsea cubeba inhibit the NLRP3 inflammasome and ameliorate dextran sulfate sodium-induced colitis in mice.

The intracellular sensor NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome controls caspase-1 activity and the maturation and release of the cytokines interleukin (IL)-1ß and IL-18. The NLRP3 inflammasome has attracted the attention of the pharmaceutical industry because it promotes the pathogenesis of many diseases, making it a promising target for drug development. Litsea cubeba (Lour.) is a plant traditionally used as a seasoning in Taiwan and in other Asian countries. In this study, we investigated the inhibitory activity of the leaves of L. cubeba against the NLRP3 inflammasome. We found that the ethanol extract of L. cubeba leaves (MLE) inhibited the NLRP3 inflammasome in macrophages by reducing caspase-1 activation and IL-1ß secretion. MLE reduced pyroptosis in macrophages and inhibited the release of NLRP3 and apoptosis-associated speck-like protein containing a CARD (ASC). In a mechanistic study, MLE reduced mitochondrial reactive oxygen species (ROS) production and preserved mitochondrial integrity, which led to reduced mitochondrial DNA release into the cytosol. MLE did not reduce the expression levels of NLRP3, IL-1ß precursor or TNF-α in lipopolysaccharide (LPS)-activated macrophages. These results indicated that MLE inhibited the NLRP3 inflammasome by suppressing the activation signals of the NLRP3 inflammasome but not by reducing the priming signal induced by LPS. In addition, oral administration of MLE (20-80 mg/kg) ameliorated dextran sulfate sodium (DSS)-induced colitis in a mouse model. Notably, mice that received MLE (1 and 2 g/kg) daily for 7 days did not exhibit visible side effects. Gas chromatography-mass spectrometry (GC-MS) analysis found that α-Terpinyl acetate (27.2%) and 1,8-Cineole (17.7%) were the major compounds in MLE. These results indicated that L. cubeba leaves have the potential to be a nutraceutical for preventing and improving NLRP3 inflammasome-related diseases.

Repositioning of the Angiotensin II Receptor Antagonist Candesartan as an Anti-Inflammatory Agent With NLRP3 Inflammasome Inhibitory Activity.

Aberrant activation of the NLRP3 inflammasome promotes the pathogenesis of many inflammatory diseases. The development of the NLRP3 inflammasome inhibitors from existing drugs for new therapeutic purposes is becoming more important. Candesartan is an angiotensin II receptor antagonist widely used as a blood pressure-lowering drug; however, the inhibitory potential of candesartan on the NLRP3 inflammasome has not yet been investigated. We demonstrated that candesartan significantly inhibited the NLRP3 inflammasome and pyroptosis in macrophages. Mechanistic analysis revealed that candesartan inhibited the expression of NLRP3 and proIL-1ß by suppressing NF-κB activation and reducing the phosphorylation of ERK1/2 and JNK1/2. Candesartan reduced mitochondrial damage and inhibited the NLRP3 inflammasome assembly by suppressing NLRP3 binding to PKR, NEK7 and ASC. In addition, candesartan inhibited IL-1ß secretion partially through autophagy induction. Furthermore, oral administration of candesartan reduced peritoneal neutrophil influx, NLRP3 and ASC expression in peritoneal cells, and lavage fluid concentrations of active caspase-1, IL-1ß, IL-6 and MCP-1 in uric acid crystal-injected mice. These results indicated that candesartan has board anti-inflammatory effects and has the potential to be repositioned to ameliorate inflammatory diseases or NLRP3-associated complications.

A GalNAc/Gal-specific lectin modulates immune responses via toll-like receptor 4 independently of carbohydrate-binding ability.

Toll-like receptor 4 (TLR4) recognizes various protein ligands; however, the protein-TLR4 binding model is unclear. Here we demonstrate a Crenomytilus grayanus lectin (CGL)-TLR4/MD2 model to show that CGL interacts with a TLR4/myeloid differentiation factor 2 (MD2) complex independently of sugar-binding properties. CGL could suppress lipopolysaccharide-induced immune responses significantly, suggesting that TLR4 itself has potential as a therapeutic target.

Ginsenoside M1 Induces Apoptosis and Inhibits the Migration of Human Oral Cancer Cells.

Oral squamous cell carcinoma (OSCC) accounts for 5.8% of all malignancies in Taiwan, and the incidence of OSCC is on the rise. OSCC is also a common malignancy worldwide, and the five-year survival rate remains poor. Therefore, new and effective treatments are needed to control OSCC. In the present study, we prepared ginsenoside M1 (20-O-beta-d-glucopyranosyl-20(S)-protopanaxadiol), a major deglycosylated metabolite of ginsenoside, through the biotransformation of Panax notoginseng leaves by the fungus SP-LSL-002. We investigated the anti-OSCC activity and associated mechanisms of ginsenoside M1 in vitro and in vivo. We demonstrated that ginsenoside M1 dose-dependently inhibited the viability of human OSCC SAS and OEC-M1 cells. To gain further insight into the mode of action of ginsenoside M1, we demonstrated that ginsenoside M1 increased the expression levels of Bak, Bad, and p53 and induced apoptotic DNA breaks, G1 phase arrest, PI/Annexin V double-positive staining, and caspase-3/9 activation. In addition, we demonstrated that ginsenoside M1 dose-dependently inhibited the colony formation and migration ability of SAS and OEC-M1 cells and reduced the expression of metastasis-related protein vimentin. Furthermore, oral administration or subcutaneous injection of ginsenoside M1 significantly reduced tumor growth in SAS xenograft mice. These results indicate that ginsenoside M1 can be translated into a potential therapeutic against OSCC.

Synthetic 4-Hydroxy Auxarconjugatin B, a Novel Autophagy Inducer, Attenuates Gouty Inflammation by Inhibiting the NLRP3 Inflammasome.

Gouty arthritis results from the generation of uric acid crystals within the joints. These uric acid crystals activate the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome, which is involved in chronic inflammatory diseases, including gouty arthritis. This study identified the polyenylpyrrole derivative 4-hydroxy auxarconjugatin B (4-HAB), a novel autophagy inducer, which attenuated uric acid crystals-mediated activation of the NLRP3 inflammasome in vitro and in vivo. 4-HAB dose-dependently reduced the release of interleukin (IL)-1ß, IL-18, active caspase-1 and apoptosis-associated speck-like protein (ASC) in uric acid crystals-activated macrophages. In a mechanistic study, 4-HAB was shown to inhibit uric acid crystals-induced mitochondrial damage, lysosomal rupture and ASC oligomerization. Additionally, 4-HAB inhibited the NLRP3 inflammasome through Sirt1-dependent autophagy induction. Furthermore, the anti-inflammatory properties of 4-HAB were confirmed in a mouse model of uric acid crystals-mediated peritonitis by the reduced levels of neutrophil influx, IL-1ß, active caspase-1, IL-6 and MCP-1 in lavage fluids. In conclusion, 4-HAB attenuates gouty inflammation, in part by attenuating activation of the NLRP3 inflammasome through the Sirt1/autophagy induction pathway.

Compound K inhibits priming and mitochondria-associated activating signals of NLRP3 inflammasome in renal tubulointerstitial lesions.

BACKGROUND: Renal tubulointerstitial lesions (TILs), a key pathological hallmark for chronic kidney disease to progress to end-stage renal disease, feature renal tubular atrophy, interstitial mononuclear leukocyte infiltration and fibrosis in the kidney. Our study tested the renoprotective and therapeutic effects of compound K (CK), as described in our US patent (US7932057B2), on renal TILs using a mouse unilateral ureteral obstruction (UUO) model. METHODS: Renal pathology was performed and renal draining lymph nodes were subjected to flow cytometry analysis. Mechanism-based experiments included the analysis of mitochondrial dysfunction, a model of tubular epithelial cells (TECs) under mechanically induced constant pressure (MICP) and tandem mass tags (TMT)-based proteomics analysis. RESULTS: Administration of CK ameliorated renal TILs by reducing urine levels of proinflammatory cytokines, and preventing mononuclear leukocyte infiltration and fibrosis in the kidney. The beneficial effects clearly correlated with its inhibition of: (i) NF-κB-associated priming and the mitochondria-associated activating signals of the NLRP3 inflammasome; (ii) STAT3 signalling, which in part prevents NLRP3 inflammasome activation; and (iii) the TGF-ß-dependent Smad2/Smad3 fibrotic pathway, in renal tissues, renal TECs under MICP and/or activated macrophages, the latter as a major inflammatory player contributing to renal TILs. Meanwhile, TMT-based proteomics analysis revealed downregulated renal NLRP3 inflammasome activation-associated signalling pathways in CK-treated UUO mice. CONCLUSIONS: The present study, for the first time, presents the potent renoprotective and therapeutic effects of CK on renal TILs by targeting the NLRP3 inflammasome and STAT3 signalling.

Ergosterol peroxide from marine fungus Phoma sp. induces ROS-dependent apoptosis and autophagy in human lung adenocarcinoma cells.

As part of our ongoing search for novel therapeutic structures from microorganism, the chemical examination of marine fungus Phoma sp. resulted in the isolation of ergosterol, ergosterol peroxide (EP), and 9,11-dehydroergosterol peroxide (DEP). The bioassay results demonstrated that the three isolates reduced the viability of various cancer cells, with EP being highest in human lung cancer cell line A549 cells. EP induced caspase-dependent apoptosis through mitochondrial damage in A549 cells. Additionally, EP-induced ROS generation and apoptosis were attenuated by ROS-generating enzymes inhibitors and antioxidant N-acetylcysteine, indicated that ROS played an important role in EP-mediated apoptosis in A549 cells. Furthermore, it was observed that EP induced ROS-dependent autophagy, which attenuated apoptosis in A549 cells. On the other hand, EP reduced the LPS/ATP-induced proliferation and migration of A549 cells through attenuated NLRP3 inflammasome activity. Additionally, EP showed synergistic cytotoxic effect with antitumor drug Sorafenib in A549 cell viability inhibition. Furthermore, Micro-Western Array and Western blot analyses demonstrated that the protein levels of EGFR, HSP27, MEK5, AKT1, mTOR, Smad2, Smad3, TAB1, NF-κB, and HIF1-α decreased, while the levels of p-p38α, p-ERK1/2, p-JNK, fibronectin and p27 increased. Collectively, the results of this study demonstrated that EP might be useful to develop a therapeutic candidate for lung cancer complications.

Repositioning of the ß-Blocker Carvedilol as a Novel Autophagy Inducer That Inhibits the NLRP3 Inflammasome.

The NLRP3 inflammasome is a multiprotein complex that plays a key role in the innate immune system, and aberrant activation of this complex is involved in the pathogenesis of inflammatory diseases. Carvedilol (CVL) is an α-, ß-blocker used to treat high blood pressure and congestive heart failure; however, some benefits beyond decreased blood pressure were observed clinically, suggesting the potential anti-inflammatory activity of CVL. In this report, the inhibitory potential of CVL toward the NLRP3 inflammasome and the possible underlying molecular mechanisms were studied. Our results showed that CVL attenuated NLRP3 inflammasome activation and pyroptosis in mouse macrophages, without affecting activation of the AIM2, NLRC4 and non-canonical inflammasomes. Mechanistic analysis revealed that CVL prevented lysosomal and mitochondrial damage and reduced ASC oligomerization. Additionally, CVL caused autophagic induction through a Sirt1-dependent pathway, which inhibited the NLRP3 inflammasome. In the in vivo mouse model of NLRP3-associated peritonitis, oral administration of CVL reduced (1) peritoneal recruitment of neutrophils; (2) the levels of IL-1ß, IL-18, active caspase-1, ASC, IL-6, TNF-α, MCP-1, and CXCL1 in the lavage fluids; and (3) the levels of NLRP3 and HO-1 in the peritoneal cells. Our results indicated that CVL is a novel autophagy inducer that inhibits the NLRP3 inflammasome and can be repositioned for ameliorating NLRP3-associated complications.

A GalNAc/Gal-specific lectin from the sea mussel Crenomytilus grayanus modulates immune response in macrophages and in mice.

A GalNAc/Gal-specific lectin (CGL) from the edible mussel Crenomytilus grayanus has been demonstrated to exhibit antibacterial properties. However, the mechanism of immune modulation by CGL in mammalian cells remains unclear. Here, we demonstrated that CGL can activate immune responses in macrophages and in mice. In the in vitro cell models, CGL induced tumour necrosis factor-α and interleukin-6 secretion in mouse RAW264.7 macrophages, mouse bone marrow-derived macrophages, human THP-1 macrophages, human peripheral blood mononuclear cells and human blood monocyte-derived macrophages. The CGL-mediated cytokine production was regulated by reactive oxygen species, mitogen-activated protein kinases, protein kinase C-α/δ and NF-κB. Interestingly, in lipopolysaccharide-activated macrophages, CGL induced endotoxin tolerance (characterized by the downregulation of nitric oxide, inducible nitric oxide synthase, interleukin-6 and cyclooxygenase II) via the downregulation of IRAK2 expression, JNK1/2 phosphorylation and NF-κB activation. CGL also slightly increased the bactericidal activity of macrophages and induced cytokine production in mouse models. Overall, our data indicate that CGL has the potential to be used as an immune modulator in mammals.

Peroxyauraptenol Inhibits Inflammation and NLRP3 Inflammasome Activation by Inhibiting Reactive Oxygen Species Generation and Preserving Mitochondrial Integrity.

Peroxyauraptenol (PXT) is a peroxide-containing coumarin compound isolated from the seeds of Cnidium monnieri. PXT exerts anti-inflammatory activities, as it reduces the levels of inducible nitric oxide synthase, nitric oxide, IL-6, and NLRP3 inflammasome-derived IL-1ß in lipopolysaccharide-activated macrophages. PXT also exerts anti-inflammatory activity by reducing reactive oxygen species generation (including mitochondrial), mitogen-activated protein kinase, protein kinase C-α/δ phosphorylation, and the release of mitochondrial DNA into the cytosol. In addition, PXT suppresses the phagocytic activity of macrophages and IL-1ß secretion by Klebsiella pneumoniae-infected macrophages. The unique peroxide group is important for the anti-inflammatory activity of PXT, as this activity is reduced when the peroxide group is replaced by a hydroxyl group. These findings suggest that PXT may be a candidate for the development of anti-inflammatory agents or a healthy supplement for preventing and ameliorating inflammation-related diseases.

Cyclooxygenase-2 regulates NLRP3 inflammasome-derived IL-1ß production.

The NLR family, pyrin domain-containing 3 (NLRP3) inflammasome is a reactive oxygen species-sensitive multiprotein complex that regulates IL-1ß maturation via caspase-1. It also plays an important role in the pathogenesis of inflammation-related disease. Cyclooxygenase-2 (COX-2) is induced by inflammatory stimuli and contributes to the pathogenesis of inflammation-related diseases. However, there is currently little known about the relationship between COX-2 and the NLRP3 inflammasome. Here, we describe a novel role for COX-2 in regulating the activation of the NLRP3 inflammasome. NLRP3 inflammasome-derived IL-1ß secretion and pyroptosis in macrophages were reduced by pharmaceutical inhibition or genetic knockdown of COX-2. COX-2 catalyzes the synthesis of prostaglandin E2 and increases IL-1ß secretion. Conversely, pharmaceutical inhibition or genetic knockdown of prostaglandin E2 receptor 3 reduced IL-1ß secretion. The underlying mechanisms for the COX-2-mediated increase in NLRP3 inflammasome activation were determined to be the following: (1) enhancement of lipopolysaccharide-induced proIL-1ß and NLRP3 expression by increasing NF-κB activation and (2) enhancement of the caspase-1 activation by increasing damaged mitochondria, mitochondrial reactive oxygen species production and release of mitochondrial DNA into cytosol. Furthermore, inhibition of COX-2 in mice in vivo with celecoxib reduced serum levels of IL-1ß and caspase-1 activity in the spleen and liver in response to lipopolysaccharide (LPS) challenge. These findings provide new insights into how COX-2 regulates the activation of the NLRP3 inflammasome and suggest that it may be a new potential therapeutic target in NLRP3 inflammasome-related diseases.