Pellino1 orchestrates gut-kidney axis to perpetuate septic acute kidney injury through activation of STING pathway and NLRP3 inflammasome.

AIMS: Intestinal barrier dysfunction is the initial and propagable factor of sepsis in which acute kidney injury (AKI) has been considered as a common life-threatening complication. Our recent study identifies the regulatory role of Pellino1 in tubular death under inflammatory conditions in vitro. The objective of our current study is to explore the impact of Pellino1 on gut-kidney axis during septic AKI and uncover the molecular mechanism (s) underlying this process. MATERIALS AND METHODS: Immunohistochemistry (IHC) was conducted to evaluate Pellino1 and NOD-like receptor thermal protein domain associated protein 3 (NLRP3) levels in renal biopsies from critically ill patients with a clinical diagnosis of sepsis. Functional and mechanistic studies were characterized in septic models of the Peli-knockout (Peli1-/-) mice by histopathological staining, enzyme-linked immunosorbent assay (ELISA), flow cytometry, immunofluorescence, biochemical detection, CRISPR/Cas9-mediated gene editing and intestinal organoid. KEY FINDINGS: Pellino1, together with NLRP3, are highly expressed in renal biopsies from critically ill patients diagnosed with sepsis and kidney tissues of septic mice. The Peli1-/- mice with sepsis become less prone to develop AKI and have markedly compromised NLRP3 activation in kidney. Loss of Peli1 endows septic mice refractory to intestinal inflammation, barrier permeability and enterocyte apoptosis that requires stimulator of interferons genes (STING) pathway. Administration of STING agonist DMXAA deteriorates AKI and mortality of septic Peli1-/- mice in the presence of kidney-specific NLRP3 reconstitution. SIGNIFICANCE: Our studies suggest that Pellino1 has a principal role in orchestrating gut homeostasis towards renal pathophysiology, thus providing a potential therapeutic target for septic AKI.

[Role of NLRP3 inflammasome in prevention and treatment of cognitive impairment-related diseases and traditional Chinese medicine intervention: a review].

Alzheimer's disease(AD), vascular dementia(VD), and traumatic brain injury(TBI) are more common cognitive impairment diseases characterized by high disability and mortality rates, imposing a heavy burden on individuals and their families. Although AD, VD, and TBI have different specific mechanisms, their pathogenesis is closely related to the nucleotide-binding oligome-rization domain-like receptor protein 3(NLRP3). The NLRP3 inflammasome is involved in neuroinflammatory responses, mediating microglial polarization, regulating the reduction of amyloid ß-protein(Aß) deposition, neurofibrillary tangles(NFTs) formation, autophagy regulation, and maintaining brain homeostasis, and synaptic stability, thereby contributing to the development of AD, VD, and TBI. Previous studies have shown that traditional Chinese medicine(TCM) can alleviate neuroinflammation, promote microglial polarization towards the M2 phenotype, reduce Aß deposition and NFTs formation, regulate autophagy, and maintain brain homeostasis by intervening in NLRP3 inflammasome, hence exerting a role in preventing and treating cognitive impairment-related diseases, reducing psychological and economic pressure on patients, and improving their quality of life. Therefore, this article elucidated the role of NLRP3 inflammasome in AD, VS, and TBI, and provided a detailed summary of the latest research results on TCM intervention in NLRP3 inflammasome for the prevention and treatment of these diseases, aiming to inherit the essence of TCM and provide references and foundations for clinical prevention and treatment of cognitive impairment-related diseases with TCM. Meanwhile, this also offers insights and directions for further research in TCM for the prevention and treatment of cognitive impairment-related diseases.

[Role of NLRP3 inflammasome in heart failure and traditional Chinese medicine intervention: a review].

Heart failure is characterized by high incidence and mortality rates, and the search for effective treatment strategies for heart failure and the improvement of clinical outcomes have always been important research directions. Imbalanced inflammation has been proven to be one of the critical pathological factors in heart failure, positively correlated with adverse events such as impaired cardiac function and myocardial fibrosis. In recent years, studies have confirmed that the activation of the NOD-like receptor thermal protein domain-associated protein 3(NLRP3) inflammasome plays a common regulatory role in the inflammation imbalance induced by various factors in heart failure. Moreover, certain traditional Chinese medicine(TCM) and active components can significantly inhibit the activation of the NLRP3 inflammasome, thereby improving heart failure. This article first overviewed the basic information about the NLRP3 inflammasome, summarized the regulatory mechanisms of the NLRP3 inflammasome in heart failure induced by various factors, introduced recent research progress on TCM and active components that inhibited the NLRP3 inflammasome to improve heart failure, aiming to provide references for innovative drug research in the field of integrated Chinese and western medicine for the prevention and treatment of heart failure.

L-AP Alleviates Liver Injury in Septic Mice by Inhibiting Macrophage Activation via Suppressing NF-κB and NLRP3 Inflammasome/Caspase-1 Signal Pathways.

Liver injury and progressive liver failure are severe life-threatening complications in sepsis, further worsening the disease and leading to death. Macrophages and their mediated inflammatory cytokine storm are critical regulators in the occurrence and progression of liver injury in sepsis, for which effective treatments are still lacking. l-Ascorbic acid 6-palmitate (L-AP), a food additive, can inhibit neuroinflammation by modulating the phenotype of the microglia, but its pharmacological action in septic liver damage has not been fully explored. We aimed to investigate L-AP's antisepticemia action and the possible pharmacological mechanisms in attenuating septic liver damage by modulating macrophage function. We observed that L-AP treatment significantly increased survival in cecal ligation and puncture-induced WT mice and attenuated hepatic inflammatory injury, including the histopathology of the liver tissues, hepatocyte apoptosis, and the liver enzyme levels in plasma, which were comparable to NLRP3-deficiency in septic mice. L-AP supplementation significantly attenuated the excessive inflammatory response in hepatic tissues of septic mice in vivo and in cultured macrophages challenged by both LPS and ATP in vitro, by reducing the levels of NLRP3, pro-IL-1ß, and pro-IL-18 mRNA expression, as well as the levels of proteins for p-I-κB-α, p-NF-κB-p65, NLRP3, cleaved-caspase-1, IL-1ß, and IL-18. Additionally, it impaired the inflammasome ASC spot activation and reduced the inflammatory factor contents, including IL-1ß and IL-18 in plasma/cultured superannuants. It also prevented the infiltration/migration of macrophages and their M1-like inflammatory polarization while improving their M2-like polarization. Overall, our findings revealed that L-AP protected against sepsis by reducing macrophage activation and inflammatory cytokine production by suppressing their activation in NF-κB and NLRP3 inflammasome signal pathways in septic liver.

LncRNA GAS5 restrains ISO-induced cardiac fibrosis by modulating mir-217 regulation of SIRT1.

Considering the effect of SIRT1 on improving myocardial fibrosis and GAS5 inhibiting occurrence and development of myocardial fibrosis at the cellular level, the aim of the present study was to investigate whether LncRNA GAS5 could attenuate cardiac fibrosis through regulating mir-217/SIRT1, and whether the NLRP3 inflammasome activation was involved in this process. Isoprenaline (ISO) was given subcutaneously to the male C57BL/6 mice to induce myocardial fibrosis and the AAV9 vectors were randomly injected into the left ventricle of each mouse to overexpress GAS5. Primary myocardial fibroblasts (MCFs) derived from neonatal C57BL/6 mice and TGF-ß1 were used to induce fibrosis. And the GAS5 overexpressed MCFs were treated with mir-217 mimics and mir-217 inhibitor respectively. Then the assays of expression levels of NLRP3, Caspase-1, IL-1ß and SIRT1 were conducted. The findings indicated that the overexpression of GAS5 reduced the expression levels of collagen, NLRP3, Capase-1, IL-1ß and SIRT1 in ISO treated mice and TGF-ß1 treated MCFs. However, this effect was significantly weakened after mir-217 overexpression, but was further enhanced after knockdown of mir-217. mir-217 down-regulates the expression of SIRT1, leading to increased activation of the NLRP3 inflammasome and subsequent pyroptosis. LncRNA GAS5 alleviates cardiac fibrosis induced via regulating mir-217/SIRT1 pathway.

Nod-like receptor protein 3 inflammasome-mediated pyroptosis contributes to chronic NaAsO2 exposure-induced fibrotic changes and dysfunction in the liver of SD rats.

The metalloid arsenic, known for its toxic properties, is widespread presence in the environment. Our previous research has confirmed that prolonged exposure to arsenic can lead to liver fibrosis injury in rats, while the precise pathogenic mechanism still requires further investigation. In the past few years, the Nod-like receptor protein 3 (NLRP3) inflammasome has been found to play a pivotal role in the occurrence and development of liver injury. In this study, we administered varying doses of sodium arsenite (NaAsO2) and 10 mg/kg.bw MCC950 (a particular tiny molecular inhibitor targeting NLRP3) to Sprague-Dawley (SD) rats for 36 weeks to explore the involvement of NLRP3 inflammasome in NaAsO2-induced liver injury. The findings suggested that prolonged exposure to NaAsO2 resulted in pyroptosis in liver tissue of SD rats, accompanied by the fibrotic injury, extracellular matrix (ECM) deposition and liver dysfunction. Moreover, long-term NaAsO2 exposure activated NLRP3 inflammasome, leading to the release of pro-inflammatory cytokines in liver tissue. After treatment with MCC950, the induction of NLRP3-mediated pyroptosis and release of pro-inflammatory cytokines were significantly attenuated, leading to a decrease in the severity of liver fibrosis and an improvement in liver function. To summarize, those results clearly indicate that hepatic fibrosis and liver dysfunction induced by NaAsO2 occur through the activation of NLRP3 inflammasome-mediated pyroptosis, shedding new light on the potential mechanisms underlying arsenic-induced liver damage.

Renoprotective effect of diacetylrhein on diclofenac-induced acute kidney injury in rats via modulating Nrf2/NF-κB/NLRP3/GSDMD signaling pathways.

Diclofenac (DF)-induced acute kidney injury (AKI) is characterized by glomerular dysfunction and acute tubular necrosis. Due to limited treatment approaches, effective and safe drug therapy to protect against such AKI is still needed. Diacetylrhein (DAR), an anthraquinone derivative, has different antioxidant and anti-inflammatory properties. Therefore, the aim of the current study was to investigate the renoprotective effect of DAR on DF-induced AKI while elucidating the potential underlying mechanism. Our results showed that DAR (50 and 100 mg/kg) markedly abrogated DF-induced kidney dysfunction decreasing SCr, BUN, serum NGAL, and serum KIM1 levels. Moreover, DAR treatment remarkably maintained renal redox balance and reduced the levels of pro-inflammatory biomarkers in the kidney. Mechanistically, DAR boosted Nrf2/HO-1 antioxidant and anti-inflammatory response in the kidney while suppressing renal TLR4/NF-κB and NLRP3/caspase-1 inflammatory signaling pathways. In addition, DAR markedly inhibited renal pyroptosis via targeting of GSDMD activation. Collectively, this study confirmed that the interplay between Nrf2/HO-1 and TLR4/NF-κB/NLRP3/Caspase-1 signaling pathways and pyroptotic cell death mediates DF-induced AKI and reported that DAR has a dose-dependent renoprotective effect on DF-induced AKI in rats. This effect is due to powerful antioxidant, anti-inflammatory, and anti-pyroptotic activities that could provide a promising treatment approach to protect against DF-induced AKI.

Truncating NFKB1 variants cause combined NLRP3 inflammasome activation and type I interferon signaling and predispose to necrotizing fasciitis.

In monogenic autoinflammatory diseases, mutations in genes regulating innate immune responses often lead to uncontrolled activation of inflammasome pathways or the type I interferon (IFN-I) response. We describe a mechanism of autoinflammation potentially predisposing patients to life-threatening necrotizing soft tissue inflammation. Six unrelated families are identified in which affected members present with necrotizing fasciitis or severe soft tissue inflammations. Exome sequencing reveals truncating monoallelic loss-of-function variants of nuclear factor κ light-chain enhancer of activated B cells (NFKB1) in affected patients. In patients' macrophages and in NFKB1-variant-bearing THP-1 cells, activation increases both interleukin (IL)-1ß secretion and IFN-I signaling. Truncation of NF-κB1 impairs autophagy, accompanied by the accumulation of reactive oxygen species and reduced degradation of inflammasome receptor nucleotide-binding oligomerization domain, leucine-rich repeat-containing protein 3 (NLRP3), and Toll/IL-1 receptor domain-containing adaptor protein inducing IFN-ß (TRIF), thus leading to combined excessive inflammasome and IFN-I activity. Many of the patients respond to anti-inflammatory treatment, and targeting IL-1ß and/or IFN-I signaling could represent a therapeutic approach for these patients.

Disordered GPR43/NLRP3 expression in peripheral leukocytes of patients with atrial fibrillation is associated with intestinal short chain fatty acids levels.

BACKGROUND: Atrial fibrillation (AF) is associated with circulating inflammation. Short-chain fatty acids (SCFAs) derived from gut microbiota (GM) regulate leukocyte function and inhibit the release of inflammatory cytokines, which are partly mediated by the G-protein-coupled receptor 43 (GPR43) signaling. This study aimed to investigate the expression of GPR43/NOD-like receptors family pyrin domain containing 3 (NLRP3) in leukocytes and the interaction with intestinal SCFAs levels in AF patients. METHODS: Expressions of GPR43 and NLRP3 mRNA in peripheral blood leukocytes from 23 AF patients and 25 non-AF controls were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Expressions of leukocyte GPR43 and NLRP3 protein were evaluated by western blot analysis. The levels of plasma IL-1ß were measured by enzyme-linked immunosorbent assay (ELISA). The fecal SCFAs levels based on GC/MS metabolome of corresponding 21 controls and 14 AF patients were acquired from our published dataset. To evaluate the expression of NLRP3 and GPR43 and the release of IL-1ß, human THP-1 cells were stimulated with or without SCFAs (acetate, propionate, and butyrate), lipopolysaccharide (LPS), and nigericin in vitro, respectively. RESULTS: Compared to the controls, the mRNA expression in peripheral leukocytes was significantly reduced in AF patients (P = 0.011) coupled with the increase in downstream leukocyte NLRP3 mRNA expression (P = 0.007) and plasma IL-1ß levels (P < 0.001), consistent with changes in GPR43 and NLRP3 protein expression. Furthermore, leukocyte GPR43 mRNA levels were positively correlated with fecal GM-derived acetic acid (P = 0.046) and negatively correlated with NLRP3 mRNA expression (P = 0.024). In contrast to the negative correlation between left atrial diameter (LAD) and GPR43 (P = 0.008), LAD was positively correlated with the leukocyte NLRP3 mRNA levels (P = 0.024). Subsequent mediation analysis showed that 68.88% of the total effect of intestinal acetic acid on AF might be mediated by leukocyte GPR43/NLRP3. The constructed GPR43-NLRP3 score might have a predictive potential for AF detection (AUC = 0.81, P < 0.001). Moreover, SCFAs treatment increased GPR43 expression and remarkably reduced LPS/nigericin-induced NLRP3 expression and IL-1ß release in human THP-1 cells in vitro. CONCLUSIONS: Disrupted interactions between GPR43 and NLRP3 expression in peripheral blood leukocytes, associated with reduced intestinal GM-derived SCFAs, especially acetic acid, may be involved in AF development and left atrial enlargement by enhancing circulating inflammation.

Dicoumarol attenuates NLRP3 inflammasome activation to inhibit inflammation and fibrosis in knee osteoarthritis.

Knee osteoarthritis (KOA) is a major cause of disability in elderly individuals. Dicoumarol is a coumarin­like compound derived from sweet clover [Melilotus officinalis (L.) Pall]. It has been suggested that dicoumarol exhibits various types of pharmacological activities, including anticoagulant, antitumor and antibacterial effects. Due to its various biological activities, dicoumarol has a potential protective effect against OA. Therefore, the present study aimed to assess the effects of dicoumarol on knee osteoarthritis. In the present study, dicoumarol was found to protect rat synoviocytes from lipopolysaccharide (LPS)­induced cell apoptosis. Western blot analysis showed that dicoumarol significantly reduced the protein expression levels of fibrosis­related markers and inflammatory cytokines (Tgfb, Timp, Col1a, Il1b and Il18). The inhibitory rates of these proteins were all >50% (P<0.01) compared with those in the LPS and ATP­induced group. Consistently, the mRNA expression levels of these markers and cytokines were decreased to normal levels by dicoumarol after the treatment of rat synovial fibroblasts with LPS and ATP. Mechanistic studies demonstrated that dicoumarol did not affect NF­κB signaling, but it did directly interact with NOD­like receptor protein 3 (NLRP3) to promote its protein degradation, which could be reversed by MG132, but not NH4Cl. The protein half­life of NLRP3 was accelerated from 26.1 to 4.3 h by dicoumarol. Subsequently, dicoumarol could alleviate KOA in vivo; knee joint diameter was decreased from 11.03 to 9.93 mm. Furthermore, the inflammation and fibrosis of the knee joints were inhibited in rats. In conclusion, the present findings demonstrated that dicoumarol could impede the progression of KOA by inhibiting NLRP3 activation, providing a potential treatment strategy for KOA.

Adipose tissue IL-18 production is independent of caspase-1 and caspase-11.

BACKGROUND: Inflammation in adipose tissue, resulting from imbalanced caloric intake and energy expenditure, contributes to the metabolic dysregulation observed in obesity. The production of inflammatory cytokines, such as IL-1ß and IL-18, plays a key role in this process. While IL-1ß promotes insulin resistance and diabetes, IL-18 regulates energy expenditure and food intake. Previous studies have suggested that caspase-1, activated by the Nlrp3 inflammasome in response to lipid excess, mediates IL-1ß production, whereas activated by the Nlrp1b inflammasome in response to energy excess, mediates IL-18 production. However, this has not been formally tested. METHODS: Wild-type and caspase-1-deficient Balb/c mice, carrying the Nlrp1b1 allele, were fed with regular chow or a high-fat diet for twelve weeks. Food intake and mass gain were recorded weekly. At the end of the twelve weeks, glucose tolerance and insulin resistance were evaluated. Mature IL-18 protein levels and the inflammatory process in the adipose tissue were determined. Fasting lipid and cytokine levels were quantified in the sera of the different experimental groups. RESULTS: We found that IL-18 production in adipose tissue is independent of caspase-1 activity, regardless of the metabolic state, while Nlrp3-mediated IL-1ß production remains caspase-1 dependent. Additionally, caspase-1 null Balb/c mice did not develop metabolic abnormalities in response to energy excess from the high-fat diet. CONCLUSION: Our findings suggest that IL-18 production in the adipose tissue is independent of Nlrp3 inflammasome and caspase-1 activation, regardless of caloric food intake. In contrast, Nlrp3-mediated IL-1ß production is caspase-1 dependent. These results provide new insights into the mechanisms underlying cytokine production in the adipose tissue during both homeostatic conditions and metabolic stress, highlighting the distinct roles of caspase-1 and the Nlrp inflammasomes in regulating inflammatory responses.

The heavy subunit of ferritin stimulates NLRP3 inflammasomes in hepatic stellate cells through ICAM-1 to drive hepatic inflammation.

Serum ferritin concentrations increase during hepatic inflammation and correlate with the severity of chronic liver disease. Here, we report a molecular mechanism whereby the heavy subunit of ferritin (FTH) contributes to hepatic inflammation. We found that FTH induced activation of the NLRP3 inflammasome and secretion of the proinflammatory cytokine interleukin-1ß (IL-1ß) in primary rat hepatic stellate cells (HSCs) through intercellular adhesion molecule-1 (ICAM-1). FTH-ICAM-1 stimulated the expression of Il1b, NLRP3 inflammasome activation, and the processing and secretion of IL-1ß in a manner that depended on plasma membrane remodeling, clathrin-mediated endocytosis, and lysosomal destabilization. FTH-ICAM-1 signaling at early endosomes stimulated Il1b expression, implying that this endosomal signaling primed inflammasome activation in HSCs. In contrast, lysosomal destabilization was required for FTH-induced IL-1ß secretion, suggesting that lysosomal damage activated inflammasomes. FTH induced IL-1ß production in liver slices from wild-type mice but not in those from Icam1-/- or Nlrp3-/- mice. Thus, FTH signals through its receptor ICAM-1 on HSCs to activate the NLRP3 inflammasome. We speculate that this pathway contributes to hepatic inflammation, a key process that stimulates hepatic fibrogenesis associated with chronic liver disease.

[Mechanism of traditional Chinese medicine in regulating NLRP3 inflammasomes to alleviate renal interstitial fibrosis in diabetic nephropathy: a review].

Diabetic nephropathy(DN), a progressive chronic kidney disease(CKD) induced by diabetes mellitus, is the main cause of end-stage renal disease. Renal interstitial fibrosis(RIF) is an irreversible factor in the progression and deterioration of the renal function in DN. Chronic inflammation has become a key link in the pathogenesis of DN-RIF. The NOD-like receptor thermal protein domain associated protein 3(NLRP3) inflammasome is an important inflammatory regulator regulated by a variety of signals. It promotes the production of pro-inflammatory cytokines and induces renal inflammatory cell infiltration to participate in the process of renal fibrosis, demonstrating a complex mechanism of action. In view of the important role of NLRP3 inflammasomes in the prevention and treatment of DN-RIF, a large number of experimental studies have demonstrated that traditional Chinese medicine(TCM) can reduce the inflammation by regulating the pathways involving NLRP3 inflammasome, thereby slowing down the progression of DN-RIF and improving the renal function. This paper reviews the relationship between NLRP3 inflammasomes and DN-RIF, and the research progress in the mechanism of TCM intervention in NLRP3 inflammasomes to alleviate DN-RIF, aiming to provide new ideas for the targeted treatment of DN-RIF.

[Mechanism of Shenling Baizhu Powder on treatment of alcoholic liver disease by regulating TLR4/NLRP3 pathway].

This study aims to investigate the regulatory effects of Shenling Baizhu Powder(SBP) on cellular autophagy in alcoholic liver disease(ALD) and its intervention effect through the TLR4/NLRP3 pathway. A rat model of chronic ALD was established by gavage of spirits. An ALD cell model was established by stimulating BRL3A cells with alcohol. High-performance liquid chromatography(HPLC) was utilized for the compositional analysis of SBP. Liver tissue from ALD rats underwent hematoxylin-eosin(HE) and oil red O staining for pathological evaluation. Enzyme-linked immunosorbent assay(ELISA) was applied to quantify lipopolysaccharides(LPS), tumor necrosis factor-alpha(TNF-α), interleukin-1 beta(IL-1ß), and interleukin-18(IL-18) levels. Quantitative reverse transcription polymerase chain reaction(qRT-PCR) was conducted to evaluate the mRNA expression of myeloid differentiation factor 88(MyD88) and Toll-like receptor 4(TLR4). The effect of different drugs on BRL3A cell proliferation activity was assessed through CCK-8 analysis. Western blot analysis was performed to examine the protein expression of NOD-like receptor pyrin domain-containing 3(NLRP3), nuclear factor-kappa B P65(NF-κB P65), phosphorylated nuclear factor-kappa B P65(p-P65), caspase-1, P62, Beclin1, and microtubule-associated protein 1 light chain 3(LC3Ⅱ). The results showed that SBP effectively ameliorated hepatic lipid accumulation, reduced liver function, mitigated hepatic tissue inflammation, and reduced levels of LPS, TNF-α, IL-1ß, and IL-18. Moreover, SBP exhibited the capacity to modulate hepatic autophagy induced by prolonged alcohol intake through the TLR4/NLRP3 signaling pathway. This modulation resulted in decreased expression of LC3Ⅱ and Beclin1, an elevation in P62 expression, and the promotion of autolysosome formation. These research findings imply that SBP can substantially enhance liver function and mitigate lipid irregularities in the context of chronic ALD. It achieves this by regulating excessive autophagic responses caused by prolonged spirit consumption, primarily through the inhibition of the TLR4/NLRP3 pathway.

Modulation and Determination of the Status of Inflammasomes in Leishmania-Infected Macrophages.

Leishmania, an intra-macrophage kinetoplastid parasite, modulates a vast array of defensive mechanisms of the host macrophages to create a comfortable environment for their survival. When the host encounters intracellular pathogens, a multimeric protein complex called NLRP3 inflammasome gets turned on, leading to caspase-1 activation-mediated maturation of IL-1ß from its pro-form. However, Leishmania often manages to neutralize inflammasome activation by manipulating negative regulatory molecules of the host itself. Exhaustion of NLRP3 and pro-IL-1ß result from decreased NF-κB activity in infection, which was attributed to increased expression of A20, a negative regulator of NF-κB signalling. Moreover, reactive oxygen species, another key requirement for inflammasome activation, are inhibited by mitochondrial uncoupling protein 2 (UCP2) which is upregulated by Leishmania. Inflammasome activation is a complex event and procedures involved in monitoring inflammasome activation need to be accurate and error-free. In this chapter, we summarize the protocol that includes various experimental procedures required for the determination of the status of inflammasomes in Leishmania-infected macrophages.

Chronic mild stress-induced dysregulation of MAPK and PI3K/AKT signaling in the hippocampus and medial prefrontal cortex of WKY female rats.

Wistar-Kyoto (WKY) rats subjected to chronic mild stress (CMS) represent a valid model of treatment-resistant depression (TRD). Considering that depression is more prevalent in women than in men, in the present study, female rats were used. We investigated the effect of CMS on behavior and different factors involved in neuroinflammatory processes and neuroplasticity in the hippocampus and medial prefrontal cortex (mPFC) of WKY female rats. The results show that unstressed WKY females exhibited hypolocomotion, decreased exploratory behavior, and an increase in the total grooming time. After exposure to CMS, WKY females displayed intensified grooming. To investigate potential neural mechanisms underlying these behavioral changes, we analyzed signaling and inflammatory pathways in the hippocampus and mPFC. The findings indicate reduced BDNF and elevated levels levels of IL-1ß in both brain structures and NLRP3 in the mPFC of unstressed WKY female rats. WKY rats subjected to CMS showed a further decrease in BDNF levels and increased IL-1ß and NLRP3 in these brain structures. WKY showed reduced pERK1/2 and increased pp38 levels in both brain structures, while CMS revealed a further increase of pp38 in WKY in these brain structures. Expressions of p110ß and pAKT were decreased in the hippocampus and mPFC of WKY rats. The CMS further suppressed p110 and the downstream AKT phosphorylation in the hippocampus, but did not affect the p110 and pAKT in the mPFC. Our findings indicate behavioral and molecular differences in genetically vulnerable WKY female rats and in their response to CMS that may be involved in TRD.

Discovery of anti-inflammatory agents from 3, 4-dihydronaphthalene-1(2H)-one derivatives by inhibiting NLRP3 inflammasome activation.

NLRP3 inflammatory vesicles are a polymer of cellular innate immunity composed of a pair of proteins. The continuous activation of NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammatory vesicles induces the occurrence and enhancement of inflammatory response. In this study, a series of 3, 4-dihydronaphthalene-1(2H)-one derivatives (DHNs, 6a-u, 7a-e, 8a-n) were synthesized and characterized by NMR and HRMS. We evaluated the cytotoxicity and anti-inflammatory activity of all compounds in vitro, and selected 7a substituted by 7-Br in A-ring and 2-pyridylaldehyde in C-ring as effective lead compounds. Specifically, 7a can block the assembly and activation of NLRP3 inflammasome by down-regulating the expression of NLPR3 and apoptosis-associated speck-like protein containing a CARD (ASC), and inhibiting the production of reactive oxygen species (ROS) and other inflammatory mediators. In addition, 7a inhibits the phosphorylation of inhibitor kappa B alpha (IκBα) and NF-κB/p65 and the nuclear translocation of p65, thereby inhibiting nuclear factor kappa-B (NF-κB) signaling. Molecular docking analysis confirmed that 7a could reasonably bind the active sites of NLRP3, ASC and p65 proteins. Therefore, 7a is predicted as a potential NLRP3 inflammatory vesicle inhibitor and deserves further research and development.

Mitochondria-targeted reactive oxygen species blockor SS-31 blocks hepatic stellate cell activation and alleviates hepatic fibrosis by regulating NLRP3 inflammasomes.

This study aimed to elucidate the effect of mitochondria-targeted reactive oxygen species (ROS) blockor SS-31 on hepatic stellate cells (HSC) activation during liver fibrosis. TGF-ß1 was employed to induce HSC activation, while MitoSOX Red was utilized to assess the presence of mitochondrial ROS. The mitochondrial membrane potential (MMP) was measured using the JC-1 probe, and the ATP level was determined using a specific kit. The proliferation of HSCs was assessed using CCK-8 and colony formation assays, whereas flow cytometry was employed to detect HSC apoptosis. Fibrotic markers (COL1A1 and α-SMA) and NLRP3 inflammasome components (NLRP3, caspase-1, and ASC) were analyzed via Western blotting. Liver fibrosis was induced in mice using CCl4, and subsequently, histopathological changes were observed through HE staining and Masson staining. In TGF-ß1-activated HSCs, mitochondrial ROS expression increased, MMP and ATP content decreased, indicating mitochondrial damage. After TGF-ß1 induction, HSC proliferation increased, apoptosis decreased, and COL1A1, α-SMA, and NLRP3 inflammasome protein expression increased. After SS-31 treatment, mitochondrial ROS expression decreased, MMP recovered, ATP level increased, HSC proliferation decreased, apoptosis increased, and the expressions of COL1A1, α-SMA, and NLRP3 inflammasome decreased. NLRP3 blockor MCC950 treatment blocked HSC activation. CCL4-induced liver fibrosis mice had inflammatory cell infiltration and significant collagen fiber deposition in the liver. After SS-31 treatment, liver inflammation and collagen deposition were significantly reduced. SS-31, as a mitochondria-targeted ROS blockor, can block HSC activation by regulating the NLRP3 inflammasome, thereby alleviating liver fibrosis.

Assessing and mitigating foodborne acetochlor exposure induced ileum toxicity in broiler chicks: The role of omega-3 polyunsaturated fatty acids supplementation and molecular pathways analysis.

Excessive acetochlor residues present ecological and food safety challenges. Here, broiler chicks were exposed to varied acetochlor doses to first assess its effects on the gut. Subsequent dietary supplementation with omega-3 was used to assess its anti-contamination effects. Pathologically, acetochlor induced notable ileal lesions including inflammation, barrier disruption, tight junction loss, and cellular anomalies. Mechanistically, acetochlor stimulated the TNFα/TNFR1 and TLR4/NF-κB/NLRP3 pathways, promoting RIPK1/RIPK3 complex formation, MLKL phosphorylation, NLRP3 inflammasome activation, Caspase-1 activation, and GSDMD shearing with inflammatory factor release. These mechanisms elucidate ileal cell death patterns essential for understanding chicken enteritis. Omega-3 supplementation showed promise in mitigating inflammation, though its precise counteractive role remains unclear. Our findings suggest early omega-3 intervention offered protective benefits against acetochlor's adverse intestinal effects, emphasizing its potential poultry health management role. Harnessing dietary interventions' therapeutic potential will be pivotal in ensuring sustainable poultry production and food safety despite persistent environmental contaminants.

Vitamin D Mitigates Hepatic Fat Accumulation and Inflammation and Increases SIRT1/AMPK Expression in AML-12 Hepatocytes.

Emerging evidence has demonstrated a strong correlation between vitamin D status and fatty liver disease. Aberrant hepatic fat infiltration contributes to oxidant overproduction, promoting metabolic dysfunction, and inflammatory responses. Vitamin D supplementation might be a good strategy for reducing hepatic lipid accumulation and inflammation in non-alcoholic fatty liver disease and its associated diseases. This study aimed to investigate the role of the most biologically active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D), in hepatic fat accumulation and inflammation in palmitic acid (PA)-treated AML-12 hepatocytes. The results indicated that treatment with 1,25(OH)2D significantly decreased triglyceride contents, lipid peroxidation, and cellular damage. In addition, mRNA levels of apoptosis-associated speck-like CARD-domain protein (ASC), thioredoxin-interacting protein (TXNIP), NOD-like receptor family pyrin domain-containing 3 (NLRP3), and interleukin-1ß (IL-1ß) involved in the NLRP3 inflammasome accompanied by caspase-1 activity and IL-1ß expression were significantly suppressed by 1,25(OH)2D in PA-treated hepatocytes. Moreover, upon PA exposure, 1,25(OH)2D-incubated AML-12 hepatocytes showed higher sirtulin 1 (SIRT1) expression and adenosine monophosphate-activated protein kinase (AMPK) phosphorylation. A SIRT1 inhibitor alleviated the beneficial effects of 1,25(OH)2D on PA-induced hepatic fat deposition, IL-1ß expression, and caspase-1 activity. These results suggest that the favorable effects of 1,25(OH)2D on hepatic fat accumulation and inflammation may be, at least in part, associated with the SIRT1.