Disconnecting prefrontal cortical neurons from the ventral midline thalamus: Loss of specificity due to progressive neural toxicity of an AAV-Cre in the rat thalamus.

BACKGROUND: The thalamic reuniens (Re) and rhomboid (Rh) nuclei are bidirectionally connected with the medial prefrontal cortex (mPFC) and the hippocampus (Hip). Fiber-sparing N-methyl-D-aspartate lesions of the ReRh disrupt cognitive functions, including persistence of certain memories. Because such lesions irremediably damage neurons interconnecting the ReRh with the mPFC and the Hip, it is impossible to know if one or both pathways contribute to memory persistence. Addressing such an issue requires selective, pathway-restricted and direction-specific disconnections. NEW METHOD: A recent method associates a retrograde adeno-associated virus (AAV) expressing Cre recombinase with an anterograde AAV expressing a Cre-dependent caspase, making such disconnection feasible by caspase-triggered apoptosis when both constructs meet intracellularly. We injected an AAVrg-Cre-GFP into the ReRh and an AAV5-taCasp into the mPFC. As expected, part of mPFC neurons died, but massive neurotoxicity of the AAVrg-Cre-GFP was found in ReRh, contrasting with normal density of DAPI staining. Other stainings demonstrated increasing density of reactive astrocytes and microglia in the neurodegeneration site. COMPARISON WITH EXISTING METHODS: Reducing the viral titer (by a 4-fold dilution) and injection volume (to half) attenuated toxicity substantially, still with evidence for partial disconnection between mPFC and ReRh. CONCLUSIONS: There is an imperative need to verify potential collateral damage inherent in this type of approach, which is likely to distort interpretation of experimental data. Therefore, controls allowing to distinguish collateral phenotypic effects from those linked to the desired disconnection is essential. It is also crucial to know for how long neurons expressing the Cre-GFP protein remain operational post-infection.

Piceatannol selectively inhibited the JNK3 enzyme and augmented apoptosis through inhibition of Bcl-2/Cyt-c/caspase-dependent pathways in the oxygen-glucose deprived SHSY-5Y cell lines: In silico and in vitro study.

JNK3, a neuronal kinase activated by stress, plays a role in stress-induced apoptosis, leading to neuronal cell death following cerebral ischemia. This study investigates the neuroprotective effects of piceatannol (PCT) in SHSY-5Y neuroblastoma cells after hypoxic injury and its interaction with JNK3. We analyzed the crystal coordinates, interaction energies, and amino acid interactions to determine PCT's selectivity for JNK3. The electrostatic potential was computed using density functional theory, while molecular dynamics assessed the stability and structural consistency of the JNK3-PCT complex. We used SP600125 (SP6), a JNK3 inhibitor, as a reference compound. Additionally, we performed cell-free JNK 1, 2, and 3 kinase assays to evaluate the isoform selectivity of PCT. Cytotoxicity and cell viability were determined by an MTT test. To assess apoptosis, we used acridine orange/ethidium bromide dual fluorescent labeling and ANNEXIN A5-FITC flow cytometry. Western blot was used to evaluate the attenuation of JNK3 and apoptotic proteins. In silico studies revealed a stronger binding affinity between PCT and JNK3 compared to JNK1 and JNK2, which was further supported by the in vitro kinase assay. PCT-treated cells exhibited a decrease in Cyt-c and caspase-3 expression and an increase in Bcl-2 level, compared to hypoxic control (p < .001). PCT also demonstrated superior efficacy over SP6 in inhibiting JNK3 phosphorylation (p < .001). Furthermore, PCT significantly increased the expression of neuronal genes, including NgN1, neuroD2, and survivin (p < .001). In conclusion, PCT is a potential JNK3 inhibitor, since it inhibited phosphorylation and the Bcl-2/Cyt-C/caspase-3-dependent apoptotic pathway after ischemic/hypoxic insult.

Natural compound library screening to identify berberine as a treatment for rheumatoid arthritis.

OBJECTIVE: Fibroblast-like synoviocytes (FLS) play a critical role on the exacerbation and deterioration of rheumatoid arthritis (RA). Aberrant activation of FLS pyroptosis signaling is responsible for the hyperplasia of synovium and destruction of cartilage of RA. This study investigated the screened traditional Chinese medicine berberine (BBR), an active alkaloid extracted from the Coptis chinensis plant, that regulates the pyroptosis of FLS and secretion of inflammatory factors in rheumatoid arthritis. METHODS: First, BBR was screened using a high-throughput drug screening strategy, and its inhibitory effect on RA-FLS was verified by in vivo and in vitro experiments. Second, BBR was intraperitoneally administrated into the collagen-induced arthritis rat model, and the clinical scores, arthritis index, and joint HE staining were evaluated. Third, synovial tissues of CIA mice were collected, and the expression of NLRP3, cleaved-caspase-1, GSDMD-N, Mst1, and YAP was detected by Western blot. RESULTS: The administration of BBR dramatically alleviated the severity of collagen-induced arthritis rat model with a decreased clinical score and inflammation reduction. In addition, BBR intervention significantly attenuates several pro-inflammatory cytokines (interleukin-1ß, interleukin-6, interleukin-17, and interleukin-18). Moreover, BBR can reduce the pyroptosis response (caspase-1, NLR family pyrin domain containing 3, and gasdermin D) of the RA-FLS in vitro, activating the Hippo signaling pathway (Mammalian sterile 20-like kinase 1, yes-associated protein, and transcriptional enhanced associate domains) so as to inhibit the pro-inflammatory effect of RA-FLS. CONCLUSION: These results support the role of BBR in RA and may have therapeutic implications by directly repressing the activation, migration of RA-FLS, which contributing to the attenuation of the progress of CIA. Therefore, targeting PU.1 might be a potential therapeutic approach for RA. Besides, BBR inhibited RA-FLS pyroptosis by downregulating of NLRP3 inflammasomes (NLRP3, caspase-1) and eased the pro-inflammatory activities via activating the Hippo signaling pathway, thereby improving the symptom of CIA.

Biomimetic-gasdermin-protein-expressing nanoplatform mediates tumor-specific pyroptosis for cancer immunotherapy.

Pyroptosis, mediated by gasdermin proteins, has shown excellent efficacy in facilitating cancer immunotherapy. The strategies commonly used to induce pyroptosis suffer from a lack of tissue specificity, resulting in the nonselective activation of pyroptosis and consequent systemic toxicity. Moreover, pyroptosis activation usually depends on caspase, which can induce inflammation and metabolic disorders. In this study, inspired by the tumor-specific expression of SRY-box transcription factor 4 (Sox4) and matrix metalloproteinase 2 (MMP2), we constructed a doubly regulated plasmid, pGMD, that expresses a biomimetic gasdermin D (GSDMD) protein to induce the caspase-independent pyroptosis of tumor cells. To deliver pGMD to tumor cells, we used a hyaluronic acid (HA)-shelled calcium carbonate nanoplatform, H-CNP@pGMD, which effectively degrades in the acidic endosomal environment, releasing pGMD into the cytoplasm of tumor cells. Upon the initiation of Sox4, biomimetic GSDMD was expressed and cleaved by MMP2 to induce tumor-cell-specific pyroptosis. H-CNP@pGMD effectively inhibited tumor growth and induced strong immune memory effects, preventing tumor recurrence. We demonstrate that H-CNP@pGMD-induced biomimetic GSDMD expression and tumor-specific pyroptosis provide a novel approach to boost cancer immunotherapy.

Combination of Doxycycline with Metronidazole Protects against Pyroptosis in Rats with Endometritis through the Modulation of TLR4/NF-κB Pathway.

BACKGROUND: Endometritis is a condition usually resulted from the bacterial infection of uterus, causing pelvic disease, sepsis, shock, uterine necrosis and even death if it is inappropriately treated. The aim of this study is to explore the pathogenesis of endometritis, and investigate whether the combination of doxycycline and metronidazole offers stronger protection against lipopolysaccharide (LPS)-induced endometritis, and decipher more about the mechanisms underlying endometritis-related pyroptosis. METHODS: Sprague-Dawley (SD) rats were divided into five groups (n = 8 per group): control, model, metronidazole, doxycycline, and combination groups. In control group, the rats were injected with saline, while in other groups, lipopolysaccharide was injected into uterus of the rats to establish endometritis. Hematoxylin-eosin (H&E) staining was performed as part of the histopathological examination of endometrium. The integrity of chromatin and pyroptosis were evaluated by terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay. Western blot and quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) were performed to ascertain the activation of toll-like receptors (TLR4)/nuclear factor-kappa B (NF-κB) pathway by detecting protein levels of phosphorylated p50 (p-p50)/p50, phosphorylated nuclear factor-kappa B (p-NF-κB)/NF-κB, phosphorylated IkappaB (p-IκB), and TLR4 protein and mRNA. Development of pyroptosis was also detected by determining the levels of caspase-1 and caspase-5 through Western blot and qRT-PCR. Enzyme-linked immunosorbent assay (ELISA) was used to detect levels of interleukin (IL)-1ß, IL-18, IL-2, IL-4, IL-6 and tumor necrosis factor alpha (TNF-α), and flow cytometry was adopted to determine T-helper (Th)1 and Th2 cell percentage to assess the extent of pyroptosis and Th1/Th2 imbalance. RESULTS: The uterine of the model group exhibited pathological alterations and higher degree of cell apoptosis. Compared with the control rats, model group showed lower protein levels of p-p50/p50 (p < 0.001), p-NF-κB/NF-κB (p < 0.001), p-IκB (p < 0.001), and TLR4 protein (p < 0.001) and mRNA (p < 0.001). Elevated levels of caspase-1 (p < 0.001), caspase-5 (p < 0.001), IL-1ß (p < 0.001), IL-18 (p < 0.001), IL-2 (p < 0.01), TNF-α (p < 0.05) and Th1/Th2 (p < 0.001) as well as reduced levels of IL-4 (p < 0.05) and IL-6 (p < 0.01) were observed in the model group, which could however be reversed by metronidazole (p < 0.01) or doxycycline (p < 0.01), with a more significant effect detected if a combination of the two drugs was administered (p < 0.01). CONCLUSIONS: The combination of doxycycline and metronidazole protects against rat endometritis by inhibiting TLR4/NF-κB pathway-mediated inflammation and suppressing pyroptosis.

Synergistic inhibitory actions of resveratrol, epigallocatechin-3-gallate, and diallyl trisulfide against skin cancer cell line A431 through mitochondrial caspase dependent pathway: a combinational drug approach.

The harmful effect of chemotherapeutic side effects has paid a way to discover a novel with curative way for skin cancer treatment. Skin cancer prevention is more viable with the use of combination of bioactive agents than using of single bioactive compounds. Present work was demonstrated to evaluate the interaction of Resveratrol (Res), Epigallocatechin-3-gallate (EGCG), and diallyl trisulfide (DATS) with each other as a binary combination on A431 cells. Nuclear fragmentation analysis of combination of bioactive agents using DAPI analysis, detection of apoptosis, analysis of cell cycle, ROS assay, antimigration assays, and western blotting were implemented to study the combination of bioactive compounds on A431 cell line. Among the selected combination EGCG + DATS had a synergetic effect reducing cellular migration, increased intercellular reactive oxygen species generation, condensation, cell phagocytosis induced by phosphatidylserine externalization, rise in sub-G1 DNA content, and S-phase were cell cycle arrest. The combinations EGCG + DATS induced apoptotic proteins in A431 cells by upregulation of proapoptotic Bax and Bad proteins, a downmodulation of anti-apoptotic proteins Bcl2 and caspases (caspase-3, and -9) activity got triggered by intrinsic pathway. The combination of EGCG + DATS showed good anticancer potential against A431 skin cancer cell line via the mitochondrial caspase dependent pathway with very strong synergism. This finding will help to produce a novel combination/chemoprevention using dietary bioactive agents (EGCG + DATS) for the treatment of skin cancer after clinical trial.

GLP-1RAs inhibit the activation of the NLRP3 inflammasome signaling pathway to regulate mouse renal podocyte pyroptosis.

OBJECTIVE: Podocytes are closely related to renal function as an important part of the glomerulus. The reduction and damage of podocytes lead to further decline of renal function and aggravate the progression of DKD. Glucagon-like peptide-1 receptor agonists (GLP-1RAS) have recently attracted great attention in improving podocyte dysfunction, but the specific mechanism remains uncertain. METHODS: We used mouse kidney podocyte MPC5 to construct a high-glucose injury model. Cell viability was detected by the MTT method; RT-qPCR and western blotting were used to detect the expressions of NF-κB p65, NLRP3, GSDMD, N-GSDMD, caspase-1 and cleaved-caspase-1, and we used ELISA to detect the expressions of inflammatory factors IL-1ß and IL-18. RESULTS: Our results showed that high glucose decreased podocyte survival, while liraglutide and semaglutide increased podocyte survival under high glucose. Liraglutide and semaglutide can inhibit the expression of pyroptosis-related genes and proteins and also inhibit the expression of inflammatory factors IL-1ß, IL-18 increase. CONCLUSION: The protective effect of liraglutide and semaglutide on podocytes may be achieved by regulating the NLRP3 inflammasome pathway and inhibiting pyroptosis, and there were no significant differences between the two GLP-1RAs (liraglutide and semaglutide) in inhibiting podocyte pyroptosis.

Caspase cleavage of gasdermin E causes neuronal pyroptosis in HIV-associated neurocognitive disorder.

Despite effective antiretroviral therapies, 20-30% of persons with treated HIV infection develop a neurodegenerative syndrome termed HIV-associated neurocognitive disorder (HAND). HAND is driven by HIV expression coupled with inflammation in the brain but the mechanisms underlying neuronal damage and death are uncertain. The inflammasome-pyroptosis axis coordinates an inflammatory type of regulated lytic cell death that is underpinned by the caspase-activated pore-forming gasdermin proteins. The mechanisms driving neuronal pyroptosis were investigated herein in models of HAND, using multi-platform molecular and morphological approaches that included brain tissues from persons with HAND and simian immunodeficiency virus (SIV)-infected non-human primates as well as cultured human neurons. Neurons in the frontal cortices from persons with HAND showed increased cleaved gasdermin E (GSDME), which was associated with ß-III tubulin degradation and increased HIV levels. Exposure of cultured human neurons to the HIV-encoded viral protein R (Vpr) elicited time-dependent cleavage of GSDME and Ninjurin-1 (NINJ1) induction with associated cell lysis that was inhibited by siRNA suppression of both proteins. Upstream of GSDME cleavage, Vpr exposure resulted in activation of caspases-1 and 3. Pretreatment of Vpr-exposed neurons with the caspase-1 inhibitor, VX-765, reduced cleavage of both caspase-3 and GSDME, resulting in diminished cell death. To validate these findings, we examined frontal cortical tissues from SIV-infected macaques, disclosing increased expression of GSDME and NINJ1 in cortical neurons, which was co-localized with caspase-3 detection in animals with neurological disease. Thus, HIV infection of the brain triggers the convergent activation of caspases-1 and -3, which results in GSDME-mediated neuronal pyroptosis in persons with HAND. These findings demonstrate a novel mechanism by which a viral infection causes pyroptotic death in neurons while also offering new diagnostic and therapeutic strategies for HAND and other neurodegenerative disorders.

Melatonin alleviates pyroptosis by regulating the SIRT3/FOXO3α/ROS axis and interacting with apoptosis in Atherosclerosis progression.

BACKGROUND: Atherosclerosis (AS), a significant contributor to cardiovascular disease (CVD), is steadily rising with the aging of the global population. Pyroptosis and apoptosis, both caspase-mediated cell death mechanisms, play an essential role in the occurrence and progression of AS. The human pineal gland primarily produces melatonin (MT), an indoleamine hormone with powerful anti-oxidative, anti-pyroptotic, and anti-apoptotic properties. This study examined MT's anti-oxidative stress and anti-pyroptotic effects on human THP-1 macrophages treated with nicotine. METHODS: In vitro, THP-1 macrophages were induced by 1 µM nicotine to form a pyroptosis model and performed 30 mM MT for treatment. In vivo, ApoE-/- mice were administered 0.1 mg/mL nicotine solution as drinking water, and 1 mg/mL MT solution was intragastric administrated at 10 mg/kg/day. The changes in pyroptosis, apoptosis, and oxidative stress were detected. RESULTS: MT downregulated pyroptosis, whose changes were paralleled by a reduction in reactive oxygen species (ROS) production, reversal of sirtuin3 (SIRT3), and Forkhead box O3 (FOXO3α) upregulation. MT also inhibited apoptosis, mainly caused by the interaction of caspase-1 and caspase-3 proteins. Vivo studies confirmed that nicotine could accelerate plaque formation. Moreover, mice treated with MT showed a reduction in AS lesion area. CONCLUSIONS: MT alleviates pyroptosis by regulating the SIRT3/FOXO3α/ROS axis and interacting with apoptosis. Importantly, our understanding of the inhibitory pathways for macrophage pyroptosis will allow us to identify other novel therapeutic targets that will help treat, prevent, and reduce AS-associated mortality.

ß-Caryophyllene decreases neuroinflammation and exerts neuroprotection of dopaminergic neurons in a model of hemiparkinsonism through inhibition of the NLRP3 inflammasome.

INTRODUCTION: Parkinson's disease represents a neurodegenerative condition characterized by the progressive loss of dopaminergic neurons within the Substantia Nigra pars compacta (SNpc), resulting in diminished dopamine levels in the striatum (STR) and chronic neuroinflammation. Recent investigations have proposed the neuroprotective potential of the endocannabinoid system in neurodegenerative disorders. ß-caryophyllene (BCP) is recognized for its antioxidant and anti-inflammatory properties, attributed to its activation of the type 2 cannabinoid receptor. This study aimed to assess the neuroprotective impact of BCP on dopaminergic neurons, with a particular focus on inhibiting the NLRP3 inflammasome. METHODS: A model of hemiparkinsonism, induced by 6-hydroxydopamine (6-OHDA), served as the experimental framework. Motor function was evaluated using the cylinder test, and inflammasome inhibition was determined by assessing the expression of NLRP3, caspase-1, and the pro-inflammatory cytokine IL-1ß in both the SNpc and STR through ELISA analysis. Furthermore, the evaluation of oxidative stress was facilitated by quantifying malondialdehyde (MDA) levels in the same regions. RESULTS: BCP treatment demonstrated significant improvements in motor dysfunction, as assessed by the cylinder test (p=0.0011) and exhibited a neuroprotective effect on dopaminergic neurons within the SNpc (p=0.0017), as well as nerve fibers in the STR (p=0.0399). In terms of its ability to inhibit the inflammasome, BCP led to decreased expression levels of NLRP3 (p=0.0401 in STR and p = 0.0139 in SNpc), caspase-1 (p=0.0004 in STR), and MDA (p=0.0085 in STR and p=0.0414 in SNpc). CONCLUSION: These results point to BCP's potential in mitigating the motor deficit, inhibiting NLRP3 inflammasome activation, and attenuating lipid peroxidation induced by 6-OHDA.

Activation of cannabinoid receptors 2 alleviates myocardial damage in cecal ligation and puncture-induced sepsis by inhibiting pyroptosis.

BACKGROUND: It has been reported that cannabinoid receptors 2 (CB2 receptors) play an important role in the pathophysiological process of sepsis, which may also be associated with the regulation of pyroptosis, an inflammatory programmed cell death. The present study aimed to investigate the protective effect of CB2 receptors on myocardial damage in a model of septic mice by inhibiting pyroptosis. METHODS: The C57BL/6 mice underwent cecal ligation and puncture (CLP) to induce sepsis. All mice were randomly divided into the sham, CLP, or CLP+HU308 group. Blood and heart tissue samples were collected 12 h after surgery. Hematoxylin and eosin staining was used for analyzing histopathological results. Creatine kinase isoenzymes (CK-MB) and IL-1ß were measured using ELISA, while lactate dehydrogenase (LDH) level was determined using photoelectric colorimetry. The expression levels of CB2 receptors and pyroptosis-associated proteins (NLRP3, caspase-1, and GSDMD) were measured using western blotting. The location and distribution of CB2 receptors and caspase-1 in myocardial tissues were assessed by immunofluorescence. TUNEL staining was used to quantify the number of dead cells in myocardial tissues. RESULTS: The CLP procedure increased CB2 receptor expression in mice. CB2 receptors were located in myocardial macrophages. Activating CB2 receptors decreased the levels of myocardial damage mediator LDH, CK-MB, and inflammatory cytokine IL-1ß. The results also showed that CLP increased the pyroptosis in myocardial tissues, while CB2 agonist HU308 inhibited pyroptosis by decreasing the level of NLRP3 and activating caspase-1 and GSDMD. CONCLUSIONS: CB2 receptor activation has a protective effect on the myocardium of mice with sepsis by inhibiting pyroptosis.

High-fat diet alters intestinal microbiota and induces endoplasmic reticulum stress via the activation of apoptosis and inflammation in blunt snout bream.

The primary organ for absorbing dietary fat is the gut. High dietary lipid intake negatively affects health and absorption by causing fat deposition in the intestine. This research explores the effect of a high-fat diet (HFD) on intestinal microbiota and its connections with endoplasmic reticulum stress and inflammation. 60 fish (average weight: 45.84 ± 0.07 g) were randomly fed a control diet (6% fat) and a high-fat diet (12 % fat) in four replicates for 12 weeks. From the result, hepatosomatic index (HSI), Visceralsomatic index (VSI), abdominal fat (ADF), Intestosomatic index (ISI), mesenteric fat (MFI), Triglycerides (TG), total cholesterol (TC), non-esterified fatty acid (NEFA) content were substantially greater on HFD compared to the control diet. Moreover, fish provided the HFD significantly obtained lower superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities. In contrast, an opposite result was seen in malondialdehyde (MDA) content in comparison to the control. HFD significantly altered intestinal microbiota in blunt snout bream, characterized by an increased abundance of Aeromonas, Plesiomonas proteobacteria, and firmicutes with a reduced abundance of Cetobacterium and ZOR0006. The transcriptional levels of glucose-regulated protein 78 (grp78), inositol requiring enzyme 1 (ire1), spliced X box-binding protein 1 (xbp1), DnaJ heat shock protein family (Hsp40) member B9 (dnajb9), tumor necrosis factor alpha (tnf-α), nuclear factor-kappa B (nf-κb), monocyte chemoattractant protein-1 (mcp-1), and interleukin-6 (il-6) in the intestine were markedly upregulated in fish fed HFD than the control group. Also, the outcome was similar in bax, caspases-3, and caspases-9, ZO-1, Occludin-1, and Occludin-2 expressions. In conclusion, HFD could alter microbiota and facilitate chronic inflammatory signals via activating endoplasmic reticulum stress.

The noncanonical inflammasome-induced pyroptosis and septic shock.

Sepsis remains one of the most common and lethal conditions globally. Currently, no proposed target specific to sepsis improves survival in clinical trials. Thus, an in-depth understanding of the pathogenesis of sepsis is needed to propel the discovery of effective treatment. Recently attention to sepsis has intensified because of a growing recognition of a non-canonical inflammasome-triggered lytic mode of cell death termed pyroptosis upon sensing cytosolic lipopolysaccharide (LPS). Although the consequences of activation of the canonical and non-canonical inflammasome are similar, the non-canonical inflammasome formation requires caspase-4/5/11, which enzymatically cleave the pore-forming protein gasdermin D (GSDMD) and thereby cause pyroptosis. The non-canonical inflammasome assembly triggers such inflammatory cell death by itself; or leverages a secondary activation of the canonical NLRP3 inflammasome pathway. Excessive cell death induced by oligomerization of GSDMD and NINJ1 leads to cytokine release and massive tissue damage, facilitating devastating consequences and death. This review summarized the updated mechanisms that initiate and regulate non-canonical inflammasome activation and pyroptosis and highlighted various endogenous or synthetic molecules as potential therapeutic targets for treating sepsis.

Effect of NLRP3 deficiency on cytotoxic and IL-1ß-producing activities of synthetic candidalysin peptide.

OBJECTIVES: Candidalysin (CL), a hydrophobic peptide toxin secreted by Candida albicans, is a key virulence factor that contributes to cytolysis, tissue damage, and immune activation. CL is thought to exert some of its biological activities, including IL-1ß production, through the activation of the NLRP3-inflammasome pathway. To date, the mechanism by which CL affects human NLRP3 is not fully understood. We investigated specific activities of synthetic CL peptides using human-derived NLRP3-deficient cells. METHODS: Two distinct synthetic CL peptide solutions were prepared: CLd, with CL completely solubilized as nanoparticles in dimethyl sulfoxide, and CLw, with CL partly solubilized in water, and including insoluble microparticles. THP-1 human monocytic cells and NLRP3-deficient THP-1 cells were differentiated into macrophages and stimulated with these peptide solutions. Cell membrane damage, lactate dehydrogenase release, IL-1ß production, and caspase-1 activation in stimulated cells were subsequently evaluated. RESULTS: Both CLd and CLw exhibited cytotoxic activities independent of NLRP3. Importantly, CLd induced IL-1ß production and caspase-1 activation in an NLRP3-independent manner, whereas these activities in CLw-stimulated cells were entirely NLRP3-dependent, suggesting that the NLRP3-dependent response might be triggered by insoluble microparticles. CONCLUSIONS: Our results demonstrate that inherent CL activities can cause cell damage and IL-1ß production in an NLRP3-independent manner. Our research advances the elucidation of the role of NLRP3 in CL biological activity, underscoring the necessity for further exploration of the precise mechanisms underlying the NLRP3-independent effects of CL and providing novel insights into the complexity of host-pathogen interactions.

Inhibition of the uric acid efflux transporter ABCG2 enhances stimulating effect of soluble uric acid on IL-1ß production in murine macrophage-like J774.1 cells.

Soluble uric acid (UA) absorbed by cells through UA transporters (UATs) accumulates intracellularly, activates the NLRP3 inflammasome and thereby increases IL-1ß secretion. ABCG2 transporter excludes intracellular UA. However, it remains unknown whether ABCG2 inhibition leads to intracellular accumulation of UA and increases IL-1ß production. In this study, we examined whether genetic and pharmacological inhibition of ABCG2 could increase IL-1ß production in mouse macrophage-like J774.1 cells especially under hyperuricemic conditions. We determined mRNA and protein levels of pro-IL-1ß, mature IL-1ß, caspase-1 and several UATs in culture supernatants and lysates of J774.1 cells with or without soluble UA pretreatment. Knockdown experiments using an shRNA against ABCG2 and pharmacological experiments with an ABCG2 inhibitor were conducted. Extracellularly applied soluble UA increased protein levels of pro-IL-1ß, mature IL-1ß and caspase-1 in the culture supernatant from lipopolysaccharide (LPS)-primed and monosodium urate crystal (MSU)-stimulated J774.1 cells. J774.1 cells expressed UATs of ABCG2, GLUT9 and MRP4, and shRNA knockdown of ABCG2 increased protein levels of pro-IL-1ß and mature IL-1ß in the culture supernatant. Soluble UA increased mRNA and protein levels of ABCG2 in J774.1 cells without either LPS or MSU treatment. An ABCG2 inhibitor, febuxostat, but not a urate reabsorption inhibitor, dotinurad, enhanced IL-1ß production in cells pretreated with soluble UA. In conclusion, genetic and pharmacological inhibition of ABCG2 enhanced IL-1ß production especially under hyperuricemic conditions by increasing intracellularly accumulated soluble UA that activates the NLRP3 inflammasome and pro-IL-1ß transcription in macrophage-like J774.1 cells.

The protective effects of EGCG was associated with HO-1 active and microglia pyroptosis inhibition in experimental intracerebral hemorrhage.

Intracerebral hemorrhage (ICH), which has high mortality and disability rate is associated with microglial pyroptosis and neuroinflammation, and the effective treatment methods are limited Epigallocatechin-3-gallate (EGCG) has been found to play a cytoprotective role by regulating the anti-inflammatory response to pyroptosis in other systemic diseases. However, the role of EGCG in microglial pyroptosis and neuroinflammation after ICH remains unclear. In this study, we investigated the effects of EGCG pretreatment on neuroinflammation-mediated neuronal pyroptosis and the underlying neuroprotective mechanisms in experimental ICH. EGCG pretreatment was found to remarkably improved neurobehavioral performance, and decreased the hematoma volume and cerebral edema in mice. We found that EGCG pretreatment attenuated the release of hemin-induced inflammatory cytokines (IL-1ß, IL-18, and TNF-α). EGCG significantly upregulated the expression of heme oxygenase-1 (HO-1), and downregulated the levels of pyroptotic molecules and inflammatory cytokines including Caspase-1, GSDMD, NLRP3, mature IL-1ß, and IL-18. EGCG pretreatment also decreased the number of Caspase-1-positive microglia and GSDMD along with NLRP3-positive microglia after ICH. Conversely, an HO-1-specific inhibitor (ZnPP), significantly inhibited the anti-pyroptosis and anti-neuroinflammation effects of EGCG. Therefore, EGCG pretreatment alleviated microglial pyroptosis and neuroinflammation, at least in part through the Caspase-1/GSDMD/NLRP3 pathway by upregulating HO-1 expression after ICH. In addition, EGCG pretreatment promoted the polarization of microglia from the M1 phenotype to M2 phenotype after ICH. The results suggest that EGCG is a potential agent to attenuate neuroinflammation via its anti-pyroptosis effect after ICH.

Mechanistic study of dual-function inhibitors targeting topoisomerase II and Rad51-mediated DNA repair pathway against castration-resistant prostate cancer.

BACKGROUND: Castration-resistant prostate cancer (CRPC) is refractory to hormone treatment and the therapeutic options are continuously advancing. This study aims to discover the anti-CRPC effects and underlying mechanisms of small-molecule compounds targeting topoisomerase (TOP) II and cellular components of DNA damage repair. METHODS: Cell proliferation was determined in CRPC PC-3 and DU-145 cells using anchorage-dependent colony formation, sulforhodamine B assay and flow cytometric analysis of CFSE staining. Flow cytometric analyses of propidium iodide staining and JC-1 staining were used to examine the population of cell-cycle phases and mitochondrial membrane potential, respectively. Nuclear extraction was performed to detect the nuclear localization of cellular components in DNA repair pathways. Protein expressions were determined using Western blot analysis. RESULTS: A series of azathioxanthone-based derivatives were synthesized and examined for bioactivities in which WC-A13, WC-A14, WC-A15, and WC-A16 displayed potent anti-CRPC activities in both PC-3 and DU-145 cell models. These WC-A compounds selectively downregulated both TOP IIα and TOP IIß but not TOP I protein expression. WC-A13, WC-A14, and WC-A15 were more potent than WC-A16 on TOP II inhibition, mitochondrial dysfunction, and induction of caspase cascades indicating the key role of amine-containing side chain of the compounds in determining anti-CRPC activities. Furthermore, WC-A compounds induced an increase of γH2AX and activated ATR-Chk1 and ATM-Chk2 signaling pathways. P21 protein expression was also upregulated by WC-A compounds in which WC-A16 showed the least activity. Notably, WC-A compounds exhibited different regulation on Rad51, a major protein in homologous recombination of DNA in double-stranded break repair. WC-A13, WC-A14, and WC-A15 inhibited, whereas WC-A16 induced, the nuclear translocation of Rad51. CONCLUSION: The data suggest that WC-A compounds exhibit anti-CRPC effects through the inhibition of TOP II activities, leading to mitochondrial stress-involved caspase activation and apoptosis. Moreover, WC-A13, WC-A14, and WC-A15 but not WC-A16 display inhibitory activities of Rad51-mediated DNA repair pathway which may increase apoptotic effect of CRPC cells.

Huoxue Jiedu Recipe represses mitochondrial fission to alleviate submandibular gland inflammation in Sjögren's syndrome.

Sjögren's syndrome (SS) is the second most common autoimmune rheumatism. Huoxue Jiedu Recipe (HXJDR) is a kind of traditional Chinese medicine with a variety of pharmacological functions; however, its biological function in SS has not been studied yet. Peripheral blood mononuclear cells (PBMCs) and serum samples were isolated from healthy controls and patients with SS. NOD/Ltj mice were used for developing the SS mouse model. The levels of inflammatory cytokines and NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome-related markers as well as dynamin-related protein 1 (Drp1) were determined by ELISA, quantitative real-time PCR, and western blot analysis, respectively. Hematoxylin and eosin and TUNEL staining detected the pathological damage. A transmission electron microscope was used to observe the mitochondrial microstructure. Inflammatory cytokines IL-18, IL-1ß, B-cell activating factor (BAFF), BAFF-receptor (BAFF-R), IL-6, and TNF-α in serum samples and NLRP3 inflammasome-related makers (NLRP3, cysteinyl aspartate-specific proteinase 1 [caspase-1], apoptosis-associated speck-like protein containing a caspase-1 recruitment domain [ASC], IL-1ß) in PBMCs were greatly upregulated in patients with SS. Furthermore, cytoplasmic phosphorylation of Drp1 and mitochondrial Drp1 level were significantly increased in PBMCs, while mitochondrial swelling and fuzzy inner ridge were observed in PBMCs of patients with SS, suggesting increased mitochondrial fission. Compared with control mice, SS mice showed decreased salivary flow rate, increased submandibular gland index, and more severe inflammatory infiltration and damage as well as mitochondrial fission in submandibular gland tissues. After HXJDR administration, these effects were significantly reversed. HXJDR treatment could alleviate the inflammatory infiltration and pathological damage in submandibular glands of SS mice by inhibiting Drp-1-dependent mitochondrial fission.

Leptin and prolactin reduce cryodamage in normozoospermic human semen samples during cryopreservation / La leptina y la prolactina reducen el criodaño en muestras de semen humano normozoospérmico durante la criopreservación

Objectives: Cryopreservation has destructive effects on the function and structure of spermatozoa. It is known that leptin and prolactin play an active role in decreasing the rates of reactive oxygen species and DNA fragmentation, as well as enhancing sperm motility. Hence, this experiment aimed to investigate the effects of leptin and prolactin as pro-survival factors on the normozoospermic human semen samples during cryopreservation. Material and methods: Semen samples were collected from 15 healthy, fertile men ranging from 25 to 40 years. Cryopreservation of the samples was performed in liquid nitrogen over a period of two weeks, using five varying concentrations of leptin/prolactin, 0, 10, 100, 500, and 1000ng/ml respectively. Sperm motility, total caspase activity, and mitochondrial and cytosolic ROS were measured by flowcytometry, TUNEL, and other appropriate tests after thawing of the samples. Results: Both hormones were observed to have positive effects on the motility of the samples post-cryopreservation, the highest improvement being in the 100ng/ml concentration leptin and prolactin in comparison to the control group (P=0.01 and P=0.041, respectively). A significant reduction of mitochondrial ROS was also observed in 100 and 1000ng/ml of leptin (P=0.042), and there was a considerable decrease in the cytosolic ROS in the 100ng/ml of prolactin in comparison to the control group (P=0.048). Total caspase activity was also highly reduced in the 100, 500, and 1000ng/ml of leptin compared to the control group (P=0.039). Interestingly, both hormones also significantly decreased DNA fragmentation in 1000ng/ml compared to the control group (P=0.042). (AU)

Objetivos: La criopreservación tiene efectos destructivos sobre la función y estructura de los espermatozoides. Se sabe que la leptina y la prolactina desempeñan un papel activo en la disminución de las tasas de especies reactivas de oxígeno (ROS) y la fragmentación del ADN, así como en la mejora de la motilidad de los espermatozoides. Por lo tanto, este experimento tuvo como objetivo investigar los efectos de la leptina y la prolactina como factores de supervivencia en las muestras de semen humano normozoospérmico durante la criopreservación. Material y métodos: Se recolectaron muestras de semen de 15 hombres sanos y fértiles de entre 25 y 40 años. La crioconservación de las muestras se realizó en nitrógeno líquido durante un período de 2 semanas, utilizando 5 concentraciones variables de leptina/prolactina: 0, 10, 100, 500 y 1000ng/ml respectivamente. La motilidad de los espermatozoides, la actividad de caspasa total y las ROS mitocondriales y citosólicas se midieron mediante citometría de flujo, TUNEL y otras pruebas apropiadas después de descongelar las muestras. Resultados: Se observó que ambas hormonas tienen efectos positivos sobre la motilidad de las muestras después de la crioconservación, la mayor mejora se encuentra en la concentración de leptina y prolactina de 100ng/ml en comparación con el grupo de control (p=0,01 y p=0,041, respectivamente). También se observó una reducción significativa de las ROS mitocondriales en 100 y 1000ng/ml de leptina (p=0,042), y hubo una disminución considerable en las ROS citosólicas en los 100ng/ml de prolactina en comparación con el grupo de control (p=0,048). La actividad de la caspasa total también se redujo considerablemente en los 100, 500 y 1000ng/ml de leptina en comparación con el grupo de control (p=0,039). Curiosamente, ambas hormonas también redujeron significativamente la fragmentación del ADN en 1000ng/ml en comparación con el grupo de control (p=0,042). (AU)

Contributions of circadian clock genes to cell survival in fibroblast models of lithium-responsive bipolar disorder.

Bipolar disorder (BD) is characterized by mood episodes, disrupted circadian rhythms and gray matter reduction in the brain. Lithium is an effective pharmacotherapy for BD, but not all patients respond to treatment. Lithium has neuroprotective properties and beneficial effects on circadian rhythms that may distinguish lithium responders (Li-R) from non-responders (Li-NR). The circadian clock regulates molecular pathways involved in apoptosis and cell survival, but how this overlap impacts BD and/or lithium responsiveness is unknown. In primary fibroblasts from Li-R/Li-NR BD patients and controls, we found patterns of co-expression among circadian clock and cell survival genes that distinguished BD vs. control, and Li-R vs. Li-NR cells. In cellular models of apoptosis using staurosporine (STS), lithium preferentially protected fibroblasts against apoptosis in BD vs. control samples, regardless of Li-R/Li-NR status. When examining the effects of lithium treatment of cells in vitro, caspase activation by lithium correlated with period alteration, but the relationship differed in control, Li-R and Li-NR samples. Knockdown of Per1 and Per3 in mouse fibroblasts altered caspase activity, cell death and circadian rhythms in an opposite manner. In BD cells, genetic variation in PER1 and PER3 predicted sensitivity to apoptosis in a manner consistent with knockdown studies. We conclude that distinct patterns of coordination between circadian clock and cell survival genes in BD may help predict lithium response.