Palladium(II)-Indenyl Complexes Bearing N-Heterocyclic Carbene (NHC) Ligands as Potent and Selective Metallodrugs toward High-Grade Serous Ovarian Cancer Models.

In this study, we synthesized novel Pd(II)-indenyl complexes using various N-heterocyclic carbene (NHC) ligands, including chelating NHC-picolyl, NHC-thioether, and diNHC ligands, and two monodentate NHCs. Transmetalation reactions between a Pd(II)-indenyl precursor and silver-NHC complexes were generally employed, except for chelating diNHC derivatives, which required direct reaction with bisimidazolium salts and potassium carbonate. Characterization included NMR, HRMS analysis, and single-crystal X-ray diffraction. In vitro on five ovarian cancer cell lines showed notable cytotoxicity, with IC50 values in the micro- and submicromolar range. Some compounds exhibited intriguing selectivity for cancer cells due to higher tumor cell uptake. Mechanistic studies revealed that monodentate NHCs induced mitochondrial damage while chelating ligands caused DNA damage. One chelating NHC-picolyl ligand showed promising cytotoxicity and selectivity in high-grade serous ovarian cancer models, supporting its consideration for preclinical study.

Blockage of the NLRP3 inflammasome by MCC950 inhibits migration and invasion in adenomyosis.

RESEARCH QUESTION: Does the NOD-like receptor protein 3 (NLRP3) inflammasome have an effect in adenomyosis? DESIGN: Fresh-frozen endometrial tissues and paraffin specimens were obtained from endometrial tissues from patients with adenomyosis and controls. Western blot, quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) were applied to assess expression of the NLRP3 inflammasome components. Primary eutopic endometrial stromal cells were isolated from the uteri of patients with adenomyosis. After NLRP3 was knocked down using small interfering RNA, proliferation, invasion and epithelial-mesenchymal transition (EMT) were evaluated using EdU, CCK8, transwell assays and western blot. Importantly, a mouse model of adenomyosis was established to evaluate the effects of the NLRP3 inhibitor MCC950 on the formation of adenomyosis. RESULTS: Expression of the NLRP3 inflammasome components was elevated in the ectopic or eutopic endometrium of patients with adenomyosis. NLRP3 knockdown inhibited migration, invasion and EMT in endometrial cells and primary endometrial cells (P < 0.0001). MCC950, which blocks the NLRP3 inflammasome, reduced migration and invasion of endometrial cells (P < 0.01) and primary endometrial cells (P < 0.0001) considerably. Importantly, in the mouse model of adenomyosis, MCC950 had a mitigating effect on the severity of adenomyosis (P < 0.01). CONCLUSIONS: NLRP3 was found to enhance migration, invasion and EMT of human endometrial cells in adenomyosis. Notably, the NLRP3 inhibitor MCC950 reduced migration and invasion of endometrial cells effectively. Furthermore, in the mouse model of adenomyosis, MCC950 exhibited a therapeutic effect by alleviating the severity of adenomyosis.

The NLRP3 inhibitor Dapansutrile improves the therapeutic action of lonafarnib on progeroid mice.

The role of the inflammasomes in aging and progeroid syndromes remain understudied. Recently, MCC950, a NLRP3 inhibitor, was used in Zmpste24-/- mice to ameliorate the phenotypes. However, the safety of MCC950 was questioned due to liver toxicity observed in humans. Nevertheless, inhibition of the inflammasomes would be a beneficial therapy for progeria. Here, we show that OLT1177 (dapansutrile), other NLRP3 inhibitor, improved cellular and animal phenotypes using progeroid fibroblasts and a LmnaG609G/G609G mouse model. In both cases dapansutrile reduced progerin accumulation, NLRP3-inflammasome activation and secretory phenotype of senescence, extended the lifespan of progeroid animals, preserved bodyweight, and reduced kyphosis, inflammation, and senescence. Interestingly, dapansutrile further improved the effect of lonafarnib, the only FDA-approved drug for the progeria. The combination of both drugs reduced the inflammation and senescence, extended survival and ameliorated various progeroid defects both in vitro and in vivo, compared with treatment using lonafarnib alone. These findings and the safety of dapansutrile demonstrated in several clinical trials proposes it as a possible co-adjuvant treatment with lonafarnid in HGPS.

Deciphering physiological and transcriptional mechanisms of maize seed germination.

Maize is a valuable raw material for feed and food production. Healthy seed germination is important for improving the yield and quality of maize. Seed aging occurs relatively fast in crops and it is a process that delays germination as well as reduces its rate and even causes total loss of seed viability. However, the physiological and transcriptional mechanisms that regulate maize seeds, especially aging seed germination remain unclear. Coronatine (COR) which is a phytotoxin produced by Pseudomonas syringae and a new type of plant growth regulator can effectively regulate plant growth and development, and regulate seed germination. In this study, the physiological and transcriptomic mechanisms of COR-induced maize seed germination under different aging degrees were analyzed. The results showed that 0.001-0.01 µmol/L COR could promote the germination of aging maize seed and the growth of primary roots and shoots. COR treatment increased the content of gibberellins (GA3) and decreased the content of abscisic acid (ABA) in B73 seeds before germination. The result of RNA-seq analysis showed 497 differentially expressed genes in COR treatment compared with the control. Three genes associated with GA biosynthesis (ZmCPPS2, ZmD3, and ZmGA2ox2), and two genes associated with GA signaling transduction (ZmGID1 and ZmBHLH158) were up-regulated. Three genes negatively regulating GA signaling transduction (ZmGRAS48, ZmGRAS54, and Zm00001d033369) and two genes involved in ABA biosynthesis (ZmVP14 and ZmPCO14472) were down-regulated. The physiological test results also showed that the effects of GA and ABA on seed germination were similar to those of high and low-concentration COR, respectively, which indicated that the effect of COR on seed germination may be carried out through GA and ABA pathways. In addition, GO and KEGG analysis suggested that COR is also highly involved in antioxidant enzyme systems and secondary metabolite synthesis to regulate maize seed germination processes. These findings provide a valuable reference for further research on the mechanisms of maize seed germination.

Dapansutrile OLT1177 suppresses foreign body response inflammation while preserving vascularisation of implanted materials.

Mitigating inflammation associated with the foreign body response (FBR) remains a significant challenge in enhancing the performance of implantable medical devices. Current anti-inflammatory approaches aim to suppress implant fibrosis, the major outcome of the FBR, but also inadvertently inhibit beneficial immune signalling necessary for tissue healing and vascularization. In a previous study, we demonstrated the feasibility of 'selective' immunosuppression targeting the NLRP3 inflammasome using the small molecule inhibitor MCC950, leading to reduced implant fibrosis without compromising healing and leading to enhanced vascularization. However, the clinical potential of MCC950 is severely limited due to its failure to pass Phase I clinical safety trials. This has triggered substantial efforts to develop safer analogues of NLRP3 inhibitors. Dapansutrile (OLT1177) is emerging as a leading candidate amongst current NLRP3 inhibitors, demonstrating both safety and effectiveness in a growing number of clinical indications and Phase 2 trials. While the anti-inflammatory effects of OLT1177 have been shown, validation of these effects in the context of implanted materials and the FBR have not yet been demonstrated. In this study, we show OLT1177 possesses beneficial effects on key cell types which drive FBR outcomes, including macrophages, fibroblasts, and smooth muscle cells. Evaluation of OLT1177 in a 28 day subcutaneous implantation model showed OLT1177 reduced fibrotic capsule formation while promoting implant vascularization. Mechanistic studies revealed that this occurred through activation of early pro-angiogenic markers while suppressing late-stage anti-angiogenic markers. These findings establish OLT1177 as a promising therapeutic approach for mitigating implant fibrosis while supporting vascularisation, suggesting a highly promising selective immunosuppressive strategy for the FBR warranting further research to explore its optimal integration into medical materials and devices.

Targeting BRD4 mitigates hepatocellular lipotoxicity by suppressing the NLRP3 inflammasome activation and GSDMD-mediated hepatocyte pyroptosis.

Nod-like receptor family pyrin-containing protein 3 (NLRP3) inflammasome plays a pathologic role in metabolic dysfunction-associated steatohepatitis (MASH), but the molecular mechanism regulating the NLRP3 inflammasome activation in hepatocellular lipotoxicity remains largely unknown. Bromodomain-containing protein 4 (BRD4) has emerged as a key epigenetic reader of acetylated lysine residues in enhancer regions that control the transcription of key genes. The aim of this study is to investigate if and how BRD4 regulated the NLRP3 inflammasome activation and pyroptosis in MASH. Using the AML12 and primary mouse hepatocytes stimulated by palmitic acid (PA) as an in vitro model of hepatocellular lipotoxicity, we found that targeting BRD4 by genetic knockdown or a selective BRD4 inhibitor MS417 protected against hepatosteatosis; and this protective effect was attributed to inhibiting the activation of NLRP3 inflammasome and reducing the expression of Caspase-1, gasdermin D (GSDMD), interleukin (IL)-1ß and IL-6. Moreover, BRD4 inhibition limited the voltage-dependent anion channel-1 (VDAC1) expression and oligomerization in PA-treated AML12 hepatocytes, thereby suppressing the NLRP3 inflammasome activation. Additionally, the expression of BRD4 enhanced in MASH livers of humans. Mechanistically, BRD4 was upregulated during hepatocellular lipotoxicity that in turn modulated the active epigenetic mark H3K27ac at the promoter regions of the Vdac and Gsdmd genes, thereby enhancing the expression of VDAC and GSDMD. Altogether, our data provide novel insights into epigenetic mechanisms underlying BRD4 activating the NLRP3 inflammasome and promoting GSDMD-mediated pyroptosis in hepatocellular lipotoxicity. Thus, BRD4 might serve as a novel therapeutic target for the treatment of MASH.

PLUNC inhibits invasion and metastasis in nasopharyngeal carcinoma by inhibiting NLRP3 inflammasome activation.

Nasopharyngeal carcinoma (NPC) is a malignant tumor that occurs in the nasopharynx. Palate, lung, and nasal epithelium clone (PLUNC) has been identified as an early secreted protein that is specifically expressed in the nasopharynx. The aim of this study was to determine the role and mechanism of PLUNC in NPC. We used mRNA sequencing (seq) combined with ribosome-nascent chain complex (RNC)-seq to determine the biological role of PLUNC. The expression of epithelial-to-mesenchymal transition (EMT)-related molecules was detected by western blotting. Then, cell migration and invasion were detected by wound healing and Transwell chamber assays. NPC cells were injected into the tail vein of nude mice to explore the biological role of PLUNC in vivo. The sequencing results showed that PLUNC inhibited the progression of NPC and its expression was correlated with that of NOD-like receptors. Experiments confirmed that PLUNC inhibited the invasion and metastasis of NPC cells by promoting the ubiquitination degradation of NLRP3. PLUNC overexpression in combination with the treatment by MCC950, an inhibitor of NLRP3 inflammasome activation, was most effective in inhibiting NPC invasion and metastasis. In vivo experiments also confirmed that the combination of PLUNC overexpression and MCC950 treatment effectively inhibited the lung metastasis of NPC cells. In summary, our research suggested that PLUNC inhibited the invasion and metastasis of NPC by inhibiting NLRP3 inflammasome activation, and targeting the PLUNC-NLRP3 inflammasome axis could provide a new strategy for the diagnosis and treatment of NPC patients.

Stress-induced NLRP3 inflammasome activation and myelin alterations in the hippocampus of PTSD rats.

Inflammatory and myelin changes may contribute to the pathophysiology of post-traumatic stress disorder (PTSD). The NOD-like receptor (NLR) family, pyrin domain-containing protein 3 (NLRP3), a brain inflammasome, is activated in the hippocampus of mice with PTSD. In other psychiatric disorders, NLRP3 expression has been associated with axonal myelination and demyelination. However, the association between NLRP3 and myelin in rats with PTSD remains unclear. Therefore, this study aims to investigate the relationship between the NLRP3 inflammasome and myelin in the hippocampus of rats with PTSD. A rat model of post-traumatic stress disorder was established using the single-prolonged stress (SPS) approach. Hippocampal tissues were collected for the detection of NLRP3 inflammasome-associated proteins and myelin basic protein at 3, 7, and 14 days after SPS. To further explore the relationship between NLRP3 and myelin, the NLRP3-specific inhibitor MCC950 was administered intraperitoneally to rats starting 72 h before SPS, and then alterations in NLRP3 inflammasome-associated proteins and myelin were observed in the PTSD and control groups. We found that NLRP3 and downstream related proteins were activated in the hippocampus of rats 3 days after SPS, and the myelin content in the hippocampus increased after SPS stress. MCC950 reduced the expression of NLRP3-related pathway proteins, improved anxiety behaviour and spatial learning memory impairment, and inhibited the increase in myelin content in the hippocampal region of rats after SPS. In conclusion the study indicates that NLRP3 has a significant role in the hippocampal region of rats with PTSD. Inhibition of the NLRP3 inflammasome could be a potential target for treating PTSD.

Inhibition of NLRP3 inflammasome ameliorates LPS-induced neuroinflammatory injury in mice via PINK1/Parkin pathway.

Parkinson's disease (PD) is characterized by the severe loss of dopaminergic neurons in the substantia nigra pars compacta, leading to motor dysfunction. The onset of PD is often accompanied by neuroinflammation and α-Synuclein aggregation, and extensive research has focused on the activation of microglial NLRP3 inflammasomes in PD, which promotes the death of dopaminergic neurons. In this study, a model of cerebral inflammatory response was constructed in wild-type and Parkin＋/－ mice through bilateral intraventricular injection of LPS. LPS-induced activation of the NLRP3 inflammasome in wild-type mice promotes the progression of PD. The use of MCC950 in wild mice injected with LPS induces activation of Parkin/PINK and improves autophagy, which in turn improves mitochondrial turnover. It also inhibits LPS-induced inflammatory responses, improves motor function, protects dopaminergic neurons, and inhibits microglia activation. Furthermore, Parkin＋/－ mice exhibited motor dysfunction, loss of dopaminergic neurons, activation of the NLRP3 inflammasome, and α-Synuclein aggregation beginning at an early age. Parkin ± mice exhibited more pronounced microglia activation, greater NLRP3 inflammasome activation, more severe autophagy dysfunction, and more pronounced motor dysfunction after LPS injection compared to wild-type mice. Notably, the use of MCC950 in Parkin ± mice did not ameliorate NLRP3 inflammasome activation, autophagy dysfunction, or α-synuclein aggregation. Thus, MCC950 can only exert its effects in the presence of Parkin/PINK1, and targeting Parkin-mediated NLRP3 inflammasome activation is expected to be a potential therapeutic strategy for Parkinson's disease.

Coronatine-treated seedlings increase the tolerance of cotton to low-temperature stress.

Coronatine, an analog of Jasmonic acid (JA), has been shown to enhance crop tolerance to abiotic stresses, including chilling stress. However, the underlying molecular mechanism remains largely unknown. In this study, we investigated the effect of Coronatine on cotton seedlings under low temperature using transcriptomic and metabolomics analysis. Twelve cDNA libraries from cotton seedlings were constructed, and pairwise comparisons revealed a total of 48,322 differentially expressed genes (DEGs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified the involvement of these unigenes in various metabolic pathways, including Starch and sucrose metabolism, Sesquiterpenoid and triterpenoid biosynthesis, Phenylpropanoid biosynthesis, alpha-Linolenic acid metabolism, ABC transporters, and Plant hormone signal transduction. Additionally, substantial accumulations of jasmonates (JAs), abscisic acid and major cell wall metabolites were observed. Transcriptome analysis revealed differential expression of regulatory genes, and qRT-PCR analysis confirmed the expression patterns of 9 selected genes. Co-expression analysis showed that the JA-responsive genes might form a network module with ABA biosynthesis genes or cell wall biosynthesis genes, suggesting the existence of a COR-JA-cellulose and COR-JA-ABA-cellulose regulatory pathway in cotton seedlings. Collectively, our findings uncover new insights into the molecular basis of coronatine--associated cold tolerance in cotton seedlings.

Ramelteon protects against social defeat stress-associated abnormal behaviors.

Psychological stress affects the neuroendocrine regulation, which modulates mental status and behaviors. Melatonin, a hormone synthesized primarily by the pineal gland, regulates many brain functions, including circadian rhythms, pain, sleep, and mood. Selective pharmacological melatonin agonist ramelteon has been clinically used to treat mood and sleep disorders. Posttraumatic stress disorder (PTSD) is a psychiatric condition associated with severe trauma; it is generally triggered by traumatic events, which lead to severe anxiety and uncontrollable trauma recall. We recently reported that repeated social defeat stress (RSDS) may induce robust anxiety-like behaviors and social avoidance in mice. In the present study, we investigated whether melatonin receptor activation by melatonin and ramelteon regulates RSDS-induced behavioral changes. Melatonin treatment improved social avoidance and anxiety-like behaviors in RSDS mice. Moreover, treatment of the non-selective MT1/MT2 receptor agonist, ramelteon, markedly ameliorated RSDS-induced social avoidance and anxiety-like behaviors. Moreover, activating melatonin receptors also balanced the expression of monoamine oxidases, glucocorticoid receptors, and endogenous antioxidants in the hippocampus. Taken together, our findings indicate that the activation of both melatonin and ramelteon regulates RSDS-induced anxiety-like behaviors and PTSD symptoms. The current study also showed that the regulatory effects of neuroendocrine mechanisms and cognitive behaviors on melatonin receptor activation in repeated social defeat stress.

Combined Insults of a MASH Diet and Alcohol Binges Activate Intercellular Communication and Neutrophil Recruitment via the NLRP3-IL-1ß Axis in the Liver.

Binge drinking in obese patients positively correlates with accelerated liver damage and liver-related death. However, the underlying mechanism and the effect of alcohol use on the progression of metabolic-dysfunction-associated steatotic liver disease (MASLD) remain unexplored. Here, we show that short-term feeding of a metabolic-dysfunction-associated steatohepatitis (MASH) diet plus daily acute alcohol binges for three days induce liver injury and activation of the NLRP3 inflammasome. We identify that a MASH diet plus acute alcohol binges promote liver inflammation via increased infiltration of monocyte-derived macrophages, neutrophil recruitment, and NET release in the liver. Our results suggest that both monocyte-derived macrophages and neutrophils are activated via NLRP3, while the administration of MCC950, an NLRP3 inhibitor, dampens these effects.In this study, we reveal important intercellular communication between hepatocytes and neutrophils. We discover that the MASH diet plus alcohol induces IL-1ß via NLRP3 activation and that IL-1ß acts on hepatocytes and promotes the production of CXCL1 and LCN2. In turn, the increase in these neutrophils recruits chemokines and causes further infiltration and activation of neutrophils in the liver. In vivo administration of the NLRP3 inhibitor, MCC950, improves the early phase of MetALD by preventing liver damage, steatosis, inflammation, and immune cells recruitment.

Activation of the ROS/TXNIP/NLRP3 pathway disrupts insulin-dependent glucose uptake in skeletal muscle of insulin-resistant obese mice.

Oxidative stress and the activation of the nucleotide-binding domain, leucine-rich-containing family, pyrin domain containing 3 (NLRP3) inflammasome have been linked to insulin resistance in skeletal muscle. In immune cells, the exacerbated generation of reactive oxygen species (ROS) activates the NLRP3 inflammasome, by facilitating the interaction between thioredoxin interacting protein (TXNIP) and NLRP3. However, the precise role of ROS/TXNIP-dependent NLRP3 inflammasome activation in skeletal muscle during obesity-induced insulin resistance remains undefined. Here, we induced insulin resistance in C57BL/6J mice by feeding them for 8 weeks with a high-fat diet (HFD) and explored whether the ROS/TXNIP/NLRP3 pathway was involved in the induction of insulin resistance in skeletal muscle. Skeletal muscle fibers from insulin-resistant mice exhibited increased oxidative stress, as evidenced by elevated malondialdehyde levels, and altered peroxiredoxin 2 dimerization. Additionally, these fibers displayed augmented activation of the NLRP3 inflammasome, accompanied by heightened ROS-dependent proximity between TXNIP and NLRP3, which was abolished by the antioxidant N-acetylcysteine (NAC). Inhibition of the NLRP3 inflammasome with MCC950 or suppressing the ROS/TXNIP/NLRP3 pathway with NAC restored insulin-dependent glucose uptake in muscle fibers from insulin-resistant mice. These findings provide insights into the mechanistic link between oxidative stress, NLRP3 inflammasome activation, and obesity-induced insulin resistance in skeletal muscle.

Inhibition of NLRP3 inflammasome by MCC950 under hypoxia alleviates photoreceptor apoptosis via inducing autophagy in Müller glia.

NLRP3 inflammasome activation has emerged as a critical initiator of inflammatory response in ischemic retinopathy. Here, we identified the effect of a potent, selective NLRP3 inhibitor, MCC950, on autophagy and apoptosis under hypoxia. Neonatal mice were exposed to hyperoxia for 5 days to establish oxygen-induced retinopathy (OIR) model. Intravitreal injection of MCC950 was given, and then autophagy and apoptosis markers were assessed. Retinal autophagy, apoptosis, and related pathways were evaluated by western blot, immunofluorescent labeling, transmission electron microscopy, and TUNEL assay. Autophagic activity in Müller glia after NLRP3 inflammasome inhibition, together with its influence on photoreceptor death, was studied using western blot, immunofluorescence staining, mRFP-GFP-LC3 adenovirus transfection, cell viability, proliferation, and apoptosis assays. Results showed that activation of NLRP3 inflammasome in Müller glia was detected in OIR model. MCC950 could improve impaired retinal autophagic flux and attenuate retinal apoptosis while it regulated the retinal AMPK/mTOR/ULK-1 pathway. Suppressed autophagy and depressed proliferation capacity resulting from hypoxia was promoted after MCC950 treatment in Müller glia. Inhibition of AMPK and ULK-1 pathway significantly interfered with the MCC950-induced autophagy activity, indicating MCC950 positively modulated autophagy through AMPK/mTOR/ULK-1 pathway in Müller cells. Furthermore, blockage of autophagy in Müller glia significantly induced apoptosis in the cocultured 661W photoreceptor cells, whereas MCC950 markedly preserved the density of photoreceptor cells. These findings substantiated the therapeutic potential of MCC950 against impaired autophagy and subsequent apoptosis under hypoxia. Such protective effect might involve the modulation of AMPK/mTOR/ULK-1 pathway. Targeting NLRP3 inflammasome in Müller glia could be beneficial for photoreceptor survival under hypoxic conditions.

Pyroptosis inhibitors MCC950 and VX-765 mitigate myocardial injury by alleviating oxidative stress, inflammation, and apoptosis in acute myocardial hypoxia.

Acute myocardial infarction (AMI) is a prevalent cardiovascular disease with high morbidity and mortality rates worldwide. Pyroptosis is an inflammatory form of programmed cell death that has been linked to various pathological conditions. However, its exact contribution to the onset and progression of heart injury in AMI has not yet fully elucidated. Herein, we established mouse AMI model by ligating the left anterior descending artery and performed transcriptome analysis during the early phase of AMI. Mouse HL-1 and human AC-16 cardiomyocytes were subjected to hypoxia to simulate ischemic injury in vitro. Our results revealed a significant activation of the inflammatory response at 3 h post-ligation, as confirmed by RNA sequencing. We identified the occurrence of NLRP3 inflammasome-mediated pyroptosis in the cardiac tissues of human cases with AMI, as well as in mouse models of AMI and hypoxia-induced cardiomyocytes, using immunohistochemistry staining and Western blotting assays. Concurrently, pharmacological inhibition of NLRP3 inflammasome-mediated pyroptosis with MCC950 and VX-765 effectively decreased hypoxia-induced cardiomyocytes injury, while mitigating myocardial oxidative stress, apoptosis and inflammation caused by hypoxia. Moreover, the circulating levels of gasdermin D (GSDMD), the pyroptosis executor, were remarkably elevated in the plasma of mice with early AMI and in the supernatant of hypoxia-exposed cardiomyocytes in a time-dependent manner using ELISA and Western blotting. Furthermore, the change in circulating GSDMD positively correlated with Creatine Kinase-MB (CK-MB) in the plasma of early-stage AMI mouse. In summary, these findings indicated a critical role for NLRP3 inflammasome-mediated pyroptosis in the progression of AMI, the administration of MCC950 and VX-765 may be attractive candidate therapeutic approaches for cardiac injury caused by acute hypoxia or even AMI. Additionally, the circulating GSDMD exhibits potential as a newly diagnostic biomarker for AMI.

Cigarette smoke impairs the hematopoietic supportive property of mesenchymal stem cells via the production of reactive oxygen species and NLRP3 activation.

BACKGROUND: Mesenchymal stem cells (MSCs) play important roles in tissue homeostasis by providing a supportive microenvironmental niche for the hematopoietic system. Cigarette smoking induces systemic abnormalities, including an impeded recovery process after hematopoietic stem cell transplantation. However, the role of cigarette smoking-mediated alterations in MSC niche function have not been investigated. METHODS: In the present study, we investigated whether exposure to cigarette smoking extract (CSE) disrupts the hematopoietic niche function of MSCs, and pathways impacted. To investigate the effects on bone marrow (BM)-derived MSCs and support of hematopoietic stem and progenitor cells (HSPCs), mice were repeatedly infused with the CSE named 3R4F, and hematopoietic stem and progenitor cells (HSPCs) supporting function was determined. The impact of 3R4F on MSCs at cellular level were screened by bulk-RNA sequencing and subsequently validated through qRT-PCR. Specific inhibitors were treated to verify the ROS or NLRP3-specific effects, and the cells were then transplanted into the animal model or subjected to coculture with HSPCs. RESULTS: Both direct ex vivo and systemic in vivo MSC exposure to 3R4F resulted in impaired engraftment in a humanized mouse model. Furthermore, transcriptomic profile analysis showed significantly upregulated signaling pathways related to reactive oxygen species (ROS), inflammation, and aging in 3R4F-treated MSCs. Notably, ingenuity pathway analysis revealed the activation of NLRP3 inflammasome signaling pathway in 3R4F-treated MSCs, and pretreatment with the NLRP3 inhibitor MCC950 rescued the HSPC-supporting ability of 3R4F-treated MSCs. CONCLUSION: In conclusion, these findings indicate that exposure to CSE reduces HSPCs supportive function of MSCs by inducing robust ROS production and subsequent NLRP3 activation.

Jasmonate mimic modulates cell elongation by regulating antagonistic bHLH transcription factors via brassinosteroid signaling.

Lodging restricts growth, development, and yield formation in maize (Zea mays L.). Shorter internode length is beneficial for lodging tolerance. However, although brassinosteroids (BRs) and jasmonic acid (JA) are known to antagonistically regulate internode growth, the underlying molecular mechanism is still unclear. In this study, application of the JA mimic coronatine (COR) inhibited basal internode elongation at the jointing stage and repressed expression of the cell wall-related gene XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE 1 (ZmXTH1), whose overexpression in maize plants promoted internode elongation. We demonstrated that the basic helix-loop-helix (bHLH) transcription factor ZmbHLH154 directly binds to the ZmXTH1 promoter and induces its expression, whereas the bHLH transcription factor ILI1 BINDING BHLH 1 (ZmIBH1) inhibits this transcriptional activation by forming a heterodimer with ZmbHLH154. Overexpressing ZmbHLH154 led to longer internodes, whereas zmbhlh154 mutants had shorter internodes than the wild type. The core JA-dependent transcription factors ZmMYC2-4 and ZmMYC2-6 interacted with BRASSINAZOLE RESISTANT 1 (ZmBZR1), a key factor in BR signaling, and these interactions eliminated the inhibitory effect of ZmBZR1 on its downstream gene ZmIBH1. Collectively, these results reveal a signaling module in which JA regulates a bHLH network by attenuating BR signaling to inhibit ZmXTH1 expression, thereby regulating cell elongation in maize.

Coronatine orchestrates ABI1-mediated stomatal opening to facilitate bacterial pathogen infection through importin ß protein SAD2.

Stomatal immunity plays an important role during bacterial pathogen invasion. Abscisic acid (ABA) induces plants to close their stomata and halt pathogen invasion, but many bacterial pathogens secrete phytotoxin coronatine (COR) to antagonize ABA signaling and reopen the stomata to promote infection at early stage of invasion. However, the underlining mechanism is not clear. SAD2 is an importin ß family protein, and the sad2 mutant shows hypersensitivity to ABA. We discovered ABI1, which negatively regulated ABA signaling and reduced plant sensitivity to ABA, was accumulated in the plant nucleus after COR treatment. This event required SAD2 to import ABI1 to the plant nucleus. Abolition of SAD2 undermined ABI1 accumulation. Our study answers the long-standing question of how bacterial COR antagonizes ABA signaling and reopens plant stomata during pathogen invasion.

Ameliorative effects of topical ramelteon on imiquimod-induced psoriasiform inflammation in mice.

Psoriasis is a long-lasting, immune-related inflammatory skin disease that affects 2-3% of the global population. It is distinguished by erythematous, silvery, and scaly patches. Ramelteon is a type of melatonin agonist that is used to treat insomnia. It has enhanced non-classical immunomodulatory and anti-inflammatory activities. The aim of the study is to assess the ameliorative effects of topical ramelteon on imiquimod (IMQ)-aggravated psoriasiform-like dermatosis in mice. The 32 albino mouse males were placed into six groups of eight animals, all of them. With the exception of the control group, all groups gained a once-a-day regimen of topical imiquimod 5% cream at a dose of 62.5 mg for eight uninterrupted days, while mice in the control group gained vaseline-based ointment alternately. Immediately after an 8-day induction period in the imiquimod group, mice in the clobetasol and ramelteon treatment groups obtained a twice-daily regimen of topical clobetasol propionate 0.05% ointment and 0.1% ointment, respectively, for a further 8 days. This extends the total duration of the experimental study to 16 continuous days. The findings of our study found that ramelteon significantly mitigated the concentrations of inflammatory cytokines in the skin tissue, including interleukin (IL)-6, IL-17A, IL-23, tumor necrosis factor-α (TNF-α), and vascular endothelial growth factor (VEGF), as well as the scores associated with psoriatic lesions, including erythema, scaling, skin thickening, ear thickness, and overall cumulative PASI scores. Additionally, the anti-inflammatory impact of ramelteon was achieved by markedly increasing IL-10 levels in the skin tissue and correcting cutaneous histopathological alterations. Ramelteon ointment (0.1%) was comparable to that of clobetasol (0.05%) ointment in alleviating a mouse model of imiquimod-induced psoriasiform inflammation; this is probably due to its potential anti-inflammatory and immunomodulatory activities. Therefore, ramelteon could be a good additive option for therapeutic management of immune-triggered inflammatory conditions such as psoriasis.

Pachymaran Alleviates Acute Gouty Arthritis by Inhibiting NLRP3 Inflammasome Activation and Pyroptosis.

Objective: Acute gouty arthritis is the most common rheumatic diseases, and leads to a heavy clinical burden, thereby to explore the treatment effects of pachymaran on acute gouty arthritis and elucidate its mechanism are meaningful. Methods: Eighteen SPF C57BL/6 mice were randomly divided into three groups: the sham group, model group, and pachymaran group (200mg/kg), with 6 mice in each group. The acute gouty arthritis model of mice was established by injecting 0.025 mL sodium urate solution into the right ankle cavity of the mice. The pachymaran group was given 200mg/kg of pachymaran intragastrically, in the sham group and model group were given the same volume of normal saline, respectively, for 7 consecutive days. Blood samples were collected from the orbital venous plexus 1 h after the last administration, all mice were killed, and ankle tissue samples were collected. The pathological changes of mouse ankle synovial tissue were observed by HE staining. The expression levels of IL-1ß and IL-18 inflammatory factors in the serum of mice were determined by ELISA. The ultrastructure of the synovial tissue of the mouse ankle joint was observed by transmission electron microscope. The protein expression levels of NLRP3, ASC, IL-1ß, IL-18, GSDMD, and Caspase-1 in synovial tissue of mouse ankle were detected by Western blot assay. Mouse chondrocytes were cultured and divided into groups I, II, III, and IV. Group I was the control group without any drug intervention. The cells in groups II, III, and IV were stimulated with sodium urate solution (100µg/mL), and groups III and IV were intervened by pachymaran (200µg/mL), among which the NLRP3 agonist Nigericin sodium salt intervened group IV. The expression levels of NLRP3, IL-1ß, GSDMD, and Caspase-1 proteins were detected by Western blot assay, and the apoptosis rate was detected by flow cytometry. Results: Compared with the sham group, the pathological injury of mice ankle synovial tissue in the model group was significantly aggravated, as the membrane was incomplete, mitochondria were swollen, the ridge was unclear or even disappeared, and the pathological injury of mice ankle synovial tissue in the pachymaran group was significantly improved vs model group; the serum levels of IL-1ß and IL-18 were increased in model group vs sham group, and pachymaran decreased these index vs model group; Compared with the sham group, protein expression levels of NLRP3, ASC, IL-1ß, IL-18, GSDMD, and Caspase-1 were significant increased in model group, and pachymaran suppressed these proteins vs model group. The TEM results showed that in model group the wide swelling of mitochondria accompanied by disappearance of mitochondrial cristae vs sham group, and the mitochondrial ridge was slightly damaged, or the mitochondria were only swollen, and the ridge was still clearly visible in pachymaran group. In vitro experiments, Compared with group I, the protein expression levels of NLRP3, IL-1ß, GSDMD, Caspase-1, and the apoptosis rate of chondrocytes in group II were significantly increased. Compared with group II, the protein expression levels of NLRP3, IL-1ß, GSDMD, Caspase-1, and the apoptosis rate of chondrocytes in group III were significantly decreased. Compared with group III, the protein expression levels of NLRP3, IL-1ß, GSDMD, Caspase-1, and the apoptosis rate of chondrocytes in group IV were significantly increased. Conclusion: Pachymaran maintain the structural integrity of joints and alleviate the progression of acute gouty arthritis by inhibiting NLRP3 inflammasome activation and pyroptosis, pachymaran may be used and applied to clinical treatment.