Osteocyte-derived exosomes regulate the DLX2/wnt pathway to alleviate osteoarthritis by mediating cartilage repair.

BACKGROUND: Chondrocyte viability, apoptosis, and migration are closely related to cartilage injury in osteoarthritis (OA) joints. Exosomes are identified as potential therapeutic agents for OA. OBJECTIVE: This study aimed to investigate the role of exosomes derived from osteocytes in OA, particularly focusing on their effects on cartilage repair and molecular mechanisms. METHODS: An injury cell model was established by treating chondrocytes with IL-1ß. Cartilage repair was evaluated using cell counting kit-8, flow cytometry, scratch test, and Western Blot. Molecular mechanisms were analyzed using quantitative real-time PCR, bioinformatic analysis, and Western Blot. An OA mouse model was established to explore the role of exosomal DLX2 in vivo. RESULTS: Osteocyte-released exosomes promoted cell viability and migration, and inhibited apoptosis and extracellular matrix (ECM) deposition. Moreover, exosomes upregulated DLX2 expression, and knockdown of DLX2 activated the Wnt pathway. Additionally, exosomes attenuated OA in mice by transmitting DLX2. CONCLUSION: Osteocyte-derived exosomal DLX2 alleviated IL-1ß-induced cartilage repair and inactivated the Wnt pathway, thereby alleviating OA progression. The findings suggested that osteocyte-derived exosomes may hold promise as a treatment for OA.

The protective effects of repetitive transcranial magnetic stimulation with different high frequencies on motor functions in MPTP/probenecid induced Parkinsonism mouse models.

BACKGROUND: High-frequency repeated transcranial magnetic stimulation (rTMS) stimulating the primary motor cortex (M1) is an alternative, adjunctive therapy for improving the motor symptoms of Parkinson's disease (PD). However, whether the high frequency of rTMS positively correlates to the improvement of motor symptoms of PD is still undecided. By controlling for other parameters, a disease animal model may be useful to compare the neuroprotective effects of different high frequencies of rTMS. OBJECTIVE: The current exploratory study was designed to compare the protective effects of four common high frequencies of rTMS (5, 10, 15, and 20 Hz) and iTBS (a special form of high-frequency rTMS) and explore the optimal high-frequency rTMS on an animal PD model. METHODS: Following high frequencies of rTMS application (twice a week for 5 weeks) in a MPTP/probenecid-induced chronic PD model, the effects of the five protocols on motor behavior as well as dopaminergic neuron degeneration levels were identified. The underlying molecular mechanisms were further explored. RESULTS: We found that all the high frequencies of rTMS had protective effects on the motor functions of PD models to varying degrees. Among them, the 10, 15, and 20 Hz rTMS interventions induced comparable preservation of motor function through the protection of nigrostriatal dopamine neurons. The enhancement of brain-derived neurotrophic factor (BDNF), dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT-2) and the suppression of TNF-α and IL-1ß in the nigrostriatum were involved in the process. The efficacy of iTBS was inferior to that of the above three protocols. The effect of 5 Hz rTMS protocol was weakest. CONCLUSIONS: Combined with the results of the present study and the possible side effects induced by rTMS, we concluded that 10 Hz might be the optimal stimulation frequency for preserving the motor functions of PD models using rTMS treatment.

Mechanism of Vaginal Epithelial Cell Pyroptosis Induced by the NLRP3 Inflammasome in Vulvovaginal Candidiasis.

PROBLEM: Vulvovaginal candidiasis (VVC) is a common mucosal fungal infection, and Candida albicans is the main causative agent. The NLRP3 inflammasome plays an important role in VVC, but the underlying mechanism is unknown. METHOD OF STUDY: Vaginal epithelial cells were divided into three groups: control, C. albicans strain SC5314 (wild-type, WT), and WT+ Matt Cooper Compound 950 (MCC950, a specific NLRP3 inhibitor). After human vaginal epithelial cells were pretreated with 1 µmol/L MCC950 for 2 h, C. albicans (MOI = 1) was cocultured with the human vaginal epithelial cells for 12 h. The cell supernatants were collected, LDH was detected, and the IL-1ß and IL-18 levels were determined by ELISA. The expression of the pyroptosis-related proteins NLRP3, Caspase-1 p20 and GSDMD was measured by Western blotting analysis. The protein expression of the pyroptosis-related N-terminus of GSDMD (GSDMD-N) was detected by immunofluorescence. RESULTS: In this study, we showed that the WT C. albicans strain induced pyroptosis in vaginal epithelial cells, as indicated by the LDH and proinflammatory cytokine levels and the upregulated levels of the pyroptosis-related proteins NLRP3, Caspase-1 p20, and GSDMD-N. MCC950 reversed the changes in the expression of these proteins and proinflammatory cytokines in vaginal epithelial cells. CONCLUSION: C. albicans activated the NLRP3 inflammasome to induce vaginal epithelial cell pyroptosis. MCC950 inhibited the NLRP3 inflammasome, reduced vaginal epithelial cell pyroptosis, and decreased the release of inflammatory cytokines.

Effect of glucocorticoid blockade on inflammatory responses to acute sleep fragmentation in male mice.

The association between sleep and the immune-endocrine system is well recognized, but the nature of that relationship is not well understood. Sleep fragmentation induces a pro-inflammatory response in peripheral tissues and brain, but it also activates the hypothalamic-pituitary-adrenal (HPA) axis, releasing glucocorticoids (GCs) (cortisol in humans and corticosterone in mice). It is unclear whether this rapid release of glucocorticoids acts to potentiate or dampen the inflammatory response in the short term. The purpose of this study was to determine whether blocking or suppressing glucocorticoid activity will affect the inflammatory response from acute sleep fragmentation (ASF). Male C57BL/6J mice were injected i.p. with either 0.9% NaCl (vehicle 1), metyrapone (a glucocorticoid synthesis inhibitor, dissolved in vehicle 1), 2% ethanol in polyethylene glycol (vehicle 2), or mifepristone (a glucocorticoid receptor antagonist, dissolved in vehicle 2) 10 min before the start of ASF or no sleep fragmentation (NSF). After 24 h, samples were collected from brain (prefrontal cortex, hypothalamus, hippocampus) and periphery (liver, spleen, heart, and epididymal white adipose tissue (EWAT)). Proinflammatory gene expression (TNF-α and IL-1ß) was measured, followed by gene expression analysis. Metyrapone treatment affected pro-inflammatory cytokine gene expression during ASF in some peripheral tissues, but not in the brain. More specifically, metyrapone treatment suppressed IL-1ß expression in EWAT during ASF, which implies a pro-inflammatory effect of GCs. However, in cardiac tissue, metyrapone treatment increased TNF-α expression in ASF mice, suggesting an anti-inflammatory effect of GCs. Mifepristone treatment yielded more significant results than metyrapone, reducing TNF-α expression in liver (only NSF mice) and cardiac tissue during ASF, indicating a pro-inflammatory role. Conversely, in the spleen of ASF-mice, mifepristone increased pro-inflammatory cytokines (TNF-α and IL-1ß), demonstrating an anti-inflammatory role. Furthermore, irrespective of sleep fragmentation, mifepristone increased pro-inflammatory cytokine gene expression in heart (IL-1ß), pre-frontal cortex (IL-1ß), and hypothalamus (IL-1ß). The results provide mixed evidence for pro- and anti-inflammatory functions of corticosterone to regulate inflammatory responses to acute sleep loss.

[Preparation of Lactobacillus paracei TK1501 postbiotic and its inhibitory effect against Helicobacter pylori infection in mice].

OBJECTIVE: To prepare a postbiotic using soybean fermentation product of Lactobacillus paracasei TK1501 and evaluate its inhibitory effect against Helicobacter pylori (Hp) infection in mice. METHODS: L. paracasei TK1501 was cultured for 32 h at 37 ℃ in an anaerobic condition for solid substrate fermentation with a solid to water ratio of 1:1.5 in the substrate and an inoculation density of 5×107 CFU/mL. The postbiotic was isolated and purified using macroporous resin XAD-16N adsorption, cation exchange chromatography and HPLC, and its stability and antibacterial activity were assessed. The inhibitory effect of this postbiotic against Hp infection was evaluated in a mouse model with gastric mucosal Hp infection, which were treated with the postbiotic via gavage for 4 weeks at the dose of 0.02 or 0.1 mL. Serum levels of TNF-α and IL-1ß of the mice were analyzed after the treatments, and gastric tissues of the mice were collected for HE staining. RESULTS: L. paracasei TK1501 postbiotic could be easily degraded by protease and had good thermal stability and tolerance to exposures to acid, base, and organic solvents. In the in vitro experiment, the postbiotic showed strong inhibitory effects in bacterial cultures of Staphylococcus aureus, Hp and other common pathogenic bacteria without obviously affecting the resident bacteria in the digestive tract. In the mouse models, treatment with the postbiotic at the dose of 0.1 mL significantly alleviated Hp infection and lowered the serum levels of TNF-α and IL-1ß of the mice. CONCLUSION: L. paracasei TK1501 postbiotic has strong inhibitory effects on Hp and Staphylococcus aureus but not on normal intestinal flora in mice.

Elevated proinflammatory cytokines in response to mechanical stimulus are associated with reduced knee loading 2 years after anterior cruciate ligament reconstruction.

BACKGROUND: The aim of this study was to test the hypothesis that proinflammatory cytokines correlate with knee loading mechanics during gait following a mechanical walking stimulus in subjects 2 years after anterior cruciate ligament reconstruction. Elevated systemic levels of proinflammatory cytokines can be sustained for years after injury. Considering roughly 50% of these patients progress to Osteoarthritis 10-15 years after injury, a better understanding of the role of proinflammatory cytokines such as tumor necrosis factor-α and Interleukin-1ß on Osteoarthritis risk is needed. METHODS: Serum proinflammatory cytokines concentrations were measured in 21 subjects 2 years after unilateral ACLR from blood drawn at rest and 3.5 h after 30 min of walking. An optoelectronic system and a force plate measured subjects' knee kinetics. Correlations were tested between inflammatory marker response and knee extension and knee adduction moments. FINDINGS: Changes in proinflammatory cytokines due to mechanical stimulus were correlated (R = 0.86) and showed substantial variation between subjects in both cytokines at 3.5 h post-walk. Knee loading correlated with 3.5-h changes in tumor necrosis factor-α concentration (Knee extension moment: R = -0.5, Knee adduction moment: R = -0.5) and Interleukin-1ß concentration (Knee extension moment: R = -0.44). However, no significant changes in concentrations were observed in tumor necrosis factor-α and Interleukin-1ß when comparing baseline and post walking stimulus conditions. INTERPRETATION: The significant associations between changes in serum proinflammatory markers following a mechanical stimulus and gait metrics in subjects at risk for developing Osteoarthritis underscore the importance of investigating the interaction between biomarkers and biomechanical factors in Osteoarthritis development.

Increased serum levels of proNGF, mature NGF and interleukins in burn-injured children.

OBJECTIVE: Burns are among the most common injuries in children. In burns of more than 20% of the total body surface area, a systemic inflammatory response involving several chemical mediators occurs. Among them, nerve growth factor (NGF) regulates the inflammatory response related to wound healing and promotes keratinocyte proliferation and angiogenesis. The aim of our study was to investigate the physiological response to injury in children with moderate-severe burns, assaying proNGF, mature NGF (mNGF), interleukins (IL)-1ß, and Il-10 serum levels. PATIENTS AND METHODS: This is a prospective observational study, including twelve children hospitalized for moderate-severe burns at the Gemelli Hospital (Rome). Their laboratory features were compared to those of patients with obstructive hydrocephalus who underwent surgery. RESULTS: Our results showed an increase in proNGF and mNGF serum levels. In burn patients, proNGF levels increased before mNGF, and serum concentrations of both were not correlated with burn extension and depth. The most significant levels of mNGF and proNGF were reported in scalds involving the face. Serum IL-1ß and IL-10 peak levels were reached with a time-course pattern similar to proNGF. CONCLUSIONS: Our preliminary results validate the hypothesis that serum levels of proNGF and mNGF may represent inflammatory biomarkers useful for monitoring burn patients and defining new strategies for their treatment.

Exploring the impact of interleukins on sarcopenia development: A systematic review and meta-analysis.

BACKGROUND: The role of interleukins in sarcopenia development has been acknowledged, yet the specifics of their involvement remain to be fully understood. This study aimed to explore alterations in interleukin levels among sarcopenia patients. METHODS: Searches were conducted in Embase, Medline, and the Cochrane Library for literature published up to May 2023. Eligible observational studies with a diagnosis of sarcopenia were included. The Newcastle-Ottawa Scale was utilized for quality assessment. For data synthesis, a random-effects model was used, and the Mantel-Haenszel method was used for pooled estimates. RESULTS: Of the 7685 articles screened, 37 met the inclusion criteria. Statistically significant differences in the levels of IL-1ß, IL-6 and IL-10 were detected in sarcopenia patients. Specifically, IL-1ß (95 % CI: 0.33 [0.12, 0.54], P < 0.05), IL-6 (95 % CI: 0.91 [0.59, 1.24], P < 0.05), and IL-10 (95 % CI: 0.11 [0.07,0.15], P < 0.05) were detected. However, no significant associations were found between serum IL-4 (95 % CI: 0.36 [-0.18, 0.42], P = 0.44), IL-8 (95 % CI: -1.05 [-3.06, 0.95], P = 0.3), IL-12 (95 % CI: -3.92 [-8.32,0.48], P = 0.08) or IL-17 (95 % CI: 0.22 [-2.43, 2.88], P = 0.87) and sarcopenia. Subgroup analysis showed no significant difference in IL-6 (95 % CI: -0.03 [-0.72, 0.66], P = 0.93) and IL-10 (95 % CI: 0.1 [-0.44, 0.64], P = 0.72) among patients with European standard sarcopenia. CONCLUSIONS: Inflammation plays a role in sarcopenia, and the serum levels of IL-1ß, IL-6, and IL-10 are associated with sarcopenia. Further research is needed to clarify these associations. CLINICAL TRIALS REGISTRATION NUMBER: CRD42024506656.

Targeting the Gut-Kidney Axis in Diarrhea with Kidney-Yang Deficiency Syndrome: The Role of Sishen Pills in Regulating TMAO-Mediated Inflammatory Response.

BACKGROUND Sishen Pills (SSPs) are commonly used to treat diarrhea with kidney-yang deficiency syndrome. Trimethylamine-N-oxide (TMAO) is produced through the metabolism of gut microbiota and can participate in diarrhea in kidney-yang deficiency syndrome by mediating the "gut-kidney axis" to transmit inflammatory factors. This study combined network pharmacology with animal experiments to explore whether SSPs can treat diarrhea with kidney-yang deficiency syndrome by affecting the interaction between TMAO and gut microbiota. MATERIAL AND METHODS A mouse model of diarrhea with kidney-yang deficiency syndrome was constructed by using adenine and Folium sennae decoction, and SSP decoction was used for treatment. This study utilized network pharmacology to predict the potential mechanisms of SSPs in treating diarrhea with kidney-yang deficiency syndrome. 16S rRNA high-throughput sequencing was used to analyze gut mucosal microbial characteristics. ELISA was used to measure TMAO, NOD-like receptor thermal protein domain associated protein 3 (NLRP3), interleukin-1ß (IL-1ß), and transforming growth factor-ß1 (TGF-ß1) levels. We performed Masson and immunohistochemical (Occludin, ZO-1) staining of kidney and small intestinal tissues. The fluorescein diacetate (FDA) hydrolysis spectrophotometric method was used to assess the microbial activity in contents of the small intestine. RESULTS Network pharmacology analysis revealed that SSPs can modulate 108 target points involved in the development of diarrhea, including IL-1ß and TNF. The experimental results demonstrated that SSP decoction significantly improved the general behavioral profiles of the mice, and also reduced TMAO, NLRP3, IL-1ß, and TGF-ß1 levels (P<0.05). Correlation analysis revealed significant positive correlations between TMAO concentrations and NLRP3, IL-1ß and TGF-ß1 levels (P<0.05). Pathological analysis revealed improvements in renal fibrosis and increased expression of the Occludin and ZO-1 proteins in intestinal tissue. In the SSP group, there was a significant increase in microbial activity (P<0.001). According to the sequencing results, the characteristic bacteria of the SSP and NR groups included Succinatimonas hippei, uncultured Solirubrobacter sp., and Clostridium tyrobutyricum. Furthermore, TMAO, NLRP3, IL-1ß, and TGF-ß1 were significantly positively correlated (P<0.05) with Succinatimonas hippei and Clostridium tyrobutyricum. By modulating Firmicutes, Succinatimonas hippei, and Clostridium tyrobutyricum, SSP decoction lowers TMAO levels to alleviate diarrhea with kidney-yang deficiency syndrome. CONCLUSIONS TMAO likely plays a significant role in the "gut-kidney axis" of diarrhea with kidney-yang deficiency syndrome. By adjusting gut microbiota to reduce the inflammatory response that is transmitted through the "gut-kidney axis" as a result of elevated TMAO levels, SSP decoction can alleviate diarrhea with kidney-yang deficiency syndrome.

miR-221-3p as a potential biomarker of chronic periodontitis and its regulatory effect on inflammatory response.

PURPOSE: To explore the function of miR-221-3p in the development and course of chronic periodontitis (CP) and offer a fresh avenue for CP diagnosis and management. METHODS: miR-221-3p expression was detected by RT-qPCR. The clinical diagnostic value of miR-221-3p in CP patients was analyzed by receiver operating characteristic (ROC). ELISA was used to determine the IL-1ß and IL-6 in CP subjects and healthy controls. Pearson correlation analysis was performed with miR-221-3p. PDLCs were induced by LPS, transfected with miR-221-3p mimics, and their expression was analyzed for the effects of IL-1ß, and IL-6. RESULTS: The miR-221-3p expression was lower in the gingival sulcus fluid GCF of CP subjects compared to healthy controls. miR-221-3p showed high potential for clinical diagnosis in CP patients by ROC analysis, with high specificity and sensitivity. miR-221-3p was negatively correlated with Probing pocket depth (PD), Attachment loss (AL), Plaque index (PI), and Bleeding index (BI), and negatively correlated with inflammatory factors IL-1ß and IL-6. In LPS-induced PDLCs, IL-1ß and IL-6 were significantly increased, whereas miR-221-3p was significantly downregulated. Overexpression of miR-221-3p inhibited the production of inflammatory factors IL-1ß and IL-6 in LPS-induced PDLCs. CLINICAL SIGNIFICANCE: miR-221-3p expression may be a potential biological marker for the diagnosis of chronic periodontitis and provide a new direction for its treatment of chronic periodontitis.

Expression of SDF-1/CXCR4 and related inflammatory factors in sodium fluoride-treated hepatocytes.

At present, the mechanism of fluorosis-induced damage to the hepatic system is unclear. Studies have shown that excess fluoride causes some degree of damage to the liver, including inflammation. The SDF-1/CXCR4 signaling axis has been reported to have an impact on the regulation of inflammation in human cells. In this study, we investigated the role of the SDF-1/CXCR4 signaling axis and related inflammatory factors in fluorosis through in vitro experiments on human hepatic astrocytes (LX-2) cultured with sodium fluoride. CCK-8 assays showed that the median lethal dose at 24 h was 2 mmol/l NaF, and these conditions were used for subsequent enzyme-linked immunosorbent assays (ELISAs) and quantitative real-time polymerase chain reaction (qPCR) analysis. The protein expression levels of SDF-1/CXCR4 and the related inflammatory factors nuclear factor-κB (NF-κB), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and interleukin 1ß (IL-1ß) were detected by ELISAs from the experimental and control groups. The mRNA expression levels of these inflammatory indicators were also determined by qPCR in both groups. Moreover, the expression levels of these factors were significantly higher in the experimental group than in the control group at both the protein and mRNA levels (P < 0.05). Excess fluorine may stimulate the SDF-1/CXCR4 signaling axis, activating the inflammatory NF-κB signaling pathway and increasing the expression levels of the related inflammatory factors IL-6, TNF-α and IL-1ß. Identification of this mechanism is important for elucidating the pathogenesis of fluorosis-induced liver injury.

Potential of cannabidiol as acne and acne scar treatment: novel insights into molecular pathways of pathophysiological factors.

Cannabidiol (CBD), which is derived from hemp, is gaining recognition because of its anti-inflammatory and lipid-modulating properties that could be utilized to treat acne. We conducted experiments to quantitatively assess the effects of CBD on acne-related cellular pathways. SEB-1 sebocytes and HaCaT keratinocytes were exposed to various CBD concentrations. CBD exhibited a concentration-dependent impact on cell viability and notably reduced SEB-1 viability; furthermore, it induced apoptosis and a significant increase in the apoptotic area at higher concentrations. Additionally, CBD remarkably reduced pro-inflammatory cytokines, including CXCL8, IL-1α, and IL-1ß. Additionally, it inhibited lipid synthesis by modulating the AMPK-SREBP-1 pathway and effectively reduced hyperkeratinization-related protein keratin 16. Simultaneously, CBD stimulated the synthesis of elastin, collagen 1, and collagen 3. These findings emphasize the potential of CBD for the management of acne because of its anti-inflammatory, apoptotic, and lipid-inhibitory effects. Notably, the modulation of the Akt/AMPK-SREBP-1 pathway revealed a novel and promising mechanism that could address the pathogenesis of acne.

Resveratrol alleviates endoplasmic reticulum stress-induced cell death and improves functional prognosis after traumatic brain injury in mice.

Resveratrol (RSV) is a polyphenol antioxidant that has been shown to have neuroprotective effects. We sought molecular mechanisms that emphasize the anti-inflammatory activity of RSV in traumatic brain injury (TBI) in mice associated with endoplasmic reticulum stress (ERS). After establishing three experimental groups (sham, TBI, and TBI+RSV), we explored the results of RSV after TBI on ERS and caspase-12 apoptotic pathways. The expression levels of C/EBP homologous protein (CHOP), glucose regulated protein 78kD (GRP78), caspase-3, and caspase-12 in cortical brain tissues were assessed by western blotting. The qPCR analysis was also performed on mRNA expression of tumor necrosis factor (TNF)-α and interleukin (IL)-1ß in cortical brain tissue. In addition, the expression of GRP78 in microglia (ionized calcium binding adaptor molecule 1; Iba-1) and neurons (neuronal nuclei; NeuN) was identified by immunofluorescence staining. The neurological function of mice was assessed by modified neurological severity scores (mNSS). After drug treatment, the expression of CHOP, GRP78, caspase-3 and caspase-12 decreased, and qPCR results showed that TNF-α and IL-1ß were down-regulated. Immunofluorescence staining showed down-regulation of Iba-1+/GRP78+ and NeuN+/GRP78+ cells after RSV treatment. The mNSS analysis confirmed improvement after RSV treatment. RSV improved apoptosis by downregulating the ERS signaling pathway and improved neurological prognosis in mice with TBI.

Dan-Lou tablets reduce inflammatory response by inhibiting the activation of NLRP3 inflammasome for coronary heart disease.

BACKGROUND: The activation of the NLRP3 inflammasome has recently been revealed as a novel pathological mechanism of coronary heart disease (CHD). The Dan-Lou tablets (DLT) is widely used in the clinical treatment of CHD and prescription characterized by multi-component and multi-target regulation. However, the anti-inflammatory mechanism of DLT in the treatment of CHD remains unclear. PURPOSE: This study aimed to evaluate the effect of DLT in the treatment of CHD on the priming and activation of the NLRP3 inflammasome and to investigate the underlying anti-inflammatory mechanisms. METHODS: First, CHD rats model were established by a high-fat diet combined with left anterior coronary artery ligation (LADCA) followed by DLT intervention. The therapeutic effect of DLT was evaluated according to cardiac function, lipid level, and cardiac histopathology. Next, data-independent acquisition (DIA) proteomics was used to identify the key differential proteins of DLT intervention in CHD rats, and bioinformatics analysis was performed. Finally, the differentially expressed proteins in the NOD-like signaling pathway were verified based on bioinformatics results, and the priming and activation steps of the NLRP3 inflammasome were detected. RESULTS: In this study, a high-fat diet combined with LADCA was utilized to generate a CHD model, and DLT alleviated myocardial ischemia injury by inhibiting lipid deposition and inflammatory response. Proteomic studies observed that the RNF31, TXN2, and GBP2 of the NOD-like receptor signaling pathway were verified as the key targets of DLT in inhibiting myocardial injury in CHD rats. Furthermore, DLT in the treatment of CHD rats may function through the downregulation of P2X7R expression, thereby interfering with the priming (TLR4/MyD88/NF-κB) and activation (NLRP3/ASC/Caspase-1) of the NLRP3 inflammasome regulated by HSP90, and may then reduce the release of the IL-1ß and IL-18 inflammatory factors to play an anti-myocardial injury effect. CONCLUSION: Our findings elucidate a novel mechanism of DLT and provide some new drug evaluation targets and therapeutic strategies for CHD. This study innovatively proposed that DLT further exerts an anti-myocardial injury effect by inhibiting P2X7R expression, thereby interfering with the priming (TLR4/MyD88/NF-κB) and activation (NLRP3/ASC/Caspase-1) of the NLRP3 inflammasome regulated by HSP90, and then downregulates the release of the IL-1ß and IL-18 inflammatory factors.

Tryptanthrin suppresses multiple inflammasome activation to regulate NASH progression by targeting ASC protein.

BACKGROUND: The adaptor protein apoptosis-associated speck-like protein (ASC) containing a caspase recruitment domain (CARD) can be activated through pyrin domain (PYD) interactions between sensors and ASC, and through CARD interactions between caspase-1 and ASC. Although the majority of ternary inflammasome complexes depend on ASC, drugs targeting ASC protein remain scarce. After screening natural compounds from Isatidis Radixin, we found that tryptanthrin (TPR) could inhibit NLRP3-induced IL-1ß and caspase-1 production, but the underlying anti-inflammatory mechanisms remain to be elucidated. PURPOSE: The purpose of this study was to determine the impact of TPR on the NLRP3, NLRC4, and AIM2 inflammasomes and the underlying mechanisms. Additionally, the efficacy of TPR was analysed in the further course of methionine- and choline-deficient (MCD)-induced NASH and lipopolysaccharide (LPS)-induced sepsis models of mice. METHODS: In vitro studies used bone marrow-derived macrophages to assess the anti-inflammatory activity of TPR, and the techniques included western blot, testing of intracellular K+ and Ca2+, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), co-immunoprecipitation, ASC oligomerization assay, surface plasmon resonance (SPR), and molecular docking. We used LPS-induced sepsis models and MCD-induced NASH models in vivo to evaluate the effectiveness of TPR in inhibiting inflammatory diseases. RESULTS: Our observations suggested that TPR could inhibit NLRP3, NLRC4, and AIM2 inflammasome activation. As shown in a mouse model of inflammatory diseases caused by MCD-induced NASH and LPS-induced sepsis, TPR significantly alleviated the progression of diseases. TPR interrupted the interactions between ASC and NLRP3/NLRC4/AIM2 in the co-immunoprecipitation experiment, and stable binding of TPR to ASC was also evident in SPR experiments. The underlying mechanisms of anti-inflammatory activities of TPR might be associated with targeting ASC, in particular, PYD domain of ASC. CONCLUSION: In general, the requirement for ASC in multiple inflammasome complexes makes TPR, as a novel broad-spectrum inflammasome inhibitor, potentially useful for treating a wide range of multifactorial inflammasome-related diseases.

Vaccarin alleviates septic cardiomyopathy by potentiating NLRP3 palmitoylation and inactivation.

BACKGROUND: Sepsis often leads to significant morbidity and mortality due to severe myocardial injury. As is known, the activation of NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome crucially contributes to septic cardiomyopathy (SCM) by facilitating the secretion of interleukin (IL)-1ß and IL-18. The removal of palmitoyl groups from NLRP3 is a crucial step in the activation of the NLRP3 inflammasome. Thus, the potential inhibitors that regulate the palmitoylation and inactivation of NLRP3 may significantly diminish sepsis-induced cardiac dysfunction. PURPOSE: The present study sought to explore the effects of the prospective flavonoid compounds targeting NLRP3 on SCM and to elucidate the associated underlying mechanisms. STUDY DESIGN: The palmitoylation and activation of NLRP3 were detected in H9c2 cells and C57BL/6 J mice. METHODS/RESULTS: Echocardiography, histological staining, western blotting, co-immunoprecipitation, qPCR, ELISA and network pharmacology were used to assess the impact of vaccarin (VAC) on SCM in mice subjected to lipopolysaccharide (LPS) injection. From the collection of 74 compounds, we identified that VAC had the strongest capability to suppress NLRP3 luciferase report gene activity in cardiomyocytes, and the anti-inflammatory characteristics of VAC were further ascertained by the network pharmacology. Exposure of LPS triggered apoptosis, inflammation, oxidative stress, mitochondrial disorder in cardiomyocytes. The detrimental alterations were significantly reversed upon VAC treatment in both septic mice and H9c2 cells exposed to LPS. In vivo experiments demonstrated that VAC treatment alleviated septic myocardial injury, indicated by enhanced cardiac function parameters, preserved cardiac structure, and reduced inflammation/oxidative response. Mechanistically, VAC induced NLRP3 palmitoylation to inactivate NLRP3 inflammasome by acting on zDHHC12. In support, the NLRP3 agonist ATP and the acylation inhibitor 2-bromopalmitate (2-BP) prevented the effects of VAC. CONCLUSION: Our findings suggest that VAC holds promise in protecting against SCM by mitigating cardiac oxidative stress and inflammation via priming NLRP3 palmitoylation and inactivation. These results lay the solid basis for further assessment of the therapeutic potential of VAC against SCM.

Hypoxia-induced activation of HIF-1alpha/IL-1beta axis in microglia promotes glioma progression via NF-κB-mediated upregulation of heparanase expression.

BACKGROUND: Glioma is a common tumor that occurs in the brain and spinal cord. Hypoxia is a crucial feature of the tumor microenvironment. Tumor-associated macrophages/microglia play a crucial role in the advancement of glioma. This study aims to illuminate the detailed mechanisms by which hypoxia regulates microglia and, consequently, influences the progression of glioma. METHODS: The glioma cell viability and proliferation were analyzed by cell counting kit-8 assay and 5-ethynyl-2'-deoxyuridine assay. Wound healing assay and transwell assay were implemented to detect glioma cell migration and invasion, respectively. Enzyme-linked immunosorbent assay was conducted to detect protein levels in cell culture medium. The protein levels in glioma cells and tumor tissues were evaluated using western blot analysis. The histological morphology of tumor tissue was determined by hematoxylin-eosin staining. The protein expression in tumor tissues was determined using immunohistochemistry. Human glioma xenograft in nude mice was employed to test the influence of hypoxic microglia-derived interleukin-1beta (IL-1ß) and heparanase (HPSE) on glioma growth in vivo. RESULTS: Hypoxic HMC3 cells promoted proliferation, migration, and invasion abilities of U251 and U87 cells by secreting IL-1ß, which was upregulated by hypoxia-induced activation of hypoxia inducible factor-1alpha (HIF-1α). Besides, IL-1ß from HMC3 cells promoted glioma progression and caused activation of nuclear factor-κB (NF-κB) and upregulation of HPSE in vivo. We also confirmed that IL-1ß facilitated HPSE expression in U251 and U87 cells by activating NF-κB. Hypoxic HMC3 cells-secreted IL-1ß facilitated the proliferation, migration, and invasion of U251 and U87 cells via NF-κB-mediated upregulation of HPSE expression. Finally, we revealed that silencing HPSE curbed the proliferation and metastasis of glioma in mice. CONCLUSION: Hypoxia-induced activation of HIF-1α/IL-1ß axis in microglia promoted glioma progression via NF-κB-mediated upregulation of HPSE expression.

Cartilage progenitor cells derived extracellular vesicles-based cell-free strategy for osteoarthritis treatment by efficient inflammation inhibition and extracellular matrix homeostasis restoration.

Osteoarthritis (OA) is a common degenerative joint disease which currently lacks of effective agents. It is therefore urgent and necessary to seek an effective approach that can inhibit inflammation and promote cartilage matrix homeostasis. Cartilage progenitor cells (CPCs) are identified as a cell population of superficial zone in articular cartilage which possess strong migration ability, proliferative capacity, and chondrogenic potential. Recently, the application of CPCs may represent a novel cell therapy strategy for OA treatment. There is growing evidence that extracellular vesicles (EVs) are primary mediators of the benefits of stem cell-based therapy. In this study, we explored the protective effects of CPCs-derived EVs (CPCs-EVs) on IL-1ß-induced chondrocytes. We found CPCs-EVs exhibited chondro-protective effects in vitro. Furthermore, our study demonstrated that CPCs-EVs promoted matrix anabolism and inhibited inflammatory response at least partially via blocking STAT3 activation. In addition, liquid chromatography-tandem mass spectrometry analysis identified 991 proteins encapsulated in CPCs-EVs. By bioinformatics analysis, we showed that STAT3 regulatory proteins were enriched in CPCs-EVs and could be transported to chondrocytes. To promoting the protective function of CPCs-EVs in vivo, CPCs-EVs were modified with cationic peptide ε-polylysine-polyethylene-distearyl phosphatidylethanolamine (PPD) for surface charge reverse. In posttraumatic OA mice, our results showed PPD modified CPCs-EVs (PPD-EVs) effectively inhibited extracellular matrix catabolism and attenuated cartilage degeneration. Moreover, PPD-EVs down-regulated inflammatory factors expressions and reduced OA-related pain in OA mice. In ex-vivo cultured OA cartilage explants, PPD-EVs successfully promoted matrix anabolism and inhibited inflammation. Collectively, CPCs-EVs-based cell-free therapy is a promising strategy for OA treatment.

Analysis of prognostic biomarker models of TXNIP/NLRP3/IL1B inflammasome pathway in patients with acute myeloid leukemia.

Background: Exploring potential biomarkers for predicting clinical outcomes and developing targeted therapies for acute myeloid leukemia (AML) is of utmost importance. This study aimed to investigate the expression pattern of the thioredoxin-interacting protein (TXNIP)/nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) pathway and its role in the prognosis of AML patients. Methods: In this study, we examined the prognostic value of TXNIP/NLRP3 pathway in AML patients using microarray data from Gene Expression Omnibus (GEO) and transcriptome data from the Cancer Genome Atlas (TCGA) to develop a prognostic model and validated the results by quantitative real-time PCR (qRT-PCR) in a validation cohort of 26 AML patients and 18 healthy individuals from Jinan University (JNU) database. Results: Analysis of the GSE13159 database revealed that TXNIP, interleukin 1 beta (IL1B) within the TXNIP/NLRP3 pathway were significantly upregulated and caspase1 (CASP1) was downregulated in AML patients (TXNIP, P = 0.031; IL1B, P = 0.042; CASP1, P = 0.038). Compared to high NLRP3 expression, AML patients with low NLRP3 expression had a longer overall survival (OS) in the GSE12417 dataset (P = 0.004). Moreover, both the training and validation results indicated that lower TXNIP, NLRP3, and IL1B expression were associated with favorable prognosis (GSE12417, P = 0.009; TCGA, P = 0.050; JNU, P = 0.026). According to the receiver operating characteristic curve analysis, this model demonstrated a sensitivity of 84% for predicting three-year survival. These data might provide novel predictors for AML outcome and direction for further investigation of the possibility of using TXNIP/NLRP3/IL1B genes in novel targeted therapies for AML.

Reynosin protects neuronal cells from microglial neuroinflammation by suppressing NLRP3 inflammasome activation mediated by NADPH oxidase.

Neuroinflammation, mediated by the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing-3 (NLRP3) inflammasome, is a significant contributor to the pathogenesis of neurodegenerative diseases (NDDs). Reynosin, a natural sesquiterpene lactone (SL), exhibits a broad spectrum of pharmacological effects, suggesting its potential therapeutic value. However, the effects and mechanism of reynosin on neuroinflammation remain elusive. The current study explores the effects and mechanisms of reynosin on neuroinflammation using mice and BV-2 microglial cells treated with lipopolysaccharide (LPS). Our findings reveal that reynosin effectively reduces microglial inflammation in vitro, as demonstrated by decreased CD11b expression and lowered interleukin-1 beta (IL-1ß) and interleukin-18 (IL-18) mRNA and protein levels. Correspondingly, in vivo, results showed a reduction in the number of Iba-1 positive cells and alleviation of morphological alterations, alongside decreased expressions of IL-1ß and IL-18. Further analysis indicates that reynosin inhibits NLRP3 inflammasome activation, evidenced by reduced transcription of NLRP3 and caspase-1, diminished NLRP3 protein expression, inhibited apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization, and decreased caspase-1 self-cleavage. Additionally, reynosin curtailed the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, demonstrated by reduced NADP+ and NADPH levels, downregulation of gp91phox mRNA, protein expression, suppression of p47phox expression and translocation to the membrane. Moreover, reynosin exhibited a neuroprotective effect against microglial inflammation in vivo and in vitro. These collective findings underscore reynosin's capacity to mitigate microglial inflammation by inhibiting the NLRP3 inflammasome, thus highlighting its potential as a therapeutic agent for managing neuroinflammation.