Role of ß-adrenergic signaling and the NLRP3 inflammasome in chronic intermittent hypoxia-induced murine lung cancer progression.

BACKGROUND: Obstructive sleep apnea (OSA), characterized by chronic intermittent hypoxia (CIH), is a prevalent condition that has been associated with various forms of cancer. Although some clinical studies suggest a potential link between OSA and lung cancer, this association remains uncertain, and the underlying mechanisms are not fully understood. This study investigated the role of the catecholamine-ß-adrenergic receptor (ßAR) and the NLRP3 inflammasome in mediating the effects of CIH on lung cancer progression in mice. METHODS: Male C57BL/6 N mice were subjected to CIH for four weeks, with Lewis lung carcinoma cells seeded subcutaneously. Propranolol (a ßAR blocker) or nepicastat (an inhibitor of catecholamine production) was administered during this period. Tumor volume and tail artery blood pressure were monitored. Immunohistochemical staining and immunofluorescence staining were employed to assess protein expression of Ki-67, CD31, VEGFR2, PD-1, PD-L1, and ASC specks in tumor tissues. ELISA was used to detect catecholamine and various cytokines, while western blot assessed the expression of cyclin D1, caspase-1, and IL-1ß. In vitro tube formation assay investigated angiogenesis. NLRP3 knockout mice were used to determine the mechanism of NLRP3 in CIH. RESULTS: CIH led to an increase in catecholamine. Catecholamine-ßAR inhibitor drugs prevented the increase in blood pressure caused by CIH. Notably, the drugs inhibited CIH-induced murine lung tumor growth, and the expression of Ki-67, cyclin D1, CD31, VEGFR2, PD-1 and PD-L1 in tumor decreased. In vitro, propranolol inhibits tube formation induced by CIH mouse serum. Moreover, CIH led to an increase in TNF-α, IL-6, IL-1ß, IFN-γ and sPD-L1 levels and a decrease in IL-10 in peripheral blood, accompanied by activation of NLRP3 inflammasomes in tumor, but these effects were also stopped by drugs. In NLRP3-knockout mice, CIH-induced upregulation of PD-1/PD-L1 in tumor was inhibited. CONCLUSIONS: Our study underscores the significant contribution of ß-adrenergic signaling and the NLRP3 inflammasome to CIH-induced lung cancer progression. These pathways represent potential therapeutic targets for mitigating the impact of OSA on lung cancer.

Paeoniflorin inhibited GSDMD to alleviate ANIT-induced cholestasis via pyroptosis signaling pathway.

BACKGROUND: Cholestasis (CT) is a group of disorders caused by impaired production, secretion or excretion of bile. This may result in the deposition of bile components in the blood and liver, which in turn causes damage to liver cells and other tissues. If untreated, CT can progress to severe complications, including cirrhosis, liver failure, and potentially life-threatening conditions. OBJECTIVE: This research was intended to elucidate the function and mechanism of Paeoniflorin (PF) in ameliorating ANIT-induced pyroptosis in CT. METHODS: CT models were established in SD rats and HepG2 cells through ANIT treatment. Histological examination was conducted using haematoxylin and eosin (HE) staining to assess the histopathological alterations in the liver. Network pharmacology was employed to identify potential PF targets in CT treatment. To evaluate pyroptosis levels, various methods were used, including serum biochemical analysis, Enzyme-Linked Immunosorbent Assay (ELISA), immunofluorescence (IF), immunohistochemistry (IHC), Western blotting, transmission electron microscopy (TEM), and scanning electron microscopy (SEM). The HuProt™ 20K Chip was utilized to pinpoint potential PF-binding targets. PF's direct mechanisms in CT treatment were explored using molecular docking (MD), molecular dynamics simulations (MDS), Cellular Thermal Shift Assay (CETSA), and Surface Plasmon Resonance (SPR). RESULTS: PF administration was found to alleviate ANIT-induced liver pathology, enhance liver function markers, and improve cell viability. Network pharmacology and pyroptosis inhibitor studies suggested that PF might mitigate CT via the NLRP3-dependent pyroptosis pathway. This hypothesis was further supported by Western blotting, IF, and IHC analyses, which indicated PF's potential to inhibit NLRP3-dependent pyroptosis in CT. GSDMD was identified as a target through HuProt™ 20K Chip screening. The binding affinity of PF to GSDMD was validated through MD, MDS, CETSA, and SPR techniques. Additionally, the regulatory impact of GSDMD on downstream inflammatory pathways was confirmed by ELISA and IHC. CONCLUSION: PF exhibited a hepatoprotective effect in ANIT-induced CT, primarily by targeting GSDMD, thereby suppressing ANIT-induced pyroptosis and the subsequent release of inflammatory mediators.

NLRP3 targets HMGB1 to exacerbate the pyroptosis of canine corneal epithelial cells infected with Staphylococcus pseudintermedius.

PURPOSE: This study focused on the mechanisms of pyroptosis and oxidative damage exacerbation by NOD-like receptor thermal protein domain associated protein 3 (NLRP3) during the infection of canine corneal epithelial cells (CCECs) with Staphylococcus pseudintermedius. METHODS: The CCECs treated with dimethyl fumarate (DMF), recombinant high mobility group protein 1 (HMGB1), or N-acetylcysteine (NAC). The gasdermin (GSDM) family and HMGB1 mRNA expression levels were detected using quantitative reverse transcription polymerase chain reaction. Lactate dehydrogenase activity, bacterial counts, the pyroptosis rate, reactive oxygen species (ROS) content, and antioxidant enzyme activity were used to reflect pyroptosis and oxidation level. RESULTS: Regulation of NLRP3 significantly affected the pyroptosis rate and GSDMD-N expression levels during S. pseudintermedius infection. Inhibition of GSDMD-N protein activation by DMF reversed the exacerbation of pyroptosis induced by NLRP3 overexpression and reduced the levels of cleaved interleukin-1ß (IL-1ß), cleaved cysteinyl aspartate-specific protease-1, and NLRP3. In addition, NLRP3 was found to target the HMGB1 promoter and regulate its protein expression, to increase ROS accumulation and GSDMD-N expression levels, and activate the NLRP3-HMGB1-ROS-GSDMD signaling axis to aggravate pyroptosis during infection. CONCLUSIONS: NLRP3 aggravates pyroptosis and oxidative damage associated with the activation of NLRP3-GSDMD and NLRP3-HMGB1-ROS-GSDMD signaling pathways during the infection of CCECs with S. pseudintermedius.

Mechanism of Biqi capsules in the treatment of gout based on network pharmacology and experimental verification.

ETHNOPHARMACOLOGICAL RELEVANCE: Gout is a crystal-related arthropathy caused by monosodium urate (MSU) deposition, resulting from purine metabolism disorders and hyperuricemia (HUA). Gout belongs to the traditional medicine category of Bi syndrome. Biqi capsules (BQ) is a traditional Chinese medicine formula used to treat Bi syndrome. The BQ prescription is derived from the ancient prescription of Hua Tuo, a famous physician in the Han Dynasty. AIM OF THE STUDY: To study the effect and mechanism of BQ in treating acute gouty arthritis (AGA) and HUA. MATERIALS AND METHODS: Analyzing BQ's signaling pathways for gout treatment via network pharmacology. The HUA model was induced orally with adenine and potassium oxonate. The rat AGA model was established by MSU injection. In vitro, MH7A and RAW 246.7 cells were treated with LPS and MSU. Serum uric acid, creatinine, and urea nitrogen levels were evaluated. Kidney and ankle joint pathology was observed via HE staining. Inflammatory signaling pathway proteins, epithelial-mesenchymal transition (EMT) pathway proteins, and uric acid metabolism-related proteins were detected by Western blot. RESULTS: 1780 potential targets for gout treatment were identified, and 1039 target proteins corresponding to BQ's active ingredients were obtained. Pathway enrichment analysis revealed BQ improved gout mainly through inflammatory pathways. Experimental results showed BQ could reduce serum uric acid level and increase uric acid clearance rate by regulating the expression of adenosine deaminase (ADA), and organic anion transporter 1 (OAT1) and glucose transporter 9 (GLUT9) in HUA mice. BQ could improve renal function and injury by inhibiting the NLRP3 pathway in HUA mice' kidneys. Additionally, BQ could alleviate ankle joint swelling and synovial injury, inhibit the TLR4/NLRP3 pathway, and reduce levels of inflammatory factors including interleukin 6 (IL-6), interleukin 1ß (IL-1ß), and tumor necrosis factor-alpha (TNF-α) in AGA rats. The main component of BQ, brucine, could inhibit the activation of NLRP3/NF-κB pathway induced by MSU and reduce the expression level of inflammatory factors (IL-6, IL-1ß, and TNF-α) in macrophages. Brucine could inhibit the activation of the EMT pathway and reduce the expression level of inflammatory factors (IL-6, TNF-α) in human fibroblast-like synoviocytes (MH7A cells) induced by MSU. CONCLUSIONS: BQ effectively reduced serum uric acid levels, improved kidney and joint damage, and ameliorated the inflammatory response caused by MSU. Its main component, brucine, effectively improved the inflammatory response and reduced the invasive ability of synoviocytes induced by MSU.

Macrophages regulate plaque progression in diabetic Apoe-/- mice dependent on Pi4p/Nlrp3 signaling pathway.

BACKGROUND AND AIMS: Atherosclerotic cardiovascular disease complicated by diabetes mellitus (DM) is the leading cause of death in diabetic patients, and it is strongly associated with macrophages and inflammasomes. It has been found that activation of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome is closely associated with phosphatidylinositol 4-phosphate (PI4P) on the trans-Golgi. However, how PI4P and NLRP3 regulate macrophage function and its role in diabetic atherosclerotic plaques is unclear. METHODS: The expression of Pi4p and Nlrp3-inflammasome-related proteins in atherosclerosis in apolipoprotein E-deficient (Apoe-/-) and Apoe-/- DM mice was investigated. Then, Pi4p levels were affected by shRNA-Pi4kb or cDNA-Sac1 plasmid to investigate the effects of changes in Pi4p-related metabolic enzymes on macrophage function. Finally, genetically modified macrophages were injected into diabetic Apoe-/- mice to explore the effects on atherosclerosis. RESULTS: DM promoted plaque progression in atherosclerotic mice and increased expression of Pi4p and Nlrp3 in plaques. In addition, impaired macrophage function induced by high glucose was reversed by transfected shRNA-Pi4kb or cDNA-Sac1 plasmid. Furthermore, decreased levels of Pi4p reduced plaque area in diabetic Apoe-/- mice. CONCLUSIONS: Our data suggests that Pi4p/Nlrp3 in macrophages play an important role in the exacerbation of atherosclerosis in diabetic mice. Pi4p-related metabolizing enzymes (PI4KB and SAC1) may be a potential therapeutic strategy for diabetic atherosclerosis, and macrophage therapy is also a potential treatment.

Mitigation of lipopolysaccharide-induced intestinal injury in rats by Chimonanthus nitens Oliv. essential oil via suppression of mitochondrial fusion protein mitofusin 2 (MFN2)-mediated mitochondrial-associated endoplasmic reticulum membranes (MAMs) formation.

ETHNOPHARMACOLOGICAL RELEVANCE: Chimonanthus nitens Oliv. is a traditional Chinese medicine with anti-inflammatory and antioxidant properties that has commonly been used for colds, fevers, and other diseases. However, its role and specific mechanism in sepsis-associated intestinal injury have not been reported. AIM OF THE STUDY: C. nitens Oliv. essential oil (CEO), an organic active compound extracted from the traditional Chinese medicine C. nitens Oliv. exhibits notable anti-inflammatory and antioxidant properties. Nevertheless, the therapeutic potential of CEO for septic intestinal injury remains undocumented. This study thus aims to elucidate the anti-inflammatory and antioxidant effects of CEO in the context of acute intestinal injury and to investigate its mechanisms of action in septic rats. MATERIALS AND METHODS: Cell and animal models were established using LPS to investigate the impact of CEO on LPS-induced intestinal pathological injury and the secretion of inflammatory factor IL-1ß. The effects of CEO on the expression of NLRP3, caspase-1, and MFN2, p-p65 protein were also examined, as well as its influence on oxidative stress injury and mitochondrial-associated endoplasmic reticulum membrane (MAM) formation. Generation of an MFN2 knockout IEC-6 cell line allowed comprehensive investigation of the protective mechanism of CEO. RESULTS: In rat models, CEO reduced IL-1ß secretion, inhibited caspase-1, ZO-1 expression and NF-κB p65 phosphorylation, while also decreasing malondialdehyde levels and enhancing superoxide dismutase activity in intestinal tissues. Cellular experiments demonstrated its ability to decrease IL-1ß secretion; NLRP3, caspase-1, and MFN2 expression; NF-κB p65 phosphorylation; reactive oxygen species (ROS) production, and mitochondrial dysfunction. MFN2 knockdown enhanced these effects synergistically with CEO, indicating potential therapeutic synergy. Further, MFN2 knockdown significantly mitigated LPS-induced NLRP3 and caspase-1 expression, IL-1ß secretion, ROS production, NF-κB p65 phosphorylation and MMP reduction in IEC-6 cells, while inhibiting MAM formation and NLRP3 localization on MAMs. Importantly, MFN2 downregulation and CEO synergistically reduced LPS-induced IL-1ß secretion and ROS production while inhibiting MAM formation in IEC-6 cells, thus inhibiting NLRP3 inflammasome activation. CONCLUSION: CEO mitigates inflammation and oxidative stress by inhibiting MAM formation and is thus a promising intervention for septic intestinal injury.

The role of inflammasomes as central inflammatory hubs in Mycobacterium tuberculosis infection.

Mycobacterium tuberculosis (Mtb) infection represents a global health problem and is characterized by formation of granuloma with a necrotic center and a systemic inflammatory response. Inflammasomes have a crucial role in the host immune response towards Mtb. These intracellular multi-protein complexes are assembled in response to pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs). Inflammasome platforms activate caspases, leading to the maturation of the proinflammatory cytokines interleukin (IL)-1 and 18 and the cleavage of gasdermin D (GSDMD), a pore-forming protein responsible for cytokine release and pyroptotic cell death. Recent in vitro and in vivo findings have highlighted the importance of inflammasome signaling and subsequent necrotic cell death in Mtb-infected innate immune cells. However, we are just beginning to understand how inflammasomes contribute to disease or to a protective immune response in tuberculosis (TB). A detailed molecular understanding of inflammasome-associated pathomechanisms may foster the development of novel host-directed therapeutics or vaccines with improved activity. In this mini-review, we discuss the regulatory and molecular aspects of inflammasome activation and the associated immunological consequences for Mtb pathogenesis.

Short-term respiratory cadmium exposure partially activates pulmonary NLRP3 inflammasome by inducing ferroptosis in mice.

Cadmium (Cd) is a common environmental metal. Previous studies indicated that long-term respiratory Cd exposure caused lung injury and airway inflammation. The purpose of this study was to evaluate whether short-term respiratory Cd exposure induces pulmonary ferroptosis and NLRP3 inflammasome activation. Adult C57BL/6J mice were exposed to Cd by inhaling CdCl2 aerosol (0, 10, or 100 ppm) for 5 days. Serum and lung Fe2+ contents were elevated in Cd-exposed mice. Oxidized AA metabolites, the major oxidized lipids during ferroptosis, were upregulated in Cd-exposed mouse lungs. Pulmonary MDA content and 4-HNE-positive cells were increased in Cd-exposed mice. ACSL4 and COX-2, two lipoxygenases, were upregulated in Cd-exposed mouse lungs. Further analyses found that phosphorylated NF-kB p65 was elevated in Cd-exposed mouse lungs. Innate immune receptor protein NLRP3 and adapter protein ASC were upregulated in Cd-exposed mouse lungs. Caspase-1 was activated and IL-1ß and IL-18 were upregulated in Cd-exposed mouse lungs. Fer-1, a specific inhibitor of ferroptosis, attenuated Cd-induced elevation of pulmonary NLRP3 and ASC, caspase-1 activation, and IL-1ß and IL-18 upregulation. Finally, mitoquinone (MitoQ), a mitochondria-target antioxidant, suppressed Cd-caused ferroptosis and NLRP3 inflammasome activation. Our results demonstrate that ferroptosis might partially mediate Cd-evoked activation of NLRP3 inflammasome in the lungs.

PROTAC based STING degrader attenuates acute colitis by inhibiting macrophage M1 polarization and intestinal epithelial cells pyroptosis mediated by STING-NLRP3 axis.

Inflammatory bowel diseases (IBDs) are chronic, relapsing, and inflammatory disorders of the gastrointestinal tract characterized by abnormal immune responses. Recently, STING has emerged as a promising therapeutic target for various autoinflammatory diseases. However, few STING-selective small molecules have been investigated as novel strategies for IBD. In this study, we sought to examine the effects of PROTAC-based STING degrader SP23 on acute colitis and explore its underlying mechanism. SP23 treatment notably alleviates dextran sulfate sodium (DSS)-induced colitis. Pharmacological degradation of STING significantly reduced the production of inflammatory cytokines, such as TNF-α, IL-1ß, and IL-6, and inhibited macrophage polarization towards the M1 type. Furthermore, SP23 administration decreased the loss of tight junction proteins, including ZO-1, occludin, and claudin-1, and downregulated STING and NLRP3 signaling pathways in intestinal inflammation. In vitro, STING activated NLRP3 inflammasome-mediated pyroptosis in intestinal epithelial cells, which could be abrogated by SP23 and STING siRNA intervention. In conclusion, these findings provide new evidence for STING as a novel therapeutic target for IBD, and reveal that hyperactivation of STING could exaggerate colitis by inducing NLRP3/Caspase-1/GSDMD axis mediated intestinal epithelial cells pyroptosis.

Butyrate and propionate are microbial danger signals that activate the NLRP3 inflammasome in human macrophages upon TLR stimulation.

Short-chain fatty acids (SCFAs) are immunomodulatory compounds produced by the microbiome through dietary fiber fermentation. Although generally considered beneficial for gut health, patients suffering from inflammatory bowel disease (IBD) display poor tolerance to fiber-rich diets, suggesting that SCFAs may have contrary effects under inflammatory conditions. To investigate this, we examined the effect of SCFAs on human macrophages in the presence of Toll-like receptor (TLR) agonists. In contrast to anti-inflammatory effects under steady-state conditions, we found that butyrate and propionate activated the NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome in the presence of TLR agonists. Mechanistically, these SCFAs prevented transcription of FLICE-like inhibitory protein (cFLIP) and interleukin-10 (IL-10) through histone deacetylase (HDAC) inhibition, triggering caspase-8-dependent NLRP3 inflammasome activation. SCFA-driven NLRP3 activation was potassium efflux independent and did not result in cell death but rather triggered hyperactivation and IL-1ß release. Our findings demonstrate that butyrate and propionate are bacterially derived danger signals that regulate NLRP3 inflammasome activation through epigenetic modulation of the inflammatory response.

Avanafil Mitigates Testicular Ischemia/Reperfusion Injury via NLRP3 Pathway Modulation in Rats.

OBJECTIVE: In our study, the effectiveness of avanafil, a second-generation phosphodiesterase-5 (PDE5) inhibitor, on testicular torsion (TT) related ischemia/reperfusion injury via NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3), which triggers inflammatory response, are studied molecularly, biochemically and histopathologically. MATERIAL AND METHOD: This study was performed on 24 male Wistar albino rats randomized into four groups. Testicular ischemia/reperfusion (I/R) model was created for groups 2, 3 and 4. Groups 3 and 4, respectively, were administered a dose of 5 and 10 mg/kg avanafil before reperfusion by gavage. The testicles which were left in ischemia for two hours, were detorsioned for four hours. All animals were sacrificed after reperfusion. Testicular tissues were examined molecularly, biochemically and histopathologically. RESULTS: The NLRP3, Interleukin-1ß (IL-1ß) and Tumor Necrosis alpha (TNF-α) mRNA expression levels were observed to be significantly increased in the I/R group compared to the healthy group (p < 0.001). After both doses of avanafil, NLRP3, IL-1ß and TNF-α mRNA expression levels, which increased as a result of I/R, decreased in both avanafil groups. (p < 0.001). The greatest decrease was seen at the dose of 10 mg/kg (p < 0.001). Increased Malondialdehyde (MDA) levels due to I/R were statistically significantly decreased in both doses of avanafil (p < 0.001). Decreased Superoxide Dismutase (SOD) levels due to I/R damage increased statistically significantly in both doses of avanafil (p < 0.001). CONCLUSION: It was found that avanafil can reduce the damage caused by testicular I/R and that it will find new applications in the future with the support of advanced experimental and clinical studies.

[Daidzein attenuates high glucose-induced inflammatory injury in macrophages by regulating NLRP3 inflammasome signaling pathway].

This study aims to explore the inhibitory effect of daidzein on macrophage inflammation induced by high glucose via regulating the NOD-like receptor protein 3(NLRP3) inflammasome signaling pathway. The cell counting kit-8(CCK-8) assay was employed to detect the effects of daidzein at different concentrations on the viability of RAW264.7 cells. Western blot was employed to determine the protein level of tumor necrosis factor(TNF)-α in macrophages exposed to different concentrations of glucose for different time periods as well as the expression levels of proteins involved in the polarization and Toll-like receptor 4(TLR4)-myeloid differentiation factor(MyD88)-NLRP3 inflammasome pathway of the macrophages exposed to high glucose. Enzyme-linked immunosorbent assay was employed to measure the levels of TNF-α, interleukin(IL)-18, and IL-1ß secreted by macrophages. The expression level of nuclear factor-kappa B(NF-κB) p65 in macrophages exposed to high glucose was detected by immunofluorescence, and the level of intracellular reactive oxygen species(ROS) was detected by the DCFH-DA fluorescent probe. The mRNA levels of NLRP3, TNF-α, and IL-18 in macrophages were determined by qRT-PCR. The results showed that treatment with 30 mmol·L~(-1) glucose for 48 h was the best condition for the modeling of macrophage injury. Compared with the blank group, the model group showed improved polarization of macrophages, increased secretion of TNF-α, IL-18, and IL-1ß, elevated ROS level, and up-regulated expression of NF-κB p65. In addition, the modeling up-regulated the mRNA levels of NLRP3, TNF-α, and IL-18 and the protein levels of TLR4, MyD88, NLRP3, NF-κB p65, p-NF-κB p65, I-κB, p-I-κB, ASC, pro-caspase-1, pro-IL-1ß, cleaved IL-1ß, and pro-IL-18. Compared with the model group, daidzein(10, 20, and 40 µmol·L~(-1)) lowered the levels of inflammatory cytokines and down-regulated the mRNA levels of NLRP3, TNF-α, and IL-18 as well as the protein levels of TLR4, MyD88, NLRP3, NF-κB p65, p-NF-κB p65, I-κB, p-I-κB, ASC, pro-caspase-1, pro-IL-1ß, cleaved IL-1ß, and pro-IL-18. In addition, daidzein reduced intracellular ROS. According to the available reports and the experimental results, high glucose can induce the polarization of macrophages and promote the secretion of inflammatory cytokines. Daidzein can inhibit the expression of ROS in macrophages by regulating the NLRP3 inflammasome signaling pathway, thereby reducing the inflammation of macrophages exposed to high glucose.

Calenduloside E Ameliorates Inflammatory Responses in Adipose Tissue via Sirtuin 2-NLRP3 Inflammasome Axis.

Obesity-related metabolic diseases are associated with a chronic inflammatory state. Calenduloside E (CE) is a triterpene saponin from sugar beet. In mouse models, CE reduced pro-inflammatory cytokines in white adipose tissue (WAT) and decreased macrophage infiltration of WAT. And CE inhibited pyroptosis in J774A.1 cells and WAT by inhibiting the activation of the nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 3 (NLRP3) inflammasome. Moreover, CE could trigger the activation of Sirtuin 2 (SIRT2), leading to a decrease in the acetylation of NLRP3, particularly at the K24 site. In addition, it has been shown that CE can reduce inflammation in adipocytes that have been induced by macrophage-conditioned medium. However, the selective SIRT2 inhibitor AGK2 hindered the beneficial effects of CE. In summary, CE has the capacity to impede NLRP3-mediated pyroptosis by triggering SIRT2 activity, thus positioning CE as a promising therapeutic avenue for combating obesity-related metabolic disorders.

Multifunctional carbomer based ferulic acid hydrogel promotes wound healing in radiation-induced skin injury by inactivating NLRP3 inflammasome.

BACKGROUND: Radiation-induced skin injury is a significant adverse reaction to radiotherapy. However, there is a lack of effective prevention and treatment methods for this complication. Ferulic acid (FA) has been identified as an effective anti-radiation agent. Conventional administrations of FA limit the reaching of it on skin. We aimed to develop a novel FA hydrogel to facilitate the use of FA in radiation-induced skin injury. METHODS: We cross-linked carbomer 940, a commonly used adjuvant, with FA at concentrations of 5%, 10%, and 15%. Sweep source optical coherence tomography system, a novel skin structure evaluation method, was applied to investigate the influence of FA on radiation-induced skin injury. Calcein-AM/PI staining, CCK8 assay, hemolysis test and scratch test were performed to investigate the biocompatibility of FA hydrogel. The reducibility of DPPH and ABTS radicals by FA hydrogel was also performed. HE staining, Masson staining, laser Doppler blood flow monitor, and OCT imaging system are used to evaluate the degree of skin tissue damage. Potential differentially expressed genes were screened via transcriptome analysis. RESULTS: Good biocompatibility and in vitro antioxidant ability of the FA hydrogels were observed. 10% FA hydrogel presented a better mechanical stability than 5% and 15% FA hydrogel. All three concentrations of FA remarkably promoted the recovery of radiation-induced skin injury by reducing inflammation, oxidative conidiation, skin blood flow, and accelerating skin tissue reconstruction, collagen deposition. FA hydrogel greatly inhibiting the levels of NLRP3, caspase-1, IL-18, pro-IL-1ß and IL-1ß in vivo and vitro levels through restraining the activation of NLRP3 inflammasome. Transcriptome analysis indicated that FA might regulate wound healing via targeting immune response, inflammatory response, cell migration, angiogenesis, hypoxia response, and cell matrix adhesion. CONCLUSIONS: These findings suggest that the novel FA hydrogel is a promising therapeutic method for the prevention and treatment of radiation-induced skin injury patients.

Mammary hydroxylated oestrogen activates the NLRP3 inflammasome in tumor-associated macrophages to promote breast cancer progression and metastasis.

Breast cancer remains one of the primary causes of cancer-related death. An imbalance of oestrogen homeostasis and an inflammatory tumor microenvironment (TME) are vital risk factors for the progression and metastasis of breast cancer. Here, we showed that oestrogen homeostasis was disrupted both in breast cancer patients and in a transgenic MMTV-PyMT mouse model of breast cancer, and significant levels of hydroxylated oestrogen accumulated in the mammary tissues of these patients and mice. We also observed that tumor-associated macrophages (TAMs) were the main population of immune cells present in the breast TME. TAM-dependent tumor metastasis could be triggered by hydroxylated oestrogen via NLRP3 inflammasome activation and IL-1ß production. Mechanistically, TAM-derived inflammatory cytokines induced the expression of matrix metalloproteinases (MMPs) in breast tumor cells, leading to breast tumor invasion and metastasis. Conceptually, our study reveals a previously unknown role of hydroxylated oestrogen in the reprogramming of the TME via NLRP3 inflammasome activation in TAMs, which ultimately facilitates breast cancer cells proliferation, migration, and invasion.

Message Transmission Between Adipocyte and Macrophage in Obesity.

Obesity is characterized by the chronic low-grade activation of the innate immune system. In this respect, macrophage-elicited metabolic inflammation and adipocyte-macrophage interaction have primary importance in obesity. Large quantity of macrophages is accumulated by different mechanisms in obese adipose tissue. Hypertrophic adipocyte-derived chemotactic monocyte chemoattractant protein-1 (MCP-1)/C-C chemokine receptor 2 (CCR2) pathway promotes more macrophage accumulation into the obese adipose tissue. However, obesity-induced changes in adipose tissue macrophage density are mainly dependent on increases in the triple-positive cluster of differentiation (CD)11b+ F4/80+ CD11c+ adipose tissue macrophage subpopulation. As epigenetic regulators, microRNAs (miRNAs) are one of the most important mediators of obesity. miRNAs are expressed by adipocytes as well as macrophages and regulate inflammation with the expression of target genes. A paracrine loop involving free fatty acids and tumor necrosis factor-alpha (TNF-α) between adipocytes and macrophages establishes a vicious cycle that aggravates inflammatory changes in the adipose tissue. Adipocyte-specific caspase-1 and production of interleukin-1beta (IL-1ß) by macrophages; both adipocyte and macrophage induction by toll-like receptor-4 (TLR4) through nuclear factor-kappaB (NF-κB) activation; free fatty acid-induced and TLR-mediated activation of c-Jun N-terminal kinase (JNK)-related pro-inflammatory pathways in CD11c+ immune cells; are effective in mutual message transmission between adipocyte and macrophage and in the development of adipose tissue inflammation. Thus, the metabolic status of adipocytes and their released exosomes are important determinants of macrophage inflammatory output. However, old adipocytes are removed by macrophages through trogocytosis or sending an "eat me" signal. As a single miRNA can be able to regulate a variety of target genes and signaling pathways, reciprocal transfer of miRNAs between adipocytes and macrophages via miRNA-loaded exosomes reorganizes the different stages of obesity. Changes in the expression of circulating miRNAs because of obesity progression or anti-obesity treatment indicate that miRNAs could be used as potential biomarkers. Therefore, it is believed that targeting macrophage-associated miRNAs with anti-obesity miRNA-loaded nano-carriers may be successful in the attenuation of both obesity and adipose tissue inflammation in clinical practice. Moreover, miRNA-containing exosomes and transferable mitochondria between the adipocyte and macrophage are investigated as new therapeutic targets for obesity-related metabolic disorders.

Endothelial Dysfunction in Obesity and Therapeutic Targets.

Parallel to the increasing prevalence of obesity in the world, the mortality from cardiovascular disease has also increased. Low-grade chronic inflammation in obesity disrupts vascular homeostasis, and the dysregulation of adipocyte-derived endocrine and paracrine effects contributes to endothelial dysfunction. Besides the adipose tissue inflammation, decreased nitric oxide (NO)-bioavailability, insulin resistance (IR), and oxidized low-density lipoproteins (oxLDLs) are the main factors contributing to endothelial dysfunction in obesity and the development of cardiorenal metabolic syndrome. While normal healthy perivascular adipose tissue (PVAT) ensures the dilation of blood vessels, obesity-associated PVAT leads to a change in the profile of the released adipo-cytokines, resulting in a decreased vasorelaxing effect. Higher stiffness parameter ß, increased oxidative stress, upregulation of pro-inflammatory cytokines, and nicotinamide adenine dinucleotide phosphate (NADP) oxidase in PVAT turn the macrophages into pro-atherogenic phenotypes by oxLDL-induced adipocyte-derived exosome-macrophage crosstalk and contribute to the endothelial dysfunction. In clinical practice, carotid ultrasound, higher leptin levels correlate with irisin over-secretion by human visceral and subcutaneous adipose tissues, and remnant cholesterol (RC) levels predict atherosclerotic disease in obesity. As a novel therapeutic strategy for cardiovascular protection, liraglutide improves vascular dysfunction by modulating a cyclic adenosine monophosphate (cAMP)-independent protein kinase A (PKA)-AMP-activated protein kinase (AMPK) pathway in PVAT in obese individuals. Because the renin-angiotensin-aldosterone system (RAAS) activity, hyperinsulinemia, and the resultant IR play key roles in the progression of cardiovascular disease in obesity, RAAS-targeted therapies contribute to improving endothelial dysfunction. By contrast, arginase reciprocally inhibits NO formation and promotes oxidative stress. Thus, targeting arginase activity as a key mediator in endothelial dysfunction has therapeutic potential in obesity-related vascular comorbidities. Obesity-related endothelial dysfunction plays a pivotal role in the progression of type 2 diabetes (T2D). The peroxisome proliferator-activated receptor gamma (PPARγ) agonist, rosiglitazone (thiazolidinedione), is a popular drug for treating diabetes; however, it leads to increased cardiovascular risk. Selective sodium-glucose co-transporter-2 (SGLT-2) inhibitor empagliflozin (EMPA) significantly improves endothelial dysfunction and mortality occurring through redox-dependent mechanisms. Although endothelial dysfunction and oxidative stress are alleviated by either metformin or EMPA, currently used drugs to treat obesity-related diabetes neither possess the same anti-inflammatory potential nor simultaneously target endothelial cell dysfunction and obesity equally. While therapeutic interventions with glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide or bariatric surgery reverse regenerative cell exhaustion, support vascular repair mechanisms, and improve cardiometabolic risk in individuals with T2D and obesity, the GLP-1 analog exendin-4 attenuates endothelial endoplasmic reticulum stress.

Bone morphogenetic protein 4 ameliorates bleomycin-induced pulmonary fibrosis in mice by repressing NLRP3 inflammasome activation and epithelial-mesenchymal transition.

Background: Idiopathic pulmonary fibrosis (IPF) is a progressive and deadly lung disease with limited therapeutic options. Bone morphogenetic protein 4 (BMP4), a multifunctional growth factor that belongs to the transforming growth factor-ß superfamily, is able to relieve pulmonary fibrosis in mice; nevertheless, the potential mechanism of action remains largely unknown. Growing evidence supports the notion that reiterant damage to the alveolar epithelial cells (AECs) is usually the "prime mover" for pulmonary fibrosis. Here, we examined the effect and mechanisms of BMP4 on bleomycin (BLM)-induced activation of NLR family pyrin domain containing 3 (NLRP3) inflammasome and epithelial-mesenchymal transition (EMT) in vivo and in vitro. Methods: The in vivo impact of BMP4 was investigated in a BLM mouse model. Histopathologic changes were analyzed by hematoxylin-eosin (H&E) and Masson's trichrome staining. The NLRP3 inflammasome activation was determined by quantitative real time polymerase chain reaction (qRT-PCR) and immunofluorescence staining. Biomarkers of EMT were measured by qRT-PCR, Western blot and immunofluorescence staining. The in vitro impact of BMP4 on BLM-induced NLRP3 inflammasome activation and EMT was explored in A549 AECs. We also evaluated whether BMP4 inhibited BLM-activated ERK1/2 signaling to address the possible molecular mechanisms. Results: BMP4 was significantly downregulated in the mouse lungs from BLM-induced pulmonary fibrosis. BMP4+/- mice presented with more severe lung fibrosis in response to BLM, and accelerated NLRP3 inflammasome activation and EMT process compared with that in BMP4+/+ mice. Whereas overexpression of BMP4 by injecting adeno-associated virus (AAV) 9 into mice attenuated BLM-induced fibrotic changes, NLRP3 inflammasome activation, and EMT in the mouse lungs, thus exerting protective efficacy against lung fibrosis. In vitro, BMP4 significantly reduced BLM-induced activation of NLRP3 inflammasome and EMT in human alveolar epithelial A549 cells. Mechanically, BMP4 repressed BLM-induced activation of ERK1/2 signaling in vivo and in vitro, suggesting that ERK1/2 inactivation contributes to BMP4-induced effects on BLM-induced activation of NLRP3 inflammasome and EMT. Conclusions: Our findings suggest that BMP4 can suppress NLRP3 inflammasome activation and EMT in AECs via inhibition of ERK1/2 signaling pathway, thus has a potential for the treatment of pulmonary fibrosis.

Function of NLRP3 inflammasome activation in multiple myeloma.

OBJECTIVE: The drug resistance of multiple myeloma (MM) cells is one of the main causes of relapse, refractory and progression of MM. METHODS: First, Western blot analysis was used to detect the expression levels of NLRP3, ASC, pro-IL-1ß and cleaved IL-1ß, and RT-qPCR was used to detect the mRNA expression levels of them. The expression levels of IL-1ß and IL-18 in the supernatant were detected by ELISA, and the expression levels of these factors in the activated group and the control group were compared to verify the activation of BMMCs and KM3. RESULT: 1. The protein expression of NLRP3 and cleavd-IL-1ß in the BMMCs cells was significantly higher than that of the control group (P < 0.05). The mRNA expression levels of caspase-1 and IL-1ß were higher than those of the control group (P = 0.03, P = 0.02). 2. The protein expression levels of NLRP3 and cleaved-IL-1ß in the KM3 cells were significantly higher than those of the control group (P < 0.05). The expressions of caspase-1 mRNA(P = 0.016) and IL-1ß mRNA(P = 0.037) were significantly increased compared with the control group. 3. The early apoptosis results of BMMCs showed that the apoptosis rate of the LPS+ATP+Dex group was lower than that of the Dex group (P = 0.017). The early apoptosis rate of the LPS+ATP+Dex+Vel group was decreased compared with the Dex+Vel group (P = 0.045). 4. The early apoptosis rate of KM3 in the LPS+ATP+Dex group was lower than that in the Dex group (P = 0.03). CONCLUSION: 1. LPS+ATP can activate NLRP3 inflammasome in multiple myeloma cells. 2. Activation of NLRP3 inflammasome inhibits the early apoptosis of myeloma cells induced by dexamethasone and bortezomib.

ROS exhaustion reverses the effects of hyperbaric oxygen on hemorrhagic transformation through reactivating microglia in post-stroke hyperglycemic mice.

Acute ischemic stroke (AIS) is a major global health concern due to its high mortality and disability rates. Hemorrhagic transformation, a common complication of AIS, leads to poor prognosis yet lacks effective treatments. Preclinical studies indicate that hyperbaric oxygen (HBO) treatment within 12 h of AIS onset alleviates ischemia/reperfusion injuries, including hemorrhagic transformation. However, clinical trials have yielded conflicting results, suggesting some underlying mechanisms remain unclear. In this study, we confirmed that HBO treatments beginning within 1 h post reperfusion significantly alleviated the haemorrhage and neurological deficits in hyperglycemic transient middle cerebral arterial occlusion (tMCAO) mice, partly due to the inhibition of the NLRP3 inflammasome-mediated pro-inflammatory response in microglia. Notably, reactive oxygen species (ROS) mediate the anti-inflammatory and protective effect of early HBO treatment, as edaravone and N-Acetyl-L-Cysteine (NAC), two commonly used antioxidants, reversed the suppressive effect of HBO treatment on NLRP3 inflammasome-mediated inflammation in microglia. Furthermore, NAC countered the protective effect of early HBO treatment in tMCAO mice with hyperglycemia. These findings support that early HBO treatment is a promising intervention for AIS, however, caution is warranted when combining antioxidants with HBO treatment. Further assessments are needed to clarify the role of antioxidants in HBO therapy for AIS.