Release Of Interleukin Research Articles

Functional interleukin-4 releasing microparticles impact THP-1 differentiated macrophage phenotype

IntroductionMacrophage cell therapies offer potential treatment in inflammatory diseases due to their ability to mobilize and stimulate their environment. However, successful treatment requires a pro-regenerative macrophage phenotype to be retained in vivo. Polymeric microparticles may provide a potential route to direct and sustain macrophage phenotype. Interleukin-4 (IL-4) is the most commonly used cytokine for in vitro modulation towards M2a macrophage phenotype. We designed IL-4 encapsulated microparticles to investigate the impact of drug release kinetics and developed a robust human peripheral blood monocyte cell (THP-1) in vitro assay to assess functional IL-4 release upon macrophage phenotype.MethodsIL-4 was encapsulated with human serum albumin (HSA) in microparticles fabricated from a blend of PLGA and a PLGA-PEG-PLGA triblock copolymer. Functional release of IL-4 and HSA over different time periods was measured using ELISAs. THP-1 differentiated macrophages were cultured either in direct contact with microparticles or indirectly through transwells. The immunomodulatory impact of microparticles on THP-1 cells were measured using ELISA and qPCR.Results and DiscussionIL-4 release kinetics fit with the first-order release kinetics model, indicating concentration dependent release. IL-4/HSA encapsulated microparticles modulated THP-1 differentiated macrophages towards pro-immunoregulatory subgroups. This strategy provides a novel approach in drug carrier development for in vitro assessments of macrophage phenotype to inform development of targeted therapies for inflammation and immune modulation.

Gypenosides alleviates HaCaT keratinocyte hyperproliferation and ameliorates imiquimod-induced psoriasis in mice

Background: Psoriasis is an autoimmune skin condition characterized by hyperproliferation of keratinocytes and chronic immune responses. Gypenosides (Gyp) exhibits anti-proliferative and anti-inflammatory effects on different diseases. However, its functioning and mechanism of Gyp on psoriasis remains unknown. Objective: To explore the effect and mechanism of Gyp on psoriasis. Material and Methods: The impact and mechanism of Gyp on psoriasis in vitro and in vivo were probed through cell counting kit-8 (CCK-8), 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay, reverse transcription quantitative polymerase chain reaction, hematoxylin and eosin staining, enzyme-linked immunosorbent serologic assay, immunofluorescence, and Western Blotting assays. Results: Gyp inhibited cell proliferation and the release of inflammatory cytokinesin interleukin (IL-22)-induced spontaneously transformed human aneuploid immortal keratinocyte cell line (HaCaT). In addition, Gyp demonstrated enhancement in erythema and scaling as well as reductions in the thickness of epidermal layers, release of inflammatory factors, and Ki-67 (a nuclear protein) level in imiquimod (IMQ)-induced mice. Mechanistically, Gyp upregulated nuclear factor erythroid 2-related factor 2 (Nrf-2) expression and diminished the level of p-p65/p65 and p-STAT3/STAT3 in skin tissues from IMQ-induced mice and IL-22-induced HaCaT cells, which were reversed with the application of ML385, an inhibitor of Nrf2. In addition, the administration of ML385 reversed decrease in cell viability and reduced the expressions of IL-1β, IL-6, and tumor necrosis factor-α (TNF-α) in IL-22-induced HaCaT cells caused by Gyp. Conclusion: In summary, Gyp reduced excessive cell growth and inflammation in psoriasis by suppressing nuclear factor kappa B (NF-κB) and signal transducer and activator of transcription 3 (STAT3) through activation of Nrf2.

Legumain deficiency halts atherogenesis by modulating T cell receptor signaling.

Atherosclerosis is an age-related pathological process associated with elevated levels of legumain in plaques and plasma. However, the underlying mechanisms remain unclear. The aim of this study was to investigate the role of legumain in the progression of atherosclerotic plaques, with a particular focus on functional and phenotypic changes in CD4+ T cells. Apolipoprotein E-deficient (Apoe-/-) mice were crossed with legumain-deficient (Lgmn-/-) mice to generate Lgmn-/-Apoe-/- mice. CD4+ T cells accumulated in the atherosclerotic plaques of Apoe-/- mice fed a high-fat diet. Deletion of legumain attenuated the deposition of CD4+ T cells in plaques and reduced the number of atherosclerotic lesions. The levels of CD4+ T cells in the blood, lymph nodes, and spleen were decreased in Lgmn-/- mice. Transcriptomic analysis revealed that the deletion of legumain decreased the differentiation, survival, and function of CD4+ memory T cells by suppressing the T cell receptor (TCR) signaling pathway. These changes are accompanied by the downregulation of the antiapoptotic protein B-cell lymphoma 2 (Bcl-2) and the reduced release of interleukin (IL)-2 and interferon (IFN)-γ. These results suggest that legumain deficiency may play a role in the development of atherosclerosis by impairing the survival, proliferation, and function of CD4+ T cells. Inhibition of legumain activity may be an innovative therapy for the treatment of atherosclerosis.

Biomimetic non-collagenous proteins-calcium phosphate complex with superior osteogenesis via regulating macrophage IL-27 secretion

Traumatic defects or non-union fractures presents a substantial challenge in the fields of tissue engineering and regenerative medicine. Although synthetic calcium phosphate-based biomaterials (CaPs) such as dibasic calcium phosphate anhydrate (DCPA) are commonly employed for bone repair, their inadequate cellular immune responses significantly impede sustained degradation and optimal osteogenesis. In this study, drawing inspiration from the key structure of an acidic non-collagenous protein-CaP complex (ANCPs-CaP) essential for natural bone formation, we prepared biomimetic mineralized dibasic calcium phosphate (MDCPA). This preparation utilized plant-derived non-collagenous protein Zein as the organic template and acidic artificial saliva as the mineralization medium. Physicochemical property analysis revealed that MDCPA is a complex of Zein and DCPA, which mimics the composite of the natural ANCP-CaP. Moreover, MDCPA exhibited enhanced biodegradability and osteogenic potential. Mechanistic insight revealed that MDCPA can be phagocytized and degraded by macrophages via the FCγRIII receptor, leading to the release of interleukin 27 (IL-27), which promotes osteogenic differentiation by osteoimmunomodulation. The critical role of IL-27 in osteogenesis is further confirmed using IL-27 gene knockout mice. Additionally, MDCPA demonstrates effective healing of critical-sized defects in rat cranial bones within only 4 w, providing a promising basis and valuable insights for critical-sized bone defects regeneration.

N-(4-methoxyphenyl) quinoline-8-sulfonamide reduces the inflammatory response of fibroblast-like synoviocytes by targeting receptor (calcitonin) activity modifying protein 1 in rheumatoid arthritis

BackgroundRheumatoid arthritis (RA) is characterized by chronic inflammation of the synovium of joints. Fibroblast-like synoviocytes (FLS) play an important role in RA pathogenesis. We aimed to investigate the effect of N-(4-methoxyphenyl) quinoline-8-sulfonamide (QS-3g) on the inflammatory response of FLS and explore the potential underlying mechanisms. MethodsWe screened and found that QS-3g exhibits the best anti-inflammatory response against FLS using the CCK8 assay. To investigate the therapeutic effects of QS-3g on K/BxN STA mice, we used H&E staining, immunofluorescence, immunohistochemistry, micro-CT, and other techniques. Additional investigations, including RNA-seq, molecular docking, and CETSA, revealed that QS-3g binds to RAMP1. ResultsAmong a series of 8-quinoline sulfonyl amide derivatives, QS-3g reduced the inflammatory response in TNF-α stimulated FLS, such as the release of interleukin (IL)-1β and IL-6. H&E staining and micro-CT showed that QS-3g inhibited synovial hypertrophy, inflammatory cell infiltration, and bone destruction. RNA-seq and CETSA analyses revealed the targeted inhibition of RAMP1 by QS-3g. Inhibition of RAMP1 expression could reduce IL-6 and IL-1β levels. Compared with RAMP1-si, combined administration of QS-3g and RAMP1-si reduced the TNF-α-induced inflammation in TNF-α stimulated FLS without statistically significant differences. Finally, the results of in vitro experiments showed that QS-3g could restore the balance of Gαi/Gαs by inhibiting Gαi and activating Gαs and up-regulate the expression of cAMP protein, thus inhibiting the RAMP1-mediated inflammatory response in FLS. ConclusionQS-3g could inhibit RAMP1 activity and mediate the Gαs/Gαi–cAMP pathway to reduce FLS inflammatory response. Therefore, QS-3g may serve as a novel anti-inflammatory compound for treating RA.

The Impact of Negative Energy Balance in Holstein-Friesian Cows on the Blood Concentrations of Interleukin-6 and Plasminogen.

Background/Objectives: The negative energy balance activaties of spontaneous lipolysis. This may promotes inflammation within the adipose tissue. The aim of the study was to explain the development of inflammation during increased lactogenesis. It was hypothesized that lipolysis contributes synthesis of interleukin-6 and plasminogen. Methods: The study was in production conditions carried out using Holstein-Friesian cows. The period studied covered time of early lactation. Results: Up to the peak of lactation, milk yield strongly influenced the rate of loss of body condition. This had an impact on with the intensity of the release of the fatty acids. In both cases this relationships strengthened to the peak of production. Oobserved tendencies towards a decrease in the concentration of glucose and an increase in that of leptin. Loss of the body condition and the release of NEFA were were influencing to affect the blood concentrations of interleukin-6 and plasminogen. We have shown that IL-6 has a relatively strong correlation with the NEFA. They correlate with IL-6 independently of EB influence. This may suggest independent associations between these variables, which could potentially be applied in practice. Conclusions: The NEFA release in the long term can increase the inflammatory response within adipose tissue and can intensify the release of interleukin-6 and plasminogen. It is likely that in the initial stage of lactogenesis, the inflammatory process developing within adipose tissue is physiologically justified. Our results can provide background to this little-described area of research.

Mouse liver sinusoidal endothelial cell responses to the glucocorticoid receptor agonist dexamethasone.

Liver sinusoidal endothelial cells (LSECs) which make up the fenestrated wall of the hepatic sinusoids, are active scavenger cells involved in blood waste clearance and liver immune functions. Dexamethasone is a synthetic glucocorticoid commonly used in the clinic and as cell culture supplement. However, the response is dependent on tissue, cell type, and cell state. The aim of this study was to investigate the effect of dexamethasone on primary mouse LSECs (C57BL/6J); their viability (live-dead, LDH release, caspase 3/7 assays), morphology (scanning electron microscopy), release of inflammatory markers (ELISA), and scavenging functions (endocytosis assays), and associated biological processes and pathways. We have characterized and catalogued the proteome of LSECs cultured for 1, 10, or 48h to elucidate time-dependent and dexamethasone-specific cell responses. More than 6,000 protein IDs were quantified using tandem mass tag technology and advanced mass spectrometry (synchronous precursor selection multi-notch MS3). Enrichment analysis showed a culture-induced upregulation of stress and inflammatory markers, and a significant shift in cell metabolism already at 10h, with enhancement of glycolysis and concomitant repression of oxidative phosphorylation. At 48h, changes in metabolic pathways were more pronounced with dexamethasone compared to time-matched controls. Dexamethasone repressed the activation of inflammatory pathways (IFN-gamma response, TNF-alpha signaling via NF-kB, Cell adhesion molecules), and culture-induced release of interleukin-6, VCAM-1, and ICAM-1, and improved cell viability partly through inhibition of apoptosis. The mouse LSECs did not proliferate in culture. Dexamethasone treated cells showed upregulation of xanthine dehydrogenase/oxidase (Xdh), and the transcription regulator Foxo1. The drug further delayed but did not block the culture-induced loss of LSEC fenestration. The LSEC capacity for endocytosis was significantly reduced at 48h, independent of dexamethasone, which correlated with diminished expression of several scavenger receptors and C-type lectins and altered expression of proteins in the endocytic machinery. The glucocorticoid receptor (NR3C1) was suppressed by dexamethasone at 48h, suggesting limited effect of the drug in prolonged LSEC culture. Conclusion: The study presents a detailed overview of biological processes and pathways affected by dexamethasone in mouse LSECs in vitro.

Coronavirus envelope protein activates TMED10-mediated unconventional secretion of inflammatory factors

The precise cellular mechanisms underlying heightened proinflammatory cytokine production during coronavirus infection remain incompletely understood. Here we identify the envelope (E) protein in severe coronaviruses (SARS-CoV-2, SARS, or MERS) as a potent inducer of interleukin-1 release, intensifying lung inflammation through the activation of TMED10-mediated unconventional protein secretion (UcPS). In contrast, the E protein of mild coronaviruses (229E, HKU1, or OC43) demonstrates a less pronounced effect. The E protein of severe coronaviruses contains an SS/DS motif, which is not present in milder strains and facilitates interaction with TMED10. This interaction enhances TMED10-oligomerization, facilitating UcPS cargo translocation into the ER-Golgi intermediate compartment (ERGIC)—a pivotal step in interleukin-1 UcPS. Progesterone analogues were identified as compounds inhibiting E-enhanced release of proinflammatory factors and lung inflammation in a Mouse Hepatitis Virus (MHV) infection model. These findings elucidate a molecular mechanism driving coronavirus-induced hyperinflammation, proposing the E-TMED10 interaction as a potential therapeutic target to counteract the adverse effects of coronavirus-induced inflammation.

7901 Clinical Challenges Of Paraneoplastic Endocrine Metabolic Syndromes: An Illustrative Example

Abstract Disclosure: S. Shankar: None. S.S. Sundar: None. M.S. Vaishnav: None. K. Thummala: None. L. Lekkala: None. S. Srikanta: None. K. Muniraj: None. T. Deepak: None. R.B. Vijay: None. V. Nath: None. C. Siddlingappa: None. P. Ravikumar: None. M.D. Chitra: None. Introduction: Paraneoplastic endocrine syndromes result from aberrant production by tumors of protein hormones, hormone precursors, or hormone like substances. In patients who are already receiving treatment for cancer, the onset of a paraneoplastic syndrome may be an indication of progression or relapse. Clinical Case: 2021: Age 81 female (hypertension). Carcinoma rectum; S/P surgery, colostomy, chemotherapy and radiotherapy. 2023: Palliative care, due to progressive metastatic (hepatic, pulmonary, bone) malignancy. 2023 Aug: Hospital admission for generalized weakness, not able to walk, severe pain and low-grade fever 10 days. A: Paraneoplastic Hyponatremia: Evaluation: S Sodium= 127, 129, 129, 137, 134 mmol/L (134-145); Spot urine sodium= 65 mmol; S Uric acid= 3.5 mg/dL (4.4-7.6); S Cortisol 8 am= 15.5 (6.7-22.6); T3 total= 0.34 ng/mL (0.97-1.69), T4 total= 6.87 mcg/dL (5.5-11), TSH= 1.87 mIU/mL (0.46-4.68); LH= 0.35 mIU/mL (16-54); FSH= 1.9 mIU/mL (23-116); S calcium= 8.9 mg/dL (8.4-10.2); S Phosphorus= 2.31mg/dL (2.5-4.5) Diagnosis: Possible paraneoplastic ADH secretion/ SIADH; pain/ narcotics-induced hypothalamic ADH secretion. Treatment: Fluid restriction, liberal salt intake, 3% saline; oral sodium bicarbonate and tolvaptan; tapentadol. B: Paraneoplastic Leukemoid reaction: Evaluation: Total leucocyte count= 29960, 36670, 44130, 58450 cells/mm3 (4000-11,000); Differential count: Neutrophils= 85-94%; Lymphocytes= 10-4%; CRP= 93.7, 24.0 mg/dL (<0.3); Procalcitonin= 0.097, 0.083 ng/mL (<0.1); Ruled out significant sepsis; no obvious laboratory or imaging evidence of any infectious or leukemic etiology. Diagnosis: Progressive leucocytosis due to Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) secretion from various neoplasms has been reported in literature. Treatment: Empirical antibiotic coverage. C: Paraneoplastic Hypokalemia: Evaluation: S Potassium= 3.3, 3.4, 3.7, 2.9, 3.0 mmol/L (3.5-5.1). Diagnosis: Hypokalemia likely due to (in vivo and in vitro) intracellular potassium uptake by rapidly proliferating leukocytes (and neoplasm- primary and metastatic). (Ectopic ACTH secretion / ruled out). Therapy: IV and oral potassium supplements. D: Paraneoplastic Fever: Cytokines (e.g., tumor necrosis factor and interleukin-1) release from tumor cells or infiltrating mononuclear cells. Clinical Lessons: Endocrine paraneoplastic syndromes can complicate patient’s clinical course, response to treatment, impact prognosis and even be confused as metastatic spread or another “non-existent” clinical entity. They can precede, occur concomitantly or present at later stage of tumour development. Recognition of the diverse clinical manifestations improves clinical outcomes (earlier cancer diagnosis, better quality of life, optimal tumour-directed therapy or decision towards non-aggressive focussed palliative care). Presentation: 6/2/2024

Milk fat globule-epidermal growth factor-VIII–derived oligopeptide 3 (MOP3) attenuates inflammation and improves survival in hepatic ischemia/reperfusion injury

IntroductionHepatic ischemia/reperfusion injury is a severe clinical condition leading to high mortality as the result of excessive inflammation, partially triggered by released damage-associated molecular patterns. Extracellular cold-inducible RNA-binding protein is a new damage-associated molecular pattern. Current clinical management of hepatic ischemia/reperfusion injury is limited to supportive therapy, necessitating the development of novel and effective treatment strategies. Milk fat globule-epidermal growth factor-VIII–derived oligopeptide 3 is a newly invented oligopeptide originating from milk fat globule-epidermal growth factor-VIII. This peptide acts as an opsonic compound that specifically binds to extracellular cold-inducible RNA-binding protein to facilitate its clearance by phagocytes, thereby attenuating inflammation. In this study, we hypothesized that milk fat globule-epidermal growth factor-VIII–derived oligopeptide 3 attenuated hepatic ischemia/reperfusion injury by inhibiting extracellular cold-inducible RNA-binding protein–induced inflammation in Kupffer cells. MethodsWe treated Kupffer cells isolated from male C57BL/6 mice with extracellular cold-inducible RNA-binding protein and various doses of milk fat globule-epidermal growth factor-VIII–derived oligopeptide 3 for 4 hours, then measured cytokines in the culture supernatants. In addition, mice underwent 70% hepatic ischemia for 60 minutes immediately followed by the intravenous administration of either vehicle or milk fat globule-epidermal growth factor-VIII–derived oligopeptide 3. Blood and ischemic liver tissues were collected 24 hours later, and inflammatory markers including cytokines, liver enzymes, chemokines, myeloperoxidase activity, and Z-DNA-binding protein 1 were measured. Hepatic tissue damage and cell death were evaluated histologically. Survival rates were monitored for 10 days posthepatic ischemia/reperfusion. ResultsThe release of interleukin-6 and tumor necrosis factor-α from extracellular cold-inducible RNA-binding protein–challenged Kupffer cells was significantly reduced by milk fat globule-epidermal growth factor-VIII–derived oligopeptide 3 in a dose-dependent manner. In hepatic ischemia/reperfusion mice, milk fat globule-epidermal growth factor-VIII–derived oligopeptide 3 treatment significantly decreased serum levels of extracellular cold-inducible RNA-binding protein, interleukin-6, tumor necrosis factor-α, aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase. Milk fat globule-epidermal growth factor-VIII–derived oligopeptide 3 treatment also significantly reduced mRNA levels of interleukin-6, tumor necrosis factor-α, interleukin-1β, Z-DNA-binding protein 1, and chemokine macrophage inflammatory protein-2, as well as myeloperoxidase activity in hepatic tissues. Histologic evaluation demonstrated that treatment with milk fat globule-epidermal growth factor-VIII–derived oligopeptide 3 significantly attenuated tissue damage and cell death in the liver of hepatic ischemia/reperfusion mice. Milk fat globule-epidermal growth factor-VIII–derived oligopeptide 3 treatment significantly improved the survival rate of hepatic ischemia/reperfusion mice. ConclusionMilk fat globule-epidermal growth factor-VIII–derived oligopeptide 3 significantly attenuated inflammation and liver tissue damage and improved survival after hepatic ischemia/reperfusion. Thus, milk fat globule-epidermal growth factor-VIII–derived oligopeptide 3 holds promise as a potential future therapeutic strategy for hepatic ischemia/reperfusion injury.

Macrophages can transmit coxsackievirus B4 to pancreatic cells and can impair these cells.

Macrophages are suspected to be involved in the pathogenesis of type 1 diabetes. The role of macrophages in the transmission of coxsackievirus B4 (CVB4) to pancreatic cells and in the alteration of these cells was investigated. Human monocytes isolated from peripheral blood were differentiated into macrophages with M-CSF (M-CSF macrophages) or GM-CSF (GM-CSF macrophages). M-CSF macrophages were inoculated with CVB4. M-CSF and GM-CSF macrophages were activated with lipopolysaccharide and interferon (IFN)-γ. Human pancreatic beta cells 1.1B4 were inoculated with CVB4 derived from M-CSF macrophages or were cocultured with CVB4-infected M-CSF macrophages. The antiviral activity of synthetic molecules in macrophage cultures was evaluated. Activated macrophages were cocultured with CVB4-persistently infected 1.1B4 cells, and the specific lysis of these cells was determined. Our study shows that CVB4 can infect M-CSF macrophages, leading to the release of interleukin-6 and tumor necrosis factor-α and later IFN-α. M-CSF macrophage-derived CVB4 can infect 1.1B4 cells, which were then altered; however, when these cells were cultured in medium containing agarose, cell layers were not altered. Fluoxetine and CUR-N373 can inhibit CVB4 replication in macrophage cultures. Supernatants of activated M-CSF and GM-CSF macrophage cultures induced lysis of CVB4-persistently infected 1.1B4 cells. The cytolytic activity of activated GM-CSF macrophages was higher towards CVB4-persistently infected 1.1B4 cells than mock-infected 1.1B4 cells. In conclusion, macrophages may play a role in CVB4 infection of pancreatic cells, and are capable of inducing lysis of infected pancreatic cells.

Vasopressin drives aberrant myeloid differentiation of hematopoietic stem cells, contributing to depression in mice.

Psychological stress is often linked to depression and can also impact the immune system, illustrating the interconnectedness of mental health and immune function. Hematopoietic stem cells (HSCs) can directly sense neuroendocrine signals in bone marrow and play a fundamental role in the maintenance of immune homeostasis. However, it is unclear how psychological stress impacts HSCs in depression. Here, we report that neuroendocrine factor arginine vasopressin (AVP) promotes myeloid-biased HSC differentiation by activating neutrophils. AVP administration increases neutrophil and Ly6Chi monocyte production by triggering HSCs that rely on intrinsic S100A9 in mice. When stimulated with AVP, neutrophils return to the bone marrow and release interleukin 36G (IL-36G), which interacts with interleukin 1 receptor-like 2 (IL-1RL2) on HSCs to produce neutrophils with high Elane expression that infiltrate the brain and induce neuroinflammation. Together, these findings define HSCs as a relay between psychological stress and myelopoiesis and identify the IL-36G-IL-1RL2 axis as a potential target for depression therapy.

A combination of carbonates and Opuntia ficus-indica extract protects esophageal cells against simulated acidic and non-acidic reflux in vitro

Buffering of stomach acid by antacids is a well-established symptomatic therapy for heartburn. In addition, preparations from prickly pear (Opuntia ficus-indica) have been shown to reduce tissue damage in experimental gastritis models and to attenuate gastrointestinal discomfort in patients. Both active principles have been included in a fixed-combination product for symptomatic treatment of heartburn containing carbonate antacids (CaCO3 and MgCO3) and an extract from Opuntia ficus-indica cladodes. The aim of the study was to characterize the acid neutralization and esophageal cell protective activities of the product and its individual active ingredients in a set of in vitro assays. Acid neutralization was assessed in a simulated stomach model. Protective activity of individual constituents and in combination was analyzed in an esophageal cell line (COLO-680 N) exposed to low pH and deoxycholic acid to simulate acidic and non-acidic reflux challenge. The combination product protected cells against low pH mediated cytotoxicity via acid neutralization by carbonates. Opuntia extract itself and the combination product attenuated bile acid-induced cell irritation as measured by reduced release of proinflammatory interleukin-6 and -8. In conclusion, addition of Opuntia extract to a mineral antacid provides dual protection against acidic and non-acidic simulated reflux challenge.

Preparation, Evaluation, and Bioinformatics Study of Hyaluronic Acid-Modified Ginsenoside Rb1 Self-Assembled Nanoparticles for Treating Cardiovascular Diseases.

(1) Objective: To optimize the preparation process of hyaluronic acid-modified ginsenoside Rb1 self-assembled nanoparticles (HA@GRb1@CS NPs), characterize and evaluate them in vitro, and investigate the mechanism of action of HA@GRb1@CS NPs in treating cardiovascular diseases (CVDs) associated with inflammation and oxidative stress. (2) Methods: The optimal preparation process was screened through Plackett-Burman and Box-Behnken designs. Physical characterization of HA@GRb1@CS NPs was conducted using transmission electron microscopy, Fourier-transform infrared spectroscopy, X-ray diffraction, and differential scanning calorimetry. Stability experiments, in vitro drug release studies, and lyophilisate selection were performed to evaluate the in vitro performance of HA@GRb1@CS NPs. The anti-inflammatory and antioxidant capabilities of HA@GRb1@CS NPs were assessed using H9c2 and RAW264.7 cells. Additionally, bioinformatics tools were employed to explore the mechanism of action of HA@GRb1@CS NPs in the treatment of CVDs associated with inflammation and oxidative stress. (3) Results: The optimal preparation process for HA@GRb1@CS NPs was achieved with a CS concentration of 2 mg/mL, a TPP concentration of 2.3 mg/mL, and a CS to TPP mass concentration ratio of 1.5:1, resulting in a particle size of 126.4 nm, a zeta potential of 36.8 mV, and a PDI of 0.243. Characterization studies confirmed successful encapsulation of the drug within the carrier, indicating successful preparation of HA@GRb1@CS NPs. In vitro evaluations demonstrated that HA@GRb1@CS NPs exhibited sustained-release effects, leading to reduced MDA (Malondialdehyde) content and increased SOD (Superoxide Dismutase) content in oxidatively damaged H9c2 cells. Furthermore, it showed enhanced DPPH (2,2-Diphenyl-1-picrylhydrazyl) and ABTS+ [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] free radical scavenging rates and inhibited the release of inflammatory factors NO (Nitric Oxide) and IL-6 (Interleukin-6) from RAW264.7 cells. (4) Conclusions: The HA@GRb1@CS NPs prepared in this study exhibit favorable properties with stable quality and significant anti-inflammatory and antioxidant capabilities. The mechanisms underlying their therapeutic effects on CVDs may involve targeting STAT3, JUN, EGFR, CASP3, and other pathways regulating cell apoptosis, autophagy, anti-lipid, and arterial sclerosis signaling pathways.

Effect of Butyrate on Food-Grade Titanium Dioxide Toxicity in Different Intestinal In Vitro Models.

Short-chain fatty acids (SCFA) are an important energy source for colonocytes and crucial messenger molecules both locally in the intestine and systemically. Butyrate, one of the most prominent and best-studied SCFA, was demonstrated to exert anti-inflammatory effects, improve barrier integrity, enhance mucus synthesis in the intestine, and promote cell differentiation of intestinal epithelial cells in vitro. While the physiological relevance is undisputed, it remains unclear if and to what extent butyrate can influence the effects of xenobiotics, such as food-grade titanium dioxide (E171, fgTiO2), in the intestine. TiO2 has been controversially discussed for its DNA-damaging potential and banned as a food additive within the European Union (EU) since 2022. First, we used enterocyte Caco-2 monocultures to test if butyrate affects the cytotoxicity and inflammatory potential of fgTiO2 in a pristine state or following pretreatment under simulated gastric and intestinal pH conditions. We then investigated pretreated fgTiO2 in intestinal triple cultures of Caco-2, HT29-MTX-E12, and THP-1 cells in homeostatic and inflamed-like state for cytotoxicity, barrier integrity, cytokine release as well as gene expression of mucins, oxidative stress markers, and DNA repair. In Caco-2 monocultures, butyrate had an ambivalent role: pretreated but not pristine fgTiO2 induced cytotoxicity in Caco-2 cells, which was not observed in the presence of butyrate. Conversely, fgTiO2 induced the release of interleukin 8 in the presence but not in the absence of butyrate. In the advanced in vitro models, butyrate did not affect the characteristics of the healthy or inflamed states and caused negligible effects in the investigated end points following fgTiO2 exposure. Taken together, the effects of fgTiO2 strongly depend on the applied testing approach. Our findings underline the importance of the experimental setup, including the choice of in vitro model and the physiological relevance of the exposure scenario, for the hazard testing of food-grade pigments like TiO2.

Cannabinol modulates the endocannabinoid system and shows TRPV1-mediated anti-inflammatory properties in human keratinocytes.

Cannabinol (CBN) is a secondary metabolite of cannabis whose beneficial activity on inflammatory diseases of human skin has attracted increasing attention. Here, we sought to investigate the possible modulation by CBN of the major elements of the endocannabinoid system (ECS), in both normal and lipopolysaccharide-inflamed human keratinocytes (HaCaT cells). CBN was found to increase the expression of cannabinoid receptor 1 (CB1) at gene level and that of vanilloid receptor 1 (TRPV1) at protein level, as well as their functional activity. In addition, CBN modulated the metabolism of anandamide (AEA) and 2-arachidonoylglicerol (2-AG), by increasing the activities of N-acyl phosphatidylethanolamines-specific phospholipase D (NAPE-PLD) and fatty acid amide hydrolase (FAAH)-the biosynthetic and degradative enzyme of AEA-and that of monoacylglycerol lipase (MAGL), the hydrolytic enzyme of 2-AG. CBN also affected keratinocyte inflammation by reducing the release of pro-inflammatory interleukin (IL)-8, IL-12, and IL-31 and increasing the release of anti-inflammatory IL-10. Of note, the release of IL-31 was mediated by TRPV1. Finally, the mitogen-activated protein kinases (MAPK) signaling pathway was investigated in inflamed keratinocytes, demonstrating a specific modulation of glycogen synthase kinase 3β (GSK3β) upon treatment with CBN, in the presence or not of distinct ECS-directed drugs. Overall, these results demonstrate that CBN modulates distinct ECS elements and exerts anti-inflammatory effects-remarkably via TRPV1-in human keratinocytes, thus holding potential for both therapeutic and cosmetic purposes.

Circulating extracellular microvesicles associated with electronic cigarette use increase endothelial cell inflammation and reduce nitric oxide production.

The purpose of this study was to determine the effect of circulating microvesicles isolated from chronic electronic (e-)cigarette users on cultured human umbilical vein endothelial cell (HUVEC) expression of nuclear factor-κB (NF-κB), cellular cytokine release, phosphorylation of endothelial nitric oxide synthase (eNOS) and NO production. The HUVECs were treated with microvesicles isolated via flow cytometry from nine non-tobacco users (five male and four female; 22±2years of age) and 10 e-cigarette users (six male and four female; 22±2years of age). Microvesicles from e-cigarette users induced significantly greater release of interleukin-6 (183.4±23.6vs. 150.6±15.4 pg/mL; P=0.002) and interleukin-8 (160.0±31.6vs. 129.4±11.2 pg/mL; P=0.01), in addition to expression of p-NF-κB p65 (Ser536) (18.8±3.4 vs. 15.6±1.5 a.u.; P=0.02) from HUVECs compared with microvesicles from non-tobacco users. Nuclear factor-κB p65 was not significantly different between microvesicles from the non-tobacco users and from the e-cigarette users (87.6±8.7vs. 90.4±24.6 a.u.; P=0.701). Neither total eNOS (71.4±21.8vs. 80.4±24.5 a.u.; P=0.413) nor p-eNOS (Thr495) (229.2±26.5vs. 222.1±22.7 a.u.; P=0.542) was significantly different between microvesicle-treated HUVECs from non-tobacco users and e-cigarette users. However, p-eNOS (Ser1177) (28.9±6.2vs. 45.8±9.0 a.u.; P<0.001) expression was significantly lower from e-cigarette users compared with non-tobacco users. Nitric oxide production was significantly lower (8.2±0.6vs. 9.7±0.9μmol/L; P=0.001) in HUVECs treated with microvesicles from e-cigarette users compared with microvesicles from non-tobacco users. This study demonstrated increased NF-κB activation and inflammatory cytokine production, in addition to diminished eNOS activity and NO production resulting from e-cigarette use. HIGHLIGHTS: What is the central question of this study? Circulating microvesicles contribute to cardiovascular health and disease via their effects on the vascular endothelium. The impact of electronic (e-)cigarette use on circulating microvesicle phenotype is not well understood. What is the main finding and its importance? Circulating microvesicles from e-cigarette users increase endothelial cell inflammation and impair endothelial nitric oxide production. Endothelial inflammation and diminished nitric oxide bioavailability are central factors underlying endothelial dysfunction and, in turn, cardiovascular disease risk. Deleterious changes in the functional phenotype of circulating microvesicles might contribute to the reported adverse effects of e-cigarette use on cardiovascular health.

Effect of exogenous free Nε-(carboxymethyl)lysine on diabetes-associated cognitive dysfunction: neuroinflammation, and metabolic disorders

Diabetes-associated cognitive dysfunction has already been attracted considerable attention. Advanced glycation end products (AGEs) from daily diets are thought to be a vital contributor to the development of this diseases. However, the effect of one of the best-characterized exogenous AGEs Nε-(carboxymethyl)lysine (CML) on cognitive function is not fully reported. In the present study, diabetical Goto-Kakizaki (GK) rats were treated with free CML for 8-weeks. It was found that oral consumption of exogenous CML significantly aggravated diabetes-associated cognitive dysfunction in behavioral test. In details, exogenous CML increased levels of oxidative stress, promoted the activation of glial cells in the brain, up-regulated the release of inflammatory cytokines interleukin-6, inhibited the protein expression of the brain-derived neurotrophic factor and thus led to neuroinflammation. Furthermore, exogenous CML promoted the amyloidogenesis in the brain of GK rats, and inhibited the expression of GLUT4. Additionally, several tricarboxylic acid cycle and glutamate-glutamine/γ-aminobutyric acid cycle intermediates including pyruvate, succinic acid, glutamine, glutamate were significantly changed in brain of GK rats treated with exogenous free CML. In conclusion, exogenous free CML is a potentially noxious compounds led to aggravated diabetes-associated cognitive dysfunction which could be possibly explained by its effects on neuroinflammation, energy and neurotransmitter amino acid homeostasis.

Nrf2 Deficiency Exacerbates Parkinson's Disease by Aggravating NLRP3 Inflammasome Activation in MPTP-Induced Mouse Models and LPS-Induced BV2 Cells.

Parkinson's disease (PD) is a movement disorder characterized by the progressive loss of dopamine neurons. Microglia-mediated neuroinflammation drives disease progression and becomes a critical factor in neuronal degeneration. Recent studies have found that nuclear factor-erythroid 2-related-2 (Nrf2) expression levels are reduced during aging and neurodegenerative diseases, but its regulatory mechanism on microglia-induced neuroinflammation has not been fully elucidated. In vivo, we used the intraperitoneal injection of the neurotoxic drug neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to establish an animal model of PD and, at the same time, administered Nrf2 inhibitors ML385 and dimethyl fumarate to regulate Nrf2 protein levels. In vitro, we used si-RNA to knock out the Nrf2 gene to intervene in BV2 cells and used lipopolysaccharide (LPS) to stimulate and induce the cell model. The study found that inhibition of Nrf2 expression aggravated the motor defects of PD mice, accompanied by a significant loss of dopaminergic neurons in the substantia nigra and striatum of the brain. In addition, after inhibition of Nrf2, the malondialdehyde (MDA) level in the substantia nigra of the midbrain of mice increased, and the levels of superoxide dismutase (SOD) and heme oxygenase-1 (HO-1) decreased, accompanied by the proliferation of microglia and astrocytes. In addition, the activation of the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome, the assembly of apoptosis-associated speck-like protein containing a CARD (ASC) protein in microglia, and the release of downstream inflammatory factors caspase-1 and interleukin (IL)-1β, were aggravated. At the cellular level, it was found that knocking out the expression of Nrf2 would aggravate the activation of NLRP3 inflammasomes and the assembly of ASC in LPS-induced BV2 cells. Inhibited Nrf2 activity can reduce the downstream antioxidant enzyme HO-1 and antioxidant levels, induce NLRP3 inflammasome activation and ASC protein assembly in microglia, and ultimately aggravate PD inflammatory response and dopamine neuron degeneration.

Interleukin-12 sustained release system promotes hematopoietic recovery after radiation injury.

The continuous production of mature blood cell lineages is maintained by hematopoietic stem cells but they are highly susceptible to damage by ionizing radiation (IR) that induces death. Thus, devising therapeutic strategies that can mitigate hematopoietic toxicity caused by IR would benefit acute radiation syndrome (ARS) victims and patients receiving radiotherapy. Herein, we describe the preparation of an injectable hydrogel formulation based on Arg-Gly-Asp-alginate (RGD-Alg) and Laponite using a simple mixing method that ensured a slow and sustained release of interleukin-12 (IL-12) (RGD-Alg/Laponite@IL-12). The local administration of RGD-Alg/Laponite@IL-12 increased survival rates and promoted the hematopoietic recovery of mice who had received sublethal-dose irradiation. Local intra-bone marrow (intra-BM) injection of RGD-Alg/Laponite@IL-12 hydrogel effectively stimulated IL12 receptor-phosphoinositide 3-kinase/protein kinase B (IL-12R-PI3K/AKT) signaling axis, which promoted proliferation and hematopoietic growth factors secretion of BM mesenchymal stem/stromal cells. This signaling axis facilitates the repair of the hematopoietic microenvironment and plays a pivotal role in hematopoietic reconstitution. In conclusion, we describe a biomaterial-sustained release of IL-12 for the treatment of irradiated hematopoietic injury and provide a new therapeutic strategy for hematopoietic ARS.