Inflammatory Signaling Pathways Research Articles

Exercise attenuates high-fat diet-induced PVAT dysfunction through improved inflammatory response and BMP4-regulated adipose tissue browning.

Perivascular adipose tissue (PVAT) dysfunction impairs vascular homeostasis. Impaired inflammation and bone morphogenetic protein-4 (BMP4) signaling are involved in thoracic PVAT dysfunction by regulating adipokine secretion and adipocyte phenotype transformation. We investigated whether aerobic exercise training could ameliorate high-fat diet (HFD)-induced PVAT dysfunction via improved inflammatory response and BMP4-mediated signaling pathways. Sprague-Dawley rats (n = 24) were divided into three groups, namely control, high-fat diet (HFD), and HFD plus exercise (HEx). After a 6-week intervention, PVAT functional efficiency and changes in inflammatory biomarkers (circulating concentrations in blood and mRNA expressions in thoracic PVAT) were assessed. Chronic HFD feeding caused obesity and dyslipidemia in rats. HFD decreased the relaxation response of PVAT-containing vascular rings and impaired PVAT-regulated vasodilatation. However, exercise training effectively reversed these diet-induced pathological changes to PVAT. This was accompanied by significantly (p < 0.05) restoring the morphological structure and the decreased lipid droplet size in PVAT. Furthermore, HFD-induced impaired inflammatory response (both in circulation and PVAT) was notably ameliorated by exercise training (p < 0.05). Specifically, exercise training substantially reversed HFD-induced WAT-like characteristics to BAT-like characteristics as evidenced by increased UCP1 and decreased FABP4 protein levels in PVAT against HFD. Exercise training promoted transcriptional activation of BMP4 and associated signaling molecules (p38/MAPK, ATF2, PGC1α, and Smad5) that are involved in browning of adipose tissue. In conjunction with gene expressions, exercise training increased BMP4 protein content and activated downstream cascades, represented by upregulated p38/MAPK and PGC1α proteins in PVAT. Regular exercise training can reverse HFD-induced obesity, dyslipidemia, and thoracic PVAT dysfunction in rats. The browning of adipose tissue through exercise appears to be modulated through improved inflammatory response and/or BMP4-mediated signaling cascades in obese rats.

Exploration on pharmacological mechanisms of YZP against neuropathic pain via inhibiting spinal inflammation and the rationality of its compatibility

Ethnopharmacological relevanceYuanhu Zhitong Prescription (YZP) is a well-known traditional Chinese medicine (TCM) formula for neuropathic pain (NP) therapy with a satisfying clinical efficacy. However, the underlying pharmacological mechanism and its compatibility principle remain unclear. Aim of the studyThis study aims to investigate the analgesic and compatibility mechanisms of YZP on neuropathic pain (NP) at the gene and biological process levels. Materials and methodsThe chronic constriction injury (CCI) rats were intragastrically administrated with extracts of YZP, YH and BZ separately, and then mechanical hypersensitivity were measured to evaluate the analgesic effects between YH and BZ before and after compatibility. Then, RNA-seq and bioinformatics analyses were performed to elucidate the potential mechanisms underlying YZP's analgesia and compatibility. Finally, the expression levels and significant differences of key genes were analyzed. ResultsBehaviorally, both YZP and YH effectively alleviated mechanical allodynia in CCI rats, with YZP being superior to YH. In contrast, we did not observe an analgesic effect of BZ. Genetically, YZP, YH, and BZ reversed the expression levels of 52, 34, and 42 aberrant genes in the spinal cord of CCI rats, respectively. Mechanically, YZP was revealed to alleviate NP mainly by modulating the inflammatory response and neuropeptide signaling pathway, which are the dominant effective processes of YH. Interestingly, the effective targets of YZP were especially enriched in leukocyte activation and cytokine-mediated signaling pathways. Moreover, BZ was found to exert an adjunctive effect in enhancing the analgesic effect of YH by promoting skeletal muscle tissue regeneration and modulating calcium ion transport. ConclusionsYH, as the monarch drug, plays a dominant role in the analgesic effect of YZP that effectively relieves NP by inhibiting the spinal inflammation and neuropeptide signaling pathway. BZ, as the minister drug, not only synergistically enhances analgesic processes of YH but also helps to alleviate the accompanying symptoms of NP. Consequently, YZP exerted a more potent analgesic effect than YH and BZ alone. In conclusion, our findings offer new insights into understanding the pharmacological mechanism and compatibility principle of YZP, which may support its clinical application in NP therapy.

DNA Nanostructures Treat Inflammatory Bowel Disease through ROS Scavenging and Gut Microbiota Modulation

AbstractInflammatory bowel disease (IBD) encompasses a collection of chronic inflammatory conditions impacting the gastrointestinal tract, with a discernible global rise in prevalence and severity. The overabundance of reactive oxygen species (ROS) in the intestinal environment leads disruption of local redox homeostasis, and perturbation of the gut microbiota, exacerbating IBD. Consequently, mitigating excess ROS and preserving gut microbiota homeostasis have emerged as effective approaches for IBD management. In this study, a triangular DNA origami nanostructure loaded with siRNA targeting tumor necrosis factor α (siTNF‐αtDON) is introduced for this purpose. This nanostructure demonstrates efficacy in eliminating ROS within cells closely associated with IBD, diminishing inflammation, and ameliorating disorders in gut microbiota, as evidenced in an IBD mouse model. Furthermore, this investigation demonstrates the effects of siTNF‐αtDON on related inflammatory signaling pathways, including NF‐κB, MAPK/ERK and Keap1/Nrf2. Collectively, this study offers insights into the potential utilization of DNA nanostructures as antioxidants and gut microbiota modulators, presenting promising avenues for the IBD treatment.

Ameliorative potential of galangin in murine model of ovalbumin-induced allergic rhinitis: a role of PI3K-PKB pathway.

To examine the potential of galangin in a mouse model of ovalbumin (OVA)-induced allergic rhinitis (AR), as chronic AR, induced by immunoglobulin-E (IgE), leads to histamine release and nasal inflammation, and although galangin exhibits antiasthmatic and anti-inflammatory potential, its effect on AR is yet to be investigated. 126 BALB/c mice. AR induction involved sensitizing female mice with OVA (5%, 500 µL, IP) for 14 days. Post OVA challenge, the mice were divided into 7 groups (n = 18/group), including normal, AR control, montelukast (10 mg/kg), galangin (5, 10, and 20 mg/kg), and per se (galangin [20 mg/kg] treatment. Various outcomes were evaluated, including nasal symptoms, histopathology, biochemistry, and nasal lavage fluid inflammatory cytokines and signaling pathways in nasal mucosal to assess galangin potential in AR. In AR mice, galangin (10 and 20 mg/kg) significantly (P < .05) reduced sneezing, rubbing, and nasal discharge post-OVA challenge. Galangin treatment attenuated (P < .05) elevated serum histamine, β-hexosaminidase, IgE, and Immunoglobulin G1 levels in AR control mice. Additionally, galangin significantly (P < .05) decreased OVA-induced alterations in IL-4, IL-6, IL-13, and interferon-γ levels in nasal lavage fluid compared to AR control mice. Western blot analysis demonstrated that galangin lowered OVA-induced AR by significantly (P < .05) downregulating the phosphorylated protein kinase B and mammalian target of rapamycin-protein expressions while markedly (P < .05) upregulating the glycogen synthase kinase-3β protein expressions in nasal mucosal. Galangin also significantly ameliorated (P < .05) the OVA-induced histological aberrations in the nasal mucosa, reflected by reduced eosinophil infiltration, hyperplasia, and edema. Galangin exhibits antihistaminic and anti-inflammatory effects in AR mice by regulating IgE-mediated histamine and inflammatory release and modulating the phosphatidylinositol 3-kinase/Ak strain transforming/mammalian target of rapamycin pathways.

Benfotiamine improves dystrophic pathology and exercise capacity in mdx mice by reducing inflammation and fibrosis.

Duchenne Muscular Dystrophy (DMD) is a progressive and fatal neuromuscular disease. Cycles of myofibre degeneration and regeneration are hallmarks of the disease where immune cells infiltrate to repair damaged skeletal muscle. Benfotiamine is a lipid soluble precursor to thiamine, shown clinically to reduce inflammation in diabetic related complications. We assessed whether benfotiamine administration could reduce inflammation related dystrophic pathology. Benfotiamine (10mg/kg/day) was fed to male mdx mice (n = 7) for 15weeks from 4weeks of age. Treated mice had an increased growth weight (5-7weeks) and myofibre size at treatment completion. Markers of dystrophic pathology (area of damaged necrotic tissue, central nuclei) were reduced in benfotiamine mdx quadriceps. Grip strength was increased and improved exercise capacity was found in mdx treated with benfotiamine for 12weeks, before being placed into individual cages and allowed access to an exercise wheel for 3weeks. Global gene expression profiling (RNAseq) in the gastrocnemius revealed benfotiamine regulated signalling pathways relevant to dystrophic pathology (Inflammatory Response, Myogenesis) and fibrotic gene markers (Col1a1, Col1a2, Col4a5, Col5a2, Col6a2, Col6a2, Col6a3, Lum) towards wildtype levels. In addition, we observed a reduction in gene expression of inflammatory gene markers in the quadriceps (Emr1, Cd163, Cd4, Cd8, Ifng). Overall, these data suggest that benfotiamine reduces dystrophic pathology by acting on inflammatory and fibrotic gene markers and signalling pathways. Given benfotiamine's excellent safety profile and current clinical use, it could be used in combination with glucocorticoids to treat DMD patients.

Cornuside alleviates psoriasis-like skin lesions in mice by relieving inflammatory effects

Psoriasis is a chronic inflammatory skin disease substantially affecting the quality of life, with no complete cure owing to its complex pathogenesis. Cornuside, a major bioactive compound present in Cornus officinalis Sieb. et Zucc., which is a well-known traditional Chinese medicine with a variety of biological and pharmacological activities, such as anti-apoptotic, antioxidant, and anti-inflammatory properties. However, its effects on psoriasis remain unclear. Our preliminary analysis of network pharmacology showed that cornuside may be involved in psoriasis by regulating the inflammatory response and IL-17 signaling pathway. Thus, we investigated the protective role and mechanism of cornuside in the pathogenesis of psoriasis in an imiquimod (IMQ)-induced psoriasis mouse model. In-vivo experiments demonstrated that cornuside-treated mice had reduced skin erythema, scales, thickness, and inflammatory infiltration. The Psoriasis Area Severity Index score was significantly lower than that of the IMQ group. Flow cytometry analysis indicated that cornuside effectively inhibited Th1- and Th17-cell infiltration and promoted aggregation of Th2 cells in skin tissues. Cornuside also inhibited the infiltration of macrophages to the skin. Furthermore, in-vitro experiments indicated that cornuside also decreased the polarization of M1 macrophages and reduced the levels of associated cytokines. Western blotting demonstrated that cornuside suppressed the phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular receptor kinase (ERK) in bone marrow-derived macrophages. Our findings indicate that cornuside has a protective effect against IMQ-induced psoriasis by inhibiting M1 macrophage polarization through the ERK and JNK signaling pathways and modulating the infiltration of immune cells as well as the expression of inflammatory factors.

Evaluation of Anti‐inflammatory Activity of Garlic Extracts in 3D Bioprinted Skin Equivalents

Ongoing obstacles in preclinical drug testing have raised significant concerns within the pharmaceutical industry. Recently, utilizing the potential of three‐dimensional (3D) bioprinting offers a solution for creating tissue models for screening of the effectiveness and safety of new drugs. In this study, the anti‐inflammatory potential of garlic extracts is assessed, specifically N‐Benzyl‐N‐methyl‐dodecan‐1‐amine (BMDA), using a 3D bioprinted artificial skin model. Comprehensive physicochemical characterization and immunocytochemical analysis demonstrate that the 3D bioprinted skin model exhibits structures and functions resembling human skin. BMDA treatment in a prepared skin model reveals its capacity to mitigate H2O2‐induced oxidative stress and trigger anti‐inflammatory responses. Notably, BMDA reduces the expression of pro‐inflammatory cytokines and chemokines by downregulating NF‐κB and mitogen‐activated protein kinase inflammatory signaling pathways. In summary, our findings highlight the potential of an artificial skin model as a robust platform for the development of new drugs.

An Amino Acids and Dipeptide Injection Inhibits the TNF-α/HMGB1 Inflammatory Signaling Pathway to Reduce Pyroptosis and M1 Microglial Polarization in POCD Mice: the Gut to the Brain.

Peripheral surgery-induced neural inflammation is a key pathogenic mechanism of postoperative cognitive dysfunction (POCD). However, the mechanism underlying neuroinflammation and associated neural injury remains elusive. Surgery itself can lead to gut damage, and the occurrence of POCD is accompanied by high levels of TNF-α in the serum and blood‒brain barrier (BBB) damage. Reductions in stress, inflammation and protein loss have been emphasized as strategies for enhanced recovery after surgery (ERAS). We designed an amino acids and dipeptide (AAD) formula for injection that could provide intestinal protection during surgery. Through the intraoperative infusion of AAD based on the ERAS concept, we aimed to explore the effect of AAD injection on POCD and its underlying mechanism from the gut to the brain. Here, we observed that AAD injection ameliorated neural injury in POCD, in addition to restoring the function of the intestinal barrier and BBB. We also found that TNF-α levels decreased in the ileum, blood and hippocampus. Intestinal barrier protectors and TNF-α inhibitors also alleviated neural damage. AAD injection treatment decreased HMGB1 production, pyroptosis, and M1 microglial polarization and increased M2 polarization. In vitro, AAD injection protected the impaired gut barrier and decreased TNF-α production, alleviating damage to the BBB by stimulating cytokine transport in the body. HMGB1 and Caspase-1 inhibitors decreased pyroptosis and M1 microglial polarization and increased M2 polarization to protect TNF-α-stimulated microglia in vitro. Collectively, these findings suggest that the gut barrier-TNF-α-BBB-HMGB1-Caspase-1 inflammasome-pyroptosis-M1 microglia pathway is a novel mechanism of POCD related to the gut-brain axis and that intraoperative AAD infusion is a potential treatment for POCD.

Sexual dimorphism in atherosclerotic plaques of aged Ldlr−/− mice

BackgroundAtherosclerosis, the main underlying pathology of cardiovascular disease, is a chronic inflammatory disease characterized by lipid accumulation and immune cell responses in the vascular wall, resulting in plaque formation. It is well-known that atherosclerosis prevalence and manifestation vary by sex. However, sexual dimorphism in the immune landscape of atherosclerotic plaques has up to date not been studied at high-resolution. In this study, we investigated sex-specific differences in atherosclerosis development and the immunological landscape of aortas at single-cell level in aged Ldlr−/− mice.MethodsWe compared plaque morphology between aged male and female chow diet-fed Ldlr−/− mice (22 months old) with histological analysis. Using single-cell RNA-sequencing and flow cytometry on CD45+ immune cells from aortas of aged Ldlr−/− mice, we explored the immune landscape in the atherosclerotic environment in males and females.ResultsWe show that plaque volume is comparable in aged male and female mice, and that plaques in aged female mice contain more collagen and cholesterol crystals, but less necrotic core and macrophage content compared to males. We reveal increased immune cell infiltration in female aortas and found that expression of pro-atherogenic markers and inflammatory signaling pathways was enriched in plaque immune cells of female mice. Particularly, female aortas show enhanced activation of B cells (Egr1, Cd83, Cd180), including age-associated B cells, in addition to an increased M1/M2 macrophage ratio, where Il1b+ M1-like macrophages display a more pro-inflammatory phenotype (Nlrp3, Cxcl2, Mmp9) compared to males. In contrast, increased numbers of age-associated Gzmk+CD8+ T cells, dendritic cells, and Trem2+ macrophages were observed in male aortas.ConclusionsAltogether, our findings highlight that sex is a variable that contributes to immunological differences in the atherosclerotic plaque environment in mice and provide valuable insights for further preclinical studies into the impact of sex on the pathophysiology of atherosclerosis.

T-bet suppresses proliferation of malignant B cells in chronic lymphocytic leukemia

AbstractThe T-box transcription factor T-bet is known as a master regulator of the T-cell response but its role in malignant B cells has not been sufficiently explored. Here, we conducted single-cell resolved multi-omics analyses of malignant B cells from patients with chronic lymphocytic leukemia (CLL) and studied a CLL mouse model with a genetic knockout of Tbx21. We found that T-bet acts as a tumor suppressor in malignant B cells by decreasing their proliferation rate. NF-κB activity, induced by inflammatory signals provided by the microenvironment, triggered T-bet expression, which affected promoter-proximal and distal chromatin coaccessibility and controlled a specific gene signature by mainly suppressing transcription. Gene set enrichment analysis identified a positive regulation of interferon signaling and negative control of proliferation by T-bet. In line, we showed that T-bet represses cell cycling and is associated with longer overall survival of patients with CLL. Our study uncovered a novel tumor suppressive role of T-bet in malignant B cells via its regulation of inflammatory processes and cell cycling, which has implications for the stratification and therapy of patients with CLL. Linking T-bet activity to inflammation explains the good prognostic role of genetic alterations in the inflammatory signaling pathways in CLL.

Abstract PO3-13-07: MUC1-C INTEGRATES CHRONIC ACTIVATION OF INTERFERON PATHWAYS WITH CHROMATIN REMODELING IN TREATMENT RESISTANCE OF TRIPLE-NEGATIVE BREAST CANCER

Abstract Chronic inflammation is intimately linked to cancer progression and resistance to treatment. Triple-negative breast cancer (TNBC) is a recalcitrant malignancy largely unresponsive to cytotoxic, targeted and immunotherapeutic agents. The Mucin1 (MUC1) transmembrane heterodimeric protein, which is aberrantly expressed in TNBCs, evolved in mammals to provide protection of epithelia from the external environment. With loss of homeostasis, the (i) MUC1 N-terminal (MUC1-N) subunit is shed and released into serum as detected by the CA15-3 assay, and (ii) MUC1 C-terminal (MUC1-C) subunit is activated and thereby induces inflammatory, proliferative and remodeling signaling pathways associated with wound healing. However, prolonged MUC1-C activation in settings of chronic inflammation promotes cancer progression and treatment resistance. The SWI/SNF PBAF chromatin remodeling complex includes the polybromo-1 (PBRM1) subunit and drives DNA damage resistance and immune evasion in certain cancer cells through mechanisms that remain unclear. Recent studies in TNBCs have demonstrated that MUC1-C drives intrinsic activation of type II interferon (IFN) pathway that is linked to chronic inflammation and immune evasion. In extending this work, we have found that MUC1-C activates the type I IFN pathway, which contributes to genomic instability and DNA damage tolerance in TNBC cells. Importantly, our results further demonstrate that MUC1-C interacts with interferon regulatory factor 1 (IRF1) as a key regulatory node in integrating activation of the type I and II IFN pathways. Here, we report that MUC1-C is necessary for PBRM1 expression and that it forms a nuclear complex with PBRM1 in TNBC cells. Analysis of global transcriptional (RNA-seq) and chromatin accessibility (ATAC-seq) profiles further demonstrated that MUC1-C and PBRM1 drive STAT1 and IRF1 expression by increasing chromatin accessibility of promoter-like signatures (PLS) on their respective genes. We also found that MUC1-C, PBRM1, and IRF1 increase the expression and chromatin accessibility on PLSs of the (i) type II IFN pathway IDO1 and WARS genes and (ii) type I IFN pathway RIG-I, MDA5, cGAS, STING and ISG15 genes that collectively contribute to DNA damage resistance and immune evasion. In support of these results, targeting MUC1-C in wild-type BRCA1/2 TNBC cells enhanced carboplatin-induced DNA damage and loss of self- renewal capacity. In addition, MUC1-C was necessary for DNA damage resistance, self-renewal and tumorigenicity of olaparib-resistant BRCA1-mutant TNBC cells. Analysis of TNBC clinical samples corroborated that (i) MUC1 and PBRM1 are associated with decreased responsiveness to chemotherapy and (ii) MUC1-C expression is associated with the depletion of tumor-infiltrating lymphocytes. These findings demonstrate that MUC1-C activates PBRM1, and thereby chromatin remodeling of IFN-stimulated genes that promote chronic inflammation, DNA damage resistance, and immune evasion. These findings have identified MUC1-C as a potential target for the treatment of TNBC that have limited therapeutic options. To that end, the generation of MAb 3D1 against the MUC1-C extracellular domain provided an opportunity for the development of agents that target MUC1-C on the cancer cell surface. As one example, an allogeneic anti-MUC1-C CAR T cell using MAb 3D1 sequences is undergoing Phase I evaluation for the treatment of MUC1-C-expressing cancers (NCT05239143: P-MUC1C-ALLO1 Allogeneic CAR-T Cells in the Treatment of Subjects with Advanced or Metastatic Solid Tumors). In addition, anti-MUC1-C huMAb3D1-MMAE ADCs are under development by the NCI NExT Program for IND-enabling studies and performing early phase clinical trials in patients with TNBC. Citation Format: Nami Yamashita, Yoshihiro Morimoto, Atsushi Fushimi, Rehan Ahmad, Atrayee Bhattacharya, Yuka Inoue, Henry Withers, Geoffrey Shapiro, Tomoharu Yoshizumi, Takayuki Ueno, Shinji Ohno, Mark Long, Donald Kufe. MUC1-C INTEGRATES CHRONIC ACTIVATION OF INTERFERON PATHWAYS WITH CHROMATIN REMODELING IN TREATMENT RESISTANCE OF TRIPLE-NEGATIVE BREAST CANCER [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-13-07.

Anti-inflammatory activities of novel heat shock protein 90 isoform selective inhibitors in BV-2 microglial cells.

Heat shock protein 90 (Hsp90) is a family of chaperone proteins that consists of four isoforms: Hsp90α, Hsp90β, glucose-regulated protein 94 (Grp94), and tumor necrosis factor type 1 receptor-associated protein (TRAP1). They are involved in modulating the folding, maturation, and activation of their client proteins to regulate numerous intracellular signaling pathways. Previous studies demonstrated that pan-Hsp90 inhibitors reduce inflammatory signaling pathways resulting in a reduction of inflammation and pain but show toxicities in cancer-related clinical trials. Further, the role of Hsp90 isoforms in inflammation remains poorly understood. This study aimed to determine anti-inflammatory activities of Hsp90 isoforms selective inhibitors on the lipopolysaccharide (LPS)-induced inflammation in BV-2 cells, a murine microglial cell line. The production of inflammatory mediators such as nitric oxide (NO), interleukin 1 beta (IL-1β), and tumor necrosis factor-alpha (TNF-α) was measured. We also investigated the impact of Hsp90 isoform inhibitors on the activation of nuclear factor kappa B (NF-κB), nuclear factor erythroid 2-related factor 2 (Nrf2), and mitogen-activated protein kinases (MAPKs). We found that selective inhibitors of Hsp90β reduced the LPS-induced production of NO, IL-1β, and TNF-α via diminishing the activation of NF-κB and Extracellular signal-regulated kinases (ERK) MAPK. The Hsp90α, Grp94, TRAP1 inhibitors had limited effect on the production of inflammatory mediators. These findings suggest that Hsp90β is the key player in LPS-induced neuroinflammation. Thereby providing a more selective drug target for development of medications involved in pain management that can potentially contribute to the reduction of adverse side effects associated with Hsp90 pan inhibitors.

Exosome miR-4738-3p-mediated regulation of COL1A2 through the NF-κB and inflammation signaling pathway alleviates osteoarthritis low-grade inflammation symptoms.

This study aimed to elucidate the roles of microRNA (miR)-4738-3p and the collagen type I alpha 2 chain (COL1A2) gene in the pathogenesis of osteoarthritis (OA) through bioinformatics analysis and cellular assays. The GSE55235 dataset was analyzed using the weighted gene co-expression network analysis (WGCNA) method to identify gene modules associated with OA. Key overlapping genes were identified from these modules and the GSE55235-differential expressed genes (DEGs). The expression levels of selected genes were determined in C28/I2 cells using the quantitative real-time polymerase chain reaction (qRT-PCR). The interaction between miR-4738-3p and COL1A2 was examined in the context of interleukin 1 beta (IL-1β) induction. Exosome characterization was achieved through transmission electron microscopy (TEM), western blotting (WB), and other analyses. The study also investigated the functional relevance of miR-4738-3p in OA pathology through various molecular and cellular assays. Our findings revealed that the green module exhibited a strong correlation with the OA phenotype in the GSE55235 dataset, with COL1A2 emerging as a hub gene and miR-4738-3p as its key downstream target. IL-1β induction suggested that COL1A2 is involved in inflammation and apoptosis, while miR-4738-3p appeared to play an antagonistic role. The analysis of exosomes underscored the significance of miR-4738-3p in cellular communication, with an enhanced level of exo-miR-4738-3p antagonizing IL-1β-induced inflammation and promoting cell survival. Conversely, a reduction in exo-miR-4738-3p led to increased cell damage. This study established a clear regulatory relationship between miR-4738-3p and COL1A2, with the nuclear factor kappa B (NF-κB) signaling pathway playing a central role in this regulation. The miR-4738-3p significantly influences the OA-associated inflammation, primarily through modulation of COL1A2 and the NF-κB pathway. Therefore, targeting miR-4738-3p offers a potential therapeutic approach for OA, with exosome miR-4738-3p presenting a promising strategy.

Elucidating the Role of SYK in the Intestinal Epithelium

Background: Inflammatory Bowel Disease (IBD) involves chronic and recurring inflammation of the gastrointestinal tract. We identified several patients presenting with immune dysregulation and systemic inflammation, including inflammation of the gut. Analysis of Whole Exome Sequencing data from pediatric IBD patients has identified five rare protein coding Spleen Tyrosine Kinase (SYK) variants. Functional studies have demonstrated increased kinase activity implicating them in disease pathophysiology. While the role of SYK is well characterized in immune cells, it is not understood what role it plays in the intestinal epithelium. Aims: The primary aim of this project is to understand the role SYK plays in the intestinal epithelium, what molecular pathways it signals through, and how its dysregulation leads to intestinal disease. Methods: To understand SYKs role in the intestinal epithelium, a BioID screen was used to identify novel SYK interacting proteins. BioID was done with wild type SYK as well as the patient derived variants in HEK293 cells. BioID hits were validated using co-immunoprecipitation (CoIP) and proximity ligation assay (PLA). Further functional analysis was done in Caco2 cells, an intestinal epithelial cell model, with western blot, ELISA, and luciferase assays to assess the effect of SYK activity on downstream inflammatory pathways. Finally, we investigated the effect of small molecule kinase inhibitors to explore possible novel treatment options. Results: The BioID screen revealed several high confidence interactors, with dysregulated interactions in the patient variants. USP25, a ubiquitin specific peptidase that is widely expressed, including in the intestinal epithelium, was identified as one of the strongest hits based on peptide counts. The interaction was validated with CoIP and PLA, and further analysis showed SYK activation decreased USP25 protein levels by up to eight-fold. The secretion of the proinflammatory chemokine, IL-8, in response to lipopolysaccharide (LPS) was found to be almost twice as high in Caco2 cells with patient SYK variants relative to WT. Cells expressing the patient variants of SYK showed increased NFkB and AP1 levels, indicating increased inflammatory gene expression when stimulated with 100ng/mL LPS. NFkB was also active for longer in cells expressing the patient variant of SYK post stimulation, with WT cells returning to baseline in under half an hour, while the patient variant stayed in an inflamed state for 36 to 48 hours after LPS was removed. Conclusions: Increased SYK activity is associated with the degradation of USP25. USP25 is known to cleave ubiquitin off TNF receptor associated factors (TRAFs). TRAFs are activated downstream of innate immune receptors such as TLR4, and need to be ubiquitinated for activation. USP25, which is responsible for the deubiquitination of these TRAFs, acts as a negative regulator of these pathways. The hyperactive SYK variants degrade USP25 and impair the cells’ ability to switch off inflammatory signaling pathways, indicated by the cells elongated period in an inflamed state. This research is funded by grants from Canadian Institute for Health Research (CIHR) and the Leona M. and Harry B. Helmsley Charitable Trust. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Lysosomal Regulation of Gasdermin D Pore Formation in Podocytes Determined by Acid Sphingomyelinase

Recent studies have shown that gasdermin D (GSDMD) pore may be involved in inflammasome product release and pyroptosis in podocytes. Nevertheless, the molecular mechanism by which GSDMD pore formation is regulated in these cells remains poorly understood. Given the important role of acid sphingomyelinase (ASM)-ceramide signaling pathway in glomerular inflammation and sclerosis under pathological conditions, the present study tested whether ASM controls lysosome-dependent autophagic degradation of GSDMD-NT and GSDMD pore repair to determine GSDMD pore formation in podocytes. WT/WT, ASM knockout (KO), and ASM overexpression (OE) podocytes were isolated from wild type, Smpd1 (gene code of ASM) gene KO, and podocyte-specific Smpd1 gene OE mice for experiments. We first demonstrated that palmitic acid (PA) as an obesity-related danger factor induced NLRP3 inflammasome activation, GSDMD pore formation on plasma membrane, and IL-1β secretion in podocytes, which was blocked by Smpd1 gene KO but exaggerated by Smpd1 gene OE. Also, it was found that PA-induced pyroptosis was prevented by ASM KO but amplified by ASM OE in podocytes. By confocal microscopy and super-resolution microscopy, we observed that PA treatment enhanced the formation of autophagosomes containing GSDMD-NT but inhibited lysosome-autophagosome interaction in podocytes. Also, FasL-induced lysosome fusion to plasma membrane was reduced by PA. All these pathological changes were remarkably attenuated by ASM KO or amitriptyline but significantly augmented by ASM OE. To further dissect the molecular mechanism by which ASM determines GSDMD pore formation, we examined whether ASM controls lysosomal TRPML1 channel-mediated Ca2+ release to regulate lysosome traffcking and associated lysosomal functions. By GCaMP3 Ca2+ imaging, we found that PA treatment significantly inhibited TRPML1 channel activity in podocytes, which was exaggerated by ASM OE. Correspondingly, PA-induced reduction of lysosome traffcking was more remarkable in ASM OE podocytes compared to WT/WT podocytes. Moreover, PA-induced decrease in lysosome-autophagosome interaction and increase in GSDMD pore formation in podocytes were hindered by ML-SA5 (TRPML1 channel agonist) but enhanced by ML-SI1 (TRPML1 channel inhibitor). In summary, our findings suggest that ASM plays a crucial role in lysosomal regulation of GSDMD pore formation via control of TRPML1 channel activity and lysosome traffcking in podocytes. This study was supported by NIH grants DK054927 and DK120491. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Reprogramming cardiac macrophages with circular RNAs to attenuate post-MI adverse remodeling

Cardiac macrophages (cMφ) play a critical role in cardiac remodeling following myocardial infarction (MI). Reprogramming cMφ towards a favorable wound-healing phenotype is emerging as a promising therapeutic strategy for MI patients. Circular RNAs (circRNAs) are a unique class of non-coding RNAs with high stability and abundance, making them attractive therapeutic targets. However, the role of circRNAs in cMφ physiology during heart failure (HF) progression is poorly understood. To identify circRNAs as key regulators of cMφ dynamics, we performed RNA sequencing and bioinformatics analysis on heart biopsies from healthy donors and patients with ischemic HF. We identified a promising circRNA, circMJ1, as a regulator of Mφ polarization and inflammatory response. CircMJ1 was highly expressed in cMφ and its expression was associated with cMφ polarization. Knockdown of circMJ1 significantly inhibited inflammatory signaling pathways in cMφ and reduced the secretion of pro-inflammatory cytokines. The secretome from circMJ1-silenced cMφ (circMJ1-KD cMφ) also significantly suppressed the migration of cardiac fibroblasts and apoptosis of cardiomyocytes. To further assess the therapeutic effcacy of circMJ1-KD Mφ in cardiac remodeling in the context of MI, we employed a novel multicellular 3D ex vivo model, called living myocardial slices (LMS). We applied circMJ1-KD Mφ to both rat LMS subjected to cryo-injury mimicking MI and HF patient-derived LMS. Interestingly, co-culture with circMJ1-KD Mφ demonstrated a remarkable reduction in inflammation and fibrosis, alongside notable improvement in cardiac function in both cryo-injured rat LMS and HF patient-derived LMS. Overall, our findings reveal circMJ1 as a crucial "switch" for Mφ dynamics and suggest the therapeutic potential of engineered circMJ1-silenced Mφ to improve post-MI healing. Funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) –465131031. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Structural identification and immunomodulatory effects of chicken egg white glycopeptides

Glycoproteins, primary proteins in egg white (EW), yield substantial EW glycopeptides (EWG) after digestion and hydrolysis. However, the structure and function of EWG have not been revealed. In this study, EWG was enriched from EW hydrolysates of simulated digestion in vitro, the structure of EWG was determined by LC-MS/MS, and their immunoregulatory effects on macrophages were investigated. The structure of EWG displayed a high degree of heterogeneity. As an example, 2 N-glycosites (N-293 and N-312), 19 peptide fragments and 45 N-glycopeptides were identified in ovalbumin. Cellular assays showed that EWG significantly increased RAW264.7 cells secretion of TNF-α (p < 0.01), indicating potent immunoactivating properties. Transcriptome sequencing analysis found that 1112 differential expression genes (DEGs) were upregulated, while 603 DEGs were downregulated in RAW264.7 cells by treatment with EWG (125 μg/mL) compared to control group. These DEGs were mainly involved in classic inflammatory signaling pathways, particularly the “TNF signaling pathway”, “NF-kappa B signaling pathway” and “MAPK signaling pathway”. Our findings provide meaningful insights for developing EWG as a potential immunoactivity ingredient and expand EW's nutritional and healthy applications.

Abstract 2066: Relationship Between Stat3 And Endoplasmic Reticulum Stress Induces Lipopolysaccharide-induced Endothelial To Mesenchymal Transition In Vascular Endothelial Cell

Background: Endothelial to mesenchymal transition (EndMT) is a process that involves the loss of endothelial cell (EC) phenotype and induced plaque instability during the progression of atherosclerosis. Primarily, EC injury, caused by an accelerated inflammatory signaling pathway, plays a crucial role in activating EC dysfunction by inducing endoplasmic reticulum (ER) stress and disrupting EC junctions. In the present study, we investigated the molecular mechanisms between EndMT and ER stress on STAT3-mdeiated inflammation under LPS-treated human umbilical vein EC (HUVEC). Methods: To explore the EndMT progression under chronic inflammation, LPS-treated HUVEC were used. Western blot, siRNA transfection, and immunofluorescence assay were employed to evaluate signaling pathways and target proteins. The phenotypic switching of ECs on the endothelium of the mouse aorta after challenging 10 mg/kg LPS was assessed by en face staining. Results: Deactivation of STAT3 by stattic pretreatment abolished not only LPS-mediated STAT3 phosphorylation, but also VCAM-1 and ICAM-1. In addition, STAT3 inhibition surprisingly reversed LPS-induced mesenchymal markers, including SM22α and vimentin, and the suppression of endothelial marker VE-cadherin. Mechanistically, stattic treatment suppressed LPS-induced EndMT progression by inhibiting protein expression and nuclear translocation of Smad2/3. Furthermore, LPS-induced chronic inflammation disrupted EC homeostasis by PERK or IRE-1α/CHOP-mediated ER stress, whereas stattic abolished LPS-mediated PERK and CHOP expression. Interestingly, depletion of CHOP by siRNA transfection significantly abolished LPS-mediated EndMT progression (p&lt;0.001. Figure 1). Notably, the absence of CHOP significantly suppressed phosphorylation of STAT3 and its nuclear expression as well. Consistent to in vitro data, STAT3 inhibition by stattic injection significantly suppressed LPS-mediated STAT3 activation as well as CHOP expression in the mouse endothelium. Conclusion: Collectively, the STAT3-CHOP axis may contribute to the loss of EC function and induce mesenchymal differentiation by aggravating chronic inflammation and EndMT.

SIRT1 exerts protective effects by inhibiting endoplasmic reticulum stress and NF-κB signaling pathways.

Silent information regulator two homolog 1 (SIRT1), an NAD + -dependent histone deacetylase, plays a pivotal regulatory role in a myriad of physiological processes. A growing body of evidence suggests that SIRT1 can exert protective effects in metabolic disorders and neurodegenerative diseases by inhibiting endoplasmic reticulum (ER) stress and the nuclear factor-κB (NF-κB) inflammatory signaling pathway. This review systematically elucidates the molecular mechanisms and biological significance of SIRT1 in regulating ER stress and the NF-κB pathway. On one hand, SIRT1 can deacetylate key molecules in the ER stress pathway, such as glucose-regulated protein 78 (GRP78), X-box binding protein 1 (XBP1), PKR-like ER kinase (PERK), inositol-requiring enzyme 1α (IRE1α), and activating transcription factor 6 (ATF6), thereby alleviating ER stress. On the other hand, SIRT1 can directly or indirectly remove the acetylation modification of the NF-κB p65 subunit, inhibiting its transcriptional activity and thus attenuating inflammatory responses. Through these mechanisms, SIRT1 can ameliorate insulin resistance in metabolic diseases, exert cardioprotective effects in ischemia-reperfusion injury, and reduce neuronal damage in neurodegenerative diseases. However, it is important to note that while these findings are promising, the complex nature of the biological systems involved warrants further investigation to fully unravel the intricacies of SIRT1's regulatory mechanisms. Nevertheless, understanding the regulatory mechanisms of SIRT1 on ER stress and the NF-κB pathway is of great significance for expanding our knowledge of the pathogenesis of related diseases and exploring new preventive and therapeutic strategies targeting SIRT1.

Effect of Shenqi Jieyu formula on inflammatory response pathway in hippocampus of postpartum depression rats

AimTo investigate whether SJF functions in similar manner as the key substance in the inflammatory process, soluble epoxide hydrolase (sEH) inhibitor, to inhibit the arachidonic acid metabolic pathway and nuclear factor kappa-B(NF-κB) signal path in the hippocampi of postpartum depression rats. MethodsThe rats were subcutaneous injected estradiol benzoate and progesterone to build PPD rat model. SJF, paroxetine hydrochloride and sEH inhibitor (AUDA) were used to treat PPD rats for 3 weeks. Then the morphological changes of hippocampi and various proteins were observed after that behavioral test were conducted in all 36 SD rats in six group: SJF, paroxetine, AUDA, PPD, sham and normal group. ResultsWeight, results of sucrose preference, upright times, total and center squares crossing decreased significantly (P < 0.01), whereas immobility time increased (P < 0.01). Results above were reversed in animals that in the SJF, paroxetine and AUDA groups. Hippocampal neurons in PPD rats partially degenerated with narrowed nuclei, increased autophagy and mitochondria bound to lysosomes were visible while the autophagy of hippocampal neurons in the paroxetine and AUDA group decreased, with a small amount of lysosomes. sEH, COX-2, 5-LOX, TNF-α, IL-1, IL-6, NF-κB p65, and Cor increased in hippocampi of PPD rats while EETs and 5-HT decreased. Protein expressions of Ibal, GFAP, p-IκBα, p65, and p-p65(S536)increased in PPD animals. Those changes were reversed by SJF, paroxetine and AUDA. Gene expressions of TNF-α, IL-1β, IL-6, 5-LOX, COX-2 and p65 increased in PPD rats and the changes of expression in these genes were reversed by paroxetine and AUDA. SJF reversed the gene expression changes of COX-2, TNF-α, and IL-1β. ConclusionSJF may have an analogous effect as sEH inhibitor to relieve depressive symptoms by suppressing inflammatory signaling pathways in hippocampi of PPD rats, which involves AA metabolic pathway and NF-κB signal pathway.