Pro-inflammatory Cytokines TNF-α Research Articles

Modulation of Gut Microbial Community and Metabolism by Bacillus licheniformis HD173 Promotes the Growth of Nursery Piglets Model.

Maintaining the balance and stability of the gut microbiota is crucial for the gut health and growth development of humans and animals. Bacillus licheniformis (B. licheniformis) has been reported to be beneficial to the gut health of humans and animals, whereas the probiotic effects of a new strain, B. licheniformis HD173, remain uncertain. In this study, nursery piglets were utilized as animal models to investigate the extensive impact of B. licheniformis HD173 on gut microbiota, metabolites, and host health. The major findings were that this probiotic enhanced the growth performance and improved the health status of the nursery piglets. Specifically, it reduced the level of pro-inflammatory cytokines IL-1β and TNF-α in the serum while increasing the level of IL-10 and SOD. In the gut, B. licheniformis HD173 reduced the abundance of pathogenic bacteria such as Mycoplasma, Vibrio, and Vibrio metschnikovii, while it increased the abundance of butyrate-producing bacteria, including Oscillospira, Coprococcus, and Roseburia faecis, leading to an enhanced production of butyric acid. Furthermore, B. licheniformis HD173 effectively improved the gut metabolic status, enabling the gut microbiota to provide the host with stronger metabolic abilities for nutrients. In summary, these findings provide scientific evidence for the utilization of B. licheniformis HD173 in the development and production of probiotic products for maintaining gut health in humans and animals.

Knockdown of trem2 promotes proinflammatory microglia and inhibits glioma progression via the JAK2/STAT3 and NF-κB pathways

BackgroundIn the tumor immune microenvironment (TIME), triggering receptor expressed on myeloid cells 2 (trem2) is widely considered to be a crucial molecule on tumor-associated macrophages(TAMs). Multiple studies have shown that trem2 may function as an immune checkpoint in various malignant tumors, mediating tumor immune evasion. However, its specific molecular mechanisms, especially in glioma, remain elusive.MethodsLentivirus was transfected to establish cells with stable knockdown of trem2. A Transwell system was used for segregated coculture of glioma cells and microglia. Western blotting, quantitative real-time polymerase chain reaction (qRT‒PCR), and immunofluorescence (IF) were used to measure the expression levels of target proteins. The proliferation, invasion, and migration of cells were detected by colony formation, cell counting kit-8 (CCK8), 5-ethynyl-2’-deoxyuridine (EdU) and transwell assays. The cell cycle, apoptosis rate and reactive oxygen species (ROS) level of cells were assessed using flow cytometry assays. The comet assay and tube formation assay were used to detect DNA damage in glioma cells and angiogenesis activity, respectively. Gl261 cell lines and C57BL/6 mice were used to construct the glioma orthotopic transplantation tumor model.ResultsTrem2 was highly overexpressed in glioma TAMs. Knocking down trem2 in microglia suppressed the growth and angiogenesis activity of glioma cells in vivo and in vitro. Mechanistically, knockdown of trem2 in microglia promoted proinflammatory microglia and inhibited anti-inflammatory microglia by activating jak2/stat1 and inhibiting the NF-κB p50 signaling pathway. The proinflammatory microglia produced high concentrations of nitric oxide (NO) and high levels of the proinflammatory cytokines TNF-α, IL-6, and IL-1β, and caused further DNA damage and promoted the apoptosis rate of tumor cells.ConclusionsOur findings revealed that trem2 in microglia plays a significant role in the TIME of gliomas. Knockdown of trem2 in microglia might help to improve the efficiency of inhibiting glioma growth and delaying tumor progression and provide new ideas for further treatment of glioma.

Weissella koreensis KJ, Which Increases Gut Tight Junction Protein Expression, Alleviates TNBS-Induced Colitis by Suppressing Inflammatory Cytokines

Inflammatory bowel disease (IBD), a chronic inflammatory disease, results from dysregulation of the immune responses. The IBD prevalence rate was 321.2 per 100,000 people in 2021 and, compared with that in 2006 (200 per 100,000 people), had increased at a rate of +46%. Therefore, the development of a safe and new treatment for IBD is urgently needed. Weissella koreensis, a strain of lactic acid bacteria (LABs), was isolated from kimchi and shown to inhibit a pro-inflammatory cytokine, tumor necrosis factor-alpha (TNF-α). Its anti-inflammatory effect was further assessed using a mouse model of colitis induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS). The administration of TNBS significantly increased myeloperoxidase (MPO) expression, macroscopic score, and colonic shortening. Oral administration of W. koreensis KJ suppressed the TNBS-induced response and significantly inhibited the expression of the pro-inflammatory cytokines TNF-α, interleukin (IL)-1β, and IL-6 in the intestinal tissues. In particular, W. koreensis KJ reversed the TNBS-induced decrease in the expression of these tight junction proteins. Therefore, since W. koreensis KJ isolated from kimchi, which increases gut tight junction proteins, attenuating colitis by suppressing inflammatory cytokines, it can be used as a therapeutic candidate for treating colitis such as IBD.

Pharmacological inhibition of STING reduces neuroinflammation-mediated damage post-traumatic brain injury.

Traumatic brain injury (TBI) remains a major public health concern worldwide with unmet effective treatment. Stimulator of interferon genes (STING) and its downstream type-I interferon (IFN) signalling are now appreciated to be involved in TBI pathogenesis. Compelling evidence have shown that STING and type-I IFNs are key in mediating the detrimental neuroinflammatory response after TBI. Therefore, pharmacological inhibition of STING presents a viable therapeutic opportunity in combating the detrimental neuroinflammatory response after TBI. This study investigated the neuroprotective effects of the small-molecule STING inhibitor n-(4-iodophenyl)-5-nitrofuran-2-carboxamide (C-176) in the controlled cortical impact mouse model of TBI in 10- to 12-week-old male mice. Thirty minutes post-controlled cortical impact surgery, a single 750-nmol dose of C-176 or saline (vehicle) was administered intravenously. Analysis was conducted 2h and 24 h post-TBI. Mice administered C-176 had significantly smaller cortical lesion area when compared to vehicle-treated mice 24 h post-TBI. Quantitative temporal gait analysis conducted using DigiGait™ showed C-176 administration attenuated TBI-induced impairments in gait symmetry, stride frequency and forelimb stance width. C-176-treated mice displayed a significant reduction in striatal gene expression of pro-inflammatory cytokines Tnf-α, Il-1β and Cxcl10 compared to their vehicle-treated counterparts 2h post-TBI. This study demonstrates the neuroprotective activity of C-176 in ameliorating acute neuroinflammation and preventing white matter neurodegeneration post-TBI. This study highlights the therapeutic potential of small-molecule inhibitors targeting STING for the treatment of trauma-induced inflammation and neuroprotective potential.

Proinflammatory cytokines IL-6, IL-1β, TNF-α in infective endocarditis

Aim. To study the features of macrophages in the tissues of resected valves in operated patients with infective endocarditis (IE), their significance and interaction with inflammatory markers to improve the effectiveness of IE diagnosis. Materials and methods. Prospectively the research included 25 adult patients with active IE (Duke criteria 2015) and 24 patients with heart defects without IE, hospitalized in a cardiosurgical hospital in Moscow (2021–2022). A standard laboratory and instrumental examination was carried out for the diagnosis of IE, including etiological diagnosis with microbiological and molecular biological methods, and echocardiographic examination of heart. Additionally, the neutrophil-to-lymphocyte ratio (NLR) was calculated. The study of macrophages was carried out in the tissues of resected valves with the determination of the expression of pro- and anti-inflammatory cytokine genes, macrophage markers (CD 68+) using real-time PCR. Results. Increased expression of proinflammatory cytokines IL-1β, TNF-α and IL-6 was revealed in the group of operated patients with IE with significant differences in IL-1β (CI [IQR] 0.00367 [0.00047–0.01553] vs 0.00018 [0.00012–0.00262]; p0.05) and IL-6 (CI [IQR] 0.00367 [0.00047–0.01553] vs 0.00018 [0.00012–0.00262]; p0.05) and IL-6 (CI [IQR] 0.00338 [0.00066–0.01674] vs 0.00054 [0.00044–0.00378]; p0.05). The expression of anti-inflammatory cytokines in valve tissues prevailed in the control group without significant differences from patients with IE. The macrophage marker CD 68+ was revealed in all examined patients with a significant quantitative predominance in the group of patients with IE. There were no differences in the expression of pro- and anti-inflammatory cytokines depending on the presence of embolic events, intracardiac complications, etiological affiliation to S. aureus, as well as hospital mortality and combined endpoint (death from all causes or recurrence of IE 6 months after surgery) in patients with IE with or without events. Cytokines IL-1β and IL-6 positively correlated with each other, with leukocytes and NLR. ROC analysis determined that IL-1β and NLR had the most favorable features for the diagnosis of IE [IL-1β AUC 0.816 (p=0.02), NLR AUC 0.807 (p=0.03)]. IL-6 did not show a diagnostic value in IE. The threshold value for IL-1β was 0.00029 (sensitivity 86.4%, specificity 60.0%, prognostic value of negative result 75.0% and positive 76.0%, AUC 0.761; p=0.008). Conclusion. The valve macrophages of patients with IE express elevated levels of proinflammatory cytokines IL-1β and IL-6, regardless of etiological affiliation or complicated course of IE. IL-1β has a high diagnostic value for determining the inflammatory activity in IE.

Exosomes derived from HUVECs alleviate ischemia-reperfusion induced inflammation in neural cells by upregulating KLF14 expression.

Neuroinflammation plays a key role in the progression of secondary brain injury after ischemic stroke, and exosomes have been increasingly recognized to eliminate inflammatory responses through various mechanisms. This study aimed to explore the effect and possible mechanism of human umbilical vein endothelial cells derived exosomes (H-EXOs) on neuroinflammation. We established a transient middle cerebral artery occlusion/reperfusion (tMCAO/R) in male rats and oxygen-glucose-deprivation/reoxygenation (OGD/R) model in cultured neurons to mimic secondary brain injury after ischemic stroke in vivo. H-EXOs were administered at the same time of reperfusion. Results showed that the production of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6, and the transcription factor Krüppel-like factor 14 (KLF14) were significantly increased both in rat brain tissue and cultured neural cells after ischemic-reperfusion (I/R) injury. H-EXOs treatment significantly improved the cultured cell viability, reduced infarct sizes, mitigated neurobehavioral defects, and alleviated the expression of pro-inflammatory cytokines compared with the control group, indicating that H-EXOs exerted anti-inflammatory effect against I/R injury. Further studies revealed that the anti-inflammatory effect of H-EXOs could be weakened by small-interfering RNA (siKLF4) transfection. KLF14 was a protective factor produced during cerebral ischemia-reperfusion injury. In conclusion, H-EXOs protect neurons from inflammation after I/R injury by enhancing KLF14 expression.

Identification of IQ Motif Containing GTPase Activating Protein 2, IQGAP-2 as an interacting partner with the Human Colonic Thiamin Pyrophosphate Transporter (hcTPPT): Effects on physiology and Cell Biology of the Transporter

Background:Humans/mammals obtain vitamin B1 from two sources: the diet and the gut-microbiota. Considerable amount of the microbiota-generated vitamin B1 exists in the form of thiamin pyrophosphate (TPP). We have previously shown that colonocytes process an effcient and specific carrier-mediated process for uptake of the generated TPP that involves the human colonic thiamine pyrophosphate transporter (hcTPPT, product of the SLC44A4 gene). We have also shown that expression of the cTPPT along the GI tract is restricted to the large intestine and have characterized different aspects of its regulation at both the transcriptional and post-transcriptional levels.Aim:To determine whether the hcTPPT has interacting protein partner(s) in human colonocytes that affect its functionality and/or cell biology.Methods: Yeast Two-Hybrid system (Y2H) was used to screen a random-primed normal human colonic cDNA library using the full-length SLC44A4 as a bait. Effect of knocking-down (with gene-specific siRNA) and over-expressing the putative interactor was examined using human-derived colonic epithelial NCM460 cells and human primary differentiated colonoid monolayers (obtained form organ donors). We also used confocal imaging, RT-qPCR and western blotting in our investigations. Results:Our Y2H screening have identified three putative interactors with the hcTPPT, namely: IQGAP-2, SNX-6, and DMXL-1. While all these putative interactors are expressed in human colonocytes, expression of the IQGAP-2 was the highest, and thus, we focused our investigation on this putative interactor. First, we verified the co-localization of IQGAP-2 with the hcTPPT in human colonic NCM460 cells using confocal imaging. We then examined the effect of expressing IQGAP-2 in NCM460 cells and in differentiated colonoids on carrier-mediated TPP uptake and observed significant induction with both models. Knock-down IQGAP-2 (with gene-specific siRNA), on the other hand, led to a significant decrease in TPP uptake by colonocytes. We also found that expressing the IQGAP-2 in colonocytes lead to a marked enhancement in hTPPT protein stability (increased protein half-life). Interestingly, we also found that expression of IQGAP-2 was suppressed by factors/conditions (e.g. lipopolysaccharide, pro-inflammatory cytokine TNF-α and hypoxia, respectively) that inhibit colonic TPP uptake.Conclusion: The accessory protein IQGAP-2 is an interacting partner with the hcTPPT in human colonocytes and that the interaction has physiological/cell biological consequences. Supported by NIH grants AA-018071 & DK-56061, and VA grants 101BX001142 & IK6BX006189. 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.

Male Offspring Exposed to Maternal Hyperandrogenemia Have Exaggerated Pressor Response to Angiotensin II That Is Partially Attenuated by Etanercept

Background: Polycystic ovary syndrome (PCOS) is the most common endocrinopathy and major cause of hyperandrogenemia in women. Our group has previously shown that male offspring (F1) exposed to maternal hyperandrogenemia are born smaller (indicative of intra-uterine growth restriction) and have exaggerated pressor response to angiotensin (Ang) II as adults. Tumor necrosis factor-α (TNF-⍺) has been shown to play a role in Ang II-mediated hypertension in different models. Increased pressor response to Ang II could signal increased risk of cardiovascular diseases later in life. Therefore, the present study was designed to test the hypothesis that exposure to maternal hyperandrogenemia programs intrarenal inflammation that plays a role in the exaggerated pressor response to Ang II in male F1s. Methods: At 4 weeks (wks) of age, female Sprague Dawley (SD) rats were fed a purified diet (10% kcal from fat) and implanted (s.c.) with either 5α-dihydrotestosterone pellets (7.5 mg/90 d; hyperandrogenemic females (HAFs)) or placebo pellets (PBO), that were replaced throughout life. At 9-12 wks of age, HAFs and PBOs were paired with SD males and allowed to get pregnant, deliver and lactate their offspring (F1-HAF and F1-PBO) until weaning (3 wks of age). Urine was collected from male F1s (n = 6-10/grp, 4-5 months (mos) of age, 1 pup/litter) which were then euthanized. Kidney cortices were used for the assay of pro-inflammatory cytokines using Bioplex. Another batch of male F1s (n = 4-7/grp, 8-10 mos of age, 1 pup/litter) were implanted with radiotelemetry transmitters. After recovery, male F1s were treated with either etanercept (a TNF-⍺ antagonist; ETA; 1.2 mg/kg, twice a week s.c. injection; ETA-F1-HAF and ETA-F1-PBO grps) or vehicle (0.9% saline; VEH; VEH-F1-HAF and VEH-F1-PBO grps) for 2 wks. Mean arterial pressure (MAP) was then measured for 6 days (Pre-enalapril), followed by treatment with enalapril (25 mg/kg/d for 9 d in drinking water) then Enalapril + Ang II (150 ng/kg/min for 6 d, s.c. minipump). Results: Male F1-HAF had significantly higher renal cortical interleukin (IL)-1α, IL-1β, IL-7 and TNF-α (203.8 ± 14 vs 141.7 ± 10, 331.4 ± 27 vs 233.7 ± 13, 524.7 ± 41 vs 379.1 ± 46, and 579.8 ± 47 vs 423.7 ± 47 pg/g tissue, respectively; p<0.05 for all), proteinuria (14.1 ± 2.4 vs 8.6 ± 0.9 mg/24 h, p<0.05), urinary kidney injury molecule-1 (1058.0 ± 123 vs 557.0 ± 122 pg/24 h, p<0.05) compared to male F1-PBO. VEH-F1-HAF had exaggerated pressor response to Ang II compared to VEH-F1-PBO starting at day 3 until day 6 of Ang II (154 ± 6 vs 128 ± 6 mmHg, p<0.05) despite similar MAP pre-enalapril (116 ± 3 vs 118 ± 2 mmHg, p=NS) and after enalapril (97 ± 3 vs 96 ± 1 mmHg, p=NS). ETA-F1-HAF had significantly lower MAP compared to VEH-F1-HAF on days 3 (119 ± 10 vs 138 ± 3 mmHg, p<0.05), 4 (131 ± 9 vs 149 ± 5 mmHg, p<0.05) and 5 (142 ± 6 vs 154 ± 5 mmHg, p<0.05) of Ang II treatment. Yet, on day 6 of Ang II treatment, MAP was similar between VEH-F1-HAF and ETA-F1-HAF (154 ± 6 vs 146 ± 5 mmHg, p=NS) and significantly higher in both groups compared to ETA-F1-PBO (133 ± 12 mmHg) and VEH-F1-PBO (128 ± 6 mmHg) [p < 0.05]. Conclusion: Exaggerated pressor response to Ang II in male F1s exposed to maternal hyperandrogenemia could be partly mediated by the pro-inflammatory cytokine TNF-α. Future studies will determine the role of different immune cells in this response and the underlying epigenetic changes in male F1s exposed to maternal hyperandrogenemia. This work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number P20GM121334 (JFR, NMS), R01HL135089 (JFR), R01AG075963 (JFR). The content is solely the responsibility of the authors and does not necessarily represent the offcial views of the National Institutes of Health. This work was also supported by the American Heart Association Career Development Award Number 938320 (NMS). 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.

Abstract 1101: Tnfa-setdb2 Pathway Regulates Fibroblast To Myofibroblast Transition In Wound Repair

Effective healing following tissue injury is predicated on an intimate coordination between immune cells and structural cells, where recruited immune cells induce or repress structural cell proliferation and gene expression. Early after tissue injury, fibroblasts begin to transition to myofibroblasts to contract and close wounds. In pathologic conditions where wounds fail to heal (such as diabetes), fibroblasts do not increase myofibroblast gene expression; however the mechanisms that control this fibroblast transition, in normal or diabetic conditions are not defined. Our group has previously identified that epigenetic alterations can control these phenotypic switches in immune and structural cells. Here, using human wound scSeq and murine transgenic models, we examined the mechanisms that may regulate this transition in the setting of injury. First, we identified that dermal fibroblasts when treated with the pro-inflammatory cytokine TNFa, significantly repressed myofibroblast genes including acta2, tgln, myl9, cald1, fibronectin and periostin. To identify possible the epigenetic mechanisms downstream of TNFa that modulate fibroblast phenotype, we performed an unbiased epigenetic superarray on TNFa-stimulated dermal fibroblasts, where SETDB2 was significantly upregulated and was also confirmed by qPCR. SiRNA-mediated knockdown of Setdb2 in murine fibroblasts prevented TNFa-mediated repression of myofibroblast genes, suggesting regulation of this transition by Setdb2. Human scSeq identified increased Setdb2 associated with increased TNFa signaling in wound fibroblasts, suggesting an interaction of these pathways. Interestingly, we found increased TNFa in macrophages and SETDB2 in fibroblasts from human diabetic wound scSeq and a murine diabetic (DIO) model, suggesting this pathway may contribute to the decreased conversion of fibroblasts to myofibroblasts seen in non-healing diabetic wounds. This work identifies setdb2 as a TNFa-mediated regulator of fibroblast to myofibroblast transition in wound repair and may identify a new therapeutic target for impaired tissue regeneration.

The role of CD20+ T cells: Insights in human peripheral blood.

CD20+ T cells constitute a small subset of T cells. These are found among CD4+, CD8+, CD4+CD8+, CD4-CD8- T, and TCRγδ+ T cells, and have been poorly characterized. The aim of this study was to characterize peripheral blood (PB) CD20+ T cells and compare them to their PB CD20- T cell counterparts. PB from 17 healthy individuals was collected. The distribution of CD20+ T cells among maturation-associated T cells compartments (naïve, central memory, transitional memory, effector memory, and effector T cells), their polarization, activation status, and expression of immune-regulatory proteins were evaluated by flow cytometry. Their function was also assessed, by measuring IFN-γ, TNF-α, and IL-17 production. Compared with CD20- T cells, CD20+ T cells represent a higher proportion of transitional memory cells. Furthermore, CD20+ T cells display a proinflammatory phenotype, characterized by the expansion of Th1, Th1/17, and Tc1 cell subsets , associated to a high expression of activation (CD25) and exhaustion (PD-1) markers. In addition, the simultaneous production of the proinflammatory cytokines IFN-γ, TNF-α, and IL-17 was also detected in CD4+CD20+ T cells. Our results show that CD20+ T cells are phenotypically and functionally different from CD20- T cells, suggesting that these cells are a distinct subset of T cells.

Mechanisms Involved in the Antihypertensive Effects of Arbequina Table Olives

The Mediterranean Diet is associated with antihypertensive properties and prevention of cardiovascular disease. This dietary pattern includes the intake of Olea europaea L. derivatives such as extra virgin olive oil and table olives, characterized by a high content of bioactive compounds. Recently, it has been demonstrated that the supplementation of Arbequina table olives (AO) reduces blood pressure (BP) in spontaneously hypertensive rats (SHR). The aim of the present study was to elucidate the possible mechanisms involved in the antihypertensive effects of AO. Consequently, we have studied different plasmatic biomarkers (angiotensin II, Ang II; malondialdehyde, MDA; interleukin 6, IL6; tumor necrosis factor α, TNFα) and the fecal microbiota composition. Experiments were performed using male SHR from 14 to 21 weeks of age. SHR strain was divided into a control (SHR-c) and a treated group (SHR-o) that received water or AO (3.85 g/kg) by gavage, respectively. At the end of the experiment, BP was measured using a non-invasive automatic analyzer; Ang II, IL6, and TNFα were determined by Elisa, and MDA by spectrophotometric analysis. Fecal microbiota was analyzed by sequencing 16S rRNA gene using the Illumina MiSeq platform. Data were analyzed by t-Student or the Mann–Whitney U test, and statistical significance was considered when p<0.05. AO supplementation decreased systolic and diastolic BP by approximately 19 mmHg in SHR-o with respect to SHR-c. The decrement in BP was accompanied by a decrease in MDA (39%) and Ang II (32%) in SHR-o without changes in the concentrations of the proinflammatory cytokines IL6 and TNFα. Moreover, regular AO intake induced changes in the microbiota associated with the normalization of BP. Our results showed variations in relative abundance in genera such as Akkermansia, Sutterella, and Peptoniphilus, that were well correlated with the decrease in BP, MDA, and Ang II. In conclusion, OA supplementation reduced oxidative stress and Ang II associated with changes in the fecal microbiota that may explain the antihypertensive effect elicited by consumption of the fruit of Olea europaea L. Supported by grants AGL2013-41188 & 2021SGR300. INSA Maria de Maeztu Unit of Excellence grant CEX2021-001234-M. 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.

Loss of Intestinal SNX27 Promotes Barrier Dysfunction and Inflammatory Responses

SNX27 belongs to the sorting nexin (SNX) family of proteins that play a crucial role in the endocytic pathway. SNX27 carries a unique PDZ domain, mediates recycling of endocytosed transmembrane proteins, and is critical for neurodevelopmental processes. However, its role in the intestine has not been explored yet. Here we aim to determine the previously unknown roles of SNX27 in regulating intestinal tissue homeostasis, epithelial barrier integrity, and related disorders. We have generated a novel mouse model of SNX27 deletion form intestinal epithelial cells (SNX27ΔIEC). We challenged the mice with DSS-induced colitis and AOM/DSS-induced colorectal cancer (CRC). We performed in vitro SNX27 loss of function studies via siRNA knockdown (KD) in SKCO15 cells. We further analyzed SNX27 expression in IBD and CRC patients via online human datasets. SNX27ΔIEC mice had shorter colon, small intestines, and heavier spleens. We found increased intestinal permeability upon SNX27 deletion. SNX27ΔIEC mice had significantly upregulated cleaved Caspase-3 (pro-apoptotic) and downregulated Bcl-xL (anti-apoptotic) protein levels. TUNEL staining also showed greater apoptotic cells in SNX27ΔIEC mice. Tight-junction proteins Claudin10 and ZO-1, as well as adherens-junction proteins ꞵ-catenin and E-cadherin, were downregulated in SNX27ΔIEC mice. The histological inflammation score and mRNA levels of pro-inflammatory cytokines IL-6 and TNF-α were higher in SNX27ΔIEC mice. Protein expression level of phospho-p65 was increased in SNX27ΔIEC mice, indicating NFkB pathway activation. Upon challenging with intestinal disorders, SNX27ΔIEC mice were more sensitive towards DSS and AOM/DSS treatments as they lost more bodyweight, had higher intestinal permeability levels, and heavier spleens. SNX27 KD in vitro reduced protein levels of PCNA and delayed wound closure in a wound healing assay. SNX27-KD cells had higher nuclear translocation of phospho-p65. Additionally, we observed cytoplasmic localization rearrangement and downregulation of ꞵ-catenin and E-cadherin, key mediators of epithelial-mesenchymal transition. Lastly, human datasets GSE11223, 6731, 8671, 21510 revealed significant downregulation of SNX27 expression in IBD and CRC patients, suggesting towards clinical relevance of SNX27 in intestinal disorders. Overall, intestinal deletion of SNX27 promotes apoptosis, inflammation, and disrupts epithelial barrier that increases susceptibility towards colonic inflammation and tumorigenesis. Our results indicate a novel role of SNX27 in regulating intestinal tissue homeostasis. Understanding the mechanisms of lower SNX27 in IBD and CRC will provide insights into new prevention and targets against these chronic diseases. We would like to acknowledge the VA Merit Award 1 I01BX004824-01, DOD CDMRP BC191198, the NIDDK/National Institutes of Health grants R01 DK105118 and R01DK114126 to Jun Sun. The study sponsors performed no role in the study design, data collection, analysis, and interpretation of data. The contents do not represent the views of the United States Department of Veterans Affairs or the United States Government. 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.

Mildronate ameliorates kidney injury and reduces inflammation in Sickle Cell Disease mice

INTRODUCTION: Sickle cell disease (SCD) is a hereditary hemoglobin disorder characterized by hemolytic anemia, vaso-occlusive crisis and chronic inflammation that affects multiple organs including kidney, liver, brain, spleen, and heart. Renal disease, the most common complication in SCD, is a progressive disease that begins in childhood as glomerular hyperfiltration, then advances to albuminuria, following by a fast decline in glomerular filtration. It may further progress to a renal failure in adults. More than 50% of SCD patients develop chronic kidney disease (CKD), and 14-18% progress to the end-stage kidney disease. Renal injury in SCD results from a cascade of events starting with chronic red blood cells sickling and hemolysis leading to increased blood viscosity, microvascular obstruction and dilation, hypoxia in the medulla, oxidative stress, and inflammation. Mildronate (meldonium) is an anti-ischemic and anti-inflammatory medication approved in Eastern Europe. HYPOTHESIS: We hypothesized that mildronate administration reduces renal medullary hypoxia and decreases oxidative stress and inflammation ameliorating renal injury in SCD mice. METHODS: The study was approved by the Howard University IACUC. Townes SCD mice, expressing human HbS, and control mice, expressing human HbA, were injected with mildronate (400 mg/kg in saline) intraperitoneally or saline as vehicle control over the course of ten days. Blood was collected and used for hematological parameters analysis. Cytokines were measured by Bio-Plex suspension array in plasma. Hypoxyprobe Biotin kit (Hypoxyprobe Inc) was used for visualization of organ hypoxia. The effect of Mildronate on systemic and renal oxidative stress was measured by malondialdehyde (MDA) using ELISA (Abcam). Urinary KIM-1 was measured by TIM-1/KIM-1 ELISA kit (R&D Systems). RESULTS: Mildornate injections did not affect hematological parameters of SCD mice including hematocrit, MCV, MCH, CMCH, erythrocytes and hemoglobin. Mildronate administration resulted in a significant reduction of the circulating pro-inflammatory cytokines IFN-γ, IL-1β and TNF-α and a trend toward reduction of IL-6, and IL-17 in SCD mice. We found higher level of hypoxia in the medulla of SCD mice compared to control mice. Mildronate injections did not reduced hypoxia in renal medulla of SCD mice. Also, mildronate injections did not change MDA levels in plasma or kidney of SCD mice. We found a significant reduction of proteinuria in SCD mice injected with mildronate compared to SCD mice injected with vehicle. Analysis of H&E-stained kidneys showed significant reduction of glomerular size and medullar congestion areas in the mildronate injected SCD mice. Urinary KIM-1 levels were reduced by mildronate administration. CONCLUSIONS: Our results demonstrate that mildronate ameliorates glomerular and tubular injury in SCD mice and reduces capillary congestion. Mildronate improves renal function, in part by reducing inflammation but not through the reduction of hypoxia or oxidative stress. In future, we plan to assess the mildronate’s impact on kidney function and vessel inflammation. This work was supported by NIH grants R01HL125005, U54MD007597, and SC1HL150685. We thank Xiaomei Niu for helping with the Bio-Plex assay. 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.

Impact of combined alpha-lipoic acid and mitoquinone supplementation on myocardial infarction in aged rats: Heart performance and molecular mechanisms

BackgroundThis study aimed to investigate the effects of combined alpha-lipoic acid (ALA) and mitoquinone (Mito Q) supplementation on cardiac function and the underlying mechanisms in aged rats with myocardial infarction (MI). MethodsThe aged rats underwent left anterior descending artery (LADA) occlusion for 30 min, followed by reperfusion for 24 h. ALA (100 mg/kg, gavage) and Mito Q (10 mg/kg, IP) were administered daily for two weeks before ischemia. Cardiac function, inflammatory, and apoptotic markers were evaluated 24 h after ischemia. ResultsThe results of this study indicated that the administration of the combination of ALA and Mito Q significantly improved cardiac function. This improvement was linked to a reduction in the expression of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β (P < 0.001) and apoptotic markers (Bax, caspase-3, and Cyt-c), as well as a decrease in the percentage of TUNEL-positive cells (P < 0.001). ConclusionThe study revealed that combined intervention synergistically mitigated cardiac dysfunction by suppressing inflammatory and apoptotic pathways in aged rats with MI. Further research is needed to validate the potential of ALA and Mito Q as therapeutic options for elderly people at risk of heart attacks.

Vitamin C alleviates rheumatoid arthritis by modulating gut microbiota balance.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic and symmetric in-flammation. Our previous research revealed an imbalance in the gut flora of RA patients and showed that certain gut microbiota can accelerate RA progression by enhancing vitamin C degradation. However, it is unclear whether vitamin C supplementation could improve the gut microbiota to prevent the development of arthritis by interfering with the gut-joint axis. In this work, we aimed to evaluate the effects of vitamin C in regulating the gut microbiota and to elucidate its potential role in the onset and progression of RA in a mouse model, thus providing a basis for the development of new intervention strategies and treatments for RA. In this study, collagen-induced arthritis (CIA) mouse models, biochemical, histological and 16S rRNA microbiological methods were used to investigate the role and possible mechanism of vitamin C in rheumatoid arthritis. The results showed that treatment of CIA mice with vitamin C effectively rescued the gut mi-crobiota imbalance and suppressed the inflammatory response associated with RA, and effectively alleviated arthritis symptoms in mice in which levels of the pro-inflammatory cytokines IL-6 and TNF-α were specifi-cally reduced. In conclusion, our results demonstrate the potential of vitamin C as a potential therapeutic choice for RA.

Naringin Inhibits Macrophage Foam Cell Formation by Regulating Lipid Homeostasis and Metabolic Phenotype.

Imbalances in lipid uptake and efflux and inflammation are major contributors to foam cell formation, which is considered a therapeutic target to protect against atherosclerosis. Naringin, a citrus flavonoid abundant in citrus fruits, has been reported to exert an antiatherogenic function, but its pharmacological mechanism is unclear. Naringin treatment effectively inhibits foam cell formation in THP-1 and RAW264.7 macrophages. In this study, mechanically, naringin maintained lipid homeostasis within macrophages through downregulation of the key genes for lipid uptake (MSR1 and CD36) and the upregulation of ABCA1, ABCG1 and SR-B1, which are responsible for cholesterol efflux. Meanwhile, naringin significantly decreased the cholesterol synthesis-related genes and increased the genes involved in cholesterol metabolism. Subsequently, the results showed that ox-LDL-induced macrophage inflammatory responses were inhibited by naringin by reducing the proinflammatory cytokines IL-1β, IL-6 and TNF-α, and increasing the anti- inflammatory cytokine IL-10, which was further verified by the downregulation of pro-inflammatory and chemokine-related genes. Additionally, we found that naringin reprogrammed the metabolic phenotypes of macrophages by suppressing glycolysis and promoting lipid oxidation metabolism to restore macrophage phenotypes and functions. These results suggest that naringin is a potential drug for the treatment of AS as it inhibits macrophage foam cell formation by regulating metabolic phenotypes and inflammation.

Discovery of novel pyrazole based Urea/Thiourea derivatives as multiple targeting VEGFR-2, EGFRWT, EGFRT790M tyrosine kinases and COX-2 Inhibitors, with anti-cancer and anti-inflammatory activities

A novel series of pyrazole derivatives with urea/thiourea scaffolds 16a-l as hybrid sorafenib/erlotinib/celecoxib analogs was designed, synthesized and tested for its VEGFR-2, EGFRWT, EGFRT790M tyrosine kinases and COX-2, pro-inflammatory cytokines TNF-α and IL-6 inhibitory activities. All the tested compounds showed excellent COX-2 selectivity index in range of 18.04–47.87 compared to celecoxib (S.I. = 26.17) and TNF-α and IL-6 inhibitory activities (IC50 = 5.0–7.50, 6.23–8.93 respectively, compared to celecoxib IC50 = 8.40 and 8.50, respectively). Screening was carried out against 60 human cancer cell lines by National Cancer Institute (NCI), compounds 16a, 16c, 16d and 16 g were the most potent inhibitors with GI% ranges of 100 %, 99.63–87.02 %, 98.98–43.10 % and 98.68–23.62 % respectively, and with GI50 values of 1.76–15.50 µM, 1.60–5.38 µM, 1.68–7.39 µM and 1.81–11.0 µM respectively, in addition, of showing good safety profile against normal cell line (F180). Moreover, compounds 16a, 16c, 16d and 16 g had cell cycle arrest at G2/M phase with induced necrotic percentage compared to sorafenib of 2.06 %, 2.47 %, 1.57 %, 0.88 % and 1.83 % respectively. Amusingly, compounds 16a, 16c, 16d and 16 g inhibited VEGFR-2 with IC50 of 25 nM, 52 nM, 324 nM and 110 nM respectively, compared to sorafenib (IC50 = 85 nM), and had excellent EGFRWT and EGFRT790M kinase inhibitory activities (IC50 = 94 nM, 128 nM, 160 nM, 297 nM), (10 nM, 25 nM, 36 nM and 48 nM) respectively, compared to both erlotinib and osimertinib (IC50 = 114 nM, 56 nM) and (70 nM, 37 nM) respectively and showed (EGFRwt/EGFRT790M S.I.) of (range: 4.44–9.40) compared to erlotinib (2.03) and osmertinib (1.89).

Pharmacological Activities of Lonicerae japonicae flos and Its Derivative-"Chrysoeriol" in Skin Diseases.

Nine electronic databases were searched, including WanFang Data, PubMed, Science Direct, Scopus, Web of Science, Springer Link, SciFinder, and China National Knowledge Infrastructure (CNKI), without regard to language constraints. The pharmacological activities of chrysoeriol from Lonicerae japonicae flos to fight against skin diseases were explained and evaluated through the literature review of either in vitro or in vivo studies. Chrysoeriol decreased the mRNA levels of proinflammatory cytokines IL-6, IL-1β, and TNF-α. These were transcriptionally regulated by NF-κB and STAT3 to combat skin inflammation. It also showed promising actions in treating many skin ailments including wound healing, depigmentation, photoprotection, and antiaging. The cutaneous route is the best delivery approach to chrysoeriol across the skin barrier. However, toxicity, dosage, and safety assessments of chrysoeriol in a formulation or nanochrysoeriol on the human epidermis for application in skin diseases must be further investigated.

Preventive and therapeutic vascular photobiomodulation decreases the inflammatory markers and enhances the muscle repair process in an animal model

Photobiomodulation therapy (PBM) has shown positive effects when applied locally to modulate the inflammatory process and facilitate muscle repair. However, the available literature on the mechanisms of action of vascular photobiomodulation (VPBM), a non-invasive method of vascular irradiation, specifically in the context of local muscle repair, is limited. Thus, this study aimed to assess the impact of vascular photobiomodulation (VPBM) using a low-level laser (LLL) on the inflammatory response and the process of skeletal muscle repair whether administered prior to or following cryoinjury-induced acute muscle damage in the tibialis anterior (TA) muscles. Wistar rats (n = 85) were organized into the following experimental groups: (1) Control (n = 5); (2) Non-Injury + VPBM (n = 20); (3) Injured (n = 20); (4) Pre-VPBM + Injury (n = 20); (5) Injury + Post-VPBM (n = 20). VPBM was administered over the vein/artery at the base of the animals' tails (wavelength: 780 nm; power: 40 mW; application area: 0.04 cm2; energy density: 80 J/cm2). Euthanasia of the animals was carried out at 1, 2, 5, and 7 days after inducing the injuries. Tibialis anterior (TA) muscles were collected for both qualitative and quantitative histological analysis using H&E staining and for assessing protein expression of TNF-α, MCP-1, IL-1β, and IL-6 via ELISA. Blood samples were collected and analyzed using an automatic hematological analyzer and a leukocyte differential counter. Data were subjected to statistical analysis (ANOVA/Tukey). The results revealed that applying VPBM prior to injury led to an increase in circulating neutrophils (granulocytes) after 1 day and a subsequent increase in monocytes after 2 and 5 days, compared to the Non-Injury + VPBM and Injured groups. Notably, an increase in erythrocytes and hemoglobin concentration was observed in the Non-Injury + VPBM group on days 1 and 2 in comparison to the Injured group. In terms of histological aspects, only the Prior VPBM + Injured group exhibited a reduction in the number of inflammatory cells after 1, 5, and 7 days, along with an increase in blood vessels at 5 days. Both the Prior VPBM + Injured and Injured + VPBM after groups displayed a decrease in myonecrosis at 1, 2, and 7 days, an increase in newly-formed and immature fibers after 5 and 7 days, and neovascularization after 1, 2, and 7 days. Regarding protein expression, there was an increase in MCP-1 after 1 and 5 days, TNF-α, IL-6, and IL-1β after 1, 2, and 5 days in the Injured + VPBM after group when compared to the other experimental groups. The Prior VPBM + Injured group exhibited increased MCP-1 production after 2 days, in comparison to the Non-Injury + VPBM and Control groups. Notably, on day 7, the Injured group continued to show elevated MCP-1 protein expression when compared to the VPBM groups. In conclusion, VPBM effectively modulated hematological parameters, circulating leukocytes, the protein expression of the chemokine MCP-1, and the proinflammatory cytokines TNF-α and IL-1β, ultimately influencing the inflammatory process. This modulation resulted in a reduction of myonecrosis, restoration of tissue architecture, increased formation of newly and immature muscle fibers, and enhanced neovascularization, with more pronounced effects when VPBM was applied prior to the muscle injury.

P162 Dimethyl fumarate modulates T cell metabolism and function in systemic lupus erythematosus patient samples

Abstract Background/Aims Activated CD4+T cells make a significant contribution to inflammation in systemic lupus erythematosus (SLE) through secretion of proinflammatory cytokines such as IFN-γ and TNF-α. CD4+T cells can also express activating NK receptors such as NKG2D that can engage with stress-induced ligands such as MICA, as noted in lupus nephritis, and mediate nonspecific tissue injury. We know that cellular metabolism regulates the activation of T cells. Evidence from patients with multiple sclerosis indicates that dimethyl fumarate (DMF), an electrophile, targets cellular metabolism to modulate CD4+T cell activation and function. Here, we investigated whether DMF modulates T cell metabolism and function in samples from patients with SLE in a series of in vitro experiments. Methods All experiments were performed using isolated T cells from freshly drawn whole blood samples from patients with SLE. T cells were isolated using negative selection using stem cell or Miltenyei magnetic bead separation kit. Isolated T cells were activated with anti-CD3 and IL-2 and incubated with either DMF at 25μM concentration or DMSO alone for three or seven days at 37 °C and 5% CO2 before harvesting. Results In Seahorse experiments, after three days of incubation, dimethyl fumarate (DMF) inhibited Figure A-i) the oxygen consumption rate (OCR) and A-ii) extra cellular acidification rate (ECAR) in isolated T cells when compared with samples incubated with vehicle, dimethyl sulfoxide (DMSO). Our results revealed that DMF significantly inhibited: 1) aerobic glycolysis and oxidative phosphorylation in activated T cells from patients with SLE (n = 4) (Figure A- i and ii) in vitro; 2) T cell activation and proliferation as assessed by a reduction in the frequency of CD69 (n = 4) and D) Ki67 (n = 2) positivity, respectively (Figure B-E); 3) Collectively, these results suggest that DMF inhibits T cell activation and proliferation in samples from patients with SLE. After 7 days of incubation, DMF significantly inhibited the expression of activating NK receptors CD158a and NKG2D on T cells whereas there was a trend toward enhancing the expression of inhibitory NK receptors NKG2A and CD158b (Figure F-I). After 7 days of incubation, DMF significantly reduced intracellular expression of IFN-γ, TNF-α, IL-17 and secretion of pro-inflammatory cytokines IFN-γ and TNF-α in supernatants (Figure J - N). Conclusion Our preliminary data indicated that, in SLE patient samples, DMF modulates metabolic programming of CD4+ T cells and inhibits glycolysis and oxidative phosphorylation of activated CD4+ T cells. In vitro, DMF inhibited: T cell activation; proliferation; the expression of activating NK receptors; and secretion of proinflammatory cytokines from T cells patients with SLE. Taken together with the existing safety data in humans, these data provide mechanistic rationale for investigating the potential of dimethyl fumarate, with a distinct mechanism of action, as a novel therapy for SLE. Disclosure L. Kell: None. K. Shah: None. S. Taylor: None. R. De Maeyer: None. D.A. Isenberg: None. D. Sen: None. M. Castelino: None. M. Leandro: None. A. Akbar: None. V. Reddy: Grants/research support; UCLH Biomedical research centre funded this work, Dr.Reddy's work was supported through MR/T024968/1 MRC-CARP Fellowship.