Concentrations Of Proinflammatory Cytokines Research Articles

Ameliorative Effect of Naringenin in 5-Fluorouracil-Induced Hepatotoxicity: An Experimental Study on Albino Wistar Rats

Background: Hepatotoxicity is an adverse complication in 5-Fluorouracil cancer treatment. Inflammation, oxidative stress, apoptosis and DNA damage are essential in the pathogenesis of liver damage. The present study evaluated the ameliorative effect of Naringenin (NG) in 5-FU at two different intervals in hepatic damage. Methods: Total (n=48) Albino Wistar rats were divided into 4 groups (N=12), Control-normal saline (group-1) per oral, 5-FU (group-2) @ 20 mg/kg b.wt was injected intraperitoneal (IP) for first 5 days, NG (group-3) @100 mg/kg b. wt/day orally for 28 days. Another group-4 was concurrently treated with NG along with 5-FU for 28 days and sacrificed on the 14th and 28th day of the experiment. Result: On exposure to 5-FU, biochemical variation of liver enzymes (ALT, AST, ALP and TP) were significantly increased while NG treatment significantly reduced these biomarkers in group 2 on 14th day and 28th day of the experiment. Furthermore, NG treatment significantly reduced lipid peroxidation and increases the antioxidant profile. It also significantly reduced elevated concentrations of pro-inflammatory cytokines like tumour necrosis factor (TNF-α), interleukin -1β (IL-1β), interleukin-6 (IL-6) with increased concentrations of interleukin-10 (IL-10) along with reduced immunohistochemical expression of nuclear factor kappa light chain enhancer of activated B cells (NF-κB) and Caspases -3 (Cas-3) by NG. On histopathology, pathological lesions were observed to support biochemical variations in group-2 rats. In group-4 rats, NG ameliorates liver enzymes, inflammatory cytokines and oxidative indices through an anti-oxidant, anti-inflammatory and anti-apoptotic property.

Abstract 6343: AnTenGagerTM, a novel “2+1” T cell engager platform, enables conditional T cell activation with reduced risk of CRS

Abstract Background: CD3+ T cell engaging bispecific antibodies (TCE) redirect T cells to attack targeted tumor cells by simultaneously binding TAA on cancer cells and CD3 complex on a T cell to form a TCR-independent immune synapse. Multiple TCEs have demonstrated promising therapeutic efficacy in the treatment of hematological malignancies. In contrast, suboptimal efficacy in the treatment of solid tumors and the risk of inducing cytokine release syndrome (CRS) continue to be major challenges. Several factors, such as format, affinity, and valency of the antibody, TAA copy number, and antigen size and epitope, can impact the therapeutic efficacy and safety of a TCE. In this study, we developed a novel “2+1” TCE platform, AnTenGagerTM, which enables conditional T cell binding and activation upon TAA-crosslinking, potent anti-tumor activity, and reduced risk of CRS. Methods: Humanized CD3 antibodies were derived from mouse hybridoma, followed by affinity optimization utilizing Computer Aided Drug Design. The Fab-scFv-Fab format was used as the backbone structure of AnTenGagers. The anti-CD3 scFv sequences and the length of peptide linkers between VH/VL, scFv/CH1, and scFv/CH2 were screened and compared in different preclinical assays. LALA mutations (L234A, L235A) were introduced to abolish the Fc receptor binding capability. In a series of in vitro and/or in vivo models, the efficacy, safety and developability of AnTenGagers targeting multiple TAAs, such as Her2, GD2, CDH6, LILRB4, FLT3 and GPRC5D, were evaluated. Results: A library of humanized parental CD3 antibodies with a broad spectrum of affinities were generated, some of which cross react with monkey CD3. AnTenGagers exhibited limited binding to CD3-positive cells before TAA-crosslinking, suggesting a reduced risk of CRS that is caused by systemic CD3+T cell activation. Four CD3 sequences with varying affinities were utilized to construct AnTenGagers with identical TAA-targeting arm. Compared to clinical benchmarks targeting the same TAA, all AnTenGagers demonstrated substantially lower binding capacity to CD3+ cells in the absence of TAA-crosslinking, while inducing increased cytotoxicity against target-positive tumor cells. In addition, they induced significantly less ex-vivo cytokine release by human PBMC than benchmarks. Notably, all AnTenGagers demonstrated enhanced in vivo efficacy compared to the benchmark, exhibiting complete response (CR) in PBMC humanized MM1S mouse CDX model while the serum concentration of proinflammatory cytokines was substantially lower in groups treated with AnTenGagers. AnTenGagers also demonstrated good developability with high expression yield, good thermostability and high stability under different stress conditions. Conclusions: These results suggested that AnTenGagerTM is a promising platform for developing the next generation of TCEs with enhanced efficacy and safety. Citation Format: Bing Hou, Hui Yuwen, Tengteng Li, Yijing Ren, Ao Sun, Zaoshun Hu, Jay Mei, Bo Shan. AnTenGagerTM, a novel “2+1” T cell engager platform, enables conditional T cell activation with reduced risk of CRS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6343.

A Mediterranean diet plan in lactating women with obesity reduces maternal energy intake and modulates human milk composition - a feasibility study.

Maternal obesity is associated with increased concentrations of human milk (HM) obesogenic hormones, pro-inflammatory cytokines, and oligosaccharides (HMOs) that have been associated with infant growth and adiposity. The objective of this pilot study was to determine if adherence to a Mediterranean meal plan during lactation modulates macronutrients and bioactive molecules in human milk from mothers with obesity. Sixteen healthy, exclusively breastfeeding women with obesity (body mass index ≥30 kg/m2) enrolled between 4 and 5 months postpartum. The women followed a 4-week Mediterranean meal plan which was provided at no cost. Maternal and infant anthropometrics, HM composition, and infant intakes were measured at enrollment and at weeks 2 and 4 of the intervention. Thirteen mother-infant dyads completed the study. Additionally, participants from an adjacent, observational cohort who had obesity and who collected milk at 5 and 6 months postpartum were compared to this cohort. Participants' healthy eating index scores improved (+27 units, p < 0.001), fat mass index decreased (-4.7%, p < 0.001), and daily energy and fat intake were lower (-423.5 kcal/day, p < 0.001 and-32.7 g/day, p < 0.001, respectively) following the intervention. While HM macronutrient concentrations did not change, HM leptin, total human milk oligosaccharides (HMOs), HMO-bound fucose, Lacto-N-fucopentaose (LNFP)-II, LNFP-III, and difucosyllacto-N-tetrose (DFLNT) concentrations were lower following the intervention. Infant intakes of leptin, tumor necrosis factor (TNF)-α, total HMOs, HMO-bound fucose, LNFP-III and DFLNT were lower following the intervention. Specific components of the maternal diet (protein and fat) and specific measures of maternal diet quality (protein, dairy, greens and beans, fruit and vegetables) were associated with infant intakes and growth. Adherence to a Mediterranean meal plan increases dietary quality while reducing total fat and caloric intake. In effect, body composition in women with obesity improved, HM composition and infants' intakes were modulated. These findings provide, for the first time, evidence-based data that enhancing maternal dietary quality during lactation may promote both maternal and child health. Longer intervention studies examining the impact of maternal diet quality on HM composition, infant growth, and infant development are warranted.

Effect of diets supplemented with coated plant essential oil on the growth performance, immunity, antioxidant activity, and fecal microbiota of weaned piglets.

This trial was conducted to compare the effect of diets supplemented with plant essential oil (PEO) and coated plant essential oil (CEO) on growth performance, immunity, antioxidant activity, and fecal microbiota of weaned piglets. A total of 360 21-day-old weaned piglets were randomly allocated into three groups, namely, CON, PEO, and CEO (basal diets supplemented with 0, 500 mg/kg PEO, and 500 mg/kg CEO, respectively) for a 4-week feeding trial. The results showed that dietary supplementation with CEO improved the average final weight and average daily gain, decreased the diarrhea rate, increased antioxidant enzyme activities, enhanced immunoglobulin concentrations, and decreased concentrations of pro-inflammatory cytokines in the serum of weaned piglets (p < 0.05). In addition, CEO addition increased the fecal concentrations of propionic acid and isovaleric acid of piglets (p < 0.05). Spearman correlation analysis showed that fecal microorganisms at the genus level were closely correlated with the volatile fatty acid concentrations. The present study indicated that PEO and CEO could improve growth performance, enhance immunity, and increase antioxidant capacity by modulating the microbial flora in weaned piglets. Moreover, CEO addition seemed to offer more positive results than of PEO addition.

Gallic acid promotes macrophage phagosome acidification and phagolysosome formation by activating NLRP3/mTOR signaling pathway

IntroductionGallic acid (GA) has a good therapeutic effect in bacteriological inhibition and plays a variety of functions in maintaining the stability of the immune system. The aim of the present study was to investigate the effect of GA on the bactericidal activity of macrophages against Vibrio vulnificus (Vv). MethodsA cell counting kit-8 (CCK-8) assay was carried out to test the cytotoxicity of GA on J774A.1 cells. Concentration of proinflammatory cytokines in J774A.1 cells were evaluated by ELISA. The internalization and degradation of Vv in the phagosomes were observed by transmission electron microscopy (TEM). The phagosome acidification and phagolysosome formation were detected to evaluate the bacteria-clearing function of J774A.1 cells. The bactericidal activity of GA in vivo was also investigated by collecting the survival time of Vv infected mice and observing the inflammatory infiltration of organs. ResultsOur results demonstrated that GA at 50 μM significantly inhibited the proinflammatory cytokines levels, promoted phagosome acidification and phagolysosome formation in J774A.1 cells with Vv infection. This may be related to the activation of NLRP3/mTOR signaling pathway. Additionally, GA treatment improves the survival and bactericidal activity of mice infected with Vv. ConclusionsIn summary, GA exerts bactericidal activity against Vv infection by regulating the formation and acidification of phagocytic lysosomes in macrophages.

Amelioration of diabetic nephropathy in mice by a single intravenous injection of human mesenchymal stromal cells at early and later disease stages is associated with restoration of autophagy

Background and aimsMesenchymal stromal cells (MSCs) a potentially effective disease-modulating therapy for diabetic nephropathy (DN) but their clinical translation has been hampered by incomplete understanding of the optimal timing of administration and in vivo mechanisms of action. This study aimed to elucidate the reno-protective potency and associated mechanisms of single intravenous injections of human umbilical cord-derived MSCs (hUC-MSCs) following shorter and longer durations of diabetes.MethodsA streptozotocin (STZ)-induced model of diabetes and DN was established in C57BL/6 mice. In groups of diabetic animals, human (h)UC-MSCs or vehicle were injected intravenously at 8 or 16 weeks after STZ along with vehicle-injected non-diabetic animals. Diabetes-related kidney abnormalities was analyzed 2 weeks later by urine and serum biochemical assays, histology, transmission electron microscopy and immunohistochemistry. Serum concentrations of pro-inflammatory and pro-fibrotic cytokines were quantified by ELISA. The expression of autophagy-related proteins within the renal cortices was investigated by immunoblotting. Bio-distribution of hUC-MSCs in kidney and other organs was evaluated in diabetic mice by injection of fluorescent-labelled cells.ResultsCompared to non-diabetic controls, diabetic mice had increases in urine albumin creatinine ratio (uACR), mesangial matrix deposition, podocyte foot process effacement, glomerular basement membrane thickening and interstitial fibrosis as well as reduced podocyte numbers at both 10 and 18 weeks after STZ. Early (8 weeks) hUC-MSC injection was associated with reduced uACR and improvements in multiple glomerular and renal interstitial abnormalities as well as reduced serum IL-6, TNF-α, and TGF-β1 compared to vehicle-injected animals. Later (16 weeks) hUC-MSC injection also resulted in reduction of diabetes-associated renal abnormalities and serum TGF-β1 but not of serum IL-6 and TNF-α. At both time-points, the kidneys of vehicle-injected diabetic mice had higher ratio of p-mTOR to mTOR, increased abundance of p62, lower abundance of ULK1 and Atg12, and reduced ratio of LC3B to LC3A compared to non-diabetic animals, consistent with diabetes-associated suppression of autophagy. These changes were largely reversed in the kidneys of hUC-MSC-injected mice. In contrast, neither early nor later hUC-MSC injection had effects on blood glucose and body weight of diabetic animals. Small numbers of CM-Dil-labeled hUC-MSCs remained detectable in kidneys, lungs and liver of diabetic mice at 14 days after intravenous injection.ConclusionsSingle intravenous injections of hUC-MSCs ameliorated glomerular abnormalities and interstitial fibrosis in a mouse model of STZ-induced diabetes without affecting hyperglycemia, whether administered at relatively short or longer duration of diabetes. At both time-points, the reno-protective effects of hUC-MSCs were associated with reduced circulating TGF-β1 and restoration of intra-renal autophagy.Graphical abstract

Neuroprotective effects of Shenghui decoction via inhibition of the JNK/p38 MAPK signaling pathway in an AlCl3-induced zebrafish (Danio rerio) model of Alzheimer's disease

Ethnopharmacological relevanceAlzheimer's disease (AD) is a multi-factorial degenerative disease, and multi-targeted therapies targeting multiple pathogenic mechanisms should be explored. Shenghui decoction (SHD) is an ancient traditional Chinese medicine (TCM) formula used clinically to alleviate AD. However, the precise mechanism of action of SHD as a therapeutic agent for AD remains unclear. Aim of the studyThis study investigated the neuroprotective properties and potential mechanisms of action of SHD in mitigating AD-like symptoms induced by AlCl3 in a zebrafish model. Materials and methodsActive components of SHD were detected using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Zebrafish were exposed to AlCl3 (200 μg/L) for 30 days to establish an AD zebrafish model. AlCl3-exposed zebrafish were treated with SHD or donepezil. Behavioral tests were used to assess learning and memory, locomotor activity, and AD-related anxiety and aggression in AlCl3-exposed zebrafish. Nissl staining and transmission electron microscopy were used to evaluate histological alterations in brain neurons. The concentrations of pro-inflammatory cytokines (tumor necrosis factor-α, TNF-α; interleukin-1β, IL-1β) were quantified using Enzyme-linked immunosorbent assay (ELISA). Markers of oxidative stress and cholinergic activity (acetylcholinesterase, AChE) were detected using biochemical assays. Western blotting and immunofluorescence were used to detect the protein expression levels of Aβ, p-tau, PSD-95, synaptophysin, TLR4, phosphorylation of NF-κB p65, p38, and JNK. ResultsFifteen SHD compounds were identified by UPLC-MS/MS analysis. SHD improved AlCl3-induced dyskinesia, learning and memory impairment, anxiety-like behavior, and aggressive behavior in zebrafish. AlCl3-exposed zebrafish showed AD-like pathology, overexpression of Aβ, hyperphosphorylated tau protein, marked neuronal damage, decreased expression of synaptic proteins, synaptophysin, and PSD-95, and impairment of synaptic structural plasticity. These effects were reversed by the SHD treatment. We also observed that SHD ameliorated oxidative stress and decreased AChE activity and inflammatory cytokine levels. These effects are similar to those observed for donepezil. Meanwhile, SHD could decrease the protein expression of TLR4 and inhibit phosphorylation of NF-κB, JNK, and p38 MAPK. These results demonstrate that SHD has the potential to exert neuroprotective effects, which may be partly mediated via inhibition of the JNK/p38 MAPK signaling pathway. ConclusionsOur findings revealed the therapeutic mechanism of SHD in mitigating AD progression and suggested that SHD is a potent neuroprotectant that contributes to the future development of TCM modernization and broader clinical applications.

Use of Monoglycerides and Diglycerides to Mitigate Poultry Production Losses: A Review.

Consumer preference dictates not only what food is consumed but also how that food is produced. Ingredients in livestock feed that are not antibiotics, not genetically modified, and not of animal origin but that are nutrient-like tend to be more acceptable to consumers, retailers, and producers. Mono- and diglycerides (MDG) fit these criteria, are commonly used in food, and are generally recognized as safe. But beyond being emulsifiers and a source of energy, MDG are also known to have antimicrobial, immune, and microbiome modulation and angiogenic activity. MDG in broiler diets have been shown to impact a variety of immune-related functions such as the regulation of circulating antibodies, as well as decreased gene expression or protein concentration of pro-inflammatory cytokines like TNF-α, IL-1β, IL-6, and IFN-γ. In addition, MDG can affect metabolic function and intestinal integrity. Results of this review show that MDG can serve as antimicrobial and growth-supporting alternatives for reducing poultry production losses, improving resource utilization and meeting consumer demand for sustainably produced and safe animal protein.

Membrane inflammasome activation by choriodecidual Ureaplasma parvum infection without intra-amniotic infection in a Non-Human Primate model†.

Intrauterine infection is a significant cause of neonatal morbidity and mortality. Ureaplasma parvum is a microorganism commonly isolated from cases of preterm birth and preterm premature rupture of membranes (pPROM). However, the mechanisms of early stage ascending reproductive tract infection remain poorly understood. To examine inflammation in fetal (chorioamnionic) membranes we utilized a non-human primate (NHP) model of choriodecidual U. parvum infection. Eight chronically catheterized pregnant rhesus macaques underwent maternal-fetal catheterization surgery at ~105-112 days gestation and choriodecidual inoculation with U. parvum (105 CFU/mL, n =4) or sterile media (controls; n = 4) starting at 115-119 days, repeated at 5-day intervals until C-section at 136-140 days (term=167 days). The average inoculation to delivery interval was 21 days, and Ureaplasma infection of the amniotic fluid (AF) was undetectable in all animals. Choriodecidual Ureaplasma infection resulted in increased fetal membrane expression of MMP-9 and PTGS2, but did not result in preterm labor or increased concentrations of AF pro-inflammatory cytokines. However, membrane expression of inflammasome sensors, NLRP3, NLRC4, AIM2, and NOD2, and adaptor ASC (PYCARD) gene expression were significantly increased. Gene expression of IL-1β, IL-18, IL-18R1 , CASPASE-1, and pro-CASPASE-1 protein increased with Ureaplasma infection. Downstream inflammatory genes MYD88 and NFκB (Nuclear factor kappa-light-chain-enhancer of activated B cells) were also significantly upregulated. These results demonstrate that choriodecidualUreaplasma infection, can cause activation of inflammasome complexes and pathways associated with pPROM and preterm labor prior to microbes being detectable in the AF.

Impact of Glucagon-Like Peptide 1 Receptor Agonists on Biochemical Markers of the Initiation of Atherosclerotic Process.

Atherosclerosis stands out as one of the leading causes of global mortality. The inflammatory response against vascular wall components plays a pivotal role in the atherogenic process. The initiation of this process is notably driven by oxidized low-density lipoprotein (oxLDL) and a range of pro-inflammatory cytokines, with interleukin-1β (Il-1β) and tumor necrosis factor α (TNFα) emerging as particularly significant in the early stages of atherosclerotic plaque formation. In recent years, researchers worldwide have been diligently exploring innovative therapeutic approaches for metabolic diseases, recognizing their impact on the atherogenesis process. Our study aimed to investigate the influence of glucagon-like peptide 1 receptor agonists (GLP-1RA) on cytokine concentrations associated with the initiation of atherosclerotic plaque formation in a group of patients with type 2 diabetes and dyslipidemia. The study encompassed 50 subjects aged 41-81 (mean: 60.7), all diagnosed with type 2 diabetes, dyslipidemia and confirmed atherosclerosis based on B-mode ultrasound. Following a 180-day treatment with dulaglutide or semaglutide, we observed a statistically significant reduction in biochemical markers (oxLDL, TNFα and Il-1β) associated with the initiation of the atherosclerotic process (p < 0.001) within our study group. In addition to the already acknowledged positive effects of GLP-1RA on the metabolic parameters of treated patients, these drugs demonstrated a notable reduction in proinflammatory cytokine concentrations and may constitute an important element of therapy aimed at reducing cardiovascular risk.

Circadian Variation of Immune Cell Populations in Preterm Human Milk: A Preliminary Study.

Introduction: Breast milk contains both nutritional and non-nutritional components for the newborn, with some of the latter exhibiting marked diurnal variations in concentration. This study aimed to analyze the circadian behavior of specific immune cell populations and proinflammatory cytokines present in the transitional milk of premature infants. Methods: The study quantified cellular components, including stem and immune cells, using flow cytometry. Additionally, ELISA assays were employed to measure proinflammatory cytokine concentrations. Results: Flow cytometry analyses revealed a diurnal rise in the percentage of CD23+, CD32+, CD36+, CD2+, and Tγδ cell populations. Conversely, nocturnal increases were observed in the percentage of CD16+, CD19+, and CD4+ populations. Notably, CD3+ and CD8+ populations did not exhibit any rhythmic variations. Proinflammatory cytokine concentrations were found to be higher in daytime milk samples compared to those collected at night. Conclusion: This study demonstrates rhythmic fluctuations in both immune cell populations and proinflammatory cytokine concentrations within the transitional milk of premature mothers.

The use of a systems approach to increase NAD+ in human participants

Reversal or mitigation against an age-related decline in NAD+ has likely benefits, and this premise has driven academic and commercial endeavour to develop dietary supplements that achieve this outcome. We used a systems-based approach to improve on current supplements by targeting multiple points in the NAD+ salvage pathway. In a double-blind, randomised, crossover trial, the supplement – Nuchido TIME+® (NT) - increased NAD+ concentration in whole blood. This was associated with an increase in SIRT1 and an increase in nicotinamide phosphoribosyltransferase (NAMPT) in peripheral blood mononucleocytes, lower concentrations of pro-inflammatory cytokines in plasma, including a reduction in interleukin 2 (IL2), a reduction in glycated serum protein and a shift in the glycosylation profile of immunoglobulin G (IgG) toward a younger biological age, all of which are likely to promote a healthier ageing trajectory.

Plasma-derived from human umbilical cord blood restores ovarian function and improves serum reproductive hormones levels in mice with premature ovarian insufficiency (POI) through cytokines and growth factors.

Premature ovarian insufficiency (POI) patients experience a decline in ovarian function and a reduction in serum reproductive hormones, leading to a significant impact on the outcomes of assisted reproductive technology. Despite the absence of an effective clinical treatment to restore fertility in POI patients, recent research has indicated that cord blood plasma (CBP) derived from human umbilical cord blood (hUCB) may offer therapeutic benefits for various degenerative diseases. The primary aim of this study is to explore approaches for enhancing ovarian function and serum reproductive hormones through the administration of CBP in a murine model. Initially, hUCB was utilized to obtain CBP (CBP), which was subsequently analyzed for cytokine and growth factor profiles in comparison to adult blood plasma (ABP) by use of flow cytometry. Subsequently, POI mouse models were established through the induction of 4-vinylcyclohexene diepoxide, followed by the injection of CBP into the tail. At 7, 14, and 21 days posttreatment, mouse ovaries and blood were collected, and their estrus cycle, body weight, and ovarian weights were evaluated using precise electronic balance. Finally, ovarian morphology and follicle number were assessed through HE staining, while serum levels of anti-Müllerian hormone (AMH), estradiol (E2) and follicle-stimulating hormone (FSH) were determined by ELISA. Our study revealed that individuals with CBP exhibited significantly lower concentrations of proinflammatory cytokines, including IL-β (p < 0.01) and IL-2 (p < 0.05), while displaying elevated levels of anti-inflammatory cytokines and chemokines, such as IL-2, IL-4, IL-6, IL-8, IL-12P70, IL-17A, IP-10, interferon-γ, and tumor necrosis factor-α (p < 0.01). Furthermore, CBP demonstrated remarkably higher levels of growth factors, including transforming growth factor-β1, vascular endothelial growth factor, and insulin-like growth factor-1 (p < 0.01) than ABP. Notably, our investigation also revealed that CBP restored the content of serum reproductive hormones, such as AMH, E2, and FSH (p < 0.05), and increased the number of primordial and primary follicles (p < 0.01) and decreased the number of luteal and atretic follicles (p < 0.01) in vivo. Our findings suggested that CBP-secreted cytokines and growth factors could be restored POI ovarian function, enhanced serum reproductive hormones and rescued follicular development in vivo. These findings further support the potential of CBP as a promising strategy in clinical applications for POI related infertility.

The relevance between abnormally elevated serum ceramide and cognitive impairment in Alzheimer's disease model mice and its mechanism.

The plasma ceramide levels in Alzheimer's disease (AD) patients are found abnormally elevated, which is related to cognitive decline. This research was aimed to investigate the mechanisms of aberrant elevated ceramides in the pathogenesis of AD. The ICR mice intracerebroventricularly injected with Aβ1-42 and APP/PS1 transgenic mice were employed as AD mice. The cognitive deficiency, impaired episodic and spatial memory were observed without altered spontaneous ability. The serum levels of p-tau and ceramide were evidently elevated. The modified expressions and activities of glycogen synthase kinase-3β (GSK-3β) and protein phosphatase 2A (PP2A) influenced the serum content of p-tau. The levels of ceramide synthesis-related genes including sptlc1, sptlc2, cers2, and cers6 in the liver of AD mice were increased, while the ceramide degradation-related gene asah2 did not significantly change. The regulations of these genes were conducted by activated nuclear factor kappa-B (NF-κB) signaling. NF-κB, promoted by free fatty acid (FFA), also increased the hepatic concentrations of proinflammatory cytokines. The FFA amount was modulated by fatty acid synthesis-related genes acc1 and srebp-1c. Besides, the decreased levels of pre-proopiomelanocortin (pomc) mRNA and increased agouti-related protein (agrp) mRNA were found in the hypothalamus without significant alteration of melanocortin receptor 4 (MC4R) mRNA. The bioinformatic analyses proved the results using GEO datasets and AlzData. Ceramide was positively related to the increased p-tau and impaired cognitive function. The increased generation of ceramide and endoplasmic reticulum stress in the hypothalamus was positively related to fatty acid synthesis and NF-κB signaling via brain-liver axis.

MiRNA-192-5p targets Dyrk1a to attenuate cerebral injury in MCAO mice by suppressing neuronal apoptosis and neuroinflammation.

Ischemic stroke (IS) is a leading cause of disability and mortality worldwide. Several studies have demonstrated the involvement of microRNAs (miRNAs) in brain diseases. miRNA-192-5p is a regulatory molecule in neurodegenerative diseases and its expression was found to be significantly downregulated in the whole blood samples of IS patients, but the specific role of miRNA-192-5p in IS not fully understood. Here, we investigated the role of miRNA-192-5p in a murine model of acute cerebral injury after IS. Male C57BL/6J mice received an intracerebroventricular (i.c.v.) injection of agomir-192-5p or antagomir-192-5p 2 h before middle cerebral artery occlusion (MCAO). Infarct volume was assessed by 2,3,5 triphenyltetrazolium chloride (TTC) staining. Brain slices were subjected to Fluoro-Jade B, TUNEL, and immunofluorescence stainings. Contents of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) were measured using enzyme-linked immunosorbent assay (ELISA) kits. In vitro, murine microglial BV-2 cells were subjected to oxygen-glucose deprivation (OGD), and the contents of pro-inflammatory cytokines were measured in cell lysates. miRNA-192-5p was downregulated in the ischemic penumbra of the cerebral cortex. Pretreatment with agomir-192-5p attenuated neurological deficits and reduced cerebral edema and infarct volume in MCAO mice. Agomir-192-5p-treated animals had fewer degenerating and apoptotic neurons in the ischemic penumbra. Additionally, agomir-192-5p significantly suppressed neuroinflammation as evidenced by decreased immunostaining for GFAP and Iba1 and decreased levels of pro-inflammatory cytokines. Antagomir-192-5p pretreatment showed the opposite effect. Furthermore, dual specificity tyrosine phosphorylation regulated kinase 1A (Dyrk1a) was identified as a target gene of miRNA-192-5p, and the elevated Dyrk1a expression in the ischemic penumbra was markedly reduced by agomir-192-5p. Dyrk1a overexpression in BV-2 microglial cells impaired miRNA-192-5p-mediated inhibition of OGD-induced activation of BV-2 microglial cells. Opposite results were obtained using miRNA-192-5p inhibitor and Dyrk1a siRNA. We found that intracerebroventricular administration of miRNA-192-5p before MCAO attenuatedacute cerebral injury by suppressing neuronal apoptosis and neuroinflammation in mice, and these protective effects might be mediated by downregulation of Dyrk1a. This study would help identify novel therapeutic targets for IS.

Phytocompounds-based therapeutic approach: Investigating curcumin and green tea extracts on MCF-7 breast cancer cell line

BackgroundBreast cancer (BC) has transcended lung cancer as the most common cancer in the world. Due to the disease's aggressiveness, rapid growth, and heterogeneity, it is crucial to investigate different therapeutic approaches for treatment. According to the World Health Organization (WHO), Plant-based therapeutics continue to be utilized as safe/non-toxic complementary or alternative treatments for cancer, even in developed countries, regardless of how cutting-edge conventional therapies are. Despite their low bioavailability, curcumin (CUR) and green tea (GT) represent safer therapeutic options. Due to their potent molecular-modulating properties on various cancer-related molecules and signaling pathways, they are considered gold-standard therapeutic agents and have been incorporated into the development of one or more therapeutic strategies of BC treatment. MethodsWe investigated the modulatory role of CUR and GT extracts on significant multi molecular targets in MCF-7 BC cell line to assess their potential as BC multi-targeting agents. We analyzed the phytocompounds in GT leaves using High-performance liquid chromatography (HPLC) and Gas chromatography-mass spectrometry (GC-MS) techniques. The mRNA expression levels of Raf-1, Telomerase, Tumor necrosis factor alpha (TNF-α) and Interleukin-8 (IL-8) genes in MCF-7 cells were quantified using quantitative real-time PCR (qRT-PCR). The cytotoxicity of the extracts was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the released Lactate dehydrogenase (LDH), a valuable marker for identifying the programmed necrosis (necroptosis). Additionally, the concentrations of the necroptosis-related proinflammatory cytokines (TNF-α and IL-8) were measured using enzyme-linked immunosorbent assay (ELISA). ResultsIn contrast to the GT, the results showed the anticancer and cytotoxic properties of CUR against MCF-7 cells, with a relatively higher level of released LDH. The CUR extract downregulated the oncogenic Raf-1, suppressed the Telomerase and upregulated the TNF-α and IL-8 genes. Results from the ELISA showed a notable increase in IL-8 and TNF-α cytokines levels after CUR treatment, which culminated after 72 h. ConclusionsAmong both extracts, only CUR effectively modulated the understudy molecular targets, achieving multi-targeting anticancer activity against MCF-7 cells. Moreover, the applied dosage significantly increased levels of the proinflammatory cytokines, which represent a component of the cytokines-targeting-based therapeutic strategy. However, further investigations are recommended to validate this therapeutic approach.

MiR-26a-5p Attenuates Oxidative Stress and Inflammation in Diabetic Retinopathy through the USP14/NF-κB Signaling Pathway.

Diabetic retinopathy (DR) is an ocular disease caused by diabetes and may lead to vision impairment and even blindness. Oxidative stress and inflammation are two key pathogenic factors of DR. Recently, regulatory roles of different microRNAs (miRNAs) in DR have been widely verified. miR-26a-5p has been confirmed to be a potential biomarker of DR. Nevertheless, the specific functions of miR-26a-5p in DR are still unclear. Primary cultured mouse retinal Müller cells in exposure to high glucose (HG) were used to establish an in vitro DR model. Müller cells were identified via morphology observation under phase contrast microscope and fluorescence staining for glutamine synthetase. The in vivo animal models for DR were constructed using streptozotocin-induced diabetic C57BL/6 mice. Western blotting was performed to quantify cytochrome c protein level in the cytoplasm and mitochondria of Müller cells and to measure protein levels of glial fibrillary acidic protein (GFAP), ubiquitin-specific peptidase 14 (USP14), as well as factors associated with NF-κB signaling (p-IκBα, IκBα, p-p65, and p65) in Müller cells or murine retinal tissues. ROS production was detected by CM-H2DCFDA staining, and the concentration of oxidative stress markers (MDA, SOD, and CAT) was estimated by using corresponding commercial kits. Quantification of mRNA expression was conducted by RT-qPCR analysis. The concentration of proinflammatory factors (TNF-α, IL-1β, and IL-6) was evaluated by ELISA. Hematoxylin-eosin staining for murine retinal tissues was performed for histopathological analysis. Immunofluorescence staining was conducted to determine NF-κB p65 nuclear translocation in Müller cells. Furthermore, the interaction between miR-26a-5p and USP14 was verified via the luciferase reporter assays. HG stimulation contributed to Müller cell dysfunction by inducing inflammation, oxidative injury, and mitochondrial damage to Müller cells. miR-26a-5p was downregulated in Müller cells under HG condition, and overexpression of miR-26a-5p relieved HG-induced Müller cell dysfunction. Moreover, miR-26a-5p targeted USP14 and inversely regulated USP14 expression. Additionally, HG-evoked activation of NF-κB signaling was suppressed by USP14 knockdown or miR-26a-5p upregulation. Rescue assays showed that the protective impact of miR-26a-5p upregulation against HG-induced Müller cell dysfunction was reversed by USP14 overexpression. Furthermore, USP14 upregulation and activation of NF-κB signaling in the retinas of DR mice were detected in animal experiments. Injection with miR-26a-5p agomir improved retinal histopathological injury and weakened the concentration of proinflammatory cytokines and oxidative stress markers in the retinas of DR mice. miR-26a-5p inhibits oxidative stress and inflammation in DR progression by targeting USP14 and inactivating the NF-κB signaling pathway.

Relationship between biomarkers of autophagy and inflammation in acute period of ischemic stroke

Postischemic neuroinflammation is a critical pathophysiological process within the entire pattern of cerebral ischemia, spanning early injury and tissue repair. According to recent experimental data, autophagy is involved in the regulation of neuroinflammation, influencing the outcome of the acute period of ischemic stroke (IS).Objective. To evaluate the relationship between autophagy biomarkers and inflammation indicators in the dynamics of the acute period of atherothrombotic IS.Materials and methods. 112 patients in the acute period of newly developed atherothrombotic IS and 56 donors (control group) were examined. Patients underwent dynamic clinical and neurological examination on the 1st, 7th and 14th days from the onset of the disease (magnetic resonance imaging, testing using the NIHSS scale, modified Rankin scale). At the same time intervals, blood was drawn for testing. The number of active autophagosomes in peripheral blood was assessed by flow cytometry using a specific Cyto-ID dye. The serum concentrations of proinflammatory cytokines IL‑1β, IL‑8, IL‑18 (interleukins‑1β, -8, -18), TNFα (tumor necrosis factor-α), autophagy biomarkers Beclin‑1, LC 3 and p62 were determined by enzyme-linked immunosorbent assay analysis. C-reactive protein was assessed by a highly sensitive immunoturbidimetric method.Results. A statistically significant increase in the studied parameters was revealed compared to the control group. The maximum increase in inflammation biomarkers was observed on the 1st day, and the maximum increase in key indicators of autophagy (LC 3, Beclin‑1, Cyto-ID) – on the 7th day after the development of ischemia. A direct relationship was established between the level of autophagy and the concentration of inflammatory biomarkers (CRP, IL‑1β, IL‑18, TNF-α) on the 1st and 7th days of acute IS.Conclusions. The identified correlations indicate the participation of activated autophagy in the regulation of post-ischemic neuroinflammation and its involvement in ischemic brain damage in the early stages of the acute period of IS (days 1–7). The results obtained confirm the literature data on the influence of autophagy on the outcome of the acute period of the disease.

Fermented calcium butyrate supplementation in post-peak laying hens improved ovarian function and tibia quality through the “gut-bone” axis

The compromised egg quality and leg abnormality during the end of the laying cycle (after 40 weeks) have been leading to poor animal welfare and substantial economic losses. Therefore, the effects of fermented calcium (Ca) butyrate, produced by fermentation by Clostridium butyricum, on production, eggshell quality, and tibial property of hens were explored. A total of 192 Hy-line brown laying hens at 50-week-old were assigned to a basal diet or the basal diet with 300 mg/kg of the fermented Ca butyrate from 50 to 58 weeks of age. Each treatment had 6 replicates with 16 hens each. The diet supplemented with 300 mg/kg fermented Ca butyrate notably increased egg weight, ovarian follicle number, and eggshell strength (P = 0.072) as compared to the basal diet, which were associated with cytokine secretion, toll-like receptor signaling pathways, and intestinal immunity based on the RNA-seq data from the granulosa. Dietary Ca butyrate inclusion decreased the expression of ileal tumor necrosis factor-alpha and serum pro-inflammatory cytokine concentration, as well as increased the content of serum immunoglobulin A when compared to the basal diet (both P < 0.05). The birds that received fermented Ca butyrate diets exhibited higher villus height (P < 0.05) and upregulated expression of tight junction proteins, whereas it did not alter the composition of cecal microbiota (P > 0.05). In addition, the diet with fermented Ca butyrate reduced the number of osteoclasts in the proximal tibia and the level of C-terminal cross-linked telopeptide of type I collagen, a bone resorption marker (P < 0.05), whereas it tended to increase the concentration of the procollagen type I N-terminal propeptide that reflects bone formation marker in serum. Moreover, the layers fed fermented Ca butyrate diets possessed higher (P < 0.05) bone area and trabecular number of the proximal tibia, yield load, and ultimate load than those that consumed basal diets. Collectively, dietary fermented Ca butyrate supplementation in post-peak layer diets improved the ovarian function and tibia quality, which might be related to enhancing intestinal integrity and consequently decreasing inflammation mediated bone resorption.

Aurantio-obtusin exerts an anti-inflammatory effect on acute kidney injury by inhibiting NF-κB pathway.

Acute kidney injury (AKI) is one of the major complications of sepsis. Aurantio-obtusin (AO) is an anthraquinone compound with antioxidant and anti-inflammatory activities. This study was developed to concentrate on the role and mechanism of AO in sepsis-induced AKI. Lipopolysaccharide (LPS)-stimulated human renal proximal tubular epithelial cells (HK-2) and BALB/c mice receiving cecal ligation and puncture (CLP) surgery were used to establish in vitro cell model and in vivo mouse model. HK-2 cell viability was measured using MTT assays. Histological alterations of mouse renal tissues were analyzed via hematoxylin and eosin staining. Renal function of mice was assessed by measuring the levels of serum creatinine (SCr) and blood urea nitrogen (BUN). The concentrations of pro-inflammatory cytokines in HK-2 cells and serum samples of mice were detected using corresponding ELISA kits. Protein levels of factors associated with nuclear factor kappa-B (NF-κB) pathway were measured in HK-2 cells and renal tissues by Western blotting. AO exerted no cytotoxic effect on HK-2 cells and AO dose-dependently rescued LPS-induced decrease in HK-2 cell viability. The concentrations of pro-inflammatory cytokines were increased in response to LPS or CLP treatment, and the alterations were reversed by AO treatment. For in vivo experiments, AO markedly ameliorated renal injury and reduced high levels of SCr and BUN in mice underwent CLP operation. In addition, AO administration inhibited the activation of NF-κB signaling pathway in vitro and in vivo. In conclusion, AO alleviates septic AKI by suppressing inflammatory responses through inhibiting the NF-κB pathway.