Relative mRNA Levels Research Articles

Protective role of ABCC drug subfamily resistance transporters (ABCC1-7) in intestinal inflammation.

The ABCC subfamily contains thirteen members. Nine of these transporters are called multidrug resistance proteins (MRPs). The MRPs have been associated with developing ulcerative colitis (UC). This study aimed to evaluate the ABCC expression in UC patients and its role in a dextran sulfate sodium (DSS)-induced colitis mice model under 5-aminosalicylates or methylprednisolone treatment and compared with control without inflammation. DSS-induced colitis mice were treated with 5-aminosalicylates (50mg/kg 24h) or methylprednisolone (2mg/kg 24h). Human rectal biopsies were obtained from UC patients. The abcc-relative mRNA levels and protein expression were determined by RT-PCR and immunohistochemistry. abcc4, abcc5, and abcc6 mRNA levels were significantly increased in DSS-induced colitis compared to the other groups. The 5-aminosalicylate treatment dramatically increased the abcc2 and abcc3 mRNA levels vs. control. Methylprednisolone treatment increased abcc1 vs. DSS-induced colitis and colitis treated with 5-aminosalicylate. Immunohistochemical analysis revealed down-regulation of ABCC1/ABCC2/ABCC5/ABCC7 in mice colitis vs. control. Treatment with 5-aminosalicylate restored ABCC5 levels, while methylprednisolone restored ABCC2/ABCC5/ABCC7 in colitis mice at similar control levels. Relative mRNA levels of mrp1-5 were increased in active UC patients vs. control. ABCC2/ABCC4/ABCC7 were conspicuously expressed in the mucosa of 5-aminosalicylate and/or methylprednisolone-treated UC patients, while ABCC2/ABCC4/ABCC5/ABCC7 in submucosa, ABCC1/ABCC5/ABCC7 in muscular, and ABCC1/ABCC4/ABCC5/ABCC7 in serosa were expressed vs. controls. This is the first report about the differential up-regulation of the ABCC subfamily gene and protein expression in DSS-induced colitis under aminosalicylates or methylprednisolone treatment.

Aluminum Induces Neurotoxicity through the MicroRNA-98-5p/Insulin-like Growth Factor 2 Axis.

Aluminum is a well-known and widely distributed environmental neurotoxin. This study aimed to investigate the effect of miR-98-5p targeting insulin-like growth factor 2 (IGF2) on aluminum neurotoxicity. Thirty-two Sprague-Dawley rats were randomly divided into four groups and administered 0, 10, 20, and 40 μmol/kg maltol aluminum [Al(mal)3], respectively. They were intraperitoneally injected every other day for three months. PC12 cells were divided into four dose groups: 0, 100, 200, and 400 μmol/L Al(mal)3, and four intervention groups: inhibitor NC, Al(mal)3 + inhibitor NC, miR-98-5p inhibitor, and Al(mal)3 + miR-98-5p inhibitor. The Morris water maze was used to test the learning and memory abilities of rats. Hematoxylin and eosin staining was used to observe the arrangement and quantity of neurons in the CA1 area of the rat hippocampus. Cell viability was detected using the Cell Counting Kit-8. Cell apoptosis was detected using flow cytometry and the 5-ethynyl-2'-deoxyuridine assay. Real-time polymerase chain reaction and Western blotting were used to detect the expression levels of miR-98-5p, IGF2 mRNA, IGF2/Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway proteins, and apoptosis-related proteins caspase3 and cleaved caspase3. The dual-luciferase assay was used to determine the targeting relationship between miR-98-5p and IGF2 mRNA. As the dose of aluminum exposure increased, the escape latency of rats gradually prolonged, and the target quadrant residence time and the number of crossing platforms gradually decreased. The arrangement of neurons in the hippocampal CA1 area was significantly loose, and their number gradually decreased. The total and early apoptosis rates of PC12 cells gradually increased, and the cell proliferation rate slowed down. Both in vivo and in vitro experimental results showed that with the increase of aluminum exposure dose, the relative expression levels of miR-98-5p and caspase3 and cleaved caspase3 proteins gradually increased, while the relative expression levels of IGF2 mRNA and IGF2, p-JAK2 (Tyr1007/1008), and p-STAT3 (Tyr705) proteins gradually decreased. After inhibiting miR-98-5p in the aluminum exposure group, the cell apoptosis rate and expression of apoptosis-related proteins decreased, and the expression of IGF2 mRNA and IGF2/JAK2/STAT3 proteins increased. These results indicate that miR-98-5p plays a vital role in aluminum-induced neurotoxicity by targeting IGF2.

The major roles of intestinal microbiota and TRAF6/NF-κB signaling pathway in acute intestinal inflammation in mice, and the improvement effect by Hippophae rhamnoides polysaccharide.

Acute enteritis, an intestinal disease with intestinal inflammation and injury as the main pathological manifestations. Inhibiting the inflammatory response is critical to the treatment of acute enteritis. Previous studies have shown that the Hippophae rhamnoides polysaccharide (HRP) has strong immune-enhancing effects. However, their functions regarding the intestines and the underlying mechanism are still unclear. In this study, the role of HRP in lipopolysaccharide (LPS)-induced acute enteritis in mice and its related mechanisms are discussed from two aspects: intestinal inflammation and intestinal microbiota. Kunming mice were inoculated with LPS to establish animal models of acute enteritis. The results showed that HRP attenuated the histological damage and maintained the intestine mucosal barrier via up-regulating the expression of occludin, claudin-1, and zona occludens-1 (ZO-1), and suppressing the levels of pro-inflammatory cytokines (tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β)). The relative mRNA and protein levels of nuclear factor-kappa B p65 (NF-κBp65) and tumor necrosis factor-receptor-associated factor 6 (TRAF6) in the intestine tissues of LPS-induced acute enteritis mice significantly increased, whereas these adverse changes were alleviated in the HRP intervention groups. Notably, HRP may regulate the expression of the TRAF6/NF-κB signaling pathway by affecting the diversity of the intestinal microbiota. Microbiota analysis showed that HRP promoted the proliferation of beneficial bacteria, including Clostridia_UCG-014, Candidatus_Saccharimonas, Lachnospiraceae_NK4A136_group, Bacteroidota, Deferribacterota, and reduced the abundance of Atopostipes, Corynebacterium, Actinobacteriota, and Desulfobacterota. The studies conformed that the gut microbiota is crucial in HRP-mediated immunity regulation. HRP shows great potential as an immune enhancer and a natural medicine for treating intestinal inflammatory diseases.

The Role of Cuproptosis Key Factor FDX1 in Gastric Cancer.

Gastric cancer is a common malignant tumor of the digestive tract, both domestically and internationally. It has high incidence and mortality rates, posing a significant threat to human health. The levels of blood copper are elevated in patients with gastric cancer. However, the exact relationship between copper overload and the malignant phenotype of gastric cancer is still unclear. This study aims to investigate the role of the Cuproptosis-related factor FDX1 in the conversion of gastric cancer to a malignant phenotype. Firstly, the relative mRNA and protein expression levels of FDX1 in gastric cancer were detected. Secondly, lentiviral transfection of gastric cancer cell lines was performed, and the effects of FDX1 functional intervention on the proliferation, invasion and migration of gastric cancer cells were assessed by CCK-8, colony formation, EdU proliferation, cell scratch and Transwell assays. Thirdly, the differential alteration of genes after overexpression of FDX1 was also analyzed by transcriptome sequencing. Finally, we assessed the tumour-forming capacity in vivo by the xenograft model. FDX1 is significantly upregulated in gastric cancer. The inhibition of FDX1 function results in the suppression of malignant phenotypic transformation in gastric cancer cells. Conversely, overexpression of FDX1 function leads to alterations in tumor-related signaling pathways and the tumor microenvironment. FDX1 plays a significant role in the malignant phenotypic transformation of gastric cancer cells. Further investigation into the regulatory mechanism of FDX1 in the malignant transformation of gastric cancer will enhance our understanding of the involvement of Cuproptosis in gastric cancer.

Thiolutin Alleviates Cardiotoxic Effects of Doxorubicin by Suppressing NLRP3 Inflammasome in the Mouse Model.

Doxorubicin (DOX) has a limitation in clinical oncology due to its dose-dependent cardiotoxicity. Thiolutin (THL) can undermine DOX-induced cardiomyocyte injury by inhibiting the NLRP3 inflammasome activation, yet the efficacy of THL in DOX-induced cardiotoxicity (DOXIC) needs to be validated in animal models. DOX-induced mice were treated with THL to evaluate the efficacy of THL. Relative NLRP3 mRNA levels were determined by quantitative PCR. Blood samples were collected from diffuse large B-cell lymphoma (DLBCL) patients with or without DOXIC to validate serum levels of cTnT, IL-1β, CRP, BNP, and IL-18 by enzyme-linked immunosorbent assay. Apoptosis and pyroptosis-related protein levels were analyzed by western blot. Cardiac function and histopathological changes were determined by echocardiography, HE, Masson's, and wheat germ agglutinin staining. In clinical samples, NLRP3 mRNA and/or protein levels were also markedly heightened in peripheral blood mononuclear cells and serum samples from DOXIC patients, along with higher concentrations of IL-18, cTnT, and IL-1β. Importantly, cTnT possessed a positive correlation with NLRP3 mRNA, IL-1β, and IL-18. Moreover, cTnT possessed a positive correlation with NLRP3 mRNA, IL-1β, and IL-18 levels, suggesting a potential link between DOXIC and NLRP3 inflammasome. The outcomes demonstrated that THL reduced LVEF and LVFS, as well as elevated LVESD and LVEDD in DOX-challenged mice, accompanied by elevated serum concentrations of cTnT, CRP, and BNP. In addition, THL attenuated DOX-induced myocardial hypertrophy and cardiac fibrosis in mice, in conjunction with attenuation of DOX-induced upregulation of C-caspase3, Bax, NLRP3, C-caspase-1/Pro-caspase, GSDMD-N/GSDMD, IL-1β, and IL-18 in heart or serum samples. In conclusion, our data supported that THL alleviates thecardiotoxic effects of DOX and suppresses NLRP3 inflammasome in the mouse model, suggesting that THL as a potential drug for DOXIC.

WTAP mediates IL-1β-induced chondrocyte injury by enhancing CA12 mRNA stability depending on m6A modification

BackgroundOsteoarthritis (OA) poses a significant risk to the mobility of patients. Carbonic anhydrase 12 (CA12) can boost apoptosis and inflammation in several cancers, but its role in OA is unknown.MethodsDifferentially expressed genes in OA were analyzed using the GEO database (GSE169077). RT-qPCR and western blot estimated relative mRNA and protein levels of CA12. Cell viability and apoptosis were estimated by cell counting and flow cytometry assays. Oxidative stress (OxS) was determined by detecting with ROS and MDA levels, as well as CAT and SOD activities. Cytokine levels of IL-6 and TNF-α were detected by ELISA. Parameters associated with apoptosis and extracellular matrix (ECM) were detected by western blot. The m6A modification profile was determined by methylated RNA immunoprecipitation assays.ResultsRelative CA12 and wilms’ tumor 1-associating protein (WTAP) mRNA and protein levels were overexpressed in OA articular cartilages and IL-1β-challenged chondrocytes CHON-001. CA12 silencing impaired IL-1β-induced cell apoptosis, inflammation, OxS, and ECM degradation in chondrocytes. Yet, CA12 overexpression exerted an opposing function. WTAP reinforced the stability of CA12 mRNA depending on the m6A modification. Furthermore, WTAP knockdown weakened cell apoptosis, inflammation, OxS, and ECM degradation in chondrocytes induced by IL-1β, but these changes were impaired after CA12 overexpression. In addition, WTAP knockdown mitigates cartilage degeneration in DMM-induced mouse models.ConclusionIL-1β-induced WTAP enhances CA12 mRNA stability depending on m6A modification, thus promoting chondrocyte apoptosis, inflammatory response, OxS, and ECM degradation, providing evidence to support the possibility of WTAP and CA12 as potential targets for OA treatment.

Deficiencies of Inducible Costimulator (ICOS) During Chronic Infection with Toxoplasma gondii Upregulate the CD28-Dependent Cytotoxicity of CD8+ T Cells and Their Effector Function Against Tissue Cysts of the Parasite.

We recently identified that the cerebral mRNA expression of inducible costimulator (ICOS) and its ligand, ICOSL, both significantly increase during the elimination of Toxoplasma gondii cysts from the brains of infected mice by the perforin-mediated cytotoxic activity of CD8+ T cells. In the present study, we examined the role of ICOS in activating the effector activity of CD8+ T cells in response to the presence of cysts in infected mice. Following the adoptive transfer of splenic CD8+ T cells from chronically infected ICOS-deficient (ICOS-/-) and wild-type (WT) mice to infected SCID mice, fewer CD8+ T cells were detected in the brains of the recipients of ICOS-/- CD8+ T cells than the recipients of WT CD8+ T cells. Interestingly, even with the lower migration rate of the ICOS-/- CD8+ T cells, those T cells eliminated T. gondii cysts more efficiently than WT CD8+ T cells did in the brains of the recipient mice. Consistently, the ICOS-/- CD8+ T cells secreted greater amounts of granzyme B in response to T. gondii antigens in vitro than WT CD8+ T cells did. We identified that CD8+ T cells of infected ICOS-/- mice express significantly greater levels of CD28 on their surface than CD8+ T cells of infected WT mice, and the relative expression of CD28 mRNA to CD8β mRNA levels in the brains of the recipients of those CD8+ T cells were strongly correlated with their relative expression levels of mRNA for T-bet transcription factors and perforin. Furthermore, blocking CD28 signaling using a combination of anti-CD80 and anti-CD86 antibodies eliminated the increased cytotoxic activity of the ICOS-/- CD8+ T cells in vitro. The present study uncovered notable compensatory interactions between ICOS and CD28, which protected the cytotoxic effector activity of CD8+ T cells against microbial infection in a murine model of chronic infection with T. gondii.

AEBP1 Silencing Protects Against Cerebral Ischemia/Reperfusion Injury by Regulating Neuron Ferroptosis and Microglia M2 Polarization Through PRKCA-PI3K-Akt Axis.

Cerebral ischemia/reperfusion injury is one of the main causes of neuronal damage. Neuron ferroptosis and microglia polarization are considered as critical processes during cerebral ischemia/reperfusion. Adipocyte enhancer-binding protein 1 (AEBP1) usually acts as a transcriptional repressor which is involved in various diseases. However, it is still remains unknown whether AEBP1 could have important roles in regulating the neuron ferroptosis and microglia polarization in cerebral ischemia/reperfusion injury. The oxygen-glucose deprivation and reperfusion (OGD/R)-treated cells and middle cerebral artery occlusion (MCAO)-treated mice were used as in vitro and in vivo models. The differentially expressed factors were analyzed according to GEO datasets. Relative mRNA and protein expression levels were detected by qRT-PCR and western blot analysis. Cell viability was measured by CCK-8 assay. ROS, GSH and iron contents were detected using specifical assay kits. CD26 and CD206 levels were measured by immunofluorescence assay. Inflammatory cytokines were detected by ELISA. The association between AEBP1 and PRKCA was assessed by luciferase reporter and ChIP analyses. The neuron damage in mice was analyzed by TTC staining and neurological deficit score. Transcription factor AEBP1 was increased in OGD/R-treated HT22 and BV2 cells. AEBP1 silencing attenuated OGD/R-induced HT22 cell ferroptosis through increasing cell viability, GSH and GPX4 levels, and decreasing ROS, iron and ACSL4 levels. AEBP1 knockdown promoted microglia M2 polarization by increasing CD206-positive cells and Arg-1 level, and reducing iNOS, TNF-α, IL-1β and IL-6 levels in BV2 cells. AEBP1 transcriptionally repressed PRKCA expression, and further regulated PI3K/Akt signaling activation. Inhibition of PRKCA or PI3K/Akt reversed the effects of AEBP1 silencing on neuron ferroptosis and microglia M2 polarization. AEBP1 downregulation attenuated neuronal damage by decreasing infarct size and deficit scores in MCAO-treated mice. AEBP1 silencing mitigated neuron ferroptosis and promoted microglia M2 polarization through increasing PRKCA and activating PI3K/Akt signaling, indicating the potentially protective action of AEBP1 knockdown in cerebral ischemia/reperfusion injury.

MAM7 from Vibrio parahaemolyticus: Expression, purification and effects on RAW264.7cells.

V. parahaemolyticus is a Gram-negative bacterium that causes gastroenteritis. Within the realm of bacterial interactions with the gut, the outer membrane protein MAM7 plays a key role. However, the precise function of MAM7 in intestinal inflammation, particularly its interactions with macrophages, remains unclear. In this study, we successfully expressed and purified recombinant MAM7. After optimization of the MAM7 expression condition, it was found that the optimal concentration and temperature were 0.75mM and 15°C, respectively, resulting in a 27-fold increase in its yield. Furthermore, RAW264.7 cytotoxicity assay was conducted. The CCK-8 results revealed that MAM7 substantially stimulated the proliferation of RAW264.7cells, with its optimal concentration determined to be 7.5μg/mL. Following this, the NO concentration of MAM7 was tested, revealing a significant increase (p<0.05) in NO levels. Additionally, the relative mRNA levels of IL-1β, IL-6, and TNF-α in RAW264.7cells were measured by qRT-PCR, showing a remarkable elevation (p<0.05). Moreover, ELISA results demonstrated that MAM7 effectively stimulated the secretion of IL-6 and TNF-α by RAW264.7cells. In summary, these findings strongly suggest that MAM7 serves as a proinflammatory adhesion factor with the capacity to modulate immune responses.

Correlation analysis of AVPR1a and AVPR2 with abnormal water and sodium and potassium metabolism in rats.

In clinical practice, an increasing number of patients exhibit concurrent cardiac and renal dysfunction, known as "cardiorenal syndrome," where each condition exacerbates the other, resulting in poorer patient prognosis. Fluid and sodium retention can lead to excessive fluid overload in the body; therefore, correcting fluid and sodium metabolic disorders is crucial for alleviating patient symptoms. This study was to investigate the abnormalities in water and sodium metabolism, as well as the expression levels of arginine vasopressin receptor 1a (AVPR1a) and arginine vasopressin receptor 2 (AVPR2), in a rat model of chronic renal failure-chronic heart failure (CRF-CHF). One hundred male Sprague-Dawley rats were randomly assigned into four groups: the CG group (normal feeding), the CRF group (3/4 nephrectomy using a "two-step surgical method"), the CHF group (subcutaneous injection of isoproterenol at 100 mg/kg), and the CRF-CHF group (3/4 nephrectomy followed by a subcutaneous injection of isoproterenol at 100 mg/kg 1 week later). 4 weeks post-surgery, urine and blood samples were collected to measure 24 h urinary protein, sodium, and potassium levels. Serum creatinine (SCr) and blood urea nitrogen (BUN) levels were determined using assay kits. Left ventricular end diastolic pressure (LVEDP) and left ventricular systolic pressure (LVSP) were measured via left ventricular catheterization. The heart was weighed to calculate the left ventricular weight to body weight ratio (LVW/BW). The renal cortex and medulla were isolated to assess the relative mRNA and protein expression levels of AVPR1a and AVPR2. Compared to the CG group, the CRF and CRF-CHF groups exhibited significantly elevated levels of 24 h urinary protein, SCr, BUN, and relative expression levels of AVPR1a and AVPR2 in the renal cortex and medulla. The CHF and CRF-CHF groups showed significant increases in LVEDP and LVW/BW (P < 0.05). Additionally, compared to the CG group, the other three groups had significantly increased urinary sodium and blood potassium levels, and significantly decreased urinary potassium and blood sodium levels (P < 0.05). Compared to the CRF and CHF groups, the CRF-CHF group exhibited significantly higher levels of 24 h urinary protein, SCr, BUN, and relative expression levels of AVPR1a and AVPR2 in the renal cortex and medulla, along with significantly increased LVEDP and LVW/BW, significantly reduced LVSP, significantly increased urinary sodium and blood potassium levels, and significantly decreased urinary potassium and blood sodium levels (P < 0.05). Rats with CRF-CHF experienced exacerbated renal and cardiac failure, characterized by significant disturbances in water and sodium metabolism and abnormal expression of AVPR1a and AVPR2.

Melatonin attenuates cardiac dysfunction and inflammation in dilated cardiomyopathy via M2 macrophage polarization.

Melatonin is a neuroendocrine hormone that exerts protective effects on the heart. Increasing evidence suggests that macrophage M2-type polarization improves myocardial regeneration and repair. Therefore, this study investigated whether melatonin ameliorates dilated cardiomyopathy (DCM) by modulating M2-type polarization. DCM mice were established by induction with doxorubicin and then treated with melatonin. Cardiac dysfunction was determined by measuring left ventricular ejection fraction (LVEF) and left ventricular internal dimensions at end-diastole and end-systole. Heart injury and fibrosis were determined by hematoxylin and eosin staining and Sirius Red staining, respectively. Serum concentrations of melatonin, tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) were measured through enzyme-linked immunosorbent assays. M2-type macrophages were analyzed by flow cytometry. Relative mRNA and protein levels were determined by reverse transcription quantitative polymerase chain reaction and western blotting, respectively. Circulating melatonin levels were significantly decreased in DCM mice and were associated with LVEF. Treatment with melatonin markedly ameliorated cardiac dysfunction, improved survival, and alleviated pathological changes and collagen deposition in DCM mice. Furthermore, melatonin-treated DCM mice displayed lower serum and cardiac levels of IL-1β, IL-6, and TNF-α, as well as higher numbers of M2-type macrophages in cardiac tissue, indicating that melatonin treatment could decrease inflammatory responses and facilitate M2 macrophage polarization in DCM mice. Thus, melatonin treatment alleviated cardiac dysfunction and inflammatory responses by promoting M2 macrophage polarization in the DCM mouse model.

CSIG-22. PHARMACOLOGIC INHIBITION AND KNOCKDOWN OF CAMK2 IMPAIR GROWTH OF PATIENT-DERIVED GLIOBLASTOMA CULTURES

Abstract INTRODUCTION Glioblastoma (GBM), a primary brain malignancy with a median survival of 15-18 months, presents a pressing challenge in neuro-oncology. Patient-derived GBM cultures (PDGCs) exhibit spontaneous calcium (Ca2+) waves, which are thought to drive tumor growth and represent a therapeutic target. The mechanisms underlying the generation and the effectors of these Ca2+ waves remain elusive. We hypothesize CAMK2, a Ca2+/calmodulin-dependent protein kinase expressed as four distinct isoforms, is a key effector of such Ca2+ waves by phosphorylating substrates that promote tumor growth. METHODS Using multiple PDGCs, CAMK2 isoform-specific mRNA levels were assessed via qRT-PCR both at baseline and after shRNA-mediated knockdown (KD). Tumorsphere formation and WST-8 assays were performed to assess PDGC clonogenic potential after either KD or pharmacologic inhibition with the cell-permeable CAMK2 inhibitor KN93. RESULTS The relative mRNA levels of CAMK2 isoforms across three PDGC lines were, in decreasing order: CAMK2D, CAMK2G, CAMK2B, and CAMK2A. Expression of CAMK2D- and CAMK2B-specific shRNAs significantly reduced tumorsphere growth compared to control shRNA. CAMK2D KD reduced sphere formation by 55.13 ± 3.54% (p=0.0001) and 47.72 ± 9.55% (p=0.008) in separate PDGC lines, while CAMK2B KD reduced sphere formation by 41.15 ± 11.51% (p=0.021) and sphere size by 41.78% ± 10.50% (p=0.049) (n=3/experiment). KN93 treatment reduced tumorsphere formation by 59.23 ± 1.40% (p=0.0006) and 42.94 ± 13.0% (p=0.032) in two PDGC lines (n=3) and PDGC viability, while CAMK2-inactive analogue KN92 and DMSO minimally impacted viability (p&amp;lt;0.0001, two-way ANOVA). CONCLUSION CAMK2D and CAMK2B KD, as well as KN93 treatment, reduce tumorsphere formation, suggesting CAMK2 has a tumorigenic role in GBM. Further investigation of CAMK2 isoforms and in vivo validation are underway. Future aims include directly testing the link between spontaneous Ca2+ waves and CAMK2 activation and identifying CAMK2 phosphorylation targets that mediate tumor growth.

Amphiregulin is overexpressed in human cardiac tissue in hypothermia deaths; associations between the transcript and stress hormone levels in cardiac deaths

Background Amphiregulin (AREG) is a growth factor linked to cardioprotection and heart pathology during myocardial stress. Our aim was to investigate cardiac AREG expression, its potential as a postmortem hypothermia marker and its possible stress hormone dependency in different types of deaths. Materials and methods Heart RNA was isolated from hypothermic, cardiac and non-cardiac deaths. Relative AREG mRNA levels and urine stress hormone concentrations were measured by qPCR and enzyme-linked immunosorbent assays from eight different death cause groups. Receiver operating characteristic curve was used to evaluate a cut-off point for AREG expression as a hypothermia marker. Regulatory elements were predicted by PROMO. Results The AREG mRNA levels were significantly higher in hypothermic deaths than in most cardiac and non-cardiac deaths. AREG expression indicated hypothermic deaths with nearly 70% sensitivity and specificity. However, high expression levels were also detected in non-ischaemic deaths. The highest concentrations of adrenaline and cortisol were detected in hypothermic deaths, while the highest noradrenaline concentrations associated with atherosclerotic heart disease (AHD) deaths with acute myocardial infarction and trauma deaths. There were no significant correlations between stress hormones and AREG mRNA in hypothermic and non-cardiac deaths, whereas moderate-to-high associations were detected in cardiac deaths. Putative response elements for cortisol and catecholamines were found in AREG. Conclusions Severe hypothermia activates cardiac AREG expression practicable as a postmortem hypothermia marker. Cortisol and catecholamines may act as transcriptional modifiers of this gene, especially in long-term ischaemic heart disease. However, the exact role of these hormones in upregulation of AREG during hypothermia remains unclear.

Maternal Supplementation of Collagen Peptide Chelated Trace Elements Enhances Skeletal Muscle Development in Chicks.

Maternal nutrition plays an important role in regulating the growth and development of offspring. Collagen peptide chelated trace elements (PTE), as a new additive, have been proven to have a positive maternal effect on the intestinal health of offspring, but its effect on the growth anddevelopment is still unclear. This study aimed to investigate the effect of maternal PTE supplementation on the skeletal muscle development of offspring. A total of 270 breeder hens were randomly divided into 3 groups, and fed basal diet (CON), basal diet + 500mg/kg PTE (L-PTE), and 1000mg/kg PTE (H-PTE) for 8weeks. A total of 180 eggs were collected from each group for incubation, and then the hatched male chicks (6 replicates, 12 chicks/replicate) were allocated according to maternal treatment for a 14-day feeding experiment. The results showed that maternal PTE supplementation significantly increased the deposition of iron (Fe), zinc (Zn), and manganese (Mn) in the egg yolk (P < 0.05). In comparison with the CON group, the body weight (days 1 and 14), breast muscle weight (day 14), and muscle fiber density (day 14) of broilers were increased in the L-PTE group (P < 0.05). The serum creatinine (CREA) levels in 1-day-old broilers were reduced in the L-PTE group and H-PTE group(P < 0.05). Additionally, maternal PTE supplementation could upregulate the relative expression level of catalase (CAT) mRNA in breast muscle (P < 0.05). The relative mRNA levels of hemeoxygenase-1 (HO-1) and superoxide dismutase 1(SOD1) in the H-PTE group were significantly upregulated (P < 0.05). Moreover, PTE treatment upregulated the mRNA expression of skeletal muscle development-related genes (Pax7 and MyoG) and the IGF signaling pathway (mTOR, IGF-1R, and IGF-2R) (P < 0.05). In conclusion, maternal PTE supplementation may improve the growth performance and skeletal muscle development of offspring by activating the IGF signaling pathway.

Preparation of New Liposomal Daunorubicin and Evaluation of its Anti-colorectal Cancer in HCT116 Cells

Background: Colorectal cancer (CRC) is the third most common cause of cancerrelated deaths worldwide. To develop more effective anti-CRC drugs, this research evaluated the impact of synthesized liposomal daunorubicin on HTC116 colon cancer cell line. Methods: Liposomal daunorubicin (LDNR) was synthesized by the thin layer hydration method and size was determined by dynamic light diffraction (DLS). MTT assay was used to determine the cytotoxicity and IC50 of LDNR against the HCT116 CRC cell line. Relative mRNA expression of the NF-κB gene and apoptosis were evaluated in 24 hours treatments of HCT116 cells by qRT-PCR and flow cytometry, respectively. Results: The hydrodynamic diameter of liposomes containing daunorubicin (DNR) was determined 25.2 nm. MTT assay showed a 38% decrease in HCT116 cells viability after 24-hour treatment with the DNR (0.5 μM). The lowest (0.125 μM) and highest (2 μM) dose of LDNR showed 20.4% and 71.6% cytotoxicity, respectively. LDNR showed dose-dependent cytotoxicity with the IC50 of 0.87 μM. The phase contrast microscope evaluation confirmed the LDNR cytotoxicity. The DNR and LDNR (0.87 μM) decreased relative mRNA levels of NF-κB 63% (P= 0.023) and 99.6% (P=0.003), respectively. The percentage of apoptotic cells in the DNR and LDNR increased by 27.1% (P&lt;0.0001) and 49.7% (P&lt;0.0001), respectively. Conclusion: DNR increases the rate of apoptosis by decreasing the NF-κB gene expression in HCT116 cells. These effects are intensified in the liposomal form. Therefore, the LDNR produced in this research can be considered in the treatment of CRC.

Low-power red laser and blue LED modulate telomere maintenance and length in human breast cancer cells.

Cancer cells have the ability to undergo an unlimited number of cell divisions, which gives them immortality. Thus, the cancer cell can extend the length of its telomeres, allowing these cells to divide unlimitedly and avoid entering the state of senescence or cellular apoptosis. One of the main effects of photobiomodulation (PBM) is the increase in the production of adenosine triphosphate (ATP) and free radicals, mainly reactive oxygen species (ROS). Existent data indicates that high levels of ROS can cause shortening and dysfunctional telomeres. Therefore, a better understanding of the effects induced by PBM on cancer cell telomere maintenance is needed. This work aimed to evaluate the effects of low-power red laser (658nm) and blue LED (470nm) on the TRF1 and TRF2 mRNA levels and telomere length in human breast cancer cells. MCF-7 and MDA-MB-231 cells were irradiated with a low-power red laser (69J cm-2, 0.77W/cm-2) and blue LED (482J cm-2, 5.35W/cm-2), alone or in combination, and the relative mRNA levels of the genes and telomere length were assessed by quantitative reverse transcription polymerase chain reaction. The results suggested that exposure to certain red laser and blue LED fluences decreased the TRF1 and TRF2 mRNA levels in both human breast cancer cells. Telomere length was increased in MCF-7 cells after exposure to red laser and blue LED. However, telomere length in MDA-MB-231 was shortened after exposure to red laser and blue LED at fluences evaluated. Our research suggests that photobiomodulation induced by red laser and low-power blue LED could alter telomere maintenance and length.

The effect and mechanism of exposure to polystyrene nanoplastics on lipid metabolism in mice liver

Objective: To investigate the effect and potential mechanism of exposure to 20 nm polystyrene nanoplastics (PS-NPs) on lipid metabolism in mice liver. Methods: An animal experimental model was designed, which was completed from September 2022 to July 2023 on the exposure omics platform of the School of Public Health at Capital Medical University and the Key Laboratory of Environment and Population Health at the Chinese Center for Disease Control and Prevention.1 mg/kg and 10 mg/kg PS-NPs tail vein mice exposure models were constructed. After exposure 7 d, serum was collected to measure the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and air flow assisted desorption electrospray ionization-mass spectrometry imaging (AFADESI-MSI) analysis were used to analyze the mRNA levels of fatty acid esterification related genes (Dgat1 and Dgat2) and lipid transport related genes (ApoB, Cd36, ApoE and Mttp) and metabolites' spatial changes in liver tissue. In vivo imaging system (IVIS) and tissue shake sections were employed to observe the fluorescence biological distribution of PS-NPs. t-test or one-way ANOVA was used to explore the difference between groups. Results: The serum ALT levels were (83.97±4.58), (91.17±13.69) and (142.43±6.09) U/L in the control group, 1 mg/kg PS-NPs exposure group and 10 mg/kg PS-NPs exposure group respectively (F=37.281, P<0.05). The relative mRNA levels of Dgat1, Dgat2, ApoB, Cd36 and ApoE were (1.49±0.63, 2.53±0.32, 2.45±0.54), (1.07±0.38, 1.86±0.83, 2.23±0.73), (1.01±0.13, 1.58±0.43, 2.03±0.52), (1.01±0.14, 1.55±0.37, 1.52±0.51), (1.01±0.17, 2.11±0.27, 2.39±0.93) in these three groups respectively. The differences were statistically significant (F=11.54, 6.95, 14.90, 5.98 and 14.68, P<0.05). AFADESI-MSI analysis found that PS-NPs exposure led to a significant decrease in the levels of glutarylcarnitine and O-Linoleoylcarnitine (t=4.12 and 3.35, P<0.05), which were associated with lipid beta oxidation. The content of triglycerides (TG) (m/z 921.726 4, t=8.69, P<0.05; m/z 919.711 4, t=3.20, P<0.05), phosphatidylic acid (PA) (m/z 895.712 3, t=3.60, P<0.05; m/z 821.526 6, t=3.36, P<0.05), lysophosphatidylcholine (LysoPC) (m/z 560.310 6, t=3.35, P<0.05; m/z 582.295 3, t=6.28, P<0.05), phosphatidylcholine (PC) (m/z 778.533 9, t=3.53, P<0.05; m/z 804.549 6, t=3.60, P<0.05; m/z 820.523 1, t=3.37, P<0.05), phosphatidylethanolamine (PE) (m/z 772.523 3, t=3.08, P<0.05) showed a significant increase in the PS-NPs exposure group. In vivo and in vitro imaging and in situ cell localization revealed that PS-NPs were mainly enriched in hepatic stellate cells and hepatic Kupffer cells in liver tissue. Conclusion: Exposure to PS-NPs induces disorder of liver lipid metabolism, which may be related to the accumulation of PS-NPs in hepatic stellate cells and hepatic Kupffer cells, providing basis for searching early biomarkers of PS-NPs exposure and further mechanism research.

Methylation of Selected CpG-Sites of the Gene CSF1 as a Factor in Regulation of Its Expression and a Marker of Human Biological Aging

—Age-associated transformation of methylation patterns is considered to be an important predictor of human biological age. Changes in the level of CpG-dinucleotide methylation contribute to a shift in the function of a number of genes, including those associated with the functioning of the immune system. One such gene is CSF1. The protein product of this gene is associated with inflammatory aging, making it an important biomarker of age-related diseases. We studied the methylation profile of the promoter-associated CpG island of the CSF1 gene by MALDI-TOF mass spectrometry. Dependences between the character of CpG-site methylation within the investigated regions and the relative level of the gene mRNA and its protein product in people of different age groups were sought. For two CpG sites, a high level of correlation with the studied parameters is shown. A search for the landing sites of transcription factor binding sites associated with gene transcription showed that these CpG dinucleotides are part of motifs for the NFI family transcription factors and the EGR1 factor. We hypothesize that these CpG sites play an important role in the regulation of CSF1 gene expression.

Analysis of Expression of the GRIPAP1, DLG4, KIF1B, NGFRAP1, and NRF1 Genes in Peripheral Blood of the Patients with Parkinson's Disease in the Early Clinical Stages.

Parkinson's disease (PD) is one of the most common progressive neurodegenerative diseases. Animportant feature of the disease is its long latent period, which necessitates search for prognostic biomarkers. One method of identifying biomarkers of PD is to study changes in gene expression in peripheral blood of the patients in early stages of the disease and have not been treated. In this study, we analyzed relative mRNA levels of the genes GRIPAP1, DLG4, KIF1B, NGFRAP1, and NRF1, which are associated with neurotransmitter transport, apoptosis, and mitochondrial dysfunction, in the peripheral blood of PD patients using reverse transcription and real-time PCR with TaqMan probes. The results of this study suggest that the GRIPAP1 and DLG4 genes could be considered as potential biomarkers for the early clinical stages of Parkinson's disease. The data obtained may indicate that NGFRAP1 is involved in pathogenesis of both PD and other neurodegenerative diseases. Furthermore, in the early clinical stages of the disease we studied, the KIF1B and NRF1 genes were found not to be involved in PD pathogenesis at the expression level.

Comparison of the effect of anticoccidial drug, probiotic, synbiotic, phytochemicals and vaccine in prevention and control of coccidiosis in broiler chickens challenged with Eimeria spp

The objective of this study was to investigate the effects of an anti-coccidiosis drug, vaccine, probiotic, symbiotic, and phytochemicals in the prevention and control of coccidia infection in broilers. A total of 525 one-day-old Ross 308 chicks were randomly allocated to 7 experimental diets with 5 replicates of 15 birds each in a completely randomized design. Experimental diets consisted of negative control (NC) without any additives and not challenged. The other 6 groups were challenged with mixed Eimeria and fed the basal diet with no additives (Positive Control, PC) or supplemented with Coxidine 100 (1 g / 1 kg), probiotic, synbiotic, Livacox T vaccine and phytobiotic additives based on the manufacturer's recommended dose. Body weight gain (WG), feed intake (FI) and feed conversion ratio (FCR) were recorded weekly. Oocysts per gram of excreta (OPG) were determined on d 25 to 33 and 42. One bird per cage was euthanized to analyze lesion score and jejunum and ileum inflammatory genes expression. Coccidial challenge reduced WG (P < 0.05) during 15 to 28 d and vaccine treatment was more effective in improving WG and FCR on d 29 to 42 and 1 to 42 (P < 0.05) than other treatments. Birds in the PC group had higher (P < 0.05) OPG than NC group for all days and the vaccine treatment resulted in the lowest rate of OPG compared to other treatments (P < 0.05) at 27, 28, 29, 30, 32, and 33 d of age and overall average. Relative mRNA levels of IFN-γ, IL-1β and IL-10 were significantly upregulated among treatments under coccidiosis challenge in jejunum and ileum except for IL-1β expression in the ileum. In conclusion, based on the results of this study the individual characteristics of feed additives for the prevention of coccidiosis can be different depending on the type and source of feed additives, duration, and amount used, levels of oocyst inoculation and Eimeria types.