Increased mRNA Levels Research Articles

#1593 ARGDRR, a novel disintegrin, attenuates AKI to CKD-related renal fibrosis

Abstract Background and Aims Acute kidney injury (AKI) can lead to progress renal fibrosis, resulting in chronic kidney disease (CKD) and end stage renal disease (ESRD). Nevertheless, the underlying pathological mechanisms remain unclear. Platelet activation has been demonstrated as one of the important factors involved in the pathophysiology of AKI, and studies show the therapeutic potential for using platelet inhibitors or antagonists in ischemia-reperfusion (IR)-induced AKI models. However, drug-induced side effects, including thrombotic microangiopathies (TMA)-related kidney damage, thrombocytopenia and bleeding, hinder clinical application in AKI or CKD treatment. Our newly developed dual effect disintegrin, ARGDRR, is derived from snake venom exert a high affinity to αIIbβ3 and αvβ3 binding affinity without causing bleeding and other thrombosis-related side effects. Method We performed a unilateral ischemia-reperfusion injury (UIRI) model to investigate integrin αIIb(Itga2b) and β3(Itgb3) mRNA levels in the injured kidney for 15 days. For exploring the therapeutic efficiency of ARGDRR in AKI, we administered ARGDRR post-ischemic injury and measured platelet activation after 24 hours using quantitative polymerase chain reaction (qPCR) and immunohistochemistry (IHC) for Itgb3, Lcn2, and CD42b. For exploring the therapeutic efficiency of ARGDRR in CKD progression, we administrated ARGDRR daily for 15 days after IRI with nephrectomy, and examined the difference of renal dysfunction, renal fibrosis, and cell death compared to sham group. Results We found that platelet activation with progressively increased mRNA level of platelet glycoprotein IIb-GPIIIa (Itga2b and Itgb3) in the UIRI kidney over time. After 24 hr of ischemic AKI, ARGDRR administration reduced Lcn2 and Itgb3 mRNA level and CD42b positive area in the kidney, indicating its efficacy in protecting the kidneys by inhibiting platelet activation and aggregation. In AKI to CKD transition, serum creatine and blood urea nitrogen (BUN) were significantly lower both in efficacious and high dose of ARGDRR administration group. ARGDRR significantly reduced CD42b expression and increased LTL positive area. Also, ARGDRR administration alleviated renal fibrosis, cell apoptosis and senescence-related molecules p21 and Ctgf expression in the AKI to CKD transition. Conclusion Our results have successfully proved our hypothesis that disintegrin-ARGDRR has the potential for renal protection in the transition of AKI to CKD through inhibition of platelet activation and aggregation during acute injury.

Nicotinic acid attenuates amyloid β1-42-induced mitochondrial dysfunction and inhibits the mitochondrial pathway of apoptosis in differentiated SH-SY5Y cells

Alzheimer's disease (AD) is a chronic neurodegenerative disease characterized by progressive memory loss and behavioral disorders. The excessive accumulation of amyloid β (Aβ) and the formation of neurofibrillary tangles (NFTs) damage synaptic connections and the death of neurons. However, the underlying mechanisms of pathogenesis of AD remain unclear. Growing evidence indicates that impaired mitochondrial function may play a crucial role in the development of AD. In the current study, we investigated whether nicotinic acid (NA) could protect against amyloid β1-42-induced cytotoxicity in differentiated SH-SY5Y cells. Our results revealed the neuroprotective effects of NA on the differentiated SH-SY5Y cells treated with Aβ1-42. In detail, the 1-h pre-incubation with NA increased cell viability and lowered LDH levels. NA pre-incubation abolished Aβ1-42 treatment-associated alterations of mRNA levels of synaptic genes and enhanced the relative β3 Tubulin fluorescence intensity. NA eliminated the Aβ1-42-induced mitochondrial dysfunction by increasing the potential of mitochondrial membranes and maintaining a balance between the fusion and fission of mitochondria. Moreover, Aβ1-42 decreased mRNA levels of anti-apoptotic bcl2 and increased mRNA levels of pro-apoptotic: bim, bak, cytochrome c, and caspase 9. At the same time, the NA pre-treatment reduced Aβ1-42-dependent apoptotic death of differentiated SH-SY5Y cells.The above data suggest that NA presents a protective activity against Aβ1-42-induced cytotoxicity in differentiated SH-SY5Y cells by inhibiting the mitochondrial pathway of apoptosis and restoring the proper function of mitochondria.

Galectin-receptor interaction: a key player in liver fibrosis induced by Schistosoma japonicum infection

BackgroundSchistosoma japonicum eggs lodge in the liver and induce a fibrotic granulomatous immune response in the liver of host. Galectin 3 (Gal-3) is a protein implicated in fibrosis in multiple organs. However, the pathology and molecular mechanisms promoting hepatic granuloma formation remain poorly understood.MethodsTo investigate the effect of blocking galectin-receptor interactions by α-lactose on liver immunopathology in mice with S. japonicum infection, C57BL/6 mice were infected with S. japonicum and alpha (α)-lactose was intraperitoneally injected to block the interactions of galectins and their receptors.ResultsCompared with S. japonicum-infected mice, there were significantly decreased Gal-3 mRNA and protein expression levels, decreased intensity of Gal-3 fluorescence in the liver, decreased serum ALT and AST levels, decreased egg numbers of S. japonicum in the liver section, attenuated hepatic and spleen pathology, and alleviated liver fibrosis accompanied with decreased protein expression levels of fibrosis markers [α-smooth muscle actin (α-SMA), collagen I, and collagen IV] in the liver of S. japonicum-infected mice blocked galectin-receptor interactions with hematoxylin-eosin staining, Masson’s trichrome staining, immunohistochemistry, or Western blot analysis. Compared with S. japonicum-infected mice, blocking galectin-receptor interactions led to increased eosinophil infiltration and higher eosinophil cationic protein (ECP) expression in the liver, accompanied by increased mRNA levels of eosinophil granule proteins [ECP and eosinophil peroxidase (EPO)], IL-5, CCL11, and CCR3 in the liver and decreased mRNA levels of Gal-3 and M2 macrophage cytokines (TGF-β, IL-10, and IL-4) in the liver and spleen by using quantitative real-time reverse transcription-polymerase chain reaction. In addition, there were increased Beclin1 protein expression and protein expression ratio of LC3B-II/LC3B-I and decreased p62 protein expression and protein expression ratios of phospho-mTOR/mTOR and phospho-AKT/AKT by Western blot; increased double-labeled F4/80+/LC3B+ cells by immunofluorescence staining; increased M1 macrophage polarization in the liver of S. japonicum-infected mice blocked galectin-receptor interactions by flow cytometric analysis and immunofluorescence staining.ConclusionsOur data found that blockage of galectin-receptor interactions downregulated Gal-3, which in turn led to reduced liver functional damage, elevated liver eosinophil recruitment, promoted macrophage autophagy through the Akt/mTOR signaling pathway, and alleviated liver pathology and fibrosis. Therefore, Gal-3 plays a pivotal role during S. japonicum infection and could be a target of pharmacologic potential for liver fibrosis induced by S. japonicum infection.Graphical

Immunomodulation of adipose-derived mesenchymal stem cells on peripheral blood mononuclear cells in colorectal cancer patients with COVID-19.

Accumulating evidence has shown that adipose tissue-derived mesenchymal stem cells (ADSCs) are an effective therapeutic approach for managing coronavirus disease 2019 (COVID-19); however, further elucidation is required to determine their underlying immunomodulatory effect on the mRNA expression of T helper cell-related transcription factors (TFs) and cytokine release in peripheral blood mononuclear cells (PBMCs). To investigate the impact of ADSCs on the mRNA expression of TFs and cytokine release in PBMCs from colorectal cancer (CRC) patients with severe COVID-19 (CRC+ patients). PBMCs from CRC+ patients (PBMCs-C+) and age-matched CRC patients (PBMCs-C) were stimulated and cultured in the presence/absence of ADSCs. The mRNA levels of T-box TF TBX21 (T-bet), GATA binding protein 3 (GATA-3), RAR-related orphan receptor C (RORC), and forkhead box P3 (FoxP3) in the PBMCs were determined by reverse transcriptase-polymerase chain reaction. Culture supernatants were evaluated for levels of interferon gamma (IFN-γ), interleukin 4 (IL-4), IL-17A, and transforming growth factor beta 1 (TGF-β1) using an enzyme-linked immunosorbent assay. Compared with PBMCs-C, PBMCs-C+ exhibited higher mRNA levels of T-bet and RORC, and increased levels of IFN-γ and IL-17A. Additionally, a significant decrease in FoxP3 mRNA and TGF-β1, as well as an increase in T-bet/GATA-3, RORC/FoxP3, IFN-γ/IL-4, and IL-17A/TGF-β1 ratios were observed in PBMCs-C+. Furthermore, ADSCs significantly induced a functional regulatory T cell (Treg) subset, as evidenced by an increase in FoxP3 mRNA and TGF-β1 release levels. This was accompanied by a significant decrease in the mRNA levels of T-bet and RORC, release of IFN-γ and IL-17A, and T-bet/GATA-3, RORC/FoxP3, IFN-γ/IL-4, and IL-17A/TGF-β1 ratios, compared with the PBMCs-C+alone. The present in vitro studies showed that ADSCs contributed to the immunosuppressive effects on PBMCs-C+, favoring Treg responses. Thus, ADSC-based cell therapy could be a beneficial approach for patients with severe COVID-19 who fail to respond to conventional therapies.

α-Pinene Improves Follicle Morphology and Increases the Expression of mRNA for Nuclear Factor Erythroid 2-Related Factor 2 and Peroxiredoxin 6 in Bovine Ovarian Tissues Cultured In Vitro.

Oxidative stress during in vitro of ovarian tissues has adverse effects on follicle survival. α-pinene is a monoterpenoid molecule with antioxidant activity that has great potential to maintain cell survival in vitro. This study investigated the effect of α-pinene (1.25, 2.5, 5.0, 10.0, or 20.0 μg/mL) on primordial follicle growth and morphology, as well as on stromal cells and collagen fibers in bovine ovarian slices cultured for six days. The effect of α-pinene on transcripts of catalase (CAT), superoxide dismutase (SOD), peroxiredoxin 6 (PRDX6), glutathione peroxidase (GPX1), and nuclear factor erythroid 2-related factor 2 (NRF2) was investigated by real-time PCR. The tissues were processed for histological analysis to evaluate follicular growth, morphology, stromal cell density, and collagen fibers. The results showed that 2.5, 5.0, or 10.0 µg/mL α-pinene increased the percentages of normal follicles but did not influence follicular growth. The α-pinene (10.0 µg/mL) kept the stromal cell density and collagen levels in cultured bovine ovarian tissue like uncultured tissues. Ovarian tissues cultured in control medium had reduced expression of mRNA for NRF2, SOD, CAT, GPX1, and PRDX6, but α-pinene (10.0 µg/mL) increased mRNA levels for NRF2 and PRDX6. In conclusion, 10.0 µg/mL α-pinene improves the follicular survival, preserves stromal cell density and collagen levels, and increases transcripts of NRF2 and PRDX6 after in vitro culture of bovine ovarian tissue.

Role of secreted frizzled-related protein 5 in granulosa cells of hu sheep ovaries

The objective of this study was to examine the expression patterns of secreted frizzled-related protein 5 (SFRP5) in the ovaries of Hu sheep and to explore the key downstream factors of SFRP5 in sheep granulosa cells (GCs) using RNA-seq. In the present study, SFRP5 was widely expressed in the ovary and localized to GCs and oocytes during various stages of follicular development. In addition, the expression of SFRP5 increased with follicular diameter. In contrast to the negative control, SFRP5 knockdown promoted the EdU-positive cell rate with an increase in PCNA mRNA and protein levels, whereas SFRP5 overexpression had the opposite effect. In addition, the cell cycle was propelled from the G0/G1 phase to the S phase with the upregulation of CCNB1, CCND1, CDK1, and CDK4 after SFRP5 knockdown. Moreover, SFRP5 overexpression enhanced the apoptosis of GCs with increased Caspase3 protein levels. Following SFRP5 knockdown, differentially expressed genes were mainly enriched in the PI3K/AKT, MAPK, Wnt, and Hippo signaling pathways, and several related candidate genes such as MMP1, MMP3, SFRP4, INHA, TGFA, and CASP3 were screened. In general, this study enhances our understanding of the expression of SFRP5 in the GCs of Hu sheep, along with its functions in follicular development.

Upregulation of mRNA Expression of ADGRD1/GPR133 and ADGRG7/GPR128 in SARS-CoV-2-Infected Lung Adenocarcinoma Calu-3 Cells.

Adhesion G protein-coupled receptors (aGPCRs) play an important role in neurodevelopment, immune defence and cancer; however, their role throughout viral infections is mostly unexplored. We have been searching for specific aGPCRs involved in SARS-CoV-2 infection of mammalian cells. In the present study, we infected human epithelial cell lines derived from lung adenocarcinoma (Calu-3) and colorectal carcinoma (Caco-2) with SARS-CoV-2 in order to analyse changes in the level of mRNA encoding individual aGPCRs at 6 and 12 h post infection. Based on significantly altered mRNA levels, we identified four aGPCR candidates-ADGRB3/BAI3, ADGRD1/GPR133, ADGRG7/GPR128 and ADGRV1/GPR98. Of these receptors, ADGRD1/GPR133 and ADGRG7/GPR128 showed the largest increase in mRNA levels in SARS-CoV-2-infected Calu-3 cells, whereas no increase was observed with heat-inactivated SARS-CoV-2 and virus-cleared conditioned media. Next, using specific siRNA, we downregulated the aGPCR candidates and analysed SARS-CoV-2 entry, replication and infectivity in both cell lines. We observed a significant decrease in the amount of SARS-CoV-2 newly released into the culture media by cells with downregulated ADGRD1/GPR133 and ADGRG7/GPR128. In addition, using a plaque assay, we observed a reduction in SARS-CoV-2 infectivity in Calu-3 cells. In summary, our data suggest that selected aGPCRs might play a role during SARS-CoV-2 infection of mammalian cells.

Disrupted biosynthesis of glycogen and PHB combined with physicochemical factors increased bioproduction of γ-aminobutyric acid and δ-aminolevulinic acid in cyanobacteria

In Synechocystis sp. PCC 6803, redirecting the metabolic flow from upstream substrate for carbon metabolism i.e. glyceraldehyde-3-phosphate (the glycogen synthesis precursor) and poly(3-hydroxybutyrate) (PHB), towards ALA or GABA has not been studied. The PHB synthesis, one of the pathways consuming carbon via glycolysis pathway, was disrupted to generate the strain, ΔP, and another adjacent carbon utilizing pathway that is responsible for glycogen synthesis was also disrupted in the earlier mutant strain to generate ΔGP with the aim to increase carbon flux towards precursor metabolites for GABA and ALA biosynthesis. The ΔP strain showed an increase in intracellular glutamate and ALA levels (∼19.5 and 2.5 ng g−1 DCW, respectively), whereas intracellular GABA levels were found increased (up to 7 ng g−1 DCW) in ΔGP strain as compared to the wild type strain (WT). Modified nutrient conditions including glucose, glutamate and LA supplementation as well as N-deprivation led to several fold increase in GABA and ALA levels in ΔP and ΔGP strains in comparison to those observed in WT, with the maximum GABA and ALA accumulation (∼262 and 48 ng g−1 DCW, respectively) noticed in ΔP. Abiotic stresses including cold and UV irradiation also significantly improved the intracellular GABA and ALA accumulation in mutant strains. Increased biosynthesis of GABA and ALA in mutant strains was found correlated with the increased mRNA levels of genes involved in GABA, ALA and TCA cycle metabolism. Further organic acids profiling revealed that the disruption of two pathways redirects carbon routes to TCA cycle without affecting the viability of cells. This strategy might be applicable in strain development for enhancing other high value bioproducts synthesized by connected metabolic pathways that utilize carbon sources diverted from non-essential metabolism.

Effects of antagonism of thromboxane-prostanoid receptor on alcohol-associated liver disease

Backgrounds: Alcohol-associated liver disease (ALD) is one of the leading causes of liver diseases. Thromboxane-prostanoid receptor (TP-R) is widely expressed in the liver and is activated by thromboxane A2 (TXA2). A previous study reported an increase in TP-R mRNA level in liver after ethanol feeding. Preclinical studies have documented that TP-R antagonists exert a protective effect against cardiovascular and liver diseases. In particular, TP-R antagonist is reported to attenuate ALD in rodents. However, the mechanism(s) by which antagonizing TP-R attenuates ALD remains unclear. Methods: Herein, we used the chronic plus binge ethanol feeding model. Briefly, male, C57BL/6 wild-type mice (8-week) were fed a Lieber-Decarli ethanol (5% v/v) diet (ET, n = 10) or isocaloric control diet (CON, n = 6) for ten days followed by a single binge administration of ethanol or maltose-dextrin via oral gavage. A cohort of ethanol-fed mice received SQ 29,548, a TP-R antagonist (ET+SQ, n = 8) by oral administration. Results: Western blot analysis showed that the protein level of thromboxane A2 synthase 1 ( P<0.05), responsible for TXA2 production, was upregulated in the mouse liver upon ethanol exposure. Of note, ethanol feeding significantly increased plasma aspartate transferase (AST) level ( P<0.05), which was abolished by SQ administration ( P<0.05). Moreover, compared with CON mice, ET mice showed an increase in liver protein levels of pro-inflammatory markers, including TNFa ( P<0.05), cluster of differentiation 68 (CD68, P<0.001), and vascular cell adhesion protein 1 ( P<0.05). In addition, the pro-fibrogenic markers including collagen type 1 alpha 1 chain (COL1A1, P<0.05) and matrix metalloproteinase 9 (MMP9, P<0.01) were also increased in ET mice. However, these alterations were not found in ET+SQ mice compared to their controls. In particular, ET+SQ mice exhibited a marked reduction in hepatic protein levels of CD68 ( P<0.001), COL1A1 ( P<0.001), and MMP9 ( P<0.01) versus ET mice. Interestingly, we noted that ethanol consumption decreases the protein level of NADH:ubiquinone oxidoreductase subunit B8 (NDUFB8), a component of mitochondrial complex I, in mouse liver ( P<0.05), which was reversed by SQ administration( P<0.05). Conclusion: These findings suggest that blockade of TP-R activity attenuates ethanol-induced liver inflammation and fibrosis possibly through improving the function of mitochondrial complex I. This study was supported by the NIH-NIAAA P50 award-Alcohol Center of Research-Nebraska (P50AA030407-5130). 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 1127: Erythrocyte Derived Extracellular Vesicles From Patients With Type 2 Diabetes Induce Endothelial Dysfunction Through Increased Vascular Oxidative Stress

Background: Red blood cells (RBCs) from patients with T2D (T2D-RBCs) induce endothelial dysfunction, but the mechanisms remain unclear. It is increasingly clear that extracellular vesicles (EVs) are actively secreted by RBCs and represent a novel mechanism of intercellular communication. However, the functional role of T2D-RBCs EVs in communication between RBCs and the cardiovascular system remains unknown. Purpose: This study aims to investigate whether EVs induce endothelial dysfunction in T2D and, if this is mediated via increased vascular oxidative stress. Material and Methods: EVs derived from T2D-RBCs and healthy RBCs (H-RBCs) were isolated using a membrane affinity column and co-incubated with wild-type mouse aortas for evaluation of the endothelium-dependent relaxation (EDR) using the wire myograph in the presence or absence of non-selective reactive oxygen species scavenger N-acetyl cysteine (NAC; 100 μM) applied to the aortas following the 18h EV incubation. The levels of the oxidative stress marker 4-hydroxynonenal (4-HNE) were quantified by immunohistochemistry in mouse aortas incubated with EVs. Additionally, T2D-RBCs EVs and H-RBCs EVs were co-incubated with human carotid artery endothelial cells (HCtAEC) for 8h and 24h to study the effects of EVs on vascular mRNA expression levels of eNOS, NOX1, and NOX4. Results: T2D-RBCs EVs but not H-RBCs EVs impaired EDR in aortas isolated from mice ( Fig. 1A ). This impairment was reversed by inhibiting oxidative stress in the vessel by NAC ( Fig. 1B ). Immunohistochemistry showed a significant increase in 4-HNE levels in aortas incubated with T2D-RBCs EVs ( Fig. 1C, D ). The T2D-RBCs EVs also induced a significant increase in NOX4 mRNA levels in HCtAEC after 24h but not 8h ( Fig. 1E, F ). However, T2D-RBCs EVs did not affect eNOS or NOX1 in endothelial cells. Conclusion: EVs derived from T2D-RBCs induce endothelial dysfunction through increased vascular oxidative stress.

Comprehensive Analysis of the Expression, Prognosis, and Immune Infiltrates for Chromodomain-Helicase-DNA-Binding Proteins in Breast Tumor.

Several recent studies suggest that chromodomain-helicase -DNA-binding domains (CHDs) are linked with cancers. We explored the association between chromodomain-Helicase-DNA-binding domain proteins and breast cancer (BrCa) and introduced potential prognostic markers using various databases. We analyzed the expression of the CHD family and their prognostic value in BrCa by mining UALCAN, TIMER, and Kaplan-Meier plotter databases. The association of CHD expression and immune infiltrating abundance was studied via the TIMER database. In addition, microRNAs related to the CHD family were identified by using the MirTarBase online database. The present study indicated that compared to normal tissues, BrCa tissues showed increased mRNA levels of CHD3/4/7 but decreased CHD2/5/9 expression. Interestingly, We also found a positive correlation between CHD gene expression and the infiltration of macrophage, neutrophil, and dendritic cells in BrCa, except CHD3/5. The Kaplan-Meier Plotter analysis suggested that high expression levels of CHD1/2/3/4/6/8/9 were significantly related to shorter relapse-free survival (RFS), while higher mRNA expression of CHD1, CHD2, CHD8, and CHD9 was significantly associated with longer overall survival of BrCa patients. The miRNAs of hsa-miR-615-3p and hsa-let-7b-5p were identified as being more correlated with the CHD family. The altered expression of some CHD members was significantly related to clinical cancer outcomes, and CHD1/2/8/9 could serve as potential prognostic biomarkers to improve the survival of BrCa patients. However, to evaluate the studied CHD members in detail are needed further investigations including experimental validation.

In vivo dynamics of hydrogen peroxide and regulation of antioxidant gene expression in mouse skeletal muscle under cooling stimulus

Introduction: The dynamics of reactive oxygen species (ROS) in the cytosol and mitochondria function as signaling factors that regulates diverse cellular adaptations. Hydrogen peroxide (H2O2) is considered one of the most influential ROS in regulating physiological function; being a ubiquitous molecule with a long half-life and high diffusing capacity compared to other ROS (e.g., superoxide and hydroxyl radicals). Temperature changes within skeletal muscle myocytes constitute an environmental factor that modulates the expression of antioxidant-related genes. Although muscle temperature reduction by icing is a typical physical therapy modality, the relationship between cooling stimuli and in vivo H2O2 dynamics remains unclear. The purpose of this investigation was to elucidate the H2O2 dynamics in the cytosol and mitochondria induced by cooling stimuli and to determine the relationship between these dynamics and the expression of antioxidant-related genes. Specifically, we tested the hypothesis that cooling would reduce [H2O2] in intracellular compartments (cytosol and mitochondria) and suppress antioxidant-related gene expression levels. Methods: Experiment 1. in vivo H2O2 dynamics observation: Cytosolic and mitochondrial H2O2-sensitive fluorescent proteins (HyPer7, n = 6 and MLS-HyPer7, n = 9) were expressed by electroporation in the anterior tibialis muscle (TA) of male C57/BL6 mice (9-14 week of age). The TA was exposed under anesthesia, and cytosolic and mitochondrial [H2O2] were measured when the muscle temperature was reduced from 35 °C to 0 °C using in vivo bioimaging techniques. Experiment 2. Mice (9-12 week of age) were divided into an icing (ICE) group that received cooling stimulation (muscle temperature reduced from 35 °C to 13 °C and back to 35°C, total 6 sets for 60 min, n = 6) and a control (CONT) group (muscle temperature 35 °C for 60 min, n = 6). At 3 hours after temperature intervention, the TA was removed and mRNA levels were measured by Real-time PCR. Results: Experiment 1. In vivo [H2O2] in each compartment were significantly lower in the cytosol (over the range 28-6°C) and mitochondria (over the range 29-1°C) compared to 35 °C conditions. A decreasing phase of [H2O2] in both compartments was observed from 35 °C to around 13 °C, but, subsequently increasing [H2O2] were observed in the lower temperature range (i.e., 12 to 0 °C). Experiment 2: The ICE group significantly increased mRNA levels of antioxidant enzyme-related genes Nfe2l2 and HSF1 compared to CONT (1.2 ± 0.1-fold and 1.1 ± 0.1-fold, respectively, P < 0.01). On the other hand, mRNA levels of mitochondria-related genes such as PGC1α and COX4 were unchanged. Conclusions: This investigation is the first to reveal the H2O2 and related gene expression impact of cooling stimuli in an in vivo model. Cooling mouse skeletal muscle produced temperature-dependent decreases in [H2O2] that reversed with more severe cooling. In both cytosolic and mitochondrial compartments, the lowest point for [H2O2] was found to be 13°C. Repeated cooling cycles (between 35 and 13 °C) with their attendant decreasing [H2O2] elevated antioxidant-related mRNA levels. These results support that icing therapy may improve the antioxidant capacity of muscle despite decreasing muscle ROS levels. This study was supported in part by a Grant-in-Aid for Japan Society for the Promotion of Science (JSPS) for Grant-in-Aid for JSPS Fellows (no. JP23KJ0967) and JSPS KAKENHI Grant (No. JP20H04074). 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.

EXPOSURE TO AIR POLLUTION FINE PARTICULATE MATTER (PM2.5) ALTERS SYSTEMIC AND TISSUE-SPECIFIC COMPONENTS OF THE RENIN-ANGIOTENSIN SYSTEM

Objective: Previous studies have implicated air pollution fine particulate matter (PM2.5) in various cardiovascular cardiometabolic and renal disease states. However, the molecular mechanisms by which these pollutants mediate these comorbidities have not been fully elucidated. Dysregulation of the renin-angiotensin system (RAS) may be one potential mechanism. This study aimed to investigate the participation of the RAS in air pollution-associated pathologies. Design and method: To study the impact of PM2.5 on systemic and tissue components of the RAS, male 8-week-old Balb/C mice were exposed to filtered air (FA) or urban air (UA) from Buenos Aires City, in whole-body exposure chambers for 14 weeks. Levels of main RAS components including angiotensin-converting enzyme (ACE) and ACE2, as well as AT1, AT2, and Mas receptors (R) abundance, were determined in kidney, heart, and lung tissue by Western Blotting (WB) and their corresponding mRNA expression was detected by RT-qPCR. Circulating angiotensin (Ang) II levels were determined by radioimmunoassay. Results: Exposure to air pollution resulted in increased mRNA levels of ACE and MasR, and increased protein levels of ACE in the kidney; upregulated mRNA and protein abundance of cardiac and pulmonary AT2R and MasR, together with increased levels of proteins nitrated at Tyr residues in both kidney and lung homogenates, indicative of nitrosative stress in these tissues. In addition, exposure to PM2.5 was associated with increased levels of circulating Ang II, indicative of an exacerbation of the RAS. Conclusions: Our findings indicate that chronic exposure to air pollution induces an altered expression of both tissue and systemic components of the RAS. Given that both the AT2R and the MasR have been ascribed to participate in tissue-repair mechanisms, the upregulation of these receptors detected in mouse heart and lung after chronic exposure to polluted air could represent a mechanism of tissue protection against damage induced by PM2.5.

PM2.5 Temporally Decreases Human Brain Endothelial Barrier Proteins and Concomitantly Increases Inflammation and Autophagy in a Dose Dependent Manner

Exposure to airborne particulate matter 2.5 (particle size <PM2.5 um) pollution is a significant co-morbidity for stroke and other cardio- and cerebrovascular diseases, causing premature mortality. However, mechanisms underlying PM2.5-mediated endothelial dysfunction and integrity loss are not known. Our goal was to elucidate how PM2.5 alters brain endothelial phenotype causing inflammation and loss of barrier integrity to provide better insight to the potential pathogenesis of PM2.5. We hypothesized that PM2.5 would alter integral endothelial barrier proteins as well as inflammatory mediators in a dose and temporal dependent manner, subsequently exacerbating the impact of acute ischemic-like injury. Primary adult male human brain microvascular endothelial cells (HBMEC) were preconditioned with PM2.5 (300, 75, or 15 μg/m3; 12, 24, or 36h) or vehicle, then exposed to normoxia (21% O2) or ischemic-like injury, hypoxia plus glucose deprivation (HGD; 1% O2), for 3h. HBMEC barrier function was assessed via trans-endothelial electrical resistance (TEER). HBMEC levels or expression of barrier markers (claudin-5, occludin, ZO-1), adhesion molecules (ICAM-1 and PECAM-1), inflammatory proteins (iNOS, COX-2, LOX-1, TNF-α, IL-1β), and autophagic protein (Beclin-1) were examined using qRT-PCR, in cell western, and standard immunoblotting. Ischemia-like injury as well as PM2.5 decreased HBMEC barrier function. PM2.5 also induced a concomitant temporal and dose dependent decrease in claudin-5 and increase in iNOS as well as TNF-α levels. Additionally, PM2.5 increased protein levels of beclin-1 and LOX-1 like that of HGD alone; however, combination of PM2.5 and HGD decreased levels of beclin-1. HGD alone or in combination with PM2.5 decreased levels of PECAM-1, but PM2.5 alone did not alter PECAM-1. PM2.5 in the presence or absence of HGD induced differential alteration in claudin-5 isoform protein levels. Intriguingly, PM2.5 decreased claudin-5 mRNA levels like that observed in HBMECs exposed to HGD alone. PM2.5 plus HGD significantly increased mRNA levels of both ICAM-1 and IL-1β. In conclusion, PM2.5 mediated inflammatory and vascular pathogenesis involves a potential complex intracellular molecular framework associated with the brain endothelium which is compounded by ischemic like injury. These findings have revealed detrimental effects of PM2.5 on the brain vascular endothelium which will help to identify targets in the endothelium to restore or preserve cerebrovascular homeostasis. University of Arizona Valley Research Partnership Grant VRP37 P2 (RJG), American Heart Association 19AIREA34480018 (RJG), UA VRP Grant VRP55 P1a (TSW). 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.

Effect of Lupeol on the Expression of Neuro Inflammatory Signalling Molecules in Brain Tissues of High Fat Diet and Sucrose Fed Type-2 Diabetic Rats

Lupeol has anti- inflammatory, antimicrobial, anti-protozoal, anti-proliferative, anti-invasion, anti-angiogenic and cholesterol-lowering properties. However, the mechanisms underlying the effect of lupeol on neuroinflammatory signalling molecules have not yet been identified. The study was designed to study the effect of lupeol on the expression of inflammatory signalling molecules in brain tissue of high-fat diet and sucrose-fed type-2 diabetic rats. Adult male albino rats of Wistar 150–180 days old with 180–200 g body weight (b.wt) were divided into four groups of six rats each. Group I: Control (vehicle-treated): Group II: High fat diet-induced type-2 diabetic rats; Group III: Type-2 diabetic rats treated with lupeol (25 mg/kg b.wt/day) orally for 30 days and Group IV: Type-2 diabetic rats treated with metformin (50 mg/kg, b.wt/day orally for 30 days. After 30 days of treatment, the animals were anaesthetized, and brain tissue was dissected and used for the assessment of mRNA expression analysis. Type-2 diabetic animals showed a significant increase (p<0.05) in TNF-α and IL-6 mRNA levels in brain tissue in high-fat diet-induced type-2 diabetic animals. However, lupeol treatment, effectively reduced (p<0.05) the neuroinflammatory signaling molecules (TNF- α and IL-6 mRNA) showing that lupeol has significant role over the control of neuroinflammatory signaling. Our present findings clearly show that lupeol has a significant role in reducing neuroinflammation via the downregulation of TNF-α and IL-6 in brain tissues and hence, lupeol can be a potential natural drug for the treatment of diabetic neuropathy.

Resiquimod Induces C-C Motif Chemokine Ligand 2 Via Nuclear Factor-Kappa B in SH-SY5Y Human Neuroblastoma Cells.

Toll-like receptor (TLR) 7 plays an important role in recognizing virus-derived nucleic acids. TLR7 signaling in astrocytes and microglia is critical for activating immune responses against neurotrophic viruses. Neurons express TLR7, similar to glial cells; however, the role of neuronal TLR7 has not yet been fully elucidated. This study sought to determine whether resiquimod, the TLR7/8 agonist, induces the expression of inflammatory chemokines in SH-SY5Y human neuroblastoma cells. Immunofluorescence microscopy revealed that TLR7 was constitutively expressed in SH-SY5Y cells. Stimulation with resiquimod induced C-C motif chemokine ligand 2 (CCL2) expression, accompanied by the activation of nuclear factor-kappa B (NF-κB) in SH-SY5Y cells. Resiquimod increased mRNA levels of C-X-C motif chemokine ligand 8 (CXCL8) and CXCL10, while the increase was slight at the protein level. Knockdown of NF-κB p65 eliminated resiquimod-induced CCL2 production. This study provides novel evidence that resiquimod has promising therapeutic potential against central nervous system viral infections through its immunostimulatory effects on neurons.

PM2.5 Extracts Induce INFγ-Independent Activation of CIITA, MHCII, and Increases Inflammation in Human Bronchial Epithelium.

The capacity of particulate matter (PM) to enhance and stimulate the expression of pro-inflammatory mediators has been previously demonstrated in non-antigen-presenting cells (human bronchial epithelia). Nonetheless, many proposed mechanisms for this are extrapolated from known canonical molecular pathways. This work evaluates a possible mechanism for inflammatory exacerbation after exposure to PM2.5 (from Puerto Rico) and CuSO4, using human bronchial epithelial cells (BEAS-2B) as a model. The induction of CIITA, MHCII genes, and various pro-inflammatory mediators was investigated. Among these, the phosphorylation of STAT1 Y701 was significantly induced after 4 h of PM2.5 exposure, concurrent with a slight increase in CIITA and HLA-DRα mRNA levels. INFγ mRNA levels remained low amidst exposure time, while IL-6 levels significantly increased at earlier times. IL-8 remained low, as expected from attenuation by IL-6 in the known INFγ-independent inflammation pathway. The effects of CuSO4 showed an increase in HLA-DRα expression after 8 h, an increase in STAT1 at 1 h, and RF1 at 8 h We hypothesize and show evidence that an inflammatory response due to PM2.5 extract exposure in human bronchial epithelia can be induced early via an alternate non-canonical pathway in the absence of INFγ.

Extract of Scutellaria baicalensis and Lonicerae flos improves growth performance, antioxidant capacity, and intestinal barrier of yellow-feather broiler chickens against Clostridium perfringens

In this study, we aimed to investigate the effect of Scutellaria baicalensis and Lonicerae Flos (SL) extract on the growth performance and intestinal health of yellow-feather broilers following a Clostridium perfringens challenge. In total, 600 one-day-old yellow-feather broilers were divided into five treatments (6 replicate pens of 20 birds per treatment), including a control (Con) group fed a basal diet and the infected group (iCon) fed a basal diet and infected with Clostridium perfringens, the other 3 groups receiving different doses of SL (150, 300, and 450 mg/kg) and infected with Clostridium perfringens. The total experimental period was 80 d. When the birds were 24-days-old, a subclinical necrotizing enteritis model was induced by orally inoculating the birds with 11,000 oocysts of mixed Eimeria species on d 24, followed by C. perfringens (108 CFU/mL) from d 28 to 30. The birds were evaluated for parameters such as average weight gain (AWG), average daily feed intake (ADFI), mortality, feed conversion ration (FCR), intestinal lesion score, intestinal C. perfringens counts, and villus histomorphometry. Results indicated that C. perfringens infection led to reduced AWG and the levels of tight junction proteins, increased the FCR, ileum E. coli load, and intestinal permeability, causing damage to the intestinal mucosal barrier (P < 0.05). Compared with the infected group, supplementing 300 mg/kg of SL significantly increased AWG at 43 to 80 d, the ratio of villus height to crypt depth in the jejunum and ileum at 35 d, and the activity of superoxide dismutase (SOD) in serum. It also significantly reduced the FCR at 22 to 42 d, intestinal lesion score, and the amount of C. perfringens in the ileum (P < 0.05). Additionally, compared with the infected group, the addition of 300 mg/kg SL significantly increased mRNA levels of claudin-2, claudin-3, mucin-2, and toll-like receptor 2 (TLR-2) in the ileum of infected birds at 35 d of age. In conclusion, supplementation with SL extract could effectively mitigate the negative effects of C. perfringens challenge by improving intestinal barrier function and histomorphology, positively influencing the growth performance of challenged birds.

Two-Month Voluntary Ethanol Consumption Promotes Mild Neuroinflammation in the Cerebellum but Not in the Prefrontal Cortex, Hippocampus, or Striatum of Mice.

Chronic ethanol exposure often triggers neuroinflammation in the brain's reward system, potentially promoting the drive for ethanol consumption. A main marker of neuroinflammation is the microglia-derived monocyte chemoattractant protein 1 (MCP1) in animal models of alcohol use disorder in which ethanol is forcefully given. However, there are conflicting findings on whether MCP1 is elevated when ethanol is taken voluntarily, which challenges its key role in promoting motivation for ethanol consumption. Here, we studied MCP1 mRNA levels in areas implicated in consumption motivation-specifically, the prefrontal cortex, hippocampus, and striatum-as well as in the cerebellum, a brain area highly sensitive to ethanol, of C57BL/6 mice subjected to intermittent and voluntary ethanol consumption for two months. We found a significant increase in MCP1 mRNA levels in the cerebellum of mice that consumed ethanol compared to controls, whereas no significant changes were observed in the prefrontal cortex, hippocampus, or striatum or in microglia isolated from the hippocampus and striatum. To further characterize cerebellar neuroinflammation, we measured the expression changes in other proinflammatory markers and chemokines, revealing a significant increase in the proinflammatory microRNA miR-155. Notably, other classical proinflammatory markers, such as TNFα, IL6, and IL-1β, remained unaltered, suggesting mild neuroinflammation. These results suggest that the onset of neuroinflammation in motivation-related areas is not required for high voluntary consumption in C57BL/6 mice. In addition, cerebellar susceptibility to neuroinflammation may be a trigger to the cerebellar degeneration that occurs after chronic ethanol consumption in humans.

Intervertebral disc injury triggers neurogenic inflammation of adjacent healthy discs

BACKGROUND CONTEXTIntervertebral disc degeneration is common and may play an important role in low back pain, but it is not well-understood. Previous studies have shown that the outer layer of the annulus fibrosus of a healthy disc is innervated by nociceptive nerve fibers. In the process of disc degeneration, it can grow into the inner annulus fibrosus or nucleus pulposus and release neuropeptides. Disc degeneration is associated with inflammation that produces inflammatory factors and potentiates nociceptor sensitization. Subsequently neurogenic inflammation is induced by neuropeptide release from activated primary afferent terminals. Because the innervation of a lumbar disc comes from multisegmental dorsal root ganglion neurons, does neurogenic inflammation in a degenerative disc initiate neurogenic inflammation in neighboring healthy discs by antidromic activity? PURPOSEThis study was based on animal experiments in Sprague-Dawley rats to investigate the role of neurogenic inflammation in adjacent healthy disc degeneration induced by disc injury. STUDY DESIGNThis was an experimental study. METHODSSeventy-five 12-week-old, male Sprague-Dawley rats were allocated to 3 groups (sham group, disc injury group and disc injury+TrkA antagonist group). The disc injury group was punctured in the tail disc between the eighth and ninth coccygeal vertebrae (Co8–9) to establish an animal model of tail intervertebral disc degeneration. The sham group underwent only skin puncture and the disc injury+TrkA antagonist group was intraperitoneally injected with GW441756 two days before disc puncture. The outcome measure included quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTSDisc injury induced an increase in aggrecan, NGF, TrkA, CGRP, SP, IL-1β, and IL-6 mRNA levels in the injured (Co8–9) and adjacent discs (Co7–8), which reached a peak on day 1, then gradually decreased, and returned to normal on day 14. After intraperitoneal injection of GW441756 prior to puncture, the mRNA levels of the above indicators were down-regulated in Co7–8 and Co8–9 intervertebral discs on the 1st and 7th days. The protein content of the above indicators in Co7–8 and Co8–9 intervertebral discs showed roughly the same trend as mRNA levels. CONCLUSIONSDegeneration of one disc can induce neurogenic inflammation of adjacent healthy discs in a rat model. CLINICAL SIGNIFICANCEThis model supports a key role of neurogenic inflammation in disc degeneration, and may play a role in the experience of low back pain.