Significant Decrease In Gene Expression Research Articles

Effect of 8 Weeks of High Intensity Interval Training (HIIT) With and Without Calorie Restriction on Gene Expression of Caspase-3 and Caspase-9 Proteins in Male Rats

Background and Aim Apoptosis or programmed death is active biological process and reversible that the balance between growth and cell death in various tissues, particularly myocardial tissue is essential role. The aim of this study was the effect of 8 weeks High Intensity Interval Training (HIIT) with and without food restriction on gene expression of Caspase 3 and 9 in rats. Methods & Materials: This study was experimental multi group design with control group which conducted on 30 rats with two months old male. Subjects were divided in to five homogenous groups including base control, control, caloric restriction, interval exercise training and caloric restriction+interval exercise training. Training groups five sessions in weeks were participated in interval exercise training for 8 weeks. The level of gene expression of the proteins Caspase 3 and 9 was evaluated by Real Time-PCR. Dates were analyzed using the 1-way ANOVA at the level of (P<0.05). Ethical Considerations: The protocol of this study was approved by the Medical Committee of Sanandaj University of Medical Sciences (Code: IR.MYK.REC.1397.5022). Results: The results showed that the training group had a significant decrease in gene expression of Caspase 3 in comparison with caloric restriction+exercise training (P<0.05). Also, exercise training and exercise training+caloric restriction significantly decrease in the gene expression of Caspase 9 compared to caloric restriction, base control and control (P<0.05) and significantly decrease in the gene expression of Caspase 3 compared to base control and control (P<0.05). Conclusion: According to the results, it seems that high intensity interval training with and without calorie restriction provides the necessary adaptations to inhibit or stop apoptosis induced by aerobic exercise.

Alzheimer’s and Parkinson’s brain tissues have reduced expression of genes for mtDNA OXPHOS Proteins, mitobiogenesis regulator PGC-1α protein and mtRNA stabilizing protein LRPPRC (LRP130)

We used RNA sequencing (RNA-seq) to quantitate gene expression in total RNA extracts of vulnerable brain tissues from Alzheimer’s disease (AD, frontal cortical ribbon) and Parkinson’s disease (PD, ventral midbrain) subjects and phenotypically negative control subjects. Paired-end sequencing files were processed with HISAT2 aligner/Cufflinks quantitation against the hg38 human genome. We observed a significant decrease in gene expression of all mtDNA OXPHOS genes in AD and PD tissues. Gene expression of the master mitochondrial biogenesis regulator PGC-1α (PPARGC1A) was significantly reduced in AD; expression of genes for mitochondrial transcription factors A (TFAM) and B1/B2 (TFB1M/TFB2M) were not significantly changed in AD and PD tissues. 2-way ANOVAs showed significant reduction in AD brain Complex I subunits’ expressions and nearly significant reductions in PD brain. We found a significant reduction in both AD and PD brain samples of expression of genes for leucine-rich pentatricopeptide repeat containing (LRPPRC, a.k.a. LRP130), a known mtRNA-stabilizing protein. Our findings suggest that AD and PD brain tissues have a reduction in mitochondrial ATP production derived from a reduction of mitobiogenesis and mtRNA stability. If true, increased brain expression of PGC-1α and/or LRPPRC may improve bioenergetics of AD and PD and alter the course of neurodegeneration in both conditions. (201 words)

Comprehensive gene expression analysis of IDH1/2 mutant biliary cancers (BC).

4598 Background: Isocitrate dehydrogenases (IDH) mutations (mut) identify a distinct subtype of BC that has yet to be fully characterized. We recently showed that IDH1/2 mutant (mIDH) BC harbor specific gene alterations involving chromatin remodeling and DNA repair, and a differential immune markers profile compared to other mIDH GI tumors. Here we aim to further dissect the molecular profile of mIDH BC through a comprehensive gene expression profiling analysis. Methods: 524 BC samples (303 intrahepatic cholangiocarcinoma, IHCC, 67 extrahepatic cholangiocarcinoma, EHCC, 141 gallbladder, 13 unspecified) collected between February to December of 2019 were included. Samples were analyzed using NextGen DNA sequencing (NextSeq, 592 gene panel), whole transcriptome RNA sequencing (NovaSeq) and immunohistochemistry (Caris Life Sciences, Phoenix, AZ). EBseq was used to identify differentially expressed genes in mIDH vs wild type (WT) tumors with control for FDR ( Q < 0.2). Pathway and functional enrichment analysis was performed using g:Profiler and Enrichr. Results: mIDH frequency in our cohort was 11.4% (60/524), with higher prevalence of IDH1 mut (8.8%). IHCC showed the highest mut prevalence: IDH1 13.5%, IDH2 4.6%. mIDH was more common in females ( P = 0.0036). A total of 774 genes were significantly differentially expressed between mIDH and WT: 582 underexpressed (Fold change, FC: 0.025~0.699); 192 overexpressed (FC: 1.43~3.3). Pathway enrichment showed a significant decrease of gene expression in cytokine-cytokine receptor interaction ( Q = 0.002) and inflammatory response genes ( Q = 0.005) in mIDH. Interferon-γ- and PD1 signaling-related genes expression was significantly lower in mIDH vs WT ( Q = 0.02) including IFNG (FC 0.32), NKG7 (FC 0.36), CD8B (FC 0.37), BATF (FC 0.40), PD1 (FC 0.53), SLAMF6 (FC 0.55) and PD-L2 (FC 0.60). Wnt and cadherin signaling were also enriched for altered expression in several genes in mIDH BC ( Q = 3.86e-7 and < 0.00001, respectively). Conclusions: To our knowledge, this is the largest and most extensive gene expression profiling study focused on mIDH BC. Our data show for the first time a distinct gene expression profile characterizing mIDH tumors which display significant downregulation of inflammatory response pathways and immune-related genes. These findings contribute to further the understanding of mIDH BC and may inform the future development of rational combination therapies.

Angiotensin AT2 receptors in the solitary tract nucleus lower blood pressure via inhibition of GABA signaling

Chronic CNS administration of angiotensin AT2 receptor (AT2R) agonists lowers blood pressure (BP) in normal and hypertensive rodents, but the location(s) and mechanism(s) of action are unknown. One locus may be the intermediate solitary tract nucleus (intNTS). Our previous studies demonstrated a high density of AT2R located on GABA neurons in the intNTS (de Kloet et al, Brain Struct. Func., 2016, 22:891–912), and decades of research have established increased GABA signaling in the intNTS as a pathophysiological mechanism contributing to increased BP and hypertension. These studies reveal that reductions in BP (~8 mmHg) induced by 2‐week intracerebroventricular (icv) infusion of the AT2R agonist C21 (7.5 ng/kg/h) in normotensive mice is associated with significant decreases in gene expression of GABA synthetic enzymes (GAD‐1 & GAD‐2) in the intNTS. Based on this, we developed the hypothesis that the depressor action of C21 is mediated through AT2R located on intNTS GABA neurons and a reduction of GABA signaling. To test this, we engineered a novel mouse line that has Cre‐recombinase expression directed to the AT2R gene (AT2R‐Cre), and demonstrated that Cre recombinase activity mirrors AT2R mRNA expression within the NTS. Next, we used the Cre‐LoxP system and virally (AAV)‐mediated gene transfer to direct expression of the light‐sensitive excitatory opsin channelrhodopsin‐2 (ChR2) and/or eYFP specifically to neurons in the intNTS that synthesize AT2R. Using these mice, we performed electrophysiological‐ or cardiovascular recordings during optogenetic manipulations (blue light) that selectively excite AT2R‐expressing neurons in the intNTS. For the electrophysiological studies, optical stimulation produced a strong inward current in voltage clamp (611.80 ± 78.55 pA; n=9), and robust frequency dependent firing of action potentials in current‐clamp, validating functional opsin expression within AT2R‐expressing neurons. Optical activation of the Chr2/eYFP‐containing AT2R‐expressing neurons in brain slices resulted in activation of adjacent (non‐eYFP‐labeled) neurons, as indicated by light‐evoked currents (n=31), an effect blocked by GABA receptor antagonists (PTX and CGP). Cardiovascular studies revealed that (i) Optogenetic stimulation of the AT2R intNTS neurons expressing ChR2 in isoflurane anesthetized mice elicited significant elevations in BP compared with control mice that express only eYFP; (ii) The AT2R‐containing neurons that were activated by light stimulation are GABAergic, as indicated by increased expression and co‐localization of c‐Fos mRNA with GAD‐1 mRNA and eYFP; (iii) Chronic icv infusion of C21 as above significantly blunted the light‐induced increases in BP, an effect that persisted across all patterns of optical stimulation. Collectively, these data indicate that AT2R‐mediated decreases in BP involve a blunting of GABA signaling in the intNTS, and suggest that neurons expressing AT2R in the intNTS may be an access point for cardiovascular control.Support or Funding InformationNIH grants HL‐125805 (AdK), HL‐145028 (AdK), HL‐136595 (EGK/CS)

Renal Fibrosis Is Significantly Attenuated Following Targeted Disruption of Cd40 in Experimental Renal Ischemia.

BackgroundRenal artery stenosis is a common cause of renal ischemia, contributing to the development of chronic kidney disease. To investigate the role of local CD40 expression in renal artery stenosis, Goldblatt 2‐kidney 1‐clip surgery was performed on hypertensive Dahl salt‐sensitive rats (S rats) and genetically modified S rats in which CD40 function is abolished (Cd40 mutant).Methods and ResultsFour weeks following the 2‐kidney 1‐clip procedure, Cd40 mutant rats demonstrated significantly reduced blood pressure and renal fibrosis in the ischemic kidneys compared with S rat controls. Similarly, disruption of Cd40 resulted in reduced 24‐hour urinary protein excretion in Cd40 mutant rats versus S rat controls (46.2±1.9 versus 118.4±5.3 mg/24 h; P<0.01), as well as protection from oxidative stress, as indicated by increased paraoxonase activity in Cd40 mutant rats versus S rat controls (P<0.01). Ischemic kidneys from Cd40 mutant rats demonstrated a significant decrease in gene expression of the profibrotic mediator, plasminogen activator inhibitor‐1 (P<0.05), and the proinflammatory mediators, C‐C motif chemokine ligand 19 (P<0.01), C‐X‐C Motif Chemokine Ligand 9 (P<0.01), and interleukin‐6 receptor (P<0.001), compared with S rat ischemic kidneys, as assessed by quantitative PCR assay. Reciprocal renal transplantation documented that CD40 exclusively expressed in the kidney contributes to ischemia‐induced renal fibrosis. Furthermore, human CD40‐knockout proximal tubule epithelial cells suggested that suppression of CD40 signaling significantly inhibited expression of proinflammatory and ‐fibrotic genes.ConclusionsTaken together, our data suggest that activation of CD40 induces a significant proinflammatory and ‐fibrotic response and represents an attractive therapeutic target for treatment of ischemic renal disease.

The effects of interval aerobic training on mesenchymal biomarker gene expression, the rate of tumor volume, and cachexia in mice with breast cancer.

Objective(s):It seems that regular exercise can have inhibitory effects on the progression of breast cancer. This study, therefore, aimed to investigate the influences of interval aerobic training on mesenchymal biomarker gene expression, muscle cachexia, and tumor volume changes in mice with breast cancer.Materials and Methods:Thirty-two female Balb/c mice were allocated to four groups: Exercise Tumor Exercise, Rest Tumor Rest (Control), Rest Tumor Exercise, and Exercise Tumor Rest. Interval aerobic training was done 6 weeks before and 4 weeks after tumor induction. Weight test and inverted screen test were carried out as muscle function tests. Data were analyzed using one-way ANOVA and HSD post hoc.Results:The results showed a significant decrease in gene expressions of Twist, Vimentin, and TGF-β in Exercise Tumor Exercise group in comparison with the Control group (P<0.05). Remarkable reduction of the rate of tumor volume was also observed in two training groups (Rest Tumor Exercise, Exercise Tumor Exercise) compared with the control group. According to function tests’ results, muscle functions were diminished due to cancer, but interval aerobic training can keep muscles in a normally-functioning state in cancer (P<0.05).Conclusion:Considering final results, a period of interval aerobic training can be used not only as a prevention method, but also help cancer treatment and impede cachexia by tumor volume reduction, decrease mesenchymal biomarker gene expression, and increase muscle strength functions.

Vitamin D Supplementation Improves Adipose Tissue Inflammation and Reduces Hepatic Steatosis in Obese C57BL/6J Mice.

The beneficial effect of vitamin D (VD) supplementation on body weight gain limitation and inflammation has been highlighted in primary prevention mice models, but the long-term effect of VD supplementation in tertiary prevention has never been reported in obesity models. The curative effect of VD supplementation on obesity and associated disorders was evaluated in high-fat- and high-sucrose (HFS)-fed mice. Morphological, histological, and molecular phenotype were characterized. The increased body mass and adiposity caused by HFS diet as well as fat cell hypertrophy and glucose homeostasis were not improved by VD supplementation. However, VD supplementation led to a decrease of HFS-induced inflammation in inguinal adipose tissue, characterized by a decreased expression of chemokine mRNA levels. Moreover, a protective effect of VD on HFS-induced hepatic steatosis was highlighted by a decrease of lipid droplets and a reduction of triglyceride accumulation in the liver. This result was associated with a significant decrease of gene expression coding for key enzymes involved in hepatic de novo lipogenesis and fatty acid oxidation. Altogether, our results show that VD supplementation could be of interest to blunt the adipose tissue inflammation and hepatic steatosis and could represent an interesting nutritional strategy to fight obesity-associated comorbidities.

45 Expression patterns of PRDM family genes in porcine pre-implantation embryos

Establishing proper levels of pluripotency is essential for normal development. The genome of gametes is remodelled upon fertilisation and pluripotency-related genes are expressed in blastocysts. Multiple pluripotency-related genes are involved in the well-orchestrated process; however, detailed mechanistic actions remain elusive. The PRDM family genes are reported to be closely related to the pluripotency. A previous report noted that PRDM14 plays an important role in the maintenance of pluripotency in human embryonic stem cells (ESCs) and potentially murine ESCs; loss of PRDM14 was found to cause abnormalities in genome-wide epigenetic status. Similarly, PRDM15 was found to be a key regulator of pluripotency in mouse ESCs. Structural similarities among the PRDM family suggest that other PRDM family genes may help to establish and maintain pluripotency in embryos. Unfortunately, little is known about the expression profile of PRDM family in porcine embryos. To expand our understanding of the role of PRDM family in porcine embryos, expression patterns of PRDM gene family were investigated using reverse transcription quantitative (RTq)-PCR. Candidate PRDM family genes were selected based on previous RNA-Seq data in porcine oocytes/embryos. To conduct this study, germinal vesicle (GV), MII, zygote, 4-cell, and blastocyst samples were collected. Complementary DNA synthesised from the samples was used for RT-qPCR to analyse the expression pattern of selected PRDM family genes: PRDM2, PRDM4, PRDM6, PRDM14, and PRDM15. The expression of target genes was normalized to the YWHAG level, an internal control. Then, GV stage was used as a control for ΔΔCT analysis. Two technical replications and three biological replications were performed. Analysis of variance was used for statistical analysis and P-values&amp;lt;0.05 were considered significant. There was a significant decrease in PRDM2 expression in 4-cell and blastocyst, PRDM4 expression in 4-cell, and PRDM6 in all stages (MII, zygote, 4-cell, and blastocyst), compared with the GV stage. Because zygotic genome activation occurs at the 4-cell stage in the pig, the significant decrease in gene expression (PRDM2, PRDM4, and PRDM6) indicates they may be maternally originated and involved in the reprogramming process following fertilisation. On the other hand, there was a significant increase in PRDM15 expression in blastocysts and the PRDM14 transcript was only detected in blastocysts in all three biological replicates, suggesting that the genes are most likely involved in pluripotency maintenance, as was found in previous human studies. These results indicate that PRDM family genes are differentially expressed during early embryo development in pigs and may play a role in maintenance of pluripotency. For further study, we intend to evaluate the role of PRDM family genes during early embryo development in pigs.

Recipient Signaling through the Cannabinoid Type 2 Receptor Regulates IL-6 Independent Neuroinflammation during Graft Versus Host Disease

Neurotoxicity is an increasingly recognized complication of cancer immunotherapy occurring after allogeneic hematopoietic stem cell transplantation (HSCT), immune checkpoint inhibitor administration, and chimeric antigen receptor T cell therapy. While inflammatory cytokines are thought to play a pivotal role, the immunological and biochemical pathways by which neuroinflammation occurs in these settings are still incompletely understood. Using well-defined murine models of graft versus host disease (GVHD) [C57BL/6 (H-2b)→Balb/c (H-2d) and B10.BR(H-2k)→B6(H-2b)] to examine T cell-mediated inflammation in the central nervous system (CNS) after allogeneic HSCT, we previously identified host IL-6 production as a pivotal cytokine responsible for inducing neuroinflammation. Notably, whereas blockade of this signaling pathway reduced donor T cell infiltration, inflammatory cytokine production, and microglial cell expansion in the brain, it did not reverse dysregulation in the tryptophan metabolic pathway and the subsequent production of neurotoxic metabolites, indicating the presence of an IL-6 independent pathway that promoted CNS inflammation. To understand the pathophysiology of this process, we examined the role of endocannabinoid signaling through the type 2 cannabinoid receptor (CB2R) since this receptor is expressed on all immune cells and the natural ligands for this receptor modulate cognitive and behavioral function. Using genetic approaches to define the role of CB2R signaling, we observed that absence of the CB2R on donor cells had no effect on T cell infiltration or expression of inflammatory cytokines (IFN-γ, GM-CSF, IL-6 or TNF-α) when compared to animals transplanted with wild type (WT) grafts. There was also no difference in the production of neurotoxic tryptophan metabolites when assessed by mass spectrometry. Conversely, absence of CB2R expression on host tissues significantly reduced donor T cell infiltration and inflammatory cytokine gene expression, indicating that host CB2R expression potentiated CNS inflammation. In addition, there was a significant decrease in gene expression of indole 2,3 dioxygenase and kynureninase which are key enzymes involved in the pathway by which tryptophan is metabolized to neurotoxic metabolites under inflammatory conditions, as well as a decreased ratio of neurotoxic (i.e. 3-hydroxykynurenine) to neuroprotective (i.e. kynurenic acid) tryptophan metabolites. Pharmacological inhibition of CB2R signaling with a peripherally restricted CB2R-selective antagonist had no effect on reducing GVHD-induced inflammation indicating that peripheral regulation of CB2R did not alter CNS inflammation and that a CB2R-expressing population resident in the brain was critical for regulating disease. To identify CB2R+ cells in the brain, we employed a novel CB2R-EGFP reporter mouse and examined regions with diverse functional roles (i.e. amygdala, brainstem, and cerebellum). These studies revealed that CB2R expression was detected only on host microglial cells. To determine whether microglial cells promoted CNS inflammation, we employed IL-34-/- mice, which specifically lack microglial cells, as recipients and demonstrated a significant reduction in donor T cells and inflammatory cytokines in the brain compared to WT controls. Thus, CB2R expression on microglial cells appeared to be critical for the regulation of inflammation in the CNS. Since we had shown that blockade of IL-6 failed to prevent tryptophan metabolite dysregulation, we examined whether this treatment had any effect on microglial CB2R expression. Treatment with an anti-IL-6 receptor antibody did not alter CB2R expression, indicating that the proinflammatory effects mediated by host CB2R+ microglial cells were IL-6 independent. Collectively, these results indicate that signaling through the CB2R expressed on microglial cells appears to constitute an IL-6 independent pathway by which neuroinflammation and concurrent tryptophan metabolite dysregulation is propagated. These data also provide an explanation for the failure of anti-IL-6R antibody administration with tocilizumab to prevent and/or treat neurotoxicity occurring after T cell-directed immunotherapy, and suggests that strategies targeting key enzymes in the tryptophan metabolic pathway or the CB2R directly may be novel therapeutic approaches to ameliorate this complication. Disclosures No relevant conflicts of interest to declare.

Omega-3 fatty acids and vitamin E supplementation can affect gene expressions of SIRT1, FOXO1 and UCP-2 in coronary artery disease patients

AimsThis randomized controlled trial (RCT) study was carried out to assess effects of omega-3 supplementation alone and in combination with vitamin E on gene expressions of SIRT1, SIRT3, SIRT6, FOXO1, FOXO3 and UCP-2 in patients with coronary artery disease (CAD). MethodsParticipants of this RCT included 60 male CAD patients who classified into three study groups using block randomization, including 1) OE receiving omega-3 fatty acids (EPA: 720 mg, DHA: 480 mg) and vitamin E (400 IU); 2) OP receiving omega3 fatty acids (EPA: 720 mg, DHA: 480 mg) and vitamin E placebo; and 3) PP receiving omega-3 and vitamin E placebos for two months. ResultsIn compared to PP group, gene expression of SIRT1 increased significantly in OE group (P = 0.037) while this group experienced significant decrease in gene expression of UCP-2 (P = 0.035). The expression of FOXO1 increased significantly in both OE and OP groups in compared to PP group (P = 0.021 and P = 0.035, respectively). However, gene expressions of SIRT3, SIRT6 and FOXO3 were not different between study groups. ConclusionThe current study has shown that omega-3 fatty acids and vitamin E supplementation can increase gene expressions of SIRT1, FOXO1 while it can decrease gene expression of UCP-2 in CAD patients.

524-P: RNA-Sequencing of Laser-Microdissected Glomeruli and Tubules Reveal Differentially Expressed Genes in Diabetic Kidney Disease

In order to identify novel pathways relevant to human Diabetic Kidney Disease (DKD), we performed transcriptome profiling of i) biopsy-proven diabetic glomerulosclerosis (N=15); ii) hypertensive glomerulosclerosis (N=12); iii) non-DM nephrectomized kidneys (N=4). Laser microdissection of kidney or biopsy specimens was performed under direct visual control. Approximately 5-10 glomerulus and 20 randomly selected tubulointerstitial areas were isolated from each specimen. Total RNA was extracted from isolated glomeruli and tubulointerstitial tissues, and sequenced using the SMARTer stranded RNA-Seq kit with Ribo Zero treatment and Novaseq6000, generating 32∼111 million 101 bp paired-end reads per sample. Overall, there was significant overlap between differentially expressed genes (DEGs) in both glomeruli and tubulointerstitial areas from diabetic glomerulosclerosis and hypertensive glomerulosclerosis. We identified 524 genes which showed significant increased gene expression in glomeruli in both diabetic kidney disease (DKD) and hypertensive glomerulosclerosis (HTN) compared to controls, with an additional 611 genes increased specifically in DKD and 37 in HTN compared to controls. 868 genes showed significant decrease in gene expression in glomeruli in both DKD and HTN compared to controls, with another 688 genes decreased in DKD and 88 in HTN vs. controls. Similar trends were observed for DEGs in renal tubuloinsterstitium in DKD and HTN. Several podocyte-specific genes showed reduced expression in DKD. HSPA1A, a heat shock protein, showed marked reduction in expression in DKD glomeruli vs. control (Log2 fold change -3.87, padj =1.29x10-10), as well as HTN vs. controls (Log2 fold change -3.65, padj =1.57x10-9). KEGG pathway analysis noted enrichment of MAPK signaling pathway in DKD and HTN. Integration of transcriptome findings with EWAS and GWAS data is currently underway. Our study identified important pathways implicated in DKD. Disclosure R.C.W. Ma: Advisory Panel; Self; Boehringer Ingelheim International GmbH. Research Support; Self; AstraZeneca, Bayer AG, Pfizer Inc. Stock/Shareholder; Self; GemVCare. Other Relationship; Self; AstraZeneca. N. Jin: None. C. Luk: None. H. Lee: None. C.K. Lim: None. W. So: None. R.C. Chan: None. H.Y. Lan: None. C. Ng: None. J.C. Chan: Board Member; Self; Asia Diabetes Foundation. Consultant; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Lilly Diabetes, Medtronic, Merck Sharp &amp; Dohme Corp., Sanofi-Aventis. Research Support; Self; Amgen Inc., AstraZeneca, Lee Powder, Lilly Diabetes, Pfizer Inc., Sanofi-Aventis. Speaker's Bureau; Self; Ascensia Diabetes Care. Stock/Shareholder; Self; GemVCare. T. Chan: Board Member; Self; Codex Genetics Limited. C. Szeto: None. Funding Research Grants Council of Hong Kong (T12-402/13N); Chinese University of Hong Kong

Role of Alpha Lipoic Acid in Oxidant /Antioxidant Status and Gene Expression of Glutathione Reductase in Hydrogen Peroxide Exposed Rats: (Part -2)

This study was designated to evaluate the protective role of alpha lipoic acid against oxidative stress resulted by hydrogen peroxide on some oxidants/ antioxidants parameters and gene expression of glutathione peroxidase in adult Wistar rats. Forty adult male rats were randomly divided into four equal groups (10 rats /group) and were handled daily as follows for 56 days : Control group were intubated distal water and received ordinary tap water ; group T1 were intubated 60mg/kg B.W of alpha lipoic acid and received ordinary tap water ; group T2 were received hydrogen peroxide in tap water at concentration of 0.5% , while group T3 were intubated 60mg/kg B.W of alpha lipoic acid and received ordinary tap water containing 0.5% hydrogen peroxide. Fasting blood samples were collected at 0, 28 and 56 days of experimental periods for measurement of serum peroxynitrite and malondialdehyde concentrations, as well catalase activity. Furthermore, gene expression of glutathione reductase in liver was investigated. Administration of 0.5% hydrogen peroxide in drinking water (group T2) manifested a significant elevation in serum peroxynitrite and malondialdehyde with significant decrease in catalase and Glutathione, concentrations. Also, a significant decrease in gene expression of glutathione reductase was observed as compared to other treated groups. Nevertheless, rats in group T3 shows a significantly improvement in oxidant /antioxidant status with increase in folds changes of gene expression of glutathione reductase as compared to control and T2. In conclusion, supplementation of alpha lipoic acid to rats significantly reduced oxidative stress –induced by hydrogen peroxide and caused improvement of gene expression of glutathione reductase in liver via its antioxidant properties.

Marine biogenics in sea spray aerosols interact with the mTOR signaling pathway

Sea spray aerosols (SSAs) have profound effects on our climate and ecosystems. They also contain microbiota and biogenic molecules which could affect human health. Yet the exposure and effects of SSAs on human health remain poorly studied. Here, we exposed human lung cancer cells to extracts of a natural sea spray aerosol collected at the seashore in Belgium, a laboratory-generated SSA, the marine algal toxin homoyessotoxin and a chemical inhibitor of the mammalian target of rapamycin (mTOR) pathway. We observed significant increased expression of genes related to the mTOR pathway and Proprotein convertase subtilisin/kexin type 9 (PCSK9) after exposure to homoyessotoxin and the laboratory-generated SSA. In contrast, we observed a significant decrease in gene expression in the mTOR pathway and of PCSK9 after exposure to the natural SSA and the mTOR inhibitor, suggesting induction of apoptosis. Our results indicate that marine biogenics in SSAs interact with PCSK9 and the mTOR pathway and can be used in new potential pharmaceutical applications. Overall, our results provide a substantial molecular evidence base for potential beneficial health effects at environmentally relevant concentrations of natural SSAs.

A6251 Periostin splicing variant regulates cardiac fibrosis after myocardial infarction

Objectives: Although mortality rate due to acute myocardial infarction (AMI) has been extremely improved by the development of early reperfusion therapy, chronic heart failure following AMI is still clinically major issue. We previously demonstrated the level of periostin increased significantly following ischemia. Periostin alternative splicing variant 1 (Pn 1) over-expression resulted in ventricular dilation with enhanced interstitial collagen deposition in rat AMI model. However, the role of alternative splicing variants of periostin is still unclear. Methods: We employed neutralizing antibody (PnAb) that selectively inhibit Pn 1 by blockade of exon-17 and tested in rat AMI model by injecting once a week (200ug ip) throughout experiments. Results: Administration of PnAb resulted in a significant decrease in infarcted and fibrotic area of myocardium preventing ventricular wall thinning and dilatation. Moreover, surviving myocytes in ischemic area was significantly larger than that of control IgG group. Inhibition of fibrosis by PnAb was associated with a significant decrease in gene expression of fibrotic markers such as collagen I, collagen III, and TGF-β1. Importantly, the number of α-SMA positive myofibroblasts was significantly reduced in the heart treated with PnAb, while cardiomyocyte proliferation and angiogenesis were comparable in both IgG and PnAb group. Additionally, the level of Pn 1 expression was correlated significantly with the severity of AMI. In vitro study revealed that Pn 1, but not Pn 2 or 4, inhibited fibroblast and myocyte attachment, leading to myocyte death which might account for cell slippage observed during cardiac remodeling. Conclusion: Specific inhibition of Pn exon-17 by neutralizing antibody without suppressing other Pn variants might offer a new class of medication for the

MSP‐dependent activation of CNS Ron Receptor Tyrosine Kinase signaling attenuates neuroinflammation through the physiological regulation of IL1B and NLRP3 inflammasome pathway

Neurodegenerative disorders, such as Alzheimer's Disease and aging associated dementia, are the leading causes of disease burden globally and are biologically very well characterized by severe central‐nervous‐system(CNS) inflammation. To date, very little is known about how chronic inflammation develops in the brain, challenging neuroimmune function and in turn regulating neural disease progression. A sentinel group of immune cells, the macrophages, closely regulate inflammation by taking on different states, classical activated(M1) or alternatively activated anti‐inflammatory (M2). The source of inflammation in many neurodegenerative diseases has been linked to a decrease in CNS M2 and increase in M1 population, associated with NLR family pyrin domain containing 3 (NLRP3) inflammasome activation and interleukin (IL)‐1β accumulation in microglia. Our lab studies a transmembrane tyrosine‐kinase receptor, Receptor D'Origine Nantais (Ron), which is expressed on tissue resident M2 macrophages including CNS microglia. Our preceding data confirmed that a loss of Ron responsiveness to its ligand, macrophage stimulating protein(MSP), exacerbates disease‐mediated neuroinflammation in murine models of multiple sclerosis and diet induced inflammation. These results demonstrated that Ron plays a key protective role in CNS health and homeostasis. However, the underlying cellular mechanism as to how Ron signaling mediates neuroprotection remains to be characterized. Herein, the objective of this study was to evaluate and identify a novel cell signaling pathway underlying Ron mediated neuroprotection, with in vitro assays and in vivo modelling. In the in vitro studies, immortalized human microglial cells (CHME‐3) were pretreated or untreated with Ron agonist MSP followed by LPS stimulation. MSP‐pretreated CHME‐3 cells had attenuated inflammation with significant decreases in gene expression and protein levels of interleukin‐IL1β following the LPS challenge. Gene profiling with qRT‐pcr revealed MSP‐dependent activation of Ron attenuated NLRP3 inflammasome activation with decreased microglial expression of AIM2, NLRP3 and its adaptor ASC and NLRP3 substrates, caspase‐1 and Interleukin‐IL18. To confirm these in vitro results, we evaluated the role of Ron signaling in an in vivo disease model of chronic neuroinflammation, whereby Apolipoprotein E knockout (ApoE−/−) and Ron receptor ApoE double knockout (DKO) transgenic mice were developed and maintained on a high fat (60% fat) high‐cholesterol diet(1.25%cholesterol) (HFHCD) for 18 weeks. DKO CNS exhibited increased protein expression of cyclooxygenase‐2(COX‐2) and the NLRP3 inflammasome complex. It is known that COX‐2 regulates NLRP3 inflammasome‐derived IL‐1β accumulation, thus this observation was a pivotal confirmation of an upstream regulator in this pathway. The tissue inflammation was associated with increased gene expression of COX‐2 effector inducible‐nitric oxide synthase (iNOS) and NLRP3 substrates IL1β and IL18. These results elucidate a neuroprotective mechanism of CNS Ron‐receptor tyrosine kinase activation induced suppression of NLRP3 inflammasome pathway, leading to decreased caspase‐1 and IL‐1β accumulation in microglial cells and CNS tissue. This is the first study to illustrate a potential mechanism underlying Ron expression in cellular neuroinflammation and has supported on‐going studies into the protective role of Ron in amyloidosis and tauopathy.Support or Funding Information1. NIH‐T32 NRSA Award NIH5T32GM108563‐02 (MPI), Pre‐Doctoral Fellowship2. American Association of Immunologists (AAI) Careers in Immunology Pre‐Doctoral Fellowship3. Penn State Department of Veterinary and Biomedical SciencesThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Astragaloside IV reverses MNNG-induced precancerous lesions of gastric carcinoma in rats: Regulation on glycolysis through miRNA-34a/LDHA pathway.

This study was designed to investigate the precancerous lesions of gastric carcinoma (PLGC)-reversing mechanisms of astragaloside IV (ASIV) in N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced PLGC rats. All rats were sacrificed after 10-week treatment. Gastric tissue was analyzed by using histopathology and electron microscope. To be fully evidenced, LDHA, p53, TIGAR, MCT1, MCT4, HIF-1α, CD147, and miRNA-34a were detected by Western blotting and Real-time Quantitative polymerase chain reaction (RT-qPCR). As histopathology and electron microscope showed, it can be clearly observed that the area of dysplasia was reduced in ASIV groups, indicating that MNNG-induced PLGC was markedly reversed by ASIV. Moreover, compared with model group, a significant decrease in gene expressions of LDHA, MCT1, MCT4, HIF-1α, CD147, and TIGAR was observed whereas miRNA-34a level was increased in ASIV groups. A significant up-regulation induced by MNNG in protein levels of LDHA, MCT1, MCT4, HIF-1α, and CD147 was attenuated in rats treated with ASIV. In contrast, the decreased expression of TIGAR was restored by ASIV. Interestingly, up-regulation of p53 expression induced by MNNG was further increased in ASIV groups. In brief, these results implied that abnormal glycolysis was relieved by ASIV via regulation of the expressions of LDHA, p53, TIGAR, MCT1, MCT4, HIF-1α, CD147, and miRNA-34a.

Expression of NK Cell Surface Receptors in Breast Cancer Tissue as Predictors of Resistance to Antineoplastic Treatment

Background:Currently, one of the most used strategies for the treatment of newly diagnosed patients with breast cancer is neoadjuvant chemotherapy based on the application of taxanes and anthracyclines. However, despite the high number of patients who develop a complete pathological clinical response, resistance and relapse following this therapy continue to be a clinical challenge. As a component of the innate immune system, the cytotoxic function of Natural Killer (NK) cells plays an important role in the elimination of tumor cells. However, the role of NK cells in resistance to systemic therapy in breast cancer remains unclear. The present project aims to evaluate the gene expression profile of human NK cells in breast cancer tissue resistant to treatment with taxanes–anthracyclines.Methods:Biopsies from tumor tissues were obtained from patients with breast cancer without prior treatment. Histopathological analysis and ex vivo exposure to antineoplastic chemotherapeutics were carried out. Alamar blue and lactate dehydrogenase release assays were performed for quantitative analysis of tumor viability. Gene expression profiles from tumor tissues without prior exposure to therapeutic drugs were analyzed by gene expression microarrays and verified by polymerase chain reaction.Results:A significant decrease in gene expression of cell-surface receptors related to NK cells was observed in tumor samples resistant to antineoplastic treatment compared with those that were sensitive to treatment.Conclusion:A decrease in NK cell infiltration into tumor tissue might be a predictive marker for failure of chemotherapeutic treatment in breast cancer.

Estrogen Modulates Corneal Nociception and Maintains Corneal Homeostasis in Rat Eye.

To evaluate the role of estrogen in corneal nociception, its influence on lacrimal secretion, and development of dry eye. Ovariectomy was performed in normal healthy female rats (OVX). Estrogen replacement was performed in a population of these rats (OVX+E). Tests for dry eye and corneal sensitivity were performed and compared with rats in proestrus (PRO) as controls. Gene expression of neuropeptides such as substance P, calcitonin gene receptor-like protein (CGRP), estrogen receptor α, TRPV1, and TRPM8 was evaluated in the cornea and trigeminal ganglion. Expression of substance P and CGRP in the cornea was also examined by immunohistochemistry. The response of the cornea to capsaicin and menthol was evaluated to identify the activity of receptors TRPV1 and TRPM8, respectively. There was a significant decrease in tear formation (4.2 ± 0.6 mm/min vs. 6.6 ± 0.42 mm/min), corneal sensitivity (2.2 ± 0.17 cm vs. 6 ± 0 cm), and increase in fluorescein staining in corneas after ovariectomy compared with controls. There was a significant decrease in gene expression of CGRP, substance P, TRPV1, and TRPM8 in the ovarioectomized cornea. A significant decrease in tear formation (3.17 ± 0.30 mm/min vs. 7.17 ± 0.87 mm/min) and eye wipe response (10.5 ± 1.99 wipes vs. 18.33 ± 1.05 wipes) after treatment with menthol and capsaicin in OVX rats was observed. Estrogen replacement significantly enhanced tear formation (4.02 ± 0.6 mm/min vs. 6.7 ± 0.80 mm/min), corneal sensitivity (2.2 ± 0.17 cm vs. 3.2 ± 0.17 cm), and response to capsaicin (10.5 ± 1.99 eye wipes vs. 24.5 ± 0.92 wipes) and menthol (3.17 ± 0.30 mm/min vs. 6.5 ± 0.22 mm/min) and increased expression of neuropeptides, TRPV1 and TRPM8. This study demonstrates the role of estrogen in corneal nociception and its deficiency as a cause of dry eye.

Association of the gene expression variation of tumor necrosis factor-α and expressions changes of dopamine receptor genes in progression of diabetic severe foot ulcers.

Objective(s):Regulation of pro-inflammatory factors such as TNF-α which are secreted by the immune cells through induction of their several receptors including dopamine receptors (especially DRD2 and DRD3) is one of the noticeable problems in diabetic severe foot ulcer healing. This study was conducted to evaluate the alteration of TNF-αin plasma as well as DRD2 and DRD3 changes in PBMCs of diabetics with severe foot ulcers.Materials and Methods:Peripheral blood samples were collected from 31 subjects with ulcers, 29 without ulcers, and 25 healthy individuals. Total mRNA was extracted from PBMCs for the study of DRD2, DRD3, and TNF-α gene expression variations. Expression patterns of these genes were evaluated by real-time PCR. Consequently, concentration of TNF-α was investigated in plasma.Results:Significant decrease in gene expression and plasma concentration of TNF-α in PBMCs was observed in both patient groups at P<0.05. These diminutions are correlated to the decrease in the expression of both DRD2 and DRD3 in PBMCs of both patient groups. Also, the same relationship is present between expressions of two new DRD3 transcripts with TNF-α downturn.Conclusion:We concluded that DRD2 and DRD3 expression alteration and presence of new DRD3 transcripts can be effective in reduction of TNF-α expression as a pro-inflammatory factor. Performing complementary studies, may explain that variations in DRD2 and DRD3 are prognostic and effective markers attributed to the development of diabetes severe foot ulcers.

Effect of Dietary Omega-3 Fatty Acids on Tumor-Associated Macrophages and Prostate Cancer Progression.

Preclinical and clinical studies suggest that a fish oil-based diet may play a role in delaying the progression of prostate cancer through a number of different mechanisms involving inflammatory pathways. Given the importance of tumor-associated macrophages (TAMs) in carcinogenesis, we hypothesized that a fish oil-based diet will inhibit TAM infiltration and delay the growth of prostate cancer. Androgen sensitive mouse prostate cancer (MycCaP) allograft tumors were grown in fully immunocompetent FVB mice fed a high- fat fish oil (omega-3) or corn oil (omega-6) diet. Gene expression of markers for immune cell populations, cytokines, chemokines, and signaling pathways were determined by real-time PCR and western blot in tumor tissue. Cell proliferation and apoptosis in vitro were measured by MTS assay and flow cytometry. Tumor volumes were significantly smaller in mice in ω-3 versus the ω-6 group (P = 0.048). Gene expression of markers for M1 and M2 macrophages (F4/80, iNOS, ARG1), associated cytokines (IL-6, TNF alpha, IL-10), and the chemokine CCL-2 were also lower in the omega-3 group. Correlative in vitro studies were performed in M1 and M2 polarized macrophages and mirrored the in vivo findings. Dietary fish oil and in vitro omega-3 fatty acid administration reduced protein expression of transcription factors in the nuclear factor kappa B pathway leading to a significant decrease in gene expression of downstream targets (Bcl-2, BCL-XL, XIAP, survivin) in MycCap cells. These findings underscore the potential of fish oil in modulating the clinical course of human prostate cancer through the immune system. Further preclinical and clinical studies are warranted evaluating fish oil-based therapies for inhibiting the recruitment and function of M1 and M2 tumor infiltrating macrophages. Prostate 76:1293-1302, 2016. © 2016 Wiley Periodicals, Inc.