bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulatory effects of persimmon vinegar and its fractions on hepatic immune response and CYP2E1 activation in chronically alcohol drinking rats (649.4)
Authors: Lee Hye Min , Kim You Jin , Do Gyeong‐Min , Kim Joohee , Kwon Oran
Published: June 2021

Our previous study has shown that persimmon vinegar (PV) had more potent effects on the prevention of the alcohol‐induced liver damage in vivo than any other vinegars tested. Here, we investigated the efficacy of PV and its fractions on hepatic immune response and alcohol metabolism in chronically alcohol drinking rats. Forty male Wistar rats were divided to 5 groups and fed each diet for 4 weeks; normal control, ethanol control (EC), PV, high molecular weight fraction (HPV), and low molecular weight fraction (LPV) group. Hepatic mRNA expressions of Interlekin‐1β, Interlekin‐12β, Toll‐like receptor 4 ,cyclooxygenase‐2 and protein expression of CYP2E1 were significantly decreased in PV, HPV and LPV compared to EC. (p=0.0076,p=0.0009, p=0.0142, p=0.0003, and p=0.0438, respectively). However, there was no significant difference among all treatment groups for all biomarkers with an exception of CYP2E1. The PV had significantly higher potency in suppressing CYP2E1 protein expression than the others. Taken together, the results of this study support the previous observations on the protective effect of PV against alcohol‐induced liver damage. Our findings also suggest that the effect of PV might be caused by more than two components, which are different with their molecular weight range.

Grant Funding Source: This study was supported by the Ministry of Education, Science, and Technology (Brain Korea 21 PLUS:
Intestinal barrier integrity diminished by hypoxia, indomethacin, and hydrocortisone in Caco2 cell line (650.12)
Authors: Corley Katherine , Carr Nick , King Jonathan
Published: June 2021

Background: Concomitant use of hydrocortisone and the nonspecific cyclooxygenase (COX)‐inhibitor indomethacin increases the risk for spontaneous intestinal perforation in extremely low birth weight preterm infants. The regulation of the eicosanoid pathway and inflammation play a large role in maintaining intestinal barrier integrity.

Objective: Test the hypothesis that the dysregulation of the PGE2/EP pathway leads to altered intestinal barrier integrity, impacting cell survival and tight junction function.

Methods: A Caco‐2 cell line was utilized in the setting of normoxia (160 torr) and hypoxia (20 torr) under varying conditions of pro‐inflammatory agents TNF (10ng/mL), IFN‐gamma (10ng/mL)) and anti‐inflammatory agents of indomethacin (50uM) and hydrocortisone (50ng/mL). Barrier function was assessed using transepithelial electrical resistance (TER) measurements and solute flux assays. Cell survivability and morphology was evaluated with direct confocal microscopy as well as indirect LDH quantification, cytotoxicity assays.

Results: The concomitant exposure of the Caco‐2 cell monolayer to hypoxia, pro‐inflammatory and anti‐inflammatory agents demonstrated abnormalities of barrier integrity, cell morphology, and impairment of tight junction formation on confocal microscopy, not seen in normoxia conditions Conclusions: Anti‐inflammatory agents may alter the regulation of intestinal epithelium cytotoxicity without improvement in tight junction integrity. These findings indicate the possible role of indomethacin, hydrocortisone in altering cell survival mechanisms leading to cellular necrosis vs. apoptosis under hypoxic, pro‐inflammatory environments.

Grant Funding Source: Supported in part by NSF‐MRI #1229702
Antimicrobial peptide and mucin expression differ between the diabetic NOD mouse and non‐diabetic NOR mouse (650.16)
Authors: Daft Joseph , Lorenz Robin
Published: June 2021

Type 1 Diabetes (T1D) is defined as the selective immune destruction of insulin producing β‐cells within the islet. Alterations in the intestinal microbiota, increased intestinal permeability, and an aberrant immune system are thought to play key roles in the development of T1D. Recent studies have shown that mice that develop T1D have a different microbiota compared to mice that do not develop T1D. However why mice that are on the same diet in the same facilities have different microbiota is unknown. We hypothesize that altered antimicrobial peptide (AMP) and mucin production early in life determines ones microbiota, protecting some, but not others from T1D. Non‐obese diabetic (NOD) female mice were compared to age and sex matched Non‐obese diabetic resistant (NOR) mice. AMP and mucin gene expression was measured in the ileum and colon of the two strains and protein expression was examined by immunohistochemistry. At 2 weeks of age there is a significant increase in the AMP, defcr‐4, in the ileum of NOD mice compared to NOR mice. In addition a decrease in the mucins, Muc 1, 2, and 3 was measured in the colons of NOD mice compared to NOR mice. This correlates with alterations that we have measured in Bacteroidetes and Firmicutes between the two strains of mice. We postulate that these differences in AMP and mucin expression between the two strains leads to alterations in microbiota, which leads to differences in disease outcome.
Targeting of Lewis‐a glycans accelerates chemotaxis and transepithelial migration by human PMNs (650.9)
Authors: Brazil Jennifer , Sumagin Ronen , Lee Goo , Cummings Richard , Parkos Charles , Louis Nancy
Published: June 2021

Pathogen‐triggered neutrophil (PMN) recruitment is critical for innate immunity, but aberrant PMN influx into intestinal crypts marks disease severity in inflammatory bowel disease (IBD). Fucosylated terminal glycans, such as Lewis‐x (Lex) and Sialyl Lewis‐x, have previously been implicated in the regulation of important PMN functions, including selectin‐mediated PMN trafficking. While such glycans based on the type 2 sequence (Galβ1‐4GlcNAc‐R) are abundant on PMNs, it was previously thought that PMNs lack type 1 glycans (Galβ1‐3GlcNAc‐R) required for the expression of Lewis‐a (Lea). Here, we demonstrate through immunoblot, flow cytometry, and immunohistochemical analyses of tissues from individuals with IBD, that Lea is in fact present on human PMNs. Further, immunoblotting revealed robust PMN expression of Fucosyltransferase 3/5, glycosyltransferases with α1/4 fucosyltransferase activity, required for the terminal step in Lea synthesis. We further report that treatment of PMN with Lea‐specific Abs increases both chemotaxis across collagen and migration across model intestinal epithelia. Thus, we identify novel expression of Lea by human PMN and demonstrate its relevance to PMN trafficking within the intestine. PMN Lea represents a novel target for regulating intestinal innate immunity and may be a rational target for modulating inflammation in IBD.

Grant Funding Source: Supported by Crohn's & Colitis Foundation of America and National Institutes of Health
Electrospun fiber mat design based on brain development: a new model for neural progenitor migration (651.17)
Authors: Czeisler Catherine , Short Aaron , Gygli Patrick , Ortiz Cristina , Catacutan Fay , Winter Jessica , Otero José
Published: June 2021

The brain is a complex organ made up by cells that are born far away from their adult positions and thus migrate large distances to form connections during development. This migration has been mostly studied on the scaffold formed by the Radial Glia at the germinal zones. However, we discovered that during normal embryonic development, migratory neural progenitors use other scaffolds such as blood vessels and the pial surface in order to migrate these large distances. Further, these different scaffolds contain different Extracellular Matrix (ECM) molecules, and the ECM composition atop the scaffolds changes dynamically during development. We hypothesize that the migration of neural progenitors is influenced by the topology (shape) as well as molecular composition (ECM) of these different scaffolds. In order to illustrate this phenomenon, we have modeled the three principal scaffolds in the brain (radial glia, pial surface and blood vessels) out of electrospun fibers. These fibers can be formed at different diameters to mimic a blood vessel (10‐15um) or radial glial process (3‐5um) topology or a flat tortuous surface like the pial surface, and they can be coated with different ECM proteins corresponding to the in vivo data. We cultured Neural Stem Cells (NSC) on top of these fibers and observed their migratory behavior.

Grant Funding Source: NCATS Grant 8UL1TR000090‐05
Role of STAT3 in the loud sound‐induced stress response (651.19)
Authors: Nuttall Alfred , Foster Sarah , Omelchenko Irina , Wilson Teresa
Published: June 2021

Background: Signal transducers and activators of transcription 3 (STAT3) is a stress responsive transcription factor. STAT3 also possesses transcription‐independent activities of including direct regulation mitochondrial respiration and complex I activity as well as inhibition of autophagy under normal physiological conditions. Objective: To determine the role of STAT3 in the loud sound‐induced stress response in the inner ear. Methods and Results: Exposure of male CBA/CaJ mice (8 weeks old, n=5/group) to a moderately damaging level of sound (110 dB SPL, 4‐48 kHz, 3 hrs) resulted in phosphorylation and nuclear translocation of STAT3 in many cell types in the inner ear including auditory hair cells. Pharmacological inhibition of STAT3 activity with JSI‐124 prior to noise exposure revealed improved recovery of outer hair cell (OHC) function following noise exposure by Distortion Product Otoacoustic Emission measurement. In STAT3OHC‐/‐ mice, the OHCs appeared morphologically normal and alterations in markers of autophagy were observed. Conclusions: Loud sound exposure causes widespread STAT3 activation in the cochlea and STAT3 dependent VEGF upregulation. Inhibition of STAT3 proved protective of OHC function following noise exposure suggesting a role for the STAT3 mediated inflammatory response in OHC damage. STAT3 inhibited autophagic procesess in OHCs may be involved in the recovery function.

Grant Funding Source: NIDCD R01 DC000105
Opioid growth factor reverses the progression of clinical disease in established relapse‐remitting experimental autoimmune encephalomyelitis ő a model for multiple sclerosis (651.2)
Authors: Hammer Leslie , Zagon Ian , McLaughlin Patricia
Published: June 2021

Relapse‐remitting multiple sclerosis (RR‐MS) is a chronic disorder involving inflammatory attacks on the CNS in 350,000 people in the U.S. RR‐MS reduces the quality of life, and eventually results in paralysis. OGF, chemically termed [Met5]‐enkephalin, was administered daily to female SJL/J mice with established relapse‐remitting experimental autoimmune encephalomyelitis (RR‐EAE). Mice were immunized with PLP139‐151 in complete Freund’s adjuvant, and beginning 2 days after initial behavioral signs received daily injections of OGF (10 mg/kg) or sterile saline. Within 9 days of treatment with OGF the mice had markedly reduced mean behavior scores relative to controls; the sum of the behavior scores was 140.1±14.4 for the RR‐EAE+Saline group in comparison to 72.2±11.5 for the RR‐EAE+OGF group. Disease severity scores were 16.3± 5 for RR‐EAE+Saline in comparison to 3.6±1.7 for OGF treated mice. No saline‐treated mouse had a complete remission, whereas 83% of OGF mice had complete remissions (behavioral score 蠄 0.5); the total length of time in complete remission was 9.1±3.4 days for the RR‐EAE+OGF animals over the course of the 40‐day experimental period. In summary, treatment with OGF initiated at the time of discernible disease reduced the intensity of the initial flair, reversed the progression of RR‐EAE, and resulted in complete clinical remission, supporting its use as a novel therapy for the treatment of MS.

Grant Funding Source: Shockey Family Foundation
Low dose naltrexone inhibits the progression of clinical disease in established relapse‐remitting experimental autoimmune encephalomyelitis ő a model for multiple sclerosis (651.3)
Authors: Zagon Ian , Hammer Leslie , McLaughlin Patricia
Published: June 2021

Relapse‐remitting multiple sclerosis (RR‐MS) is a chronic disorder involving inflammatory attacks on the CNS. RR‐MS affects about 350,000 people in the U.S. significantly reducing the quality of life. The Opioid Growth Factor (OGF)‐Opioid Growth Factor Receptor (OGFr) pathway has been reported to be involved in experimental autoimmune encephalomyelitis (EAE). Blockade of OGF‐OGFr interaction using low dosages of the opioid antagonist naltrexone (LDN) alters the course of disease. SJL/J mice were immunized with PLP139‐151, and were injected (ip) daily with 0.1 mg/kg naltrexone or saline beginning 2 days after initial signs of disease. Within 9 days of LDN treatment mice had markedly reduced mean behavioral scores compared to controls, with the sum of behavioral scores being 142.9±11.5 for the RR‐EAE+Saline group in comparison to 78.8±9.1for the RR‐EAE+LDN group. In the LDN group, 80% of the mice had complete remission (behavioral scores of 0.5 or less) during the 40 day observation period. Moreover, LDN‐treated mice demonstrated a 4‐fold increase in mild disease scores relative to controls. The results indicate that modulation of the OGF‐OGFr pathway by LDN in established RR‐EAE inhibited the severity of initial flair and limited the progression of clinical disease, with many LDN‐treated mice showing complete remission. These observations support LDN as a non‐toxic/safe biotherapy for the treatment of RR‐MS.

Grant Funding Source: Shockey Family Foundation
Expression of p24α2 in mammalian brain (651.4)
Authors: Nishimura Masaki , Liu Lei
Published: June 2021

Dysregulated metabolism and accumulation of amyloid‐β (Aβ) peptides in brain underlies the pathogenesis of Alzheimer's disease. Aβ is predominantly generated in neuronal cells and is considered to be secreted from the presynaptic terminals in a synaptic activity‐dependent manner. The p24 family member p24α2 has been reported to attenuate Aβ generation through inhibiting γ‐secretase processing of amyloid precursor protein (APP). However, the expression and localization of p24α2 in brain has not been examined. Here, we performed immunohistochemical staining and subcellular fractionation for p24α2 using mouse brain. Immunostaining showed that p24α2 is broadly distributed in the grey matter of the central nervous system and is predominantly localized at the synapses. Subcellular fractionation indicated that p24α2 is condensed at the active zone‐docked synaptic vesicles in the presynaptic terminals. During development, p24α2 is expressed in postnatal period at the highest level and gradually declines. We also confirmed that APP and γ‐secretase components are localized at the active zone‐docked synaptic vesicles. Our results suggest a novel functional implication of p24α2 in regulation of synaptic transmission and synaptogenesis and provides supportive evidence for a role of p24α2 in regulation of Aβ generation and secretion in brain.
Alzheimer’s‐like behavior and pathology in a transgenic mouse model of Down syndrome (651.9)
Authors: Noterman Maria , Deyo Richard , Mann Francis
Published: June 2021

Down syndrome (DS) is caused by trisomy of human chromosome 21 (Hsa21) and is correlated with various comorbidities, including Alzheimer’s disease in 50% of affected individuals. The Tc1 mouse model contains a copy of Hsa21, producing an animal model of DS. In an effort to understand the correlation between DS and Alzheimer’s disease, this study evaluated the learning, memory, and hippocampal and cortical brain morphology in the Tc1 mouse model. Genotyping of Tc1 mice indicated that Hsa21 was transmitted with a 16.67% frequency. Tc1 mice underwent open field, dark‐light, and step down test of inhibitory avoidance tests at set timepoints. Increased hypomotor and anxiety‐related activity in was identified in Hsa21 positive animals. Acquisition of hippocampal‐dependent short‐term memory was intact while long‐term memory trended towards impairment. Relative brain weight of Hsa21 positive subjects was significantly higher than control littermates with lateral ventricle area profoundly increased. Histological screening for Alzheimer’s‐like neuropathology showed neurofibrillary tangles in the cortex of all Hsa21+ mice and a diffuse plaque in one Hsa21+ subject. Immunohistochemistry was used to analyze beta‐amyloid 1‐42 levels in control and Hsa21+ mice. This heterogeneity in pathology is consistent with the spectrum of neuropathological deficits, including Alzheimer’s, seen in patients with DS.
Saxagliptin and tadalafil differentially alter left ventricular mechanics in a translational miniature swine model of heart failure with preserved ejection fraction (652.15)
Authors: Hiemstra Jessica , Cobb Melissa , Ivey Jan , Emter Craig
Published: June 2021

We previously demonstrated pathological cardiac remodeling in a swine model of heart failure with preserved ejection fraction (HFpEF) was associated with altered left ventricular (LV) mechanics. The inhibitory effect of cGMP (cyclic guanosine 3',5'‐monophosphate) on pathological hypertrophic signaling is well established. Reduced cGMP signaling in HFpEF may occur as a result of dysfunctional production or enhanced catabolism via increased phosphodiesterase (PDE) activity. We hypothesized preservation of cGMP expression would attenuate pathological remodeling and improve LV mechanics in HFpEF. Thus, the purpose of this study was to promote cGMP signaling via two mechanisms: 1) the DPP4 inhibitor saxagliptin; and 2) the PDE5 inhibitor tadalafil. We assessed LV mechanics with 2D strain echocardiography 1 and 3 months post‐aortic banding in mini‐swine divided into four groups (n=5); control non‐banded (CON), HFpEF (HF), HFpEF saxagliptin‐treated (HF‐SAX) and HFpEF tadalafil‐treated (HF‐TAD). Tadalafil attenuated compensatory increases in systolic strain and early diastolic strain rate observed in the HF and HF‐SAX groups. In contrast, saxagliptin prevented increases in late diastolic strain rate associated with enhanced atrial systole observed in HF and HF‐TAD animals. Saxagliptin increased torsion transiently (1 mo.), although at 3 mo. both apical and early diastolic rotation rate were increased only in the HF‐TAD group. In conclusion, LV mechanics were distinctly altered in response to separate methods of pharmacological cGMP regulation. Our results suggest the mechanism by which cGMP signaling is promoted may play a role in LV mechanical adaptations to developing HFpEF.
Nongenomic effects of estrogen mediate the dose‐related myocardial oxidative stress and dysfunction caused by acute ethanol in female rats (652.19)
Authors: El‐Mas Mahmoud , Abdel‐Rahman Abdel
Published: June 2021

Acute ethanol lowers blood pressure (BP) and cardiac output in proestrus and after chronic estrogen (E2) replacement in ovariectomized (OVX) female rats. However, whether rapid nongenomic effects of E2 mediate these hemodynamic effects of ethanol remained unanswered. To test this hypothesis, we investigated the effect of ethanol (0.5, 1 or 1.5 g/kg; i.v.) on left ventricular (LV) function and oxidative markers in OVX rats pretreated 30 min earlier with 1 μg/kg E2 (OVXE2) or vehicle (OVX) and in proestrus sham‐operated (SO) rats. In SO rats, ethanol caused significant and dose‐ related reductions in BP, rate of rise in LV pressure (LV dP/dtmax), and LV developed pressure (LVDP). The ethanol effects disappeared in OVX rats and were restored in OVXE2 rats suggesting rapid estrogen receptor signaling mediates the detrimental LV effects of ethanol. The E2‐dependent LV dysfunction caused by ethanol was associated with higher LV: (i) generation of reactive oxygen species, (ii) expression of the malondialdehyde and 4‐hydroxynonenal protein adducts, (iii) Akt and ERK1/2, and (iv) catalase activity. Collectively, it is concluded that rapid estrogen receptor signaling is implicated in the cellular events that lead to the generation of aldehyde protein adducts and Akt/ERK1/2 phosphorylation, which ultimately mediate the estrogen‐dependent LV oxidative stress and dysfunction caused by ethanol in female rats.

Grant Funding Source: Supported by NIAAA [2R01 AA014441‐07]
Membrane cholesterol and caveolar modulation of cardiac function, ischemic tolerance and opioidergic protection (652.21)
Authors: See Hoe Louise , Schilling Jan , Kellerhals Sarah , Headrick John , Patel Hemal , Peart Jason
Published: June 2021

Studies support a role for cholesterol‐rich membrane microdomains, caveolae, and caveolins in cardiac tolerance and conditioning to ischemia‐reperfusion (IR). The importance of membrane cholesterol in intrinsic IR tolerance and preconditioning is unclear. We assessed effects of cholesterol depletion vs. lifelong modulation of caveolin‐3 (deletion and overexpression) on myocardial function, IR tolerance and opioid preconditioning. Hearts from male C57/Bl6 mice received methyl‐β‐cyclodextrin (MβCD, 2 μM ‐ 1 mM), depleting membrane cholesterol, prior to ex vivo IR ± acute or sustained opioid receptor preconditioning (SLP). Additionally, cardiac caveolin‐3 (cav‐3) knockout (KO) and overexpressor (OE) hearts were exposed to IR ± SLP. MβCD significantly reduced sarcolemmal cholesterol content by >10‐30%, and induced significant pre‐ and post‐ischemic contractile dysfunction. Cardioprotection with acute morphine (10 μM) was abolished with >20 μM MβCD, yet SLP remained efficacious until 200 μM MβCD. Cav‐3 KO reduced, while cav‐3 OE enhanced IR tolerance. SLP improved post‐ischemic recovery in cav‐3 KO, and protection was additive in cav‐3 OE hearts. Membrane cholesterol plays a key role in intrinsic IR tolerance and conventional and novel preconditioning. Novel SLP is further delineated from conventional preconditioning stimuli, as this phenotype appears independent of cav‐3 protein.

Grant Funding Source: Supported by National Heart Foundation Australia (LS) and Australian Research Council (JP)
Screening and identification of the specific microRNAs involved in the protection of morphine preconditioning against hypoxia/reoxygenation injury in rat failing cardiomyocytes (652.6)
Authors: He Shu‐fang , Zhu Hai‐juan , Zhang Ye
Published: June 2021

This study was designed to investigate the protective effects of morphine preconditioning (MPC) against hypoxia/reoxygenation (H/R) injury in rat failing cardiomyocytes, and to screen and identify the involved microRNAs (miRNAs). The cardiomyocytes isolated from adriamycin‐induced failing rat heart received MPC at different concentrations followed by H/R and then cellular injury was measured. In another experiment, total RNA was extracted from the failing cardiomyocytes after MPC and subjected to miRNA microarray and subsequent bioinfornatics analysis and real‐time RT‐PCR validation. Typblue exclusion and LDH assay results showed that MPC significantly attenuated cell injury caused by H/R in failing cardiomyocytes. The miRNA microarray data showed that many of miRNAs differentially expressed among groups. By Gene Ontology and KEGG pathways analysis, the predicted target genes were enriched in calcium ion transport, apoptosis and MAPK signal pathway. Notably, miR‐133b was obviously downregulated in failing cardiomyocytes while highly expressed under MPC; miR‐6216 and miR‐let‐7 were upregulated in failing cardiomyocytes but decreased by MPC. The miRNA microarray data was further confirmed by real‐time RT‐PCR. In conclusion, miRNAs, especially miR‐133b, miR‐6216 and miR‐let‐7 might be involved in the protection of morphine preconditioning against H/R injury in failing cardiomyocytes.

Grant Funding Source: Supported by National Natural Science Foundation of China（81200171）
Chronic Bendavia therapy improves post‐infarction cardiac function and prevents adverse left ventricular remodeling in rats (652.8)
Authors: Dai Wangde , Shi Jianru , Hale Sharon , Kloner Robert
Published: June 2021

Bendavia (Ben), a mitochondria‐targeting peptide, protects mitochondrial function by enhancing bioenergetics under conditions of oxidative stress, thus inhibiting ROS formation. We hypothesized that Ben therapy would benefit post‐myocardial infarction (MI) cardiac function and remodeling in rats. At 2 hours after left coronary ligation, rats were randomized to receive Ben that was delivered subcutaneously by an Alzet Osmotic Pump (3 mg/kg/day, n=28) or water (n=26). Six weeks later, scar circumference assessed by histology and expressed as percentage of total left ventricular (LV) circumference, was significantly smaller in the Ben group (39.7 ± 2.2 %) versus the water group (47.4 ± 2.5 %, p=0.024). Post‐mortem LV volume was 9.6% smaller in the Ben group (p=0.019). LV fractional shortening by echo was significantly greater in the Ben group (28.8 ± 1.7 %) than in the water group (23.8 ± 1.8 %, p=0.047). LV stroke volume was greater in the Ben group (0.257 ± 0.008 ml) versus the water group (0.231 ± 0.008 ml, p=0.029), and LV ejection fraction assessed by LV ventriculography was greater with Ben (55.3 ± 1.4%) versus water (49.3 ± 1.4%, p=0.005). Ben treatment reversed the down‐regulated expression of mitochondrial energy metabolism‐related genes at the noninfarcted infarct border assessed by PCR array. Chronic Ben therapy improved post‐MI cardiac function, prevented stretching of the scar and adverse remodeling in rats.

Grant Funding Source: Supported by Stealth Peptides, Inc., Newton, Mass.
5‐HT disturbs interaction between gut epithelium and commensal bacteria (653.10)
Authors: Regmi Sushil , Shadfar Sina , Neupane Ganesh , Lee Jintae , Lee Jin‐Hyung , Kim Jung‐Ae
Published: June 2021

The gut is considered as one of immune privilege sites, which is developed by complex interplay between gut microbiome and the intestinal epithelial barrier. The mechanisms regulating gut tolerance to the commensal bacteria also play an important role in the protection of intestine against epithelial injury. Human studies in patients with inflammatory bowel disease (IBD) have implicated aberrant communication between host and gut flora such as inappropriate immune responses against their own flora. In addition to the excessive production of proinflammatory cytokines, serotonin (5‐hydroxytryptamine, 5‐HT), an enteric neurotransmitter and paracrine hormone, is also increased in the gut of IBD patients, suggesting its role in the pathogenesis of IBD. In the present study, we examined the role of 5‐HT in aberrant communication between gut epithelial cells and commensal bacteria (E coli). 5‐HT enhanced adherence of commensal E coli to HT29 colonic epithelial cells without affecting the adherence of pathogenic E coli. At the same time, 5‐HT treatment synergistically increased mRNA expression of IL‐8, TLR‐2, and Nox2 in commensal E coli‐treated‐HT29 cells. Either pretreatment with NADPH oxidase inhibitor (DPI) and antioxidant (vitamin C) or transfection with Nox2 siRNA significantly suppressed the 5‐HT‐induced adhesion of commensal E coli to HT‐29 cells. Taken together, 5‐HT disturbs the interaction between gut epithelium and commensal bacteria, which may alter gut tolerance to commensal bacteria.

Grant Funding Source: Supported by Basic Science Research Program through the National Research Foundati
Analysis of ligand bias in functional studies involving the allosteric modulation of G protein‐coupled receptors (654.6)
Authors: Ehlert Frederick , Griffin Michael
Published: June 2021

The affinity constants of a ligand for active and inactive states of a receptor ultimately determine its ability to activate downstream signaling events. In this report, we describe a reverse‐engineering strategy for estimating these microscopic constants. Our approach involves analyzing responses measured downstream in the signaling pathway of a G protein‐coupled receptor under conditions of partial receptor inactivation and allosteric modulation. The analysis also yields estimates of the isomerization constant of the unoccupied receptor, the sensitivity constant of the signaling pathway, and the more empirical parameters of the receptor population including the observed affinities and efficacies of allosteric and orthosteric ligands ‐ including inverse agonists ‐ and the efficacy of the unoccupied receptor (i.e., constitutive activity). We validate our approach with an analytical proof and by analysis of simulated data. We also use our method to analyze data from the literature. We show that the values of the microscopic constants of orthosteric and allosteric ligands are constant regardless of the allosteric interaction and the nature of the receptor‐signaling pathway as long as the same active state mediates the response. Our analysis is useful for quantifying probe‐dependent allosteric interactions and the selectivity of agonists for different signaling pathways.

Grant Funding Source: NIH grant GM 69829
Deoxynyboquinone and its derivatives upregulate glutathione‐specific genes in MDA‐MB‐231 breast cancer cells (655.10)
Authors: Ponnuraj Nagendraprabhu , Kolossov Vladimir , Parkinson Elizabeth , Benefiel Ann , Hergenrother Paul , Gaskins H.
Published: June 2021

Pharmacological activation of genes encoding antioxidant and/or phase 2 detoxifying enzymes offer a promising approach to increase the effectiveness of anti‐cancer drugs. Deoxynyboquinone (DNQ), an anti‐cancer agent, induces lethality in cancer cells through NQO1‐dependent redox cycling that consumes oxygen and generates extensive reactive oxygen species (ROS). Here, we investigated the effect of DNQ and its less toxic derivatives, IB DNQ‐2/23, on the expression of glutathione‐specific genes in MDA‐MB‐231 breast cancer cells transfected with (NQO1+) or without (NQO1‐) using qRT‐PCR analysis. A significant increase in the mRNA expression of glutamate‐cysteine ligase catalytic subunit (GCLC, 4‐fold), and glutamate‐cysteine ligase regulatory subunit (GCLM, 5‐fold) was observed in NQO1‐ cells exposed to DNQ for different time intervals. Similarly, GCLC and GCLM expression was induced 3‐fold in NQO1‐ cells exposed to IB DNQ‐2/23 with increasing time of exposure. NQO1+ cells exhibited only 2‐fold activation of glutathione specific genes with IB DNQ‐23 treatment as compared to control or DNQ treatment, whereas, the expression of glutathione reductase (GSR) was unaltered in DNQ, IB DNQ‐2/23 treatment in cells transfected with or without NQO1. Together, the results demonstrate that an intriguing link may exist between NQO1‐dependent redox cycling and glutathione responsiveness in breast cancer cells treated with DNQ. Verification of such a relationship would identify an additional mechanism that might be exploited to increase the effectiveness of ROS‐generating anti‐cancer drugs.
Reversal effects of tetramethylpyrazine on multidrug resistance in hepatocellular carcinoma cells BEL‐7402/ADM by inhibiting calcium pathway (655.6)
Authors: Wang Xuanbin , Liu Ying , Li Hongliang , Liu Ming , Ouyang Lu , Wang Linhai , Xie Yong
Published: June 2021

P‐glycoprotein (P‐gp) was one of mechanisms of Multidrug resistant (MDR) hepatocellular carcinoma (HCC) . Its activation, at least partially depends on protein kinase Cα (PKCα). Tetramethylpyrazine (TMP) is a bioactive constituent isolated from the root of Ligusticum chuanxiong Hort, a Chinese herb. The present study was to investigate if TMP reverses Human drug resistant hepatocellular carcinoma cells BEL‐7402/ADM by inhibiting calcium pathway. We found that the microscopy showed that the average immunofluorescent intensity of the PKCα level in Bel‐7402/ADM cells treated with TMP was (33.33 ± 1.7) % compared to that of without TMP (P<0.001). The fluorescent intensity of intracellular Ca2+ for BEL‐7402/ADM treated with TMP was 541.67±8.15 versus that of without TMP (665.16±2.89) (P<0.05); CCK‐8 assay showed that when TMP combined with UO126 (ERK blocker) or SP600125(JNK blocker), the IC50 value of doxorubicin on Bel‐7402/ADM was increased. However, when TMP combined with SB203580 (p38 blocker) was treated in Bel‐7402/ADM, the IC50 value was reduced. Furthermore, compared with the control group, the level of intracellular reactive oxygen species (ROS) of TMP group was increased. These suggested that TMP might reversal MDR HCC by downregulating calcium pathway which may be associated with ROS level and MAPK pathway.

Grant Funding Source: Supported by Department of Health of Hubei Province, NO. JX5A10;
Metformin with radiotherapy enforces growth retardation of head and neck squamous cell carcinoma (655.9)
Authors: Yeo Jiyoung , Park Eun‐Ji , Kim Kyungmin , Jang Hyung‐Jun , Cho Kwang‐Jae , Kim Min‐Sik
Published: June 2021

Head and neck (HN) cancer accounts for approximately tenth of all cancers worldwide and radiotherapy is also a common cancer treatment for patients. We investigated synergistic effects of metformin, one of type 2 diabetes mellitus (T2DM) drugs, when treated with radiation treatment in HN cancer models. Metformin was treated as a dose‐dependent manner with/without 8Gy‐irradiation to Fadu, a HN squamous cell line, and supplemented in diet for 2 weeks in human HN cancer xenograft‐bearing Balb/c‐nude mice under radiotherapy. In the metformin‐treated Fadu after irradiation, AMPK was significantly activated compared with each single treatment. With the reduction of Bcl‐2/Bax ratio, cleaved caspases and p53 were up‐regulated and Akt, Erk, and p38MAPK were down‐regulated. Besides, colony formation was strongly attenuated in HUVECs on an AMPK‐dependent manner and effectively inhibited Fadu tumor growth in Balb/c‐nude mice. Therefore, to conclude, combined treatment metformin with radiotherapy contributes to tumor growth retardation via improved apoptosis and inhibited proliferation of HN cancer cell. These finding may describe practical potential to further translational research and clinical trials in HN cancer.

Grant Funding Source: supported by the NRF of Korea (No. 2011‐0012555) and Ministry of Health & Welfare (A091205)
A novel nociceptin‐1 receptor antagonist produces antidepressant‐ and anxiolytic‐related effects in rodents (656.3)
Authors: Witkin Jeffrey , Statnick Michael , Rorick‐Kehn Linda , Barth Vanessa , Wafford Keith , Pintar John , Perry Kenneth , Toledo Miguel , Diaz Nuria , Lafuente Celia , Jiménez Alma , Benito Ana , Martínez‐Grau Maria Angeles , Pedregal Conception
Published: June 2021

We utilized a potent, selective, and orally‐bio‐available antagonist with documented engagement with nociceptin receptors (NOP) to assess antidepressant‐ and anxioltyic‐related effects of blockade of NOP receptors. The NOP receptor antagonist, Cpd 1, displayed antidepressant‐like neurochemical and behavioral effects in rats and mice. Serotonin outflow in rat prefrontal cortex was enhanced without affecting norepinephrine or dopamine. Correspondingly Cpd 1 decreased immobility in the forced swim test (FST). Cpd 1 augmented the effects of fluoxetine without changing target occupancies of either drug. Anti‐immobility effects of Cpd 1 were absent in mice without NOP receptors. Cpd 1 also produced anxiolytic‐like effects in some rodent models: decreased stimulus‐elicited freezing in mice and decreased stress‐induced hyperthermia in rats. Cdp 1 was not active in: marble‐burying in mice, novelty‐suppressed feeding in mice, conditioned suppression in rats, punished food‐maintained responding in rats, and the 4‐plate test in mice. Cpd 1 did not disrupte performance in 5‐choice serial reaction time tests or delayed matching‐to‐position in rats. Cpd 1 was mildly wake promoting in rats. These data suggest that Cpd 1 has unique antidepressant‐ and anxiolytic‐related pharmacological effects in rodents.
A novel N/OFQ antagonist that is potent, selective, orally‐bioavailable, and has demonstrable on‐target actions (656.4)
Authors: Barth Vanessa , Statnick Michael , McKinzie David , Rorick‐Kehn Linda , Kahl Steven , Toledo Miguel , Diaz Nuria , Lafuente Celia , Benito Ana , Jimenez Alma , Martinez‐Grau Maria , Pedregal Concepcion , Witkin Jeffrey
Published: June 2021

Background: Nociceptin/Orphanin FQ (N/OFQ) is a 17 amino acid peptide whose receptor is designated ORL1 or NOP. Agonists and antagonists for the receptor have been designed and employed to help elucidate its biological functions. We now report on a highly potent, selective, and orally‐bio‐available antagonist with documented engagement of NOP receptors in vivo.

Methods: Compound 1 was evaluated in in vitro, ex vivo, and in vivo assays to characterize its value as a NOP receptor antagonist research tool. These assays included filtration‐based [3H]‐Noc/OFQ receptor binding, blockade of Noc/OFQ stimulated GTP‐‐[35S] binding, and in vivo measures of N/OFQ receptor occupancy and blockade of Ro64,6198 induced hypothermia in Sprague‐Dawley rats.

Results: Compound 1 has a Kb of 0.17nM as an antagonist of N/OFQ‐stimulated G protein activation and a binding affinity (Ki) for N/OFQ receptors of 0.1nM. No intrinsic agonist efficacy was observed in vitro at concentrations up to 10µM. Compound 1 occupies N/OFQ receptors in the rat brain following oral administration; ED80 = 0.3 mg/kg, p.o. In vivo activity was demonstrated by its potent inhibition (minimal effective dose = 0.1 mg/kg, p.o.) of hypothermic effects of an N/OFQ agonist.

Conclusions: These data suggest that Compound 1 is as an orally‐active tool for assessments of the functional significance of NOP receptors.
Pharmacokinetic alternation of mitiglinide in depression model rats (657.3)
Authors: Xu Feng
Published: June 2021

Many diabetes patients suffer from wild to severe depression. It is unclear that depression might alter the drug metabolic process of the hypoglycemic agents or not. We conducted an animal experiment to study the impact. Eighteen Sprague‐Dawley rats were randomized into depression model group and control group. The 9 depression rats were established by exposing to chronic unpredicted mild stresses (CUMS) for 8 weeks. Open‐field test and 5‐HT levels were assayed to evaluate the depression. All rats were given 2.5mg/kg of mitiglinide (MGN) per os. MGN plasma concentration was measured by HPLC‐MS/MS. Statistical moment analysis was processed with DAS software. Open‐field test and 5‐HT levels measurement results confirmed the depression model. Cmax, Tmax, AUC0→∞, MRT0→∞, and T1/2z were reduced while CLz/F was increased in depression model rats compared to control rats. These preliminary results suggest that depression might alter the drug metabolic process of MGN.

Grant Funding Source: Supported by Shanghai Fengxian Science and Technology Research Project
From the research on anti‐inflammatory and immunopharmacology of a natural herbaceous peony to discovery of paeoniflorin ‐6'‐O‐ benzene sulfonate (657.4)
Authors: Wei Wei
Published: June 2021

Objective. Total glucosides of paeony (TGP) were main active constituents of Paeonia lactiﬂora Pall. we have disclosed its antiinﬂammatory and immune effects via regulating balance of cytokines and cellular signaling. TGP was approved for treating rheumatoid arthritis (RA) by SFDA in 1998. TGP is more secure than DMARDs but with weak efficacy, which caused by poor absorption of paeoniflorin(Pae) that is one of main monomer of TGP .Therefore, series derivatives of Pae were screened based on PK/PD study. Pae‐6′‐O‐benzene sulfonate (CP‐25) was selected as a potential medicine. G protein‐coupled receptor (GPCRs) signaling such as EPs are negatively regulated byGRK2, and play an important role in dysfunction of fibroblast‐like synoviocytes (FLS) and immunocytes in RA. The objective is investigating effects of CP‐25 on inflammation and immune response, including the role of GPCR desensitization in functions and interaction of DC, FLS, T and B cells. Methods. Agonist, antagonist, siRNA and site mutation were used as intervention, and molecular biological technology were utilized. Results. We found that CP‐25 inhibited activation of immunocyte and FLS by binding to active sites of GRK2, inhibiting excessive transmembrane of GRK2, and modulating abnormal desensitization of GPCRs. Conclusion. This research offered a foundation for developing CP‐25 as a novel drugs for RA.

Grant Funding Source: NSFC81330081
Antinociceptive effects of imidazoline I2 receptor agonists in the formalin test in rats (658.2)
Authors: Thorn David , Zhang Yanan , Li Jun‐Xu
Published: June 2021

Pain is a significant global public health problem and currently available analgesics are not adequate to meet the clinical needs, leaving a big population with undertreated pain. The imidazoline I2 receptor is an emerging drug target for analgesics as I2 receptor agonists demonstrate antinociceptive effects in rodent models of acute and chronic pain. However, little is known of the neuroanatomical mechanism mediating I2 receptor agonist‐induced antinociception. This study examined the effects of the selective I2 receptor agonists 2‐BFI, BU224, and CR4056 using the formalin test in rats. 2‐BFI (1‐10 mg/kg, i.p.) and BU224 (1‐10 mg/kg, i.p.) dose‐dependently attenuated the spontaneous flinching behavior observed during 10min (phase 1) and 20‐60min (phase 2) following formalin (2%, 50µl) treatment, while CR4056 (1‐32 mg/kg, i.p.) only decreased phase 2 flinching. Local administration of 2‐BFI (1‐10 mg/kg, s.c.) to the hindpaw of rats had no antinociceptive effects. In contrast, centrally delivered 2‐BFI (10‐100 µg, i.c.v.) dose‐dependently attenuated phase 1 and phase 2 flinching. Together, these data suggest that I2 receptor agonists produce significant antinociception via a central mechanism and further supports that I2 receptors may represent a novel drug target to treat pain. (DA034806)

Grant Funding Source: Supported by R01 DA034806/DA/NIDA NIH HHS/United States
Effect of non‐contingent ethanol administration on self‐administration of ethanol in cynomolgus macaques (658.7)
Authors: Allen Daicia , Helms Christa , Grant Kathleen
Published: June 2021

After chronic ethanol self‐administration animals show decreased variability in daily drinking, suggesting titration of dose. Previous studies found that self‐administration with limited access to ethanol was resistant to experimenter‐administered ethanol, resulting in intake of a greater‐than‐average daily dose. The current study examined the effects of ethanol gavage on daily intake with continuous access. Ten adult male cynomolgus macaques were induced to drink 4% (w/v) ethanol using a schedule‐induction procedure, followed by 22 hours/day continuous access to ethanol and water. Meals were given 3 times per day at 0, 2, and 4 hours after session start. After 6 and 12 months of daily access, animals were placed in a primate chair and administered ethanol (1.0 g/kg and 1.5 g/kg, i.g.) or saline (i.m.) 2 hours before a daily session. After 6 months of ethanol consumption, there was a robust dose‐dependent decrease in drinking after the challenges (p<0.001), indicating that dose was titrated. Interestingly, at the 12‐month time point, drinking behavior was resistant to the pretreatment, and significant differences from saline were only observed in the 1.5g/kg dose (p<0.05). The dose of self‐administered ethanol was not correlated with blood ethanol concentration after i.g. ethanol, measured immediately before the session. These data suggest that extended access to ethanol self‐administration in monkeys results in intakes that are less sensitive to pharmacological feedback.

Grant Funding Source: Supported by AA013510 (Grant)
Oral ivermectin treatment significantly reduces ethanol intake in male C57BL/6 mice (658.8)
Authors: Arabian Natalie , Yardley Megan , Asatryan Liana , Huynh Nhat , Louie Stan , Neely Mike , Alkana Ronald , Davies Daryl
Published: June 2021

Alcohol use disorders (AUD) are a health crisis of unmet need. We propose that ivermectin (IVM), an FDA approved drug, can be repurposed to address this problem. Our group recently reported that acute administration of IVM (1.25 ‐ 10.0 mg/kg i.p.) significantly reduces murine ethanol intake without causing any overt signs of toxicity, suggesting that IVM represents a safe, tolerable, and effective agent for the treatment of AUDs. However, AUDs are chronic in nature. Hence, efficacious treatment regimens would require longer term dosing strategies that would be most convenient if effective in an oral dose formulation. The current study tested the hypothesis that long term oral administration of IVM reduces ethanol (10% v/v) intake. We examined the effects of orally delivered IVM (3.1mg/kg/day) in male alcohol preferring C57BL/6 over a long term (14 day) treatment period. This dose was selected based on allometric scaling where we identified 3.1 mg/kg as one that correlates to an already established safe dosage tested in humans (30 mg). We found that long term orally delivered IVM significantly reduces ethanol intake and preference without exerting any overt signs of toxicity. Taken together, the findings provide support for the development of IVM for the prevention and/or treatment of AUDs.
Delta opioid receptor ‐ kappa opioid receptor heteromer signaling in peripheral nociceptors (659.1)
Authors: McGuire Blaine , Sullivan Laura , Chavera Teresa , Clarke William , Berg Kelly
Published: June 2021

We have found that DOR‐KOR heteromers form in peripheral nociceptors (i.e., pain‐sensing neurons) (Berg et al., 2012, Mol Pharmacol 81:264‐272). Here we sought to better understand DOR‐KOR heteromer function in peripheral nociceptors. We compared the signaling characteristics of the putative DOR‐KOR heteromer selective agonist, 6’‐GNTI, to that of the selective KOR agonist, U50488. In a rat behavioral model of thermal allodynia, intraplantar (i.pl.) injection of U50488 produced a biphasic (inverted “U”) dose response curve (DRC). The descending limb of the DRC was blocked by the ERK inhibitor, U0126 (10μg; i.pl.) suggesting that ERK mediates the downward phase. By contrast, 6’‐GNTI produced a robust, monotonic DRC that was unaffected by U0126. In primary cultures of peripheral nociceptors, U50488‐mediated activation of ERK was pertussis toxin (PTx) sensitive, whereas 6’‐GNTI‐mediated ERK activation was PTx insensitive. Although both U50488 and 6’‐GNTI inhibit adenylyl cyclase (AC) with similar potency and efficacy, U50488‐mediated inhibition of AC desensitized with repeated U50488 exposure, an effect that was blocked by U0126. Interestingly, 6’‐GNTI‐mediated inhibition of AC did not desensitize with repeated 6’‐GNTI exposure. These data suggest that DOR‐KOR heteromers may have unique signaling characteristics in peripheral nociceptors that make them promising therapeutic targets for pain.

Grant Funding Source: DA024865 and COSTAR 2T32DE014318‐12A1
Hyperbaric oxygen treatment suppresses withdrawal signs in opioid‐dependent mice (659.12)
Authors: Nicoara Daniel , Zhang Yangmiao , Nelson Jordan , Brewer Abigail , Shirachi Donald , Quock Raymond
Published: June 2021

HBO2 therapy has been approved by the Food and Drug Administration for limited clinical conditions (Gesell, The Hyperbaric Oxygen Therapy Committee Report: Indications and Results, 12th Ed, 2008). It has been reported that HBO2 treatments reduce opioid withdrawal in human subjects (Epifanova, Anesteziol Reanimatol, May‐June 1995 issue, 34‐39). However, no further research has been conducted to investigate the mechanism of HBO2 in reducing opioid withdrawal. The purpose of this research was to determine whether HBO2 treatment could suppress signs of withdrawal in opioid‐dependent mice. Male NIH Swiss mice received repeated s.c. injections of morphine sulfate or saline twice a day for 4 days. The daily dose of morphine sulfate was increased progressively from 50 mg/kg on day 1 to 125 mg/kg on day 4. On day 5, opioid withdrawal was precipitated by intraperitoneal injection of 5.0 mg/kg naloxone. Withdrawal signs in mice, including jumping, forepaw tremor and wet dog shakes, were recorded for 30 min and scored. Sixty min prior to the naloxone injection, different groups of mice received a 30‐min or 60‐min HBO2 treatment at 3.5 atmospheres absolute. The results show that HBO2 treatment caused a dose‐dependent reduction in withdrawal jumping, forepaw tremor and wet dog shakes in opioid‐dependent mice.

Grant Funding Source: Supported by NIH Grant AT‐007222 and the Allen I. White Distinguished Professorship.