bluuebunny/crossref_metadata_2025 huggingface dataset:
LW‐AFC Attenuates LPS‐Induced Neuroinflammation via Inhibiting Nuclear Factor‐ Kappa B Signaling
Authors: Liu Gang , Wang Jianhui , Zhou Wenxia , Zhang Yongxiang
Published: June 2021

Neuroinflammation and activation of microglia have been recognized as important pathological events in Alzheimer's disease (AD). LW‐active fraction combination (LW‐AFC) is extracted from Liuwei Dihuang decoction (LW), a classic traditional Chinese medicinal prescription. It has been demonstrated that LW‐AFC had neuroprotective effects on AD animal models. However, whether anti‐inflammatory effect contributes to the neuroprotection of LW‐AFC has not been fully elucidated. The present study aimed to evaluate the effects of LW‐AFC on neuroinflammation and its underlying mechanisms. C57 mice were intraperitoneally injected with lipopolysaccharide (LPS) and treated with LW‐AFC (i.g.). The results revealed that LW‐AFC significantly improved the hippocampal long‐term potentiation (LTP) and decreased the contents of tumor necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) in the serum and hippocampus of the LPS‐treated mice. In vitro study showed that LW‐AFC inhibited LPS‐induced activation of BV‐2 cells and markedly reduced the increases of TNF‐α and IL‐1β levels in the supernatant of LPS‐treated BV‐2 cells. In addition, LW‐AFC downregulated the expression level of NF‐κB in LPS‐treated BV‐2 cells. The present findings indicate that LW‐AFC might attenuate neuroinflammation both in animal and cell model, which may contribute to its neuroprotective effect in AD treatment.

Support or Funding Information

Project supported by the National Natural Science Foundation of China (81503078)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Melanin‐concentrating hormone‐R1 in the locus coeruleus may be involved in the regulation of depressive‐like behavior
Authors: Zhang Yong‐He , Ye Hui , Cui Xiang‐Yu , Cui Su‐Ying
Published: June 2021

Background

Previous anatomical and behavioral studies have shown that melanin‐concentrating hormone (MCH) is involved in the modulation of emotional states. However, little is known about brain regions other than the dorsal raphe nucleus that relate the MCH‐ergic system to depressive states. Numerous studies have shown that the locus coeruleus (LC) is involved in the regulation of depression and sleep. Although direct physiological evidence is lacking, previous studies suggest that MCH release in the LC decreases neuronal discharge. However, remaining unclear is whether the MCH‐ergic system in the LC is related to depressive‐like behavior.

Method

We treated rats with an intra‐LC injection of MCH, intracerebroventricular injection of MCH, or chronic subcutaneous injections of corticosterone to induce different depressive‐like phenotypes. We then assessed the effects of the MCH receptor 1 antagonist SNAP‐94847 on depressive‐like behavior in the forced swim test (FST) and the sucrose preference test (SPT).

Results

The intra‐LC and intracerebroventricular injections of MCH and chronic injections of corticosterone increased immobility time in the FST and decreased sucrose preference in the SPT. All of these depressive‐like behaviors were reversed by an intra‐LC microinjection of SNAP‐94847.

Conclusions

These results suggest that the MCH‐ergic system in the LC might play an important role in the regulation of depressive‐like behavior.

Support or Funding Information

This study was funded by grants from the National Natural Science Foundation of China (No. 81573407, 81872851, 81871038 and 81302746).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Annexin a1 and formyl peptide receptor 2/3 like: Interaction between hypothalamic–pituitary–adrenal axis and hippocampal memory in a model of corticosterone‐induced depressive like behavior
Authors: Crupi Rosalia , Peritore Alessio Filippo , Gugliandolo Enrico , Siracusa Rosalba , Impellizzeri Daniela , Fusco Roberta , Cordaro Marika , D'Amico Ramona , Di Paola Rosanna , Cuzzocrea Salvatore
Published: June 2021

The activity of the hypothalamic‐pituitary‐adrenal axis is commonly disregulated in stress‐related psychiatric disorders. Formyl peptide receptors (FPR) belong to a family of sensors of the immune system that detect microbe‐associated molecules. It was demonstrated that Fpr2/3 deficient mice show a distinct profile of behaviour characterised by reduced anxiety. Annexin 1 (ANXA1), an endogenous ligand of Formyl Peptide receptor 2/3 like (FPRL 2/3) is a member of the family of phospholipids and calcium annexes proteins with a well‐defined role in the delayed early inhibitory feedback of glucocorticoids in the pituitary gland. The aim of study was first, to evaluate the potential role of ANXA1 and FPRL 2/3 as mediator cell‐mediator of the inhibitory effects of glucocrticoids on corticotrophin secretion (ACTH), subsequently the possible correlation between depression like behaviour and memory impairment in the hippocampus studying Ras/ERK pathway. C57BL/6 (CORT +), Anx a1 −/− and Fpr 2/3 −/− mice were exposed to corticosterone (35 μ/ml/day) to induce depression like state via drinking water for 28 days and then behavioural test was performed to measure immobility time in the forced swimming test, locomotor activity in the open field test, sucrose consumption in the sucrose preference test, anxiety in the novelty suppressed feeding and short‐term memory, intermediate‐term memory and long‐term memory in the novel object recognition test. Anx a1−/− mice showed an improvement in depression‐like behavior compared to Fpr 2/3 like −/− and CORT + mice. Histological analyses were performed to evaluate the number of neurons in the dentate gyrus, as well as the degree of neurogenesis through the neurotrophic factors of BDNF and GDNF. In conclusion, we can state that the absence of the anx a1 protein, even more than the absence of FPR 2/3, improves the depressive behavior induced by chronic administration of glucocorticoids, as it appears to have a neuroprotective role in the hippocampus following a damage caused from a chronic condition of depression

Support or Funding Information

no funding

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Non‐coding RNAs as Markers of Neuropathic Pain
Authors: Wilkerson Jenny , Jiang Jinmai , Felix Jasmine , Bray Julie , da Silva Lais , McMahon Lance , Schmittgen Thomas
Published: June 2021

Pain is one of the most common reasons to seek medical attention and chronic pain is a worldwide epidemic. Under some circumstances, incoming protective nociceptive signaling is prolonged leading to clinical manifestations of pathological neuronal signaling. The chronic constriction injury (CCI) of the sciatic nerve is a widely used preclinical model of pathological neuropathic pain. Over the past decade, investigators have come to appreciate the extracellular actions of RNA located within cells such as mRNA, micro (mi) RNA, and other long and short noncoding (nc) RNAs. The development and/or maintenance of chronic pain could be controlled epigenetically through ncRNAs. Here we seek to characterize ncRNAs in blood, sciatic nerve, and spinal cord in a mouse model of neuropathic pain as this may serve to elucidate potential biomarkers relevant to pathological pain in humans. Male C57BL6/J mice (6 CCI and 6 sham procedure) underwent surgery for sciatic nerve ligation with chromic gut sutures. Following 7 days, mechanical allodynia was quantified using the von Frey assay. Mice were then euthanized for collection of spinal cord, sciatic nerve, and blood. cDNA was synthesized to 322 unique mature miRNAs from the total RNA. Of the 627 miRNAs examined, 414 and 376 were detectable (CT ≤ 35) in the spinal column and sciatic nerve, respectively. In the CCI mice that displayed mechanical allodynia, 11 and 126 miRNAs were differentially expressed (i.e., greater than 1.5‐fold increase or decrease; P < 0.05) in the spinal column and sciatic nerve, respectively, as compared to sham controls. Three members of the miR‐183 cluster (miR‐183, miR‐182 and miR‐96) were reduced in both the spinal column and sciatic nerve. Our data support the use of miRNAs as biomarkers to identify subpopulations of patients with neuropathic pain. Further, targeting miRNAs may hold promise for the treatment of pathological pain.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anxiolytic Effects of Overexpressing Angiotensin Converting Enzyme 2 (ACE2) in Female Mice
Authors: Cahill Karlena Maria , Alleyne Amy , Johnson Dominique , Scott Karen , Raizada Mohan , De Kloet Annette , Krause Eric
Published: June 2021

Considerable evidence has implicated the renin‐angiotensin system (RAS) as an important target for the therapeutic alleviation of stress‐related mood disorders. Along these lines, the ‘pro‐stress’ actions of Angiotensin‐II are largely thought to be mediated by the angiotensin type‐1 receptor (AT1R). On the other hand, the protective limb of the RAS that depends on the conversion of angiotensin‐II to angiotensin‐(1–7) by angiotensin‐converting enzyme 2 (ACE2) has been postulated to dampen stress and relieve anxiety. We have previously found that augmenting ACE2 activity has potent anxiolytic and hypothalamic‐pituitary‐adrenal (HPA) axis‐dampening effects in male mice. Relative to males, females are more susceptible to stress‐related mood disorders; however, whether increasing ACE2 activity in females also blunts stress responses has not been determined. This study tests the hypothesis that ACE2 overexpression decreases anxiety‐like behavior and attenuates stress‐induced HPA axis activation in female mice. We used the Cre/Lox system to generate female mice that ubiquitously overexpress ACE2. Female ACE2 knock‐in (ACEKI) mice and their wild‐type littermate controls were tested in the elevated‐plus maze (EPM), Light‐Dark Box (LDB), and Graham Cracker Test to evaluate the impact that augmenting ACE2 expression has on anxiety‐like behavior. Female mice were also subjected to an acute 30 min restraint stress challenge and blood samples were collected in order to evaluate HPA (i.e., corticosterone) reactivity. As expected, Cre‐Lox mediated overexpression of ACE2 resulted in elevated ACE2 mRNA and enzymatic activity in all tissues examined (i.e., hypothalamus, pituitary, blood, and brain). Relative to controls, ACE2KI mice spent significantly (p < 0.05) more time in the open arms of the EPM and in the light side of the LDB. When placed in a novel environment, ACEKI mice exhibited a significantly (p<0.05) decreased latency to consume graham crackers relative to controls. Importantly, total distance traveled in the EPM was not different between the groups, indicating that the locomotor activity of the female mice is unchanged by ACE2 overexpression. Interestingly, basal and acute stress‐induced corticosterone levels did not differ between ACE2KI and WT female mice. These results suggest that although neuroendocrine response to acute restraint was unaffected in females, ACE2 overexpression has anxiolytic effects on both male and female ACE2KI mice. Furthermore, our findings support the idea that the RAS may be a worthy target in the search for novel therapeutic interventions for stress‐related disorders.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of a Cell‐based Assay for the Discovery of KCa3.1 Inhibitors by Highthroughput Screening
Authors: Jakakul Chanon , Muanprasat Chatchai
Published: June 2021

Inhibition of KCa3.1 potassium channel has been demonstrated to have beneficial effects in a wide variety of human diseases including inflammation‐associated disorders and cancers. However, there is currently no therapeutic KCa3.1 inhibitor available for human use. This study aimed to establish a high‐throughput screening assay for identifying modulators of KCa3.1 in native cells, and to identify and study KCa3.1 inhibitors from a library of compounds derived from natural resources in Thailand. The screening platform was successfully developed based on thallium flux assay. Control groups treated with NS309 (KCa3.1 activator) or TRAM‐34 (KCa3.1 inhibitor) produced significantly different fluorescent signals. A primary screening of 1,352 compounds using the developed thallium flux assays identified 23 active compounds. A secondary screening using electrophysiological analyses has verified the inhibitory effect of 9 compounds. Dose‐response analysis of these compounds has identified several KCa3.1 inhibitors with low micromolar potency. The most potent compound (N134) had the IC50 of ~ 0.1 μM. These results indicate that the high‐throughput screening assay based on endogenous expression of KCa3.1 is capable of identifying small molecule inhibitors. These novel KCa3.1 inhibitors might be further developed into novel therapy of inflammation‐associated disorders and cancers.

Support or Funding Information

This work was supported by the Thailand Research Fund (DBG5980001), Faculty of Science Mahidol University, Faculty of Graduate Studies Mahidol University and Medical Scholars Program, Mahidol University.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cardiovascular effects of the scorpions Centruroides margaritatus, Centruroides limpidus and Centruroides noxius crude venom
Authors: Santos Cláudia Ferreira , Céron Jessica Dayana Galíndez , Bindá Alexandre Havt , Possani Lourival D , Vidal José Béltran , Fonteles Manassés Claudino , Nascimento Nilberto Robson Falcão
Published: June 2021

Scorpions from the Centruroides genus have high toxicity and it is potentially dangerous to humans. In this study we compare the effect of the crude venom of a colombiam scorpion Centruroides margaritatus (Cm) with two Mexican Scorpions, Centruroides limpidus (Cl) e Centruroides noxius (Cn). The venom was injected intravenously in bolus to pentobarbital anesthetized normotensive rats in the doses of 1, 10, 30, 60, 100 e 300 (μg/kg) (n=6/each group). The hemodynamic parameters were evaluted by using a pressure‐volume cateter implanted into the left ventricle (millar pressure‐volume microcatheter) and the direct effect of the venoms on cardiac output, arterial blood pressure, vascular resitsance, ejection fraction, stroke work, maximal and minimal dP/dt and tau. A lead II electrocardiogram was also evaluated. Cm venom injection did not promote significant alterations. Th venom from Cl decreased all hemodynamic parameters such as dP/dt min (dP/dt minct=−10780±2153; dP/dt min300=−5972±2921 (mmHg/s), p<0,05*) and heart rate (FCct=−403±31; FC300=−250±66 (bpm), p<0,05*). The Cn venom significantle decreased the stroke work (TCct=5957±1509; TC100=2611±1583 (mmHg*μL), p<0,05) and all other hemodynamic parameters evaluated. The structural analysis by optical microscopy showed that Cn venom induced lung hemorragy and pulmonary edema. The venom from Cn and Cl induced severe arrhythmias with ventricular extrasystoles and ventricular tachycardia. The venom of Cm is of low toxicity while the venom from Mexican Scorpions such as Cl and especially Cn have severe cardiovascular toxicity.

Support or Funding Information

CAPES, CNPq and FUNCAP

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Prenatal Exposure to Bisphenol A and Diethylhexyl Phthalate Induces Apoptosis in the Thymus of Male and Female Offspring
Authors: Dagher Josephine Bou , Al Mansi Maryam , Jacob Elyssa , Kaimal Amrita , Chuang Yen‐Jun , Mohankumar Puliyur S , MohanKumar Sheba M. J.
Published: June 2021

Bisphenol A (BPA) and Diethylhexyl Phthalate (DEHP) are common environmental endocrine disrupting chemicals (EDCs) that exert a range of potential adverse health effects. EDC exposure can occur in utero and during early postnatal life, when organ systems are differentiating resulting in a number of disorders in adulthood. The aim of this study was to evaluate the immunotoxic effect of prenatal exposure to individual and combinations of BPA and DEHP on male and female rat thymus. From gestational day 6 till 21, Sprague Dawley dams were orally administered either saline (control; n=7), BPA (5μg/Kg BW; n=7), DEHP (7.5mg/Kg BW; n=7), or a mixture of BPA and DEHP (B+D; n=7). Male and female offspring were sacrificed at 16 weeks of age. Thymus and spleen were dissected, weighed, and stored for further processing. Our data showed that spleen weight to body weight (BW) ratio of EDC treated offspring were comparable to those from age‐matched control rats. However, male offspring (but not females) that were prenatally exposed to BPA alone exhibited a 40% decrease (p<0.01) and those exposed to DEHP alone had a 27% reduction in thymus/BW ratio (p<0.05). To understand if prenatal EDC exposure was promoting early thymus involution through apoptosis, we performed Terminal deoxynucleotidyl transferase‐dUTP nick end labeling (TUNEL) staining of nicked DNA on thymic sections. There was significant apoptosis in the thymic cortex in both male and female offspring that were prenatally exposed to individual EDCs. Apoptosis was hardly detected in the medullary region. Apoptosis in the B+D group was markedly reduced compared to individual EDC exposures. These results suggest that prenatal exposure to even low BPA and moderate DEHP levels can promote apoptosis in the thymus. This could possibly affect immune functions in adulthood. Interestingly, exposure to a mixture of these EDCs did not produce any significant change in apoptosis in the thymus.

Support or Funding Information

Supported by UGA Research Foundation and start‐up funds.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Chlorogenic Acid Increases Basal Tone with No Effect on Relaxation of Rat Ileal Smooth Muscles by Mechanisms Independent of PKG Mediated Effects on Myosin Light Chain Phosphorylation
Authors: Berman Sarah E , Huston Jamie L , Lail Austin W , Piquette Nicole B , Injeti Elisha R
Published: June 2021

Chlorogenic acid (CGA) is a dietary polyphenolic compound commonly present in coffee, fruits and vegetables. The pharmacological properties of CGA include antioxidant, antibacterial, anti‐inflammatory, and antithrombotic effects. Previous experimental studies have shown that CGA induces nitric oxide‐sGC‐PKG pathway to cause relaxation of the vascular smooth muscles. But the effects of CGA on ileal smooth muscles are still unknown. The present study is designed to test the effect of CGA on PKG mediated relaxation of rat ileal smooth muscle.

Effects of CGA on basal tension and maximum contractile tension induced by physiological salt solution (PSS) containing 140 mM potassium in isolated rat ileal rings (3mm) were determined using tissue bath apparatus. CGA effects on myosin light chain phosphorylation at maximum contractile tension were also measured by freezing the ileal rings and extracting the protein and running Western blots. The role of PKG in regulating smooth muscle relaxation is tested by incubating ileal rings with specific PKG inhibitor Rp‐8‐Br‐cGMPS and measuring the basal tension, maximum contractile tension and myosin light chain phosphorylation as described above.

The results show that basal tension increases from 0.27±0.1 (control group) to 0.91±0.3 and 0.87±0.2 (mV) when treated with CGA (10μM) and PKG inhibitor (4μM) respectively. The peak contractile tension in response depolarization by 140 mM K+ PSS changes from 100% (control group) to 101±12.2 % and 117±17.2 % when treated with CGA and PKG inhibitor respectively. The results from Western blots show that myosin light chain phosphorylation didn't change significantly between CGA (36.3±0.8 %) and PKG inhibitor (35.8±0.5 %) treatments.

These results suggest that CGA is involved in increasing the basal tone of ileal smooth muscles when compared with the control group. In addition, CGA did not have any effect on the peak contractile tension and myosin light chain phosphorylation, suggesting that it may not be involved in ileal smooth muscle relaxation. However, PKG inhibitor in presence of CGA did increase basal and maximum contractile tension without any corresponding changes in myosin light chain phosphorylation indicating that other mechanisms that doesn't include PKG mediated effects on myosin light chain phosphorylation may be important in regulating basal tone of ileal smooth muscle by CGA.

Support or Funding Information

Supported by internal grant of Cedarville University School of Pharmacy

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Taurine Protects Against the Neurotoxic Effects of Manganese on the Dopaminergic Control of Lateral Cell Membrane Potential and Ciliary Response in Gill of Crassostrea virginica
Authors: Johnson Alexcia J. , Howard Aliyah , Carroll Margaret A. , Catapane Edward J.
Published: June 2021

Manganese is a neurotoxin causing manganism, a Parkinson's‐like disease involving disruption of dopaminergic neurotransmission. The mechanism by which manganese produces this dysfunction is not fully resolved. Unlike Parkinson's disease, reports postulate that manganese neurotoxicity is more likely related to downstream neuronal pathways rather than deficits in nigrostriatal function. Lack of effective treatment for manganism has been a major obstacle in its clinical management. The cilia of lateral cell in gill of Crassostrea virginica are controlled by serotonergic‐dopaminergic innervations from their ganglia. Dopamine (DA) hyperpolarizes the cell and causes cilio‐inhibition, while serotonin depolarizes and causes cilio‐excitation. Previous work of our lab showed acute and short‐term manganese treatments blocked the cilio‐inhibitory effect of DA. This neurotoxic action of manganese could be prevented by the drug p‐aminosalicylic acid or by taurine, an amino acid with efficacy in other neurodegenerative disorders. In this study we hypothesize that taurine would effectively prevent the toxic actions of manganese on both dopaminergic membrane hyperpolarization and lateral cilia inhibition. To test this we conducted acute experiments on DA dose responses (10−6 – 10−3 M) of manganese treated excised gill in the presence or absence of taurine. Ciliary activity of gill lateral cells was measured by stroboscopic microscopy and expressed as beats/min ± sem. Cell membrane potentials were simultaneously measured using the fluorescent dye DiBAC4(3). In control gill, the lateral cells responded normally to the DA dose response showing the appropriate decrease in cilia beating rates and membrane hyperpolarization. Gills treated with manganese (2 × 10−4M) had a disrupted DA response, cilio‐inhibition was impaired and the membrane did not hyperpolarize. Taurine treatment (2 × 10−4M) effectively prevented these neurotoxic effects in manganese treated gill. This physiological study in C. virginica shows that manganese disrupts the dopaminergic post‐synaptic response of lateral cells preventing membrane hyperpolarization as well disrupting cilio‐inhibition, and that taurine effectively blocked these neurotoxic actions. These findings are helpful in furthering the understanding of the mechanism of manganese neurotoxicity and provides evidence suggesting taurine be further investigated as a potential therapeutic agent for manganism.

Support or Funding Information

This work was supported in part by grant 2R25GM06003 of the Bridge Program of NIGMS, NIH grant K12GM093854‐07A1 IRACDA Program of Rutgers University and 604060048 of PSC‐CUNY.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Nitrite Disruption in Olfaction and Internal Physiology in Goldfish (Carassius auratus)
Authors: Martinez Melody , Huertas Mar
Published: June 2021

Freshwater ecosystems have experienced a dramatic increase of nitrogenous compounds as a result of farming and industrial activity. Nitrite accumulates by bacterial denitrification/nitrification in freshwater and saltwater ecosystems, reaching concentrations of 1 mM and 0.5 μM respectively. Exposure to high nitrite concentrations can disrupt the endocrine system and physiological processes such as ion homeostasis, respiratory, and cardiovascular activity. However, the accumulation of nitrite in different internal tissues and its effect on external sensitive organs, such as the olfactory epithelium, is unknown. This study aimed to determine the physiological effects of nitrite and its accumulation in several organs in goldfish (Carassius auratus). Goldfish were exposed to different nitrite treatments (0 mM, 0.3 mM, 1.0 mM, and 10 mM) for five days. Upon completion of experiment, brain, nose, liver, kidney, and gill samples were obtained for biological and histological analysis. Goldfish in two treatments (1.0 mM and 10 mM) experienced irreversible oxidation of hemoglobin known as metahemoglobemia, marked by browning of the blood and darker tissues. Biochemical analysis showed an increase of nitrite in brain, nose, liver, and gill tissues. Brain and nose tissues (240 ±0.004 μM and 260 ±0.002 μM, respectively) accumulated more nitrite than gill tissue (190 ±0.002 μM) in the lowest treatment (0.3 mM), and this trend continued as treatment concentrations increased. Preliminary statistical analysis demonstrated that both the brain and nose tissue accumulated >30% more nitrite than gill tissue in the 0.3 mM treatment. Histological analysis of the olfactory epithelium showed hyperplasia, an increase in cell size and number; furthermore, we observed an increase in the number of mucus cells and mucus production, collapse of columnar cells, and lamella constriction in both the 1.0 mM and 10 mM treatments. Histological analysis of gills showed lamella fusion and vascular congestion. Histological liver samples exhibited melanomacrophage aggregate, nucleus degeneration, and cytoplasmic vacuolation in 10 mM. Preliminary findings from electro‐olfactograms show that nitrite reduced the ability of a goldfish to detect odors at nitrite exposure as low as 0.1 mM. These results suggest that nitrite is accumulating faster in external sensory tissues than internal organs, affecting essential physiological processes such as olfactory food detection and possibly acting as a neurotoxin.

Support or Funding Information

Research support: Texas State University start‐up funding to Dr. Mar Huertas.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanisms of a Function‐Selective ERK Inhibitor and Induction of Apoptosis in Melanoma Cells
Authors: Martinez Ramon , Huang Weiliang , Mackowiak Bryan , Shapiro Paul
Published: June 2021

Various cancer cell types can contribute their proliferative phenotypes to mutations conferring constitutive activity of the extracellular signal‐regulated kinase (ERK1/2) signaling pathway. Recent trends in drug design have suggested that shifting the design of ERK1/2 pathway inhibitors from targeting the catalytic core of the kinase to allosteric inhibition may prove as a promising alternative to therapeutic development. We previously identified a novel thienyl naphthalene sulfonate compound that shows functionally‐selective inhibitory activity against ERK2 by blocking F‐site containing substrates, including Fos family proteins. Proteomic analysis of drug‐treated melanoma cell lysates suggests that treatment with this novel compound results in defects in mitochondrial function as well as upregulation of markers suggestive of activating an oxidative stress response. As well, further mass spectrometry analysis has suggested that the novel compound may generate covalent modifications on cysteine residues of the ERK2 protein. Additional studies with inhibitors of various reactive oxygen species (ROS) have shown that while the generation of ROS may be evident with compound treatment, further cell viability study has indicated that ROS is only partially responsible for the growth inhibition of melanoma cells. In these studies, we provide further insight into the interactions this novel compound makes with the ERK2 protein, and the transcriptomic and proteomic responses that ultimately result in oxidative stress and melanoma cell death.

Support or Funding Information

This work was supported by an NIGMS Institutional Training Grant (T32 GM066706).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phycocyanin Enhances the Cytotoxic Profile of Cisplatin in Platinum‐Resistant Ovarian Cancer Cells by Targeting Focal Adhesion Kinase
Authors: Levy Arkene , Joseph Shona , Hussain Syed , Saleem Mir , Rathinavelu Appu
Published: June 2021

Platinum‐based therapy is one of the major treatment approaches in ovarian cancer. Although greater than 80% of advanced stage ovarian cancer patients initially achieve remission with platinum therapy; the majority of this cohort eventually relapse, and develop drug‐resistant disease. Resistance to chemotherapeutic drugs such as cisplatin has been attributed in part to the overexpression of Focal Adhesion Kinase (FAK), which is known to promote cancer cell survival and metastasis. In recent years, there has been heightened interest in targeting focal adhesion kinase in platinum resistant cancer as a new modality in ovarian cancer treatment. In previous studies, we have reported that Phycocyanin (C‐PC), a major accessory pigment in the Limnothrix sp. 37‐2‐1, demonstrates cytotoxicity towards multiple cancer cell lines. Therefore, in the present study we evaluated the ability of C‐PC to sensitize platinum resistant OVCAR3 cells to cisplatin treatment. We also examined the response of FAK activation by phosphorylation, to treatment with cisplatin and CP‐C alone, and in combination (C‐PC +cisplatin). Cell viability was determined by MTT assay, and DNA fragmentation was used to confirm apoptotic cell death. The expression of phosphorylated FAK (pFAK), total FAK, pAKT, as well as the key apoptotic regulators p21, p53, Bax, and Bcl‐2 in response to the treatments were determined through western blotting. The results from our study confirm that C‐PC significantly enhances the cytotoxic profile of cisplatin in OVCAR‐3 cells through the induction of apoptosis as confirmed by the presence of DNA fragmentation patterns in all three treatment groups. The expression of activated pFAK was also significantly reduced in the CPC and CPC + cisplatin treatment groups and this corresponded with increased p53, p21 and Bax expression, while pAkt and Bcl‐2 expression was downregulated. These results confirm a novel effect of CPC on FAK phosphorylation in platinum resistant ovarian cancer, which can be further explored towards the treatment of advanced stage disease through combination strategies.

Support or Funding Information

This project was supported by The Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Boswellic Acid Composition of Frankincense Dietary Supplements and Correlation to Cytotoxic Efficacy against Treatment‐Resistant Triple Negative Breast Cancer Cells
Authors: Schmiech Michael , Lang Sophia , Werner Katharina , Schmidt Christoph Q , Syrovets Tatiana , Simmet Thomas
Published: June 2021

Frankincense is an oleogum resin from Boswellia trees of the Burseraceae family. The oleogum resin contains 15–20 % triterpenic acids, including therapeutically active boswellic acids [1]. Extracts of oleogum resins obtained from Boswellia serrata have been traditionally used in Indian folk medicine (Ayurveda) for the treatment of various chronic inflammatory diseases such as rheumatoid arthritis. Furthermore, boswellic acids are known to possess anticancer efficacy against various human cancer types, e.g. gliomas, prostate cancer, or leukemia [2–4]. The aim of the present study was to analyze the contents of boswellic acids in dietary supplements from various Boswellia species grown in different areas and to correlate the concentrations of distinct boswellic acids to the extracts' cytotoxic efficacy against chemoresistant triple‐negative human breast cancer cells. Accordingly, a new method to quantify a large variety of pentacyclic triterpenic acids was developed. Dietary supplements from various Boswellia species Boswellia serrata, Boswellia sacra, Boswellia carterii, and others were extracted with methanol and analyzed by high performance reversed‐phase liquid chromatography coupled with tandem mass spectrometric detection (HPLC‐MS/MS). Besides, the effects of the extracts against the treatment‐resistant, triple negative human breast cancer cell line MDA‐MB‐231 were investigated in vitro by using a cell viability assay. The extracts exhibited potent cytotoxic activity against the human breast cancer cell line. Correlations between the boswellic acid contents and the cytotoxic efficacy were determined by multiple linear regression and principal component regression. This approach allowed not only the correlation of complex phytochemical mixtures to desired biomedical effects, but it likewise provided evidence for key compounds governing the therapeutic efficacy. Hence, the study provides new insights into differential chemical composition patterns of frankincense dietary supplements from Boswellia species grown in different regions. Besides, the study identified distinct components correlating positively with the desired cytotoxic activity. Thus, the identified key compounds deserve further studies either as chemically pure substances or as defined chemical mixtures permitting development of the most active ingredients as potential anticancer drug candidates.

Support or Funding Information

This work was supported by the Academic Center for Complementary and Integrative Medicine (AZKIM), State Ministry of Baden‐Württemberg for Sciences, Research and Arts.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Constituents of Artemisia annua Dietary Supplements Induce ROS Elevation, ERK Activation, and Apoptosis in Treatment‐Resistant Triple Negative Human Breast Cancer Cells
Authors: Lang Sophia , Schmiech Michael , Hafner Susanne , Paetz Christian , Schmidt Christoph Q , Syrovets Tatiana , Simmet Thomas
Published: June 2021

In recent years, Artemisia annua L. and especially artemisinin and its semisynthetic derivatives became subject of intense investigations for their potential anticancer activity. Yet, available evidence suggests that artemisinin might not be the most active ingredient of Artemisia annua. For this reason, we have analyzed different Artemisia annua extracts, which did not contain any detectable artemisinin marketed as dietary supplements for their activity against the triple negative human breast cancer cell line MDA‐MB‐231. A bioguided fractionation by semi‐preparative HPLC was conducted and the structures of individual compounds were identified by mass spectrometry and 1H‐ and 13C‐NMR spectroscopy. Extracts as well as the isolated compounds 6,7‐dimethoxy‐coumarin, chrysosplenol D, casticin, arteannuic acid and arteannuin B were further analyzed in vitro and in vivo. The extracts and the individual compounds inhibited the viability of several cancer cell lines. Interestingly, the viability of peripheral blood mononuclear cells was not inhibited at equivalent concentrations. Moreover, treatment either with extract or the individual compounds induced cell cycle arrest in the S‐phase or the G2/M‐phase. Apoptotic cell death was identified by DNA‐fragmentation, activation of caspase 3/7, and annexin V/propidium iodide staining. Moreover, the extract and individual compounds increased the phosphorylation of the extracellular regulated kinase ERK in the presence of sustained high ROS levels, which are required for the ERK‐mediated cell death. Pretreatment with the ERK ‐ inhibitor U‐0126 was able to inhibit chrysosplenol D induced toxicity towards MDA‐MB‐231 cells. Furthermore, antiproliferative efficacies of the extract and individual compounds were confirmed in vivo using 3D breast cancer xenografts grown on the chorioallantoic membrane of fertilized chick eggs, where the Artemisia annua extract and the isolated substances significantly reduced the MDA‐MB‐231 breast cancer tumor volume as well as the expression of the Ki‐67 proliferation marker. Importantly, no systemic toxic effects on the chick embryos could be detected. This study provides new insights into a potential rational use of Artemisia annua‐derived compounds in anticancer therapy and identifies compounds beside artemisinin with potent anticancer activity. Furthermore, these findings provide evidence for the implication of the MEK‐ERK‐pathway in apoptotic cell death induced by chrysosplenol D.

Support or Funding Information

This work was supported by the Academic Center for Complementary and Integrative Medicine (AZKIM), State Ministry of Baden‐Württemberg for Sciences, Research and Arts.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pharmacological inhibition of Human Antigen R (HuR) blunts fibroblast activation and cardiac fibrosis
Authors: Green Lisa C , Anthony Sarah R , Slone Samuel , Lanzillotta Lindsey , Lorenz John , Tranter Michael
Published: June 2021

Heart failure is one of the leading causes of death globally, and the physiology of a failing heart is often worsened by the presence of cardiac fibrosis. Cardiac fibrosis is marked by the chronic activation of fibroblasts into myofibroblasts and excessive extracellular matrix (ECM) protein deposition, leading to decreased contractility and compliance of the myocardium. In this work we show that Human Antigen R (HuR), a stabilizing RNA binding protein, plays a critical role in the activation of fibroblasts and the subsequent fibrosis. In vivo, we used the transverse aortic constriction (TAC) model of heart failure and started treatment with a small molecule inhibitor of HuR four weeks after TAC surgeries. At the end of the eleven‐week study, the mice treated with the HuR inhibitor had markedly reduced fibrosis shown by Masson's Trichrome staining. Western blot and qRT‐PCR analysis also revealed a decreased expression of fibrotic markers such as fibronectin and periostin in the hearts from mice treated with HuR inhibitor. In vitro, we also saw that pharmacologically inhibiting HuR in fibroblasts isolated from adult mouse hearts blunts the activation of fibroblasts using assays such as the scratch assay. To identify a potential underlying mechanism for direct reduction in fibrosis via HuR inhibition, we interrogated the connection between HuR and TGFβ, a well‐known driving factor in fibrotic initiation and progression. Using an Actinomycin D stability assay on isolated primary myocytes, we showed a decrease in TGFβ mRNA stability upon inhibition of HuR. We also performed RNA immunoprecipitation and the results support a direct binding of HuR to TGFβ mRNA. Taken together, these suggest HuR may play a central role in the regulating the stability of TGFβ, thereby affecting pro‐fibrotic signaling. In conclusion, these results suggest that HuR is a promising therapeutic target for the reduction of cardiac fibrosis.

Support or Funding Information

This work was partially funded by an AHA Scientist Development Grant (MT), NIH R01 HL132111 (MT), and T32HL125204‐4 (JM, EK)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Screening of Metabolic Markers of Qi‐Yin deficiency in Chronic Heart Failure Mice by Untargeted Metabolomics Method
Authors: Lai Qiong , Li Fang , Liu Liang Ze , Yu Yang Bo
Published: June 2021

Qi‐Yin deficiency is the main syndrome of chronic heart failure that one of the major heart disease that threatening human health. Clarifying the time‐history of Qi‐Yin deficiency in chronic heart failure disease, and further exploring the metabolic markers of Qi‐Yin deficiency can help to explain the pathological mechanism and Precise treatment of Qi‐Yin deficiency in chronic heart failure. This study adopted chronic heart failure model induced by coronary artery ligation in mice. We investigated the changes in the appearance of clinical indications corresponding to symptoms of Qi‐Yin deficiency at different time points, which aimed to clear the time‐history of Qi‐Yin deficiency appeared in chronic heart failure model mice. Positive (Yiqifumai) and negative (Danhong) control group were set up to assistant define the time‐history of Qi‐Yin deficiency. We found that Chronic heart failure mice appeared significant weight loss, decreased food intake, spontaneous activity , accelerated heart rate, and increased T wave amplitude in the first 2–3 weeks after modeling. What's more, Yiqifumai had better effects than Danhong in the improvement of Qi‐Yin deficiency in the first 2–3 weeks, which assistant proved that this period was Qi‐Yin deficiency. The coagulation function test results also suggest that it may turn into blood stasis syndrome around 4–5 weeks. In addition, LC‐MS/MS technology was used to determine the endogenous metabolites in serum and urine of mice on the positive ion mode. The data of accurate molecular weight and fragment ion peak were checked on HMDB database, and the identification methods of PCA and OPLS‐DA were used to analyze the multivariable data. Metabolites of the chronic heart failure model mice during the period of Qi‐Yin deficiency and non Qi‐Yin deficiency were compared to explore specific metabolite groups significant related to Qi‐Yin deficiency. The untargeted metabolomic workflow is showed in Figure 1. The results of metabolomic analysis of serum and urine of model mice showed that metabolites were well‐differentiated between the two groups at 2–3 weeks and 4–5 weeks. 29 significant difference metabolites in serum and 19 significant difference metabolites in urine were identified in this study. Hence, these results demonstrated that ischemia‐induced chronic heart failure may be mainly manifested as Qi‐Yin deficiency syndrome in the first 2–3 weeks, followed by conversion to blood stasis syndrome. Metabolites of chronic heart failure mice also changed as the syndrome transformed.

Support or Funding Information

National Natural Science Foundation of China (NO. 81774150)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Towards Identifying the Binding Site for 125I‐Angiotensin 1‐7
Authors: Stoyell‐Conti Filipe Fernandes , Itty Sarin , Stewart Hanna , Asif Mahnoor , Abraham Christy , Vadhan Jason , West Crystal A , Speth Robert C
Published: June 2021

Angiotensin 1–7 (Ang 1‐7) is a product of the renin‐angiotensin system (RAS) that exhibits central actions to reduce blood pressure and improve baroreflex sensitivity. For more than a decade Mas has been considered the receptor for Ang 1‐7, however, to date, no published studies have characterized Mas receptor radioligand binding in tissue membrane preparations. Our laboratory has demonstrated high affinity (low nanomolar KD) binding of a preparation of 125/127I‐Ang 1‐7 in liver membranes, however, this binding is not pharmacologically specific in that the IC50 of Ang 1‐7 is in the micromolar range and all angiotensin peptides compete for 125/127I‐Ang 1‐7 binding equivalently. 125/127I‐Ang 1‐7 binding to liver, kidney, brain and testes membrane preparations from both rats and mice has also failed to demonstrate a high affinity, pharmacologically specific binding site for 125/127I‐Ang 1‐7. Considering that rapid degradation of Ang 1‐7 could be adversely affecting the binding assay, we tested different peptidase inhibitors (JMV‐390, O‐phenanthroline and a proprietary combination of peptidase inhibitors Biotools®) to prevent degradation of the radioligand. Although we were able to reduce Ang 1‐7 degradation with these inhibitors (~80% preservation), we also reduced the total and specific binding of 125I‐Ang 1‐7. Indeed, competition binding assays for 125I‐Ang‐(1–7) with JMV‐390, O‐phenanthroline and Biotools® showed that 125I‐Ang‐(1–7) and all the inhibitors compete for the same binding site with JMV‐390 having a nanomolar IC50. It is important to note that the radioligand used for receptor binding studies contains an iodine125 on the tyrosine in position four of Ang 1‐7, and may have different characteristics for enzymatic metabolism. To assess this possibility, we determined the metabolism of 127I‐Ang 1‐7 (non‐radioactive iodine isotope) and indeed, the metabolism of Ang 1‐7 and 127I‐Ang 1‐7 in liver appear to differ, with 127I‐Ang 1‐7 being more resistant to metabolism. In addition, competition binding assays showed that 127I‐Ang 1‐7 is ~1000‐fold more potent than Ang 1‐7 in competing for 125I‐Ang 1‐7 binding. In conclusion, our results suggest that 125I‐Ang‐(1–7) may be binding to a peptidase rather than a receptor and the addition of the iodine molecule to the tyrosine in position four attenuates its degradation. Studies are underway to identify this peptidase and determine its potential therapeutic importance as a druggable target.

Support or Funding Information

Cardiovascular Neuroscience Research Fund 337667 Speth (PI) 03/31/15‐12/31/18 ‐ Nova Southeastern Univ.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.