Receptor mRNA Expression Research Articles

Acute caffeine differently affects risk-taking and the expression of BDNF and of adenosine and opioid receptors in rats with high or low anxiety-like behavior

Anxiety disorders are common psychiatric conditions with a partially elucidated neurobiology. Caffeine, an unspecific adenosine receptor antagonist, is a common psychostimulant with anxiogenic effects in sensitive individuals. High doses of caffeine produce anxiety-like behavior in rats but it is not known if this is specific for rats with high baseline anxiety-like behavior. Thus, the aim of this study was to investigate general behavior, risk-taking, and anxiety-like behavior, as well as mRNA expression (adenosine A2A and A1, dopamine D2, and, μ, κ, δ opioid, receptors, BDNF, c-fos, IGF-1) in amygdala, caudate putamen, frontal cortex, hippocampus, hypothalamus, after an acute dose of caffeine. Untreated rats were screened using the elevated plus maze (EPM), giving each rat a score on anxiety-like behavior based on their time spent in the open arms, and categorized into a high or low anxiety-like behavior group accordingly. Three weeks after categorization, the rats were treated with 50 mg/kg caffeine and their behavior profile was studied in the multivariate concentric square field (MCSF) test, and one week later in the EPM. qPCR was performed on selected genes and corticosterone plasma levels were measured using ELISA. The results demonstrated that the high anxiety-like behavior rats treated with caffeine spent less time in risk areas of the MCSF and resituated towards the sheltered areas, a behavior accompanied by lower mRNA expression of adenosine A2A receptors in caudate putamen and increased BDNF expression in hippocampus. These results support the hypothesis that caffeine affects individuals differently depending on their baseline anxiety-like behavior, possibly involving adenosine receptors. This highlights the importance of adenosine receptors as a possible drug target for anxiety disorders, although further research is needed to fully elucidate the neurobiological mechanisms of caffeine on anxiety disorders.

Stuxnet fine-tunes Notch dose during development using a functional Polycomb response element.

Evolutionarily conserved Notch signaling is highly sensitive to changes in Notch receptor dose caused by intrinsic and environmental fluctuations. It is well known that epigenetic regulation responds dynamically to genetic, cellular and environmental stresses. However, it is unclear whether the Notch receptor dose is directly regulated at the epigenetic level. Here, by studying the role of the upstream epigenetic regulator Stuxnet (Stx) in Drosophila developmental signaling, we find that Stx promotes Notch receptor mRNA expression by counteracting the activity of Polycomb repressive complex 1 (PRC1). In addition, we provide evidence that Notch is a direct PRC1 target by identifying and validating in vivo the only bona fide Polycomb response element (PRE) among the seven Polycomb group (PcG)-binding sites revealed by DamID-seq and ChIP-seq analysis. Importantly, in situ deletion of this PRE results in increased Notch expression and phenotypes resembling Notch hyperactivation in cell fate specification. These results not only underscore the importance of epigenetic regulation in fine-tuning the Notch activity dose, but also the need to assess the physiological significance of omics-based PcG binding in development.

Anticonvulsant Role of Adenosine is Blunted During Alcohol Withdrawal Syndrome in an Adult Zebrafish Model.

Alcohol (ethanol) dependence and related disorders are life-threatening conditions and source of suffering for the user, family members and society. Alcohol withdrawal syndrome (AWS) is a little-known dynamic process associated with a high frequency of relapses. A state of hyperglutamatergic neurotransmission and imbalanced GABAergic function is related to an increased susceptibility to seizures during alcohol withdrawal. Adenosine signaling display an important role in endogenous response to decrease seizure and related damages. Here, an intermittent alcohol exposure regimen (1h daily of 0.5% ethanol solution) for 16 days or 8 days of the same ethanol exposure regimen followed by 1 or 8 days of ethanol withdrawal was used to assess adenosine signaling in the context of seizure susceptibility using adult zebrafish. In both abstainer groups, a sub-convulsant dose of pentylenetetrazol (2.5 mM) was able to increase the frequency of animals reaching a clonic seizure-like state, while continuous-treated animals had no seizure, as did control animals. The total brain mRNA expression of A1 adenosine receptor was decreased in animals with 1day of ethanol withdrawal. The agonism of A1 adenosine receptor induced an anticonvulsant effect in animals with 1day of ethanol withdrawal after the injection of the specific agonist (N6-cyclopentyladenosine, 10mg.Kg- 1; i.p.). These findings reinforce A1 adenosine receptor as a key target in acute alcohol withdrawal syndrome and zebrafish as an excellent platform to study biological mechanism of AWS.

Differential expression of pro- and anti-angiogenic factors in the endometrium between repeat breeder and normally fertile cows

In cattle, the establishment of appropriate endometrial vasculature during the estrous cycle is required for preparing a receptive endometrium. This study aimed to investigate 1) mRNA expression of potent pro- and anti-angiogenic factors, 2) protein localization of the anti-angiogenic factor thrombospondin (TSP), and 3) vascularity in the endometrium of repeat breeder (RB) and normally fertile (non-RB) cows. Caruncular and intercaruncular endometrium was collected from RB and non-RB cows during the luteal phase of the estrous cycle. RB cows had greater mRNA expression levels of TSP ligands (TSP1 and TSP2) and receptors (CD36 and CD47) than non-RB cows. Although the mRNA expression levels of most angiogenic factors did not change by repeat breeding, RB cows had greater mRNA expression of fibroblast growth factor receptor 1 (FGFR1), angiopoietin 1 (ANGPT1), and ANGPT2 and a less mRNA expression of vascular endothelial growth factor B (VEGFB) than non-RB cows. By immunohistochemistry, TSP1, TSP2, CD36, and CD47 were detected in the luminal epithelium, glandular epithelium, stromal cells, and blood vessels of the endometrium. Two indexes of vascularity, the number of blood vessels and the percentage of area stained positive for the von Willebrand factor, were lower in the endometrium of RB than in that of non-RB cows. These results demonstrate that RB cows have a greater expression of both ligands and receptors for the anti-angiogenic factor TSP and a reduced vascular distribution in the endometrium compared with non-RB cows, suggesting suppressed endometrial angiogenesis.

Orchiectomy sensitizes cortical bone in male mice to the harmful effects of kynurenine

Kynurenine (Kyn) is a tryptophan metabolite that increases with age and promotes musculoskeletal dysfunction. We previously found a sexually dimorphic pattern in how Kyn affects bone, with harmful effects more prevalent in females than males. This raises the possibility that male sex steroids might exert a protective effect that blunts the effects of Kyn in males. To test this, orchiectomy (ORX) or sham surgeries were performed on 6-month-old C57BL/6 mice, after which mice received Kyn (10 mg/kg) or vehicle via intraperitoneal injection, once daily, 5×/week, for four weeks. Bone histomorphometry, DXA, microCT, and serum marker analyses were performed after sacrifice. In vitro studies were performed to specifically test the effect of testosterone on activation of aryl hydrocarbon receptor (AhR)-mediated signaling by Kyn in mesenchymal-lineage cells. Kyn treatment reduced cortical bone mass in ORX- but not sham-operated mice. Trabecular bone was unaffected. Kyn's effects on cortical bone in ORX mice were attributed primarily to enhanced endosteal bone resorption activity. Bone marrow adipose tissue was increased in Kyn-treated ORX animals but was unchanged by Kyn in sham-operated mice. ORX surgery increased mRNA expression of the aryl hydrocarbon receptor (AhR) and its target gene Cyp1a1 in the bone, suggesting a priming and/or amplification of AhR signaling pathways. Mechanistic in vitro studies revealed that testosterone blunted Kyn-stimulated AhR transcriptional activity and Cyp1a1 expression in mesenchymal-linage cells. These data suggest a protective role for male sex steroids in blunting the harmful effects of Kyn in cortical bone. Therefore, testosterone may play an important role in regulating Kyn/AhR signaling in musculoskeletal tissues, suggesting crosstalk between male sex steroids and Kyn signaling may influence age-associated musculoskeletal frailty.

Specific dilation pattern in placental circulation and the NO/sGC role in preeclampsia placental vessels.

Endothelial functions in controlling blood flow in placental circulation are still unclear. The present study compares vascular dilations between placental circulation and other vessels, as well as between normal and preeclampsia placental vessels. Placental, umbilical, and other vessels (cerebral and mesenteric arteries) were collected from humans, sheep, and rats. Vasodilation was tested by JZ101 and DMT. Q-PCR, Western blot, and Elisa were used for molecular experiments. Endothelium-dependent/derived vasodilators, including acetylcholine, bradykinin, prostacyclin, and histamine, mediated no or minimal dilation in placental circulation, which was different from that in other vessels in sheep and rats. There were lower mRNA expressions of muscarinic receptors, histamine receptors, bradykinin receptor 2, endothelial nitric oxide synthesis (eNOS), and less nitric oxide (NO) in human umbilical vessels when compared with placental vessels. Exogenous NO donors (sodium nitroprusside, SNP) and soluble guanylate cyclase (sGC) activators (Bay41-2272) decreased the baseline of vessel tone in placental circulation in humans, sheep, and rats, but not in other arteries. The sGC inhibitor ODQ suppressed the reduced baseline caused by the SNP. The decreased baseline by SNP or Bay41-2272 was higher in placental vessels than in umbilical vessels, suggesting that the role of NO/sGC is more important in the placenta. NO concentrations in preeclampsia placental vessels were lower than those in control, while no significant change was found in umbilical plasma between the two groups. eNOS expression was similar between normal and preeclampsia placental vessels, but phosphorylated eNOS levels were significantly lower in preeclampsia. Following serotonin, SNP or Bay41-2272-mediated dilations were weaker in preeclampsia placental vessels. The decreased amplitude of SNP- or Bay41-2272 at baseline was smaller in preeclampsia. The decreased amplitudes of ODQ + SNP were comparable between the two groups. Despite higher beta sGC expression, sGC activity in the preeclampsia placenta was lower. This study demonstrated that receptor-mediated endothelium-dependent dilation in placental circulation was significantly weaker than other vessels in various species. The results, showed firstly, that exogenous NO played a role in regulating the baseline tone of placental circulation via sGC. Lower NO production and decreased NO/sGC could be one of the reasons for preeclampsia. The findings contribute to understanding specific features of placental circulation and provide information about preeclampsia in placental vessels.

Effects of dietary supplementation of different herbs on relative expression of Toll-like Receptors (TLRs) in poultry

The paucity of novel antibiotics for treating human and animal infections has heightened global fears about growing drug-resistant superbugs. Antibiotic replacement approaches for food-producing animals, particularly poultry and livestock, are urgently needed by the agricultural business. The need of the hour is to develop and exploit promising antibiotic-alternative phytochemicals in order to minimize Antibiotic Growth Promoters and develop an antibiotic-free animal production system. Phytogenic plant dietary supplements have positive effects on poultry performance and immunity. Toll-like receptors (TLRs) play a key role in the innate immune system. TLRs recognize microbes that have broken through physical barriers such as the skin or the mucosa of the digestive tract, triggering immune cell responses. Supplementation of herbs in the dietary regimen of poultry had shown stimulatory effects on the immune system. Herbs such as Amla, Ashwagandha, Garlic powder, Giloy, Turmeric, Shatavari, Aloe vera, Moringa and holy basil have been shown to increase the relative mRNA expression of different toll-like receptors, thereby enhancing broiler immunological status by modulating their immune response.

Review: Gestational and postnatal nutritional effects on the neuroendocrine control of puberty and subsequent reproductive performance in heifers.

Pubertal attainment is an intricate biological process that involves maturation of the reproductive neuroendocrine axis and increased pulsatile release of gonadotropin-releasing hormone (GnRH) and luteinizing hormone. Nutrition is a critical environmental factor controlling the timing of puberty attainment. Nutrient restriction during early postnatal development delays puberty, whereas increased feed intake and adiposity during this period hasten pubertal maturation by imprinting the hypothalamus. Moreover, the dam's nutrition during gestation can program the neuroendocrine system in the developing fetus and has the potential to advance or delay puberty in the offspring. Leptin, a hormone produced primarily by adipose cells, plays an important role in communicating energy status to the brain and regulating sexual maturation. Leptin's regulation of GnRH release is mediated by an upstream neuronal network since GnRH neurons do not contain the leptin receptor. Two groups of neurons located in the arcuate nucleus of the hypothalamus that express neuropeptide Y (NPY), an orexigenic peptide, and alpha melanocyte-stimulating hormone (αMSH), an anorexigenic peptide, are central elements of the neural circuitry that relay inhibitory (NPY) and excitatory (αMSH) inputs to GnRH neurons. Moreover, KNDy neurons, neurons in the arcuate nucleus that co-express kisspeptin, neurokinin B (NKB), and dynorphin, also play a role in the metabolic regulation of puberty. Our studies in beef heifers demonstrate that increased rates of BW gain during early postweaning (4-9 mo of age) result in reduced expression of NPY mRNA, increased expression of proopiomelanocortin and kisspeptin receptor mRNA, reduced NPY inhibitory inputs to GnRH neurons, and increased excitatory αMSH inputs to KNDy neurons. Finally, our most recent data demonstrate that nutrition of the cow during the last two trimesters of gestation can also induce transcriptional and structural changes in hypothalamic neurocircuitries in the heifer progeny that likely persist long-term after birth. Managerial approaches, such as supplementation of the dam during gestation (fetal programming), creep feeding, early weaning, and stair-step nutritional regimens have been developed to exploit brain plasticity and advance pubertal maturation in heifers.

Impact of IH-induced sympathetic hyperactivity and metabolic dysfunctions on ischemic cardiomyopathy progression

Patients with obstructive sleep apnea (OSA) exhibit poor prognosis after myocardial infarction (MI). Intermittent hypoxia (IH), the hallmark feature of OSA, promotes sympathetic hyperactivity and systemic insulin resistance, and has been identified as a major contributor to post-MI cardiac remodeling and contractile dysfunction. We hypothesize that sympathetic hyperactivity and metabolic alterations induced by IH promote desensitization of cardiac adrenergic and insulin pathways via activation of G protein-coupled receptors (GRK2), and subsequently participate in the aggravation of ischemic cardiomyopathy. MI is induced in C57bl6 mice by permanent ligation of the left coronary artery (MI). Mice are then randomized to IH (21–5% FiO2, 60 s cycle, 8 h/day) or normoxia for up to 6 weeks. After two weeks, mice are treated with a GRK2 inhibitor, Paroxetine (5 mg/kg/d), or 25% DMSO (Alzet® pumps). Longitudinal follow-up of mice includes evaluation of sympathetic activity (spectral analysis of heart rate variability (HRV), systemic insulin sensitivity (dynamic insulin tolerance test) and determination of cardiac function/remodeling (echocardiography). At the end of the protocol, cardiac interstitial fibrosis and hypertrophy are evaluated by RT-qPCR and histology (Sirius Red and WGA staining, respectively). Assessment of insulin signaling pathway is performed by Western blot. IH induces sympathetic hyperactivity in MI mice, demonstrated by a modification of the sympathovagal balance (LF increase). Paroxetine treatment abolishes the increase in LF in MI-IH condition and restores mRNA expression of β1 and β2 adrenergic receptors. MI-IH induces systemic insulin resistance compared to MI-N condition, which is prevented by Paroxetine treatment. In addition, Paroxetine treatment improves insulin signaling in the liver of MI-IH mice (pIRβ and pAKT). IH exacerbates cardiac interstitial fibrosis and worsens contractile dysfunction in MI mice, resulting in a significant decrease in ejection fraction (19.4 ± 2.1% vs. 29.9 ± 3.6% in IH vs. N respectively, P < 0.05). The effect of paroxetine on cardiac remodeling and function is currently being analyzed. These findings demonstrate that IH induces cardiac sympathetic hyperactivity and insulin resistance, which could contribute to the worsening of ischemic heart disease via GRK2.

Differential innate immune response of endometrial cells to porcine reproductive and respiratory syndrome virus type 1 versus type 2.

Modification of cellular and immunological events due to porcine reproductive and respiratory syndrome virus (PRRSV) infection is associated with pathogenesis in lungs. PRRSV also causes female reproductive dysfunction and persistent infection which can spread to fetus, stillbirth, and offspring. In this study, changes in cellular and innate immune responses to PRRSV type 1 or type 2 infection, including expression of PRRSV mediators, mRNA expression of Toll-like receptors (TLRs) and cytokine, and cytokine secretion, were examined in primary porcine glandular endometrial cells (PGE). Cell infectivity as observed by cytopathic effect (CPE), PRRSV nucleocapsid proteins, and viral nucleic acids was detected as early as two days post-infection (2 dpi) and persisted until 6 dpi. A higher percentage of CPE and PRRSV-positive cells were observed in type 2 infections. PRRSV mediator proteins, CD151, CD163, sialoadhesin (Sn), integrin and vimentin, were upregulated following type 1 and type 2 infection. CD151, CD163 and Sn were upregulated by type 2. In both PRRSV types, mRNA expression of TLR1 and TLR6 was upregulated. However, TLR3 was upregulated by type 1, but TLR4 and TLR8 mRNA and protein were downregulated by type 2 only. Interleukin (IL)-1β, IL-6 and tumor necrotic factor (TNF)-α were upregulated by type 2, but IL-8 was upregulated by type 1. Both PRRSV type 1 and 2 stimulated IL-6 but suppressed TNF-α secretion. In addition, IL-1β secretion was suppressed only by type 2. These findings reveal an important mechanism underlying the strategy of PRRSV infection in the endometrium and associated with the viral persistence.

Providing or receiving alloparental care promote partner preference and alter central oxytocin and dopamine systems in adult mandarin voles

Juveniles of cooperative breeding species usually remain in the natal area and provide care to younger siblings, a behavior considered one form of alloparenting in the natural condition. Previous studies have demonstrated the effects of providing or receiving alloparental care on adult behaviors, including anxiety-like behavior, social interaction, and parental behavior, but little is known about the influences on species-typical bonding behaviors, such as pair-bond formation. In this study, we explored this concept using socially monogamous mandarin voles (Lasiopodomys mandarinus). As the oxytocin (OT) and dopamine systems are involved in alloparental and pair-bonding behaviors, we also examined the levels of central OT and tyrosine hydroxylase (TH), as well as OT receptor (OTR) and dopamine D1-type and D2-type receptors (D1R and D2R) mRNA expression in the nucleus accumbens (NAcc) and amygdala to investigate the underlying mechanisms. Our results show that mandarin voles providing alloparental care to younger siblings displayed facilitation of partner preference formation, lower levels of OT expression in the paraventricular nucleus of the hypothalamus (PVN) and lateral hypothalamus (LH), and increased OTR and D2R mRNA expression in the NAcc compared to controls. Individuals receiving alloparental care also demonstrated facilitation of partner preference formation in adult voles. Additionally, alloparental care enhanced OT expression in the PVN, anterior medial preoptic nucleus (MPOAa), medial amygdala (MeA), and TH expression in the ventral tegmental area (VTA) and zona incerta (ZI). Furthermore, males displayed decreased D1R mRNA expression in the NAcc, whereas females showed slightly increased D2R expression in the amygdala. These results demonstrate that providing or received alloparental care can promote partner preference formation in monogamous species and that these changes are associated with altered OT and dopamine levels and their receptors in specific brain regions.

Drinking water disinfection byproduct iodoacetic acid affects thyroid hormone synthesis in Nthy-ori 3–1 cells

Iodoacetic acid (IAA) is an emerging and the most genotoxic iodinated disinfection byproduct to date. IAA can disrupt the thyroid endocrine function in vivo and in vitro, but the underlying mechanisms remain unclear. In this work, transcriptome sequencing was used to investigate the effect of IAA on the cellular pathways of human thyroid follicular epithelial cell line Nthy-ori 3–1 and determine the mechanism of IAA on the synthesis and secretion of thyroid hormone (TH) in Nthy-ori 3–1 cells. Results of transcriptome sequencing indicated that IAA affected the TH synthesis pathway in Nthy-ori 3–1 cells. IAA reduced the mRNA expression of thyroid stimulating hormone receptor, sodium iodide symporter, thyroid peroxidase, thyroglobulin, paired box 8 and thyroid transcription factor-2, inhibited the cAMP/PKA pathway and Na+-K+-ATPase, and decreased the iodine intake. The results were confirmed by our previous findings in vivo. Additionally, IAA downregulated glutathione and the mRNA expression of glutathione peroxidase 1, leading to increased reactive oxygen species production. This study is the first to elucidate the mechanisms of IAA on TH synthesis in vitro. The mechanisms are associated with down-regulating the expression of genes related to TH synthesis, inhibiting iodine uptake, and inducing oxidative stress. These findings may improve future health risk assessment of IAA on thyroid in human.

Alterations in gonadotropin, prolactin, androgen and estrogen receptor and steroidogenesis-associated gene expression in gander testes in relation to the annual period

Physiological mechanisms of seasonal changes in testicular function in birds are not fully elucidated. The balance between androgens and estrogens and testis sensitivity for gonadotropin and gonadal steroids are still unclear. The aim of the study was to examine: (1) the changes in circulating and intra-testicular steroid hormone levels and their relationship; (2) the mRNA expression of testicular gonadotropin, prolactin (PRL), progesterone (P4), androgen, and estrogen receptors, and (3) key steroidogenesis processes-related genes with immunofluorescent localization of aromatase in gander testes during the annual period. Testes from ganders (n = 25) in the first reproduction season were obtained at five breeding stages, i.e., prebreeding (PrB), peak of reproduction (PR), postbreeding (PoB), nonbreeding (NB), and onset of reproduction (OR). Males were kept under breeding conditions. It was found that plasma P4 levels decreased at the PoB and NB stages, whereas intra-testicular P4 was the highest in the NB stage. Intra-testicular estradiol (E2) levels were higher at the PoB and NB stages than the other stages, whereas testosterone (T) levels showed a nearly opposite pattern. The plasma estradiol–to–testosterone ratios were higher at the PrB, PoB and NB stages compared to other stages. The transcript abundances for luteinizing hormone receptor (LHR), PRL receptor (PRLR), estrogen receptor alpha (ERα), and estrogen receptor beta (ERβ) also change in testicular tissue during the annual period. Moreover, StAR mRNA expression was upregulated at the PoB and NB stages, and CYP11A1 transcript level was the highest at the PoB stage. Stage-dependent changes in the CYP19A1 mRNA and aromatase protein levels with higher abundances of transcript at PoB and NB stages and protein at the NB stage were observed. Localization and immunofluorescent signal intensity for aromatase also differed in relation to the examined stages. It may be suggested that differential E2 levels, as well as aromatase expression and localization across annual stages are responsible for the seasonal activation/inactivation stages of testis spermatogenesis in domestic ganders. These data strongly suggest a role of aromatase in the control of gander steroidogenesis as changes in this enzyme level are associated with alternation in gonadal steroid hormones. In addition, joint action with others hormones, like PRL and LH, seems to be important in the final effect of seasonal reproduction potential.

D-Chiro-Inositol and Myo-Inositol Induce WAT/BAT Trans-Differentiation in Two Different Human Adipocyte Models (SGBS and LiSa-2).

White adipose tissue/brown adipose tissue trans-differentiation is one of the main study targets for therapies against obesity and metabolic diseases. In recent years, numerous molecules able to induce such trans-differentiation have been identified; however, their effect in obesity therapies has not been as expected. In the present study, we investigated whether myo-inositol and its stereoisomer D-chiro-inositol could be involved in the browning of white adipose tissue. Our preliminary results clearly indicate that both, at 60 μM concentration, induce the upregulation of uncoupling protein 1 mRNA expression, the main brown adipose tissue marker, and increase mitochondrial copy number as well as oxygen consumption ratio. These changes demonstrate an activation of cell metabolism. Therefore, our results show that human differentiated adipocytes (SGBS and LiSa-2), assume the features typical of brown adipose tissue after both treatments. Furthermore, in the cell lines examined, we proved that D-chiro-inositol and myo-Inositol induce an increase in the expression of estrogen receptor mRNAs, suggesting a possible modulation by these isomers. We also found an increase in the mRNA of peroxisome proliferator-activated receptor gamma, a very important target in lipid metabolism and metabolic diseases. Our results open new opportunities for the use of inositols in therapeutic strategies to counteract obesity and its metabolic complications.

NLRP3 mediates the neuroprotective effects of SVHRSP derived from scorpion venom in rotenone-induced experimental Parkinson’s disease model

Ethnopharmacological relevanceIn traditional Chinese medicine, scorpion is used to treat diseases with symptoms such as trembling, convulsion and dementia. Our laboratory employs patented technology to extract and purify the active single component from scorpion venom. We then utilize mass spectrometry to determine the amino acid sequence of the polypeptide and synthesize it artificially to acquire the polypeptide with a purity of 99.3%, named SVHRSP (Scorpion Venom Heat-Resistant Peptide). SVHRSP has been demonstrated to display potent neuroprotective efficacy in Parkinson’s disease. Aim of the studyTo explore the molecular mechanisms and potential molecular targets of SVHRSP-afforded neuroprotection in PD mouse models, as well as to investigate the role of NLRP3 in SVHRSP-mediated neuroprotection. Materials and methodsThe PD mouse model was induced by rotenone and the neuroprotective role of SVHRSP on the PD mouse model was measured using the gait test, rotarod test, the number of dopaminergic neurons, and the activation of microglia. RNA sequencing and GSEA analysis were performed to find the differentially biological pathways regulated by SVHRSP. Primary mid-brain neuron-glial cultures and NLRP3−/− mice were applied to verify the role of NLRP3 by using qRT-PCR, western blotting, enzyme-linked immunosorbent assay (ELISA) and immunostaining. ResultsSVHRSP-afforded dopaminergic neuroprotection was accompanied with inhibition of microglia-mediated neuroinflammatory pathways. Importantly, depletion of microglia markedly reduced the neuroprotective efficacy of SVHRSP against rotenone-induced dopaminergic neurotoxicity in vitro. SVHRSP inhibited microglial NOD-like receptor pathway, mRNA expression and protein level of NLRP3 in rotenone PD mice. SVHRSP also reduced rotenone-induced caspse-1 activation and IL-1β maturation, indicating that SVHRSP mitigated activation of NLRP3 inflammasome. Moreover, inactivation of NLRP3 inflammasome by MCC950 or genetic deletion of NLRP3 almost abolished SVHRSP-afforded anti-inflammatory, neuroprotective effects and improvement of motor performance in response to rotenone. ConclusionsNLRP3 mediated the neuroprotective effects of SVHRSP in rotenone-induced experimental PD model, providing additional evidence for the mechanisms of SVHRSP-afforded anti-inflammatory and neuroprotective effects in PD.

Lambda-cyhalothrin enhances inflammation in nigrostriatal region in rats: Regulatory role of NF-κβ and JAK-STAT signaling

The risk to develop neurobehavioural abnormalities in humans on exposure to lambda-cyhalothrin (LCT) - a type II synthetic pyrethroid has enhanced significantly due to its extensive uses in agriculture, homes, veterinary practices and public health programs. Earlier, we found that the brain dopaminergic system is vulnerable to LCT and affects motor functions in rats. In continuation to this, the present study is focused to unravel the role of neuroinflammation in LCT-induced neurotoxicity in substantia nigra and corpus striatum in rats. Increase in the mRNA expression of proinflammatory cytokines (TNF- α, IL-1β, IL-6) and iNOS whereas decrease in anti-inflammatory cytokine (IL-10) was distinct both in substantia nigra and corpus striatum of rats treated with LCT (0.5, 1.0, 3.0 mg/kg body weight, p.o, for 45 days) as compared to control rats. Further, LCT-treated rats exhibited increased levels of glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule 1 (Iba-1), the glial marker proteins both in substantia nigra and corpus striatum as compared to controls. Exposure of rats to LCT also caused alterations in the levels of heat shock protein 60 (HSP60) and mRNA expression of toll-like receptors (TLR2 and TLR4) in the substantia nigra and corpus striatum. An increase in the phosphorylation of key proteins involved in NF-kβ (P65, Iκβ, IKKα, IKKβ) and JAK/STAT (STAT1, STAT3) signaling and alteration in the protein levels of JAK1 and JAK2 was prominent in LCT-treated rats. Histological studies revealed damage of dopaminergic neurons and reactive gliosis as evidenced by the presence of darkly stained pyknotic neurons and decrease in Nissl substance and an increase in infiltration of immune cells both in substantia nigra and corpus striatum of LCT-treated rats. Presence of reactive microglia and astrocytes in LCT-treated rats was also distinct in ultrastructural studies. The results exhibit that LCT may damage dopaminergic neurons in the substantia nigra and corpus striatum by inducing inflammation as a result of stimulation of neuroglial cells involving activation of NF-κβ and JAK/STAT signaling.

Rhoifolin protects cecal ligation and puncture induced sepsis mice model by regulating inflammatory pathway

Sepsis is a systemic infection affects several organs, which needs novel therapy for the management of it, thus protective effect of Rhoifolin was estimated against sepsis. Cecal ligation and puncture (CLP) method was used to induce sepsis and thereafter mice were treated with rhoifolin (20 and 40 mg/kg, i.p.) for one week. Food intake and survival rate was determined sepsis mice, moreover liver function test and cytokines was estimated in the serum of sepsis mice. In the lung tissue homogenate, oxidative stress parameters were determined, histopathological analysis was performed in liver and lung tissue of sepsis mice. Food intake and percentage of survival was improved in rhoifolin treated group than sham group. Level of liver function enzyme and cytokine was reduced significantly in the serum of rhoifolin treated sepsis mice. Treatment with rhoifolin ameliorates the altered oxidative stress parameters, and mRNA expression of Toll-like receptor 4 (TLR-4) in lung tissue of sepsis mice. Histopathological changes were also reverse in rhoifolin treated group than sham group of mice. In conclusion, result of report indicates Rhoifolin treatment reduces oxidative stress and inflammation in CLP induced sepsis mice, as it regulates TLR4/MyD88/NF-κB pathway.

A Longitudinal Study of the Relationship of Adiponectin with Reproduction in Infertile Women Undergoing IVF/ICSI Treatment, and an Experimental Study in Human Granulosa Cells.

This study investigated the roles of adiponectin in IVF treatment during Phase I (the basal stage before gonadotropin administration), Phase II (approximately 8 days after gonadotropin administration), and Phase III (on the ovum pick-up day), as well as the effects of adiponectin on CYP19A1 and the FSH receptor (FSHR) mRNA expression in a human granulosa-like tumor cell line (KGN). In human subjects (a longitudinal study, n = 30), blood samples were collected in all phases, while follicular fluid (FF) was only collected in Phase III. The participants were classified into successful and unsuccessful groups based on the determination of fetal heartbeats. KGN cells were treated with adiponectin/FSH/IGF-1 (an experimental study, n = 3). There was no difference in the adiponectin levels between successful and unsuccessful pregnancies in the FF (Phase III) and in serum (all phases), as well as among the three phases in both groups. Serum FSH (Phase I) was positively associated with serum adiponectin in the unsuccessful group, but it had a negative association in the successful group (all phases). Serum adiponectin and serum FSH (Phase I) were positively correlated in the unsuccessful group, whereas they were negatively correlated (all phases) in the successful group. The serum adiponectin levels (Phase III) were significantly higher than in the FF in unsuccessful pregnancies, but there was no difference in successful pregnancies. FF adiponectin concentrations were negatively correlated with serum LH in successful subjects. In KGN cells, adiponectin had no influence on CYP19A1 and FSHR mRNA expression. High adiponectin levels in serum compared to FF (Phase III) in unsuccessful subjects might negatively impact IVF treatment.

Chemical compositions and sleep-promoting activities of hop (Humulus lupulus L.) varieties.

Among natural products with sleep-promoting activity, hops have been used since ancient times as a tranquilizer and hypnotic agent. This study investigated the sleep-promoting effects of extracts of various hop (Humulus lupulus L.) varieties in invertebrate and vertebrate models. The content of α-acids, β-acids, and xanthohumol was higher in hop 70% alcohol extracts than in hop hot water extracts. Among the alcohol extracts, Citra contained a high α-acid content (229.32µg/mg), while Saphir showed high β-acid and xanthohumol content (66.37 and 4.23µg/mg, respectively). In Drosophila melanogaster, Simcoe and Mosaic water extracts and Saphir and Simcoe alcohol extracts significantly increased total nighttime sleep. Total sleep time of mice with pentobarbital-induced sleep was significantly increased by Simcoe and Mosaic water extracts and Saphir and Simcoe ethanol extracts compared to the normal group. Oral administration of Simcoe water extract and Saphir alcohol extract improved sleep in the caffeine-induced insomnia model and upregulated the mRNA expression of GABAA (gamma 2 subunit) and GABAB receptors in mouse brains. Additionally, Saphir alcohol extract significantly increased the GABA content in mouse brains. Simcoe water extract and Saphir ethanol extract modulated GABAergic signaling to improve sleep-related behaviors, including sleep duration.

Abstract 2358: NR0B2 re-educates myeloid cells within the tumor microenvironment: Potential novel strategy for breast cancer immunotherapy

Abstract Breast cancer remains the second leading cause of cancer-related deaths among women. In recent years, immunotherapy has been tremendously successful in some metastatic cancers such as melanoma. However, a majority of breast cancer patients do not benefit from existing immunotherapy treatments, leaving many with an unmet need. Although undoubtedly multifactorial, one major obstacle to anti-cancer therapies, is the highly immunosuppressive breast tumor microenvironment. This phenomenon is strongly maintained by myeloid immune cells and immunosuppressive regulatory T cells (Tregs), which hinder anti-tumor immunosurveillance and promote tumor progression. Thus, strategies to ‘re-educate’ myeloid cells to inhibit Tregs is a potentially promising anti-cancer strategy. Mining clinical data, we have found that elevated mRNA expression of the nuclear receptor, NR0B2 within breast tumors is associated with an increased time to recurrence. Single cell RNA-sequencing indicates that NR0B2 is expressed within the macrophage populations of normal breast tissue, and various dendritic cell (DC) types in PBMCs. Overexpression of NR0B2 or activation with a small molecule agonist in murine bone marrow derived macrophages (BMDMs) or DCs resulted in a dichotomous T cell expansion - away from Tregs. Conversely, Treg expansion increased when NR0B2 was knocked-down. Tumor growth was markedly increased in mice lacking myeloid specific NR0B2 expression. We further investigated the downstream targets of NR0B2 mediating this anti-tumor phenotype and identified that NLRP3 inflammasome-IL1β activity is a likely modulator in re-educating myeloid cell-Treg function. Importantly, a putative small molecule agonist decreased established metastatic lesions and increased the efficacy of αPD-L1. Subsequent medicinal chemistry was used to develop a novel NR0B2 agonist with strong anti-metastatic properties when used as a single agent in a preclinical mouse model. Collectively, our data implicates NR0B2 within myeloid cells as a modulator of Tregs, a cell population that has thus far been therapeutically intractable. Therefore, NR0B2 may prove to be a promising therapeutic target to reshape the tumor microenvironment and improve breast cancer immunotherapy. This work was supported by the Era of Hope Scholar Award from the Department of Defense Breast Cancer Research Program grant (BC200206), National Cancer Institute (R01CA234025), and NIH Chemistry-Biology Interface Training Grant (T32-GM136629). Citation Format: Hashni Epa Vidana Gamage, Sayyed Hamed Shahoei, Tiffany Nguyen, Rachel Farmer, Samuel Albright, Erin Weisser, Rafael O. Bautista, Claire P. Schane, Yu Wang, Adam Nelczyk, Liqian Ma, Srishti Tiwari, Anasuya Das Gupta, Shruti Bendre, Lionel Apetoh, Paul J. Hergenrother, Erik R. Nelson. NR0B2 re-educates myeloid cells within the tumor microenvironment: Potential novel strategy for breast cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2358.