Integrating network pharmacology and transcriptomic omics reveals that akebia saponin D attenuates neutrophil extracellular traps-induced neuroinflammation via NTSR1/PKAc/PAD4 pathway after intracerebral hemorrhage.

Neutrophils and their production of neutrophil extracellular traps (NETs) significantly contribute to neuroinflammation and brain damage after intracerebral hemorrhage (ICH). Although Akebia saponin D (ASD) demonstrates strong anti-inflammatory activities and blood-brain barrier permeability, its role in regulating NETs formation and neuroinflammation following ICH is uncharted. Our research focused on unraveling the influence of ASD on neuroinflammation mediated by NETs and the mechanisms involved. We found that increased levels of peripheral blood neutrophils post-ICH are correlated with worse prognostic outcomes. Through network pharmacology, we identified ASD as a promising therapeutic target for ICH. ASD administration significantly improved neurobehavioral performance and decreased NETs production in neutrophils. Furthermore, ASD was shown to upregulate the membrane protein NTSR1 and activate the cAMP signaling pathway, confirmed through transcriptome sequencing, western blot, and immunofluorescence. Interestingly, the NTSR1 inhibitor SR48692 significantly nullified ASD's anti-NETs effects and dampened cAMP pathway activation. Mechanistically, suppression of PKAc via H89 negated ASD's anti-NETs effects but did not affect NTSR1. Our study suggests that ASD may reduce NETs formation and neuroinflammation, potentially involving the NTSR1/PKAc/PAD4 pathway post-ICH, underlining the potential of ASD in mitigating neuroinflammation through its anti-NETs properties.

Influence of formaldehyde exposure on the molecules of the NO/cGMP-cAMP signaling pathway in different brain regions of Balb/c mice.

This toxicology study was conducted to assess the impact of formaldehyde, a common air pollutant found in Chinese gymnasiums, on the brain function of athletes. In this research, a total of 24 Balb/c male mice of SPF-grade were divided into four groups, each consisting of six mice. The mice were exposed to formaldehyde at different concentrations, including 0 mg/m3, 0.5 mg/m3, 3.0 mg/m3, and 3.0 mg/m3 in combination with an injection of L-NMMA (NG-monomethyl-L-arginine), which is a nitric oxide synthase antagonist. Following a one-week test period (8 h per day, over 7 days), measurements of biomarkers related to the nitric oxide (NO)/cGMP-cAMP signaling pathway were carried out on the experimental animals post-treatment. The study found that: (1) Exposure to formaldehyde can lead to brain cell apoptosis and neurotoxicity; (2) Additionally, formaldehyde exposure was found to alter the biomarkers of the NO/cGMP-cAMP signaling pathway, with some changes being statistically significant (p < 0.05 or p < 0.01); (3) The use of L-NMMA, an antagonist of the NO/cGMP-cAMP signaling pathway, was found to prevent these biomarker changes and had a protective effect on brain cells. The study suggests that the negative impact of formaldehyde on the brain function of mice is linked to the regulation of the NO/cGMP-cAMP signaling pathway.

The N-Linked Glycosylation Site N191 Is Necessary for PKA Signal Transduction in Eel Follicle-Stimulating Hormone Receptor.

The follicle-stimulating hormone receptor (FSHR) contains several N-linked glycosylation sites in its extracellular region. We conducted the present study to determine whether conserved glycosylated sites in eel FSHR are necessary for cyclic adenosine monophosphate (cAMP) signal transduction. We used site-directed mutagenesis to induce four mutations (N120Q, N191Q, N272Q, and N288Q) in the N-linked glycosylation sites of eel FSHR. In the eel FSHR wild-type (wt), the cAMP response was gradually increased in a dose-dependent manner (0.01-1500 ng/mL), displaying a high response (approximately 57.5 nM/104 cells) at the Rmax level. Three mutants (N120Q, N272Q, and N288Q) showed a considerably decreased signal transduction as a result of high-ligand treatment, whereas one mutant (N191Q) exhibited a completely impaired signal transduction. The expression level of the N191Q mutant was only 9.2% relative to that of the eel FSHR-wt, indicating a negligible expression level. The expression levels of the N120Q and N272Q mutants were approximately 35.9% and 24% of the FSHG-wt, respectively. The N288Q mutant had an expression level similar to that of the eel FSHR-wt, despite the mostly impaired cAMP responsiveness. The loss of the cell surface agonist-receptor complexes was very rapid in the cells expressing eel FSHR-wt and FSHR-N288Q mutants. Specifically, the N191Q mutant was completely impaired by the loss of cell surface receptors, despite treatment with a high concentration of the agonist. Therefore, we suggest that the N191 site is necessary for cAMP signal transduction. This finding implies that the cAMP response, mediated by G proteins, is directly related to the loss of cell surface receptors as a result of high-agonist treatment.

microRNA-454-mediated NEDD4-2/TrkA/cAMP axis in heart failure: Mechanisms and cardioprotective implications.

The current study aimed to investigate the mechanism by which miR-454 influences the progression of heart failure (HF) in relation to the neural precursor cell expressed, developmentally downregulated 4-2 (NEDD4-2)/tropomyosin receptor kinase A (TrkA)/cyclic adenosine 3',5'-monophosphate (cAMP) axis. Sprague-Dawley rats were used to establish a HF animal model via ligation of the left anterior descending branch of the coronary artery. The cardiomyocyte H9c2 cells were treated with H2 O2 to stimulate oxidative stress injury in vitro. RT-qPCR and Western blot assay were subsequently performed to determine the expression patterns of miR-454, NEDD4-2, TrkA, apoptosis-related proteins and cAMP pathway markers. Dual-luciferase reporter gene assay coupled with co-immunoprecipitation was performed to elucidate the relationship between miR-454, NEDD4-2 and TrkA. Gain- or loss-of-function experiments as well as rescue experiments were conducted via transient transfection (in vitro) and adenovirus infection (in vivo) to examine their respective functions on H9c2 cell apoptosis and myocardial damage. Our results suggested that miR-454 was aberrantly downregulated in the context of HF, while evidence was obtained suggesting that it targeted NEDD4-2 to downregulate NEDD4-2 in cardiomyocytes. miR-454 exerted anti-apoptotic and protective effects on cardiomyocytes through inhibition of NEDD4-2, while NEDD4-2 stimulated ubiquitination and degradation of TrkA protein. Furthermore, miR-454 activated the cAMP pathway via the NEDD4-2/TrkA axis, which ultimately suppressed cardiomyocyte apoptosis and attenuated myocardial damage. Taken together, the key findings of the current study highlight the cardioprotective role of miR-454, which is achieved through activation of the cAMP pathway by impairing NEDD4-2-induced TrkA ubiquitination.

Effects of sorafenib and an adenylyl cyclase activator on in vitro growth of well-differentiated thyroid cancer cells.

Well-differentiated thyroid carcinomas have driver mutations involving growth factor receptor-tyrosine kinases (RTKs) or their intracellular signaling pathway, that is, the mitogen-activated protein kinase (MAPK) pathway. Sorafenib is a multikinase inhibitor of RTKs and the MAPK pathway and has recently been used for the treatment of unresectable well-differentiated thyroid carcinoma. In normal thyroid follicular cells, stimulation of the thyroid-stimulating hormone (TSH) receptor activates the cyclic adenosine monophosphate (cAMP) pathway and promotes cell growth as well as hormonal secretion. However, an adenylyl cyclase (AC) activator, forskolin, has been reported to suppress the growth of thyroid carcinoma cells. To clarify the roles of the MAPK and cAMP pathways in proliferation of well-differentiated thyroid carcinoma cells, we compared the effects of sorafenib and forskolin in in vitro models. Sorafenib inhibited constitutive activation of the MAPK pathway, cyclin-dependent kinase 4 (CDK4), and phosphorylated retinoblastoma protein (RB) in 3 well-differentiated carcinoma cell lines, but it did not show sufficiently effective suppression of cell growth. Forskolin significantly suppressed the growth of all 3 cell lines and also activated the cAMP pathway and inhibited expression of cyclin D1. Our results suggest that activation of the cAMP pathway could be more potent than activation of the MAPK pathway in suppressing proliferation of well-differentiated thyroid cancer cells. We postulate that the AC activator suppresses growth of thyroid carcinoma cells through undetermined mechanisms.

The human CFTR protein expressed in CHO cells activates aquaporin-3 in a cAMP-dependent pathway: study by digital holographic microscopy.

The transmembrane water movements during cellular processes and their relationship to ionic channel activity remain largely unknown. As an example, in epithelial cells it was proposed that the movement of water could be directly linked to cystic fibrosis transmembrane conductance regulator (CFTR) protein activity through a cAMP-stimulated aqueous pore, or be dependent on aquaporin. Here, we used digital holographic microscopy (DHM) an interferometric technique to quantify in situ the transmembrane water fluxes during the activity of the epithelial chloride channel, CFTR, measured by patch-clamp and iodide efflux techniques. We showed that the water transport measured by DHM is fully inhibited by the selective CFTR blocker CFTRinh172 and is absent in cells lacking CFTR. Of note, in cells expressing the mutated version of CFTR (F508del-CFTR), which mimics the most common genetic alteration encountered in cystic fibrosis, we also show that the water movement is profoundly altered but restored by pharmacological manipulation of F508del-CFTR-defective trafficking. Importantly, whereas activation of this endogenous water channel required a cAMP-dependent stimulation of CFTR, activation of CFTR or F508del-CFTR by two cAMP-independent CFTR activators, genistein and MPB91, failed to trigger water movements. Finally, using a specific small-interfering RNA against the endogenous aquaporin AQP3, the water transport accompanying CFTR activity decreased. We conclude that water fluxes accompanying CFTR activity are linked to AQP3 but not to a cAMP-stimulated aqueous pore in the CFTR protein.

Role of Embinin in the reabsorption of nucleus pulposus in lumbar disc herniation: Promotion of nucleus pulposus neovascularization and apoptosis of nucleus pulposus cells.

Reabsorption of the nucleus pulposus (NP) in lumbar disc herniation (LDH) refers to the natural absorption or even complete disappearance of LDH. In order to better treat LDH, it is necessary to further study its mechanism and develop new therapeutic drugs. Clematidis Radix Et Rhizoma is a ranunculus family plant which has multiple biological activities, and Embinin is one of its bioactive ingredients. However, its effects on LDH were unclear. In this study, the role of Embinin was investigated in LDH rat models. LDH model was established by lumbar epidural insertion of tail disc. Our results showed that Embinin promoted lumbar disc neovascularization, induced apoptosis of NP cells in LDH rats, and promoted lumbar disc resorption. Furthermore, mechanistic study showed that Embinin activated the cAMP pathway in the rat models. In conclusion, Embinin has the potential to serve as a drug for the treatment of LDH.

1.8-cineole prevents platelet activation and aggregation by activating the cAMP pathway via the adenosine A2A receptor.

AIMS: Dysregulated platelet aggregation is a fatal condition in many bacterial- and virus-induced diseases. However, classical antithrombotics cannot completely prevent immunothrombosis, due to the unaddressed mechanisms towards inflammation. Thus, targeting platelet hyperactivation together with inflammation might provide new treatment options in diseases, characterized by immunothrombosis, such as COVID-19 and sepsis. The aim of this study was to investigate the antiaggregatory effect and mode of action of 1.8-cineole, a monoterpene derived from the essential oil of eucalyptus leaves, known for its anti-inflammatory proprieties. MAIN METHODS: Platelet activity was monitored by measuring the expression and release of platelet activation markers, i.e., P-selectin, CD63 and CCL5, as well as platelet aggregation, upon treatment with 1.8-cineole and stimulation with several classical stimuli and bacteria. A kinase activity assay was used to elucidate the mode of action, followed by a detailed analysis of the involvement of the adenylyl-cyclase (AC)-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway by Western blot and ELISA. KEY FINDINGS: 1.8-cineole prevented the expression and release of platelet activation markers, as well as platelet aggregation, upon induction of aggregation with classical stimuli and immunological agonists. Mechanistically, 1.8- cineole influences the activation of the AC-cAMP-PKA pathway, leading to higher cAMP levels and vasodilator-stimulated phosphoprotein (VASP) phosphorylation. Finally, blocking the adenosine A2A receptor reversed the antithrombotic effect of 1.8-cineole. SIGNIFICANCE: Given the recognized anti-inflammatory attributes of 1.8-cineole, coupled with our findings, 1.8-cineole might emerge as a promising candidate for treating conditions marked by platelet activation and abnormal inflammation.

Temporal and Spatial Gene Expression Profile of Stroke Recovery Genes in Mice.

Stroke patients show some degree of spontaneous functional recovery, but this is not sufficient to prevent long-term disability. One promising approach is to characterize the dynamics of stroke recovery genes in the lesion and distant areas. We induced sensorimotor cortex lesions in adult C57BL/6J mice using photothrombosis and performed qPCR on selected brain areas at 14, 28, and 56 days post-stroke (P14-56). Based on the grid walk and rotating beam test, the mice were classified into two groups. The expression of cAMP pathway genes Adora2a, Pde10a, and Drd2, was higher in poor- compared to well-recovered mice in contralesional primary motor cortex (cl-MOp) at P14&56 and cl-thalamus (cl-TH), but lower in cl-striatum (cl-Str) at P14 and cl-primary somatosensory cortex (cl-SSp) at P28. Plasticity and axonal sprouting genes, Lingo1 and BDNF, were decreased in cl-MOp at P14 and cl-Str at P28 and increased in cl-SSp at P28 and cl-Str at P14, respectively. In the cl-TH, Lingo1 was increased, and BDNF decreased at P14. Atrx, also involved in axonal sprouting, was only increased in poor-recovered mice in cl-MOp at P28. The results underline the gene expression dynamics and spatial variability and challenge existing theories of restricted neural plasticity.

(20R)-panaxadiol improves obesity by promoting white fat beigeing.

Introduction: Obesity is an important cause of a range of metabolic diseases. However, the complex mechanisms of obesity and its related diseases make some weight loss methods ineffective or have safety issues. Ginseng, a specialty of Jilin Province in China with both edible and medicinal value, contains mainly ginsenosides and other components. In order to study the anti-obesity effect of ginseng, network pharmacology was used to predict and screen the active ingredients, action targets and signaling pathways of ginseng. We found (20R)-panaxadiol (PD) is a more desirable active ingredient due to its high drug-like properties and high bioavailability. Moreover, it is closely related to cAMP pathway which is more important in metabolism regulation. The corresponding pharmacodynamic targets of PD include ADRB2 (the gene encoding the ß2-adrenoceptor receptor). Our study aimed to investigate whether Panaxadiol can promote white adipocyte beigeing and increase thermogenesis through modulating the ß2/cAMP pathway to exert anti-obesity effects. Methods: In vivo, we established high-fat feeding obesity model, genotypically obese mice (ob/ob) model, and administered PD (10 mg/kg). PD treatment in ob/ob mice along with ß2 receptor inhibitor ICI118551. In vitro, differentiated mature 3T3-L1 cells were given palmitate (PA) to induce hypertrophy model along with PD (20 µM). Results: The results of this study demonstrated that PD significantly reduced body weight, improved glucose tolerance and lipid levels in high-fat-induced obese mice and ob/ob mice, and also reduced lipid droplet size in PA-treated hypertrophic adipocytes in vitro. Molecular biology assays confirmed that cAMP response element binding protein (CREB) phosphorylation was increased after PD administration, and the expression of thermogenesis-related proteins UCP1, PRDM16 and mitochondrial biosynthesis-related proteins PGC-1α, TFAM and NRF1 were increased. Molecular docking results showed a low binding energy between ß2 receptors and PD, indicating an affinity between the ß2 receptor and PD. In addition, the ß2 receptor inhibition, reversed the anti-obesity effect of PD on the body weight, lipid droplets, the expression of thermogenesis-related proteins and CREB phosphorylation in ob/ob mice. Discussion: These results suggest that PD may promote the expression of thermogenic proteins through phosphorylation of CREB via ß2 receptor activation, and thus exert anti-obesity effects.

Gut microbiota and metabolic profiles in chronic intermittent hypoxia-induced rats: disease-associated dysbiosis and metabolic disturbances.

Aim: Chronic intermittent hypoxia (CIH) is a key characteristic of obstructive sleep apnea (OSA) syndrome, a chronic respiratory disorder. The mechanisms of CIH-induced metabolic disturbance and histopathological damage remain unclear. Methods: CIH-induced rats underwent daily 8-h CIH, characterized by oxygen levels decreasing from 21% to 8.5% over 4 min, remaining for 2 min, and quickly returning to 21% for 1 min. The control rats received a continuous 21% oxygen supply. The levels of hypersensitive C reactive protein (h-CRP), tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), interleukin 8 (IL-8), and nuclear factor kappa-B (NF-κB) were measured by ELISA. Histological analysis of the soft palates was conducted using HE staining. The microbial profiling of fecal samples was carried out by Accu16STM assay. Untargeted metabolomics of serum and soft palate tissue samples were analyzed by UPLC-MS. The protein expression of cAMP-related pathways in the soft palate was determined by Western blot. Results: After 28 h of CIH induction, a significant increase in pro-inflammatory cytokines was observed in the serum, along with mucosal layer thickening and soft palate tissue hypertrophy. CIH induction altered the diversity and composition of fecal microbiota, specifically reducing beneficial bacteria while increasing harmful bacteria/opportunistic pathogens. Notably, CIH induction led to a significant enrichment of genera such as Dorea, Oscillibacter, Enteractinococcus, Paenibacillus, Globicatella, and Flaviflexus genera. Meanwhile, Additionally, CIH induction had a notable impact on 108 serum marker metabolites. These marker metabolites, primarily involving amino acids, organic acids, and a limited number of flavonoids or sterols, were associated with protein transport, digestion and absorption, amino acid synthesis and metabolism, as well as cancer development. Furthermore, these differential serum metabolites significantly affected 175 differential metabolites in soft palate tissue, mainly related to cancer development, signaling pathways, amino acid metabolism, nucleotide precursor or intermediate metabolism, respiratory processes, and disease. Importantly, CIH induction could significantly affect the expression of the cAMP pathway in soft palate tissue. Conclusions: Our findings suggest that targeting differential metabolites in serum and soft palate tissue may represent a new approach to clinical intervention and treatment of OSA simulated by the CIH.

Arginine Promotes the Expression of Aquaporin-3 and Water Transport in Porcine Trophectoderm Cells Through NO- and cAMP-Dependent Mechanisms.

BACKGROUND: Dietary supplementation with L-arginine (Arg) has been shown to increase the volume of fetal fluids in gestating swine. Aquaporins (AQPs), known as water channel proteins, are essential for embryonic growth and development. It was not known if Arg mediates water transport through AQPs in porcine conceptus trophectoderm (pTr2) cells. METHODS: pTr2 cells derived from pregnant gilts on day 12 of gestation were cultured in customized Arg-free Dulbecco's modified Eagle's Ham medium (DMEM) supplemented with either 0.00, 0.25, or 0.50 mM Arg. RESULTS: Arg treatment increased water transport and the expression of AQP3, which was abundantly expressed in pTr2 cells at both the mRNA and protein levels. Arg also increased the expression of iNOS and the synthesis of nitric oxide (NO) in pTr2 cells. The presence of Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME; an inhibitor of NO synthase) significantly attenuated the Arg-induced expression of AQP3. Furthermore, 0.50 mM Arg increased the concentrations of cAMP and the abundances of phosphorylated cAMP-dependent protein kinase A (PKA), phosphorylated PKA α/ß/γ, and phosphorylated CREB. These effects of Arg were mimicked by Forskolin (a cell-permeable activator of adenylyl cyclase), but inhibited by H-89 (an inhibitor of cAMP-dependent protein kinase). CONCLUSIONS: The results of this study demonstrate that Arg regulates AQP3 expression and promotes water transport in pTr2 cells through NO- and cAMP-dependent signaling pathways.

Endocytic protein Pal1 regulates appressorium formation and is required for full virulence of Magnaporthe oryzae.

Endocytosis plays key roles during infection of plant-pathogenic fungi, but its regulatory mechanisms are still largely unknown. Here, we identified a putative endocytosis-related gene, PAL1, which was highly expressed in appressorium of Magnaporthe oryzae, and was found to be important for appressorium formation and maturation. Deletion of PAL1 significantly reduced the virulence of M. oryzae due to defects in appressorial penetration and invasive growth in host cells. The Pal1 protein interacted and colocalized with the endocytosis protein Sla1, suggesting it is involved in endocytosis. The Δpal1 mutant was significantly reduced in appressorium formation, which was recovered by adding exogenous cAMP and 3-isobutyl-1-methylxanthine (IBMX). Moreover, the phosphorylation level of Pmk1 in Δpal1 was also reduced, suggesting Pal1 functions upstream of both the cAMP and Pmk1 signalling pathways. As a consequence, the utilization of glycogen and lipid, appressorial autophagy, actin ring formation, localization of septin proteins, as well as turgor accumulation were all affected in the Δpal1 mutant. Taken together, Pal1 regulates cAMP and the Pmk1 signalling pathway for appressorium formation and maturation to facilitate infection of M. oryzae.

The impact of different feeds on DNA methylation, glycolysis/gluconeogenesis signaling pathway, and gene expression of sheep muscle.

DNA methylation is an important epigenetic regulatory form that regulates gene expression and tissue development. This study compared the effects of high fiber, low protein (HFLP) and low fiber, high protein (LFHP) diets on the DNA methylation profile of twin lambs' muscles, their effect on glycolysis/gluconeogenesis and related pathways by transcriptome and deep whole-genome bisulfite sequencing (WGBS). Results identified 1,945 differentially methylated regions (DMRs) and 1,471 differentially methylated genes (DMGs). Also, 487 differentially expressed transcripts belonging to 368 differentially expressed genes (DEGs) were discovered between the twin lambs under different diets. Eleven overlapped genes were detected between the DEGs and the DMGs. FKBP5 and FOXO1 were detected to be significantly different. The FOXO1 regulated cAMP and the glycolysis/gluconeogenesis pathways. The glycolysis/gluconeogenesis, and the FOXO pathways were significantly enriched. The expressions of HOMER1 and FOXO1 in the HFLP group were significantly higher than those in the LFHP group. There is a significant correlation between the upregulated gene expression and hypomethylation of HOMER1 and FOXO1 gene in HFLP group. The results showed that FOXO1 induces PDK4 expression in muscle while regulating FKBP5 activity, which stimulates glucose production by activating specific gluconeogenesis target genes. The FOXO1 was able to regulate the glucose metabolism, the cAMP and the occurrence of glycolysis/gluconeogenesis pathways. This study showed that feed type can affect the methylation levels of the glycolysis related gluconeogenesis genes and interaction pathways, providing new ideas for a better understanding of the regulation of muscle energy metabolism and feed development.

Antifungal Mechanisms of a Chinese Herbal Medicine, Cao Huang Gui Xiang, Against Candida Species.

Cao Huang Gui Xiang (CHGX) formula, a Chinese herbal medicine, has been empirically used for the treatment of Candida infections. In the present study, we discovered that the CHGX showed potent antifungal activities against the major human fungal pathogen Candida albicans and other clinical Candida species. Besides, we indicated that CHGX had in vivo efficacy on treating C. albicans infection in mice without noticeable toxicity at the clinical therapeutic concentration. We then set out to investigate the antifungal mechanisms of CHGX against C. albicans. We found that CHGX played an important role in inhibiting biofilm formation and filament development, two critical virulence factors of C. albicans. We further demonstrated that CHGX disrupted cell membrane integrity, triggered the accumulation of reactive oxygen species (ROS) and consumption of adenosine triphosphate (ATP), followed by a rapid fungal cell death in C. albicans. Multiple pathways, including the conserved Ras1-cAMP pathway and mitochondrial protein Mcu1 are involved in CHGX-induced cell death. Our finding expands the understanding of antifungal mechanism of CHGX against C. albicans, and provides new insights in treating patients with Candida infections in clinical practice.

Corrigendum: Antifungal mechanisms of a Chinese herbal medicine, Cao Huang Gui Xiang, against Candida species.

[This corrects the article DOI: 10.3389/fphar.2022.813818.].

Dual Inhibition of Ornithine Decarboxylase and A1 Adenosine Receptor Efficiently Suppresses Breast Tumor Cells.

Personized treatment of breast cancer is still a challenge, and more treatment options for breast cancer are warranted. Combination therapies have been a highly appreciated strategy for breast cancer treatment in recent years, and the development of new combination therapies could improve patient outcomes. Adenosine and polyamines are both endogenous metabolites with indispensable biological functions. Adenosine binds with the A1 adenosine receptor (A1AR) to downregulate cAMP concentration, and both low cAMP content and high polyamine levels stimulate the growth and proliferation of cancer cells. In this work, we initially used a polyamine synthesis inhibitor, DFMO (α-difluoromethylornithine), and an A1AR inhibitor, DPCPX (8-cyclopentyl-1,3-dipropylxanthine) to investigate if simultaneously inhibiting A1AR and polyamine synthesis has synergistical antitumor effects. Next, we investigated a dual inhibitor (ODC-MPI-2) of A1AR and ODC (ornithine decarboxylase 1), the rate-limiting enzyme in polyamine biosynthesis. We investigated if ODC-MPI-2 could inhibit the proliferation and growth of breast cancer cells. Our data showed that DFMO and DPCPX synergistically inhibit the growth and proliferation of MCF-7 cells. We also demonstrated that ODC-MPI-2 reduces cellular polyamine levels and elevates cAMP concentration. We further showed that ODC-MPI-2 inhibits the growth, proliferation, and migration/invasion of MCF-7 cells. Finally, ODC-MPI-2 showed a preference for inhibiting triple-negative breast cancer cells. The dual inhibition of ODC and A1AR is a new combination therapy strategy for treating breast cancer, and dual inhibitors of ODC and A1AR may be effective future drugs for treating breast cancer.

GPx6 is involved in the in vitro induced capacitation and acrosome reaction in porcine sperm.

Glutathione peroxidases (GPxs) are regarded as important protectors against oxidative stress. Some members of this protein family were reported to play key roles in protecting sperm against oxidative stress. Whether GPx6 a member of the GPx family also plays a role in protection against oxidative stress is not known to date. The objective of the present study was to evaluate the localization and function of glutathione peroxidase 6 (GPx6) in boar accessory sex glands, seminal plasma, and sperm, as well as the effect of GPx6 on vitality and capacitation in boar sperm. qPCR and Western blot analysis demonstrated the presence of GPx6 in testis, epididymis, bulbourethral glands, prostate, seminal vesicle, sperm and seminal plasma. Incubation of sperm with an GPx6 antibody had no significant effect on the viability of boar sperm prior to capacitation. Surprisingly, when capacitated sperm was incubated with the GPx6 antibody for 240 min, sperm vitality was significantly improved. Western blotting showed that in capacitated sperm without prior pretreatment, GPx6 protein content was reduced compared to sperm before capacitation. To further confirm a role for GPx6 in sperm capacitation, we tested sperm acrosome reaction by ACR.2 and FITC-PSA. The results showed that treatment of sperm with the GPx6 antibody significantly increased sperm capacitation and acrosome reaction. Furthermore, we examined the concentration of cAMP in sperm after capacitation. ELISA demonstrated that the cAMP concentration in the sperm exposed to the GPx6 antibody was significantly higher than that of the control group. In addition, the exposure of sperm to the GPx6 antibody significantly increased the concentration of H2O2, while the expression of SOD3 and CAT were decreased. Based on these observations we would like to postulate that in the boar reproductive tract the GPx6 protein becomes attached to the sperm head preventing the sperm to undergo premature capacitation by affecting components of the antioxidant pathway. How GPx6 expression following ejaculation becomes suppressed to allow sperm capacitation to take place needs further investigation.

GPR64 promotes cAMP pathway in tumor aggressiveness in sparsely granulated growth hormone cell adenomas.

PURPOSE: There is an increasing agreement that acromegaly caused by growth hormone (GH) cell adenoma has two distinct subtypes: densely granulated (DG) and sparsely granulated (SG). We hypothesized that differential molecular signatures may explain their behavior. METHODS: Total transcriptome sequencing was performed on ten DG and seven SG adenomas. The differentially expressed RNAs were identified by bioinformatic analyses, and a candidate RNA was verified by quantitative real-time PCR. Immunohistochemical staining was also performed to detect the protein expression of the candidate. Clinical parameters were correlated with protein expression. Subsequently, cell proliferation, colony formation, and cell cycle progression were analyzed after knockdown of the candidate in pituitary GH3 cells. Activation of the cAMP pathway was assessed by ELISA and Western blot. RESULTS: We confirmed that there were obvious differentially expressed genes between the subtypes. Through gene profiling, we discovered that an orphan adhesion G protein-coupled receptor, GPR64, was overexpressed in more aggressive SG adenomas. Noticeably, GPR64 knockdown significantly inhibited the proliferation of GH3 tumor cells and decreased colony formation. The knockdown also induced cell cycle arrest in GH3 tumor cells. Further studies revealed that GPR64 knockdown decreased cAMP levels and the ratios of p-CREB/CREB, indicating that it suppressed the cAMP/CREB pathway. CONCLUSIONS: Our results indicated that GPR64 may promote aggressiveness in SG-type GH cell adenomas and that it is a key factor regulating the cAMP pathway to promote aggressiveness of GH cell adenomas.

Rosiglitazone ameliorates palmitic acid-induced endoplasmic reticulum stress and steroidogenic capacity in granulosa cells.

Granulosa cells play essential roles in follicular development, oocyte maturation and sex hormone secretion. The exposure of granulosa cells to palmitic acid (PA), the main component of dietary saturated fat, inhibits cell viability. However, the mechanism underlying PA-induced cytotoxicity in granulosa cells has not been deeply investigated. Rosiglitazone (RSG) is a member of the thiazolidinedione family and is reported to protect cells from cytotoxicity and endoplasmic reticulum (ER) stress in other cell types, but whether RSG protects granulosa cells remain unknown. In this study, KGN cell line and primary granulosa cells were used as models of granulosa cells to explore the effects of PA and RSG and the underlying mechanisms. The results showed that PA inhibits cell viability and estradiol secretion through inducing ER stress and cAMP/PKA/CREB pathway. CCAAT/enhancer-binding protein homologous protein (CHOP), an ER stress marker, was demonstrated to participate in PA-induced cytotoxicity. RSG treatment rescued granulosa cells from PA-induced cell death and ER stress. Moreover, RSG was identified to ameliorate ER stress induced by tunicamycin in granulosa cells. In addition, RSG treatment rescued granulosa cells from PA-induced decrease of estrogen secretion by cAMP/PKA/CREB pathway. In conclusion, RSG can protect granulosa cells against PA-induced cytotoxicity by inhibiting ER stress, and can recover steroidogenic capacity, indicating a potential use of RSG in the treatment of granulosa cell dysfunction.