GPR30 Research Articles

GPER1 Activation Upregulates Renal Purinergic P2Y2 Receptor Expression and Lowers Blood Pressure in Ovariectomized Rats Fed High Salt Diet

Renal purinergic P2Y2 receptor promotes urinary Na+ excretion via inhibiting epithelial Na+ channel (ENaC) activity. Recent evidence implicates that ovarian hormones regulate P2Y2/ENaC natriuretic pathway, however the underlying mechanism remains unclear. Estradiol activates the G protein-coupled estrogen receptor 1 (GPER1), which promotes natriuresis in females. Taken together, we hypothesized that GPER1 activation regulates renal P2Y2 receptor expression and maintains blood pressure in ovariectomized (OVX) rats. To test our hypothesis, we determined the effect of pharmacologic activation GPER1 on (i) blood pressure and (ii) mRNA expression of renal P2Y2 receptor. In particular, female (16–20 weeks of age) Sprague Dawley rats were implanted with telemeters. After recording baseline blood pressure, rats were ovariectomized and simultaneously implanted with an osmotic minipump to deliver either G1, selective GPER1 agonist (OVX+G1), or Vehicle (OVX+Veh) for 3 weeks. Rats were fed a normal salt (NS, 0.49% NaCl) throughout the study and challenged with a high salt (HS, 4% NaCl) during the last week. Finally, animals were euthanized, renal cortex and medulla were assessed for P2Y2 and αENaC mRNA expression. We found that OVX+Veh rats fed a HS diet had greater mean arterial pressure (MAP) when compared to their baseline values prior to ovariectomy (113 ± 1 vs. 105 ± 1 mmHg; p < 0.01; n = 6). Interestingly, OVX+G1 rats fed a HS diet did not elicit significant changes in MAP when compared to their baseline values (106 ± 3 vs. 104 ± 2 mmHg; p = 0.25; n = 6). Moreover, renal cortical P2Y2 mRNA expression in OVX+G1 rats was higher compared to corresponding vehicle-treated rats (1.95 ± 0.21 vs. 1.00 ± 0.17 fold change from OVX+Veh; p < 0.01; n = 6-7/group). Renal outer and inner medullary P2Y2 mRNA expression elicited similar trends; however, it did not reach statistical significance. No differences were evident in the renal αENaC mRNA expression in response to G1 treatment. We also extended the study in female mice to determine the effect of genetic deletion of GPER1 on renal P2Y2 receptor mRNA expression. We found that P2Y2 mRNA expression in kidneys obtained from GPER1 KO mice was markedly lower when compared to wildtype litter mates. However, no differences were detected in the mRNA expression of αENaC in response to GPER1 deletion. The current results indicate that GPER1 activity upregulates the renal P2Y2 expression which may contribute to preventing the increase in blood pressure in HS-fed OVX animals, possibly by promoting natriuresis. Additional studies are required to improve our understanding for GPER1/P2Y2/ENaC signaling pathway in the postmenopausal female population. NIH/NIDDK R00DK119413 to EYG. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Unraveling the molecular landscape of prostate cancer: A focus on G-Protein-Coupled Estrogen Receptor (GPER) in migration and invasion processes

Unraveling the molecular landscape of prostate cancer: A focus on G-Protein-Coupled Estrogen Receptor (GPER) in migration and invasion processes

GPR55 activation improves anxiety- and depression-like behaviors of mice during methamphetamine withdrawal

Methamphetamine is a potent and highly addictive neurotoxic psychostimulant that triggers a spectrum of adverse emotional responses during withdrawal. G-protein coupled receptor 55 (GPR55), a novel endocannabinoid receptor, is closely associated with mood regulation. Herein, we developed a murine model of methamphetamine-induced anxiety- and depressive-like behavior during abstinence which showed a decreased GPR55 expression in the hippocampus. Activation of GPR55 mitigated these behavioral symptoms, concomitantly ameliorating impairments in hippocampal neurogenesis and reducing neuroinflammation. These findings underscore the pivotal role of GPR55 in mediating the neuropsychological consequences of methamphetamine withdrawal, potentially via mechanisms involving the modulation of hippocampal neurogenesis and inflammation.

Analysis of Binding Modes between Three Perfluorosulfonates and GPER Based on Computational Simulation and Multiple Spectral Methods.

Perfluorinated compounds (PFCs) belong to a significant category of global environmental pollutants. Investigating the toxicological effects of PFCs within biological systems is of critical significance in various disciplines such as life sciences, environmental science, chemistry, and ecotoxicology. In this study, under simulated human physiological conditions (pH = 7.4), a combination of multiple spectroscopic techniques and computational simulations was employed to investigate the impact of perfluorinated compounds (PFCs) on the G protein-coupled estrogen receptor (GPER). Additionally, the research focused on exploring the binding modes and toxicological mechanisms between PFCs and GPER at the molecular level. All three perfluorinated sulfonic acids (PFSAs) can induce quenching of GPER fluorescence through static quenching and non-radiative energy transfer. Steady-state fluorescence calculations at different temperatures revealed apparent binding constants in the order of 106, confirming a strong binding affinity between the three PFSAs and GPER. Molecular docking studies indicated that the binding sites of PFSAs are located within the largest hydrophobic cavity in the head region of GPER, where they can engage in hydrogen bonding and hydrophobic interactions with amino acid residues within the cavity. Fourier transform infrared spectroscopy, three-dimensional fluorescence, and molecular dynamics simulations collectively indicate that proteins become more stable upon binding with small molecules. There is an overall increase in hydrophobicity, and alterations in the secondary structure of the protein are observed. This study deepens the comprehension of the effects of PFCs on the endocrine system, aiding in evaluating their potential impact on human health. It provides a basis for policy-making and environmental management while also offering insights for developing new pollution monitoring methods and drug therapies.

17β-Estradiol (E2) Activates Matrix Mineralization through Genomic/Nongenomic Pathways in MC3T3-E1 Cells.

Estrogen plays an important role in osteoporosis prevention. We herein report the possible novel signaling pathway of 17β-estradiol (E2) in the matrix mineralization of MC3T3-E1, an osteoblast-like cell line. In the culture media-containing stripped serum, in which small lipophilic molecules such as steroid hormones including E2 were depleted, matrix mineralization was significantly reduced. However, the E2 treatment induced this. The E2 effects were suppressed by ICI182,780, the estrogen receptor (ER)α, and the ERβ antagonist, as well as their mRNA knockdown, whereas Raloxifene, an inhibitor of estrogen-induced transcription, and G15, a G-protein-coupled estrogen receptor (GPER) 1 inhibitor, had little or no effect. Furthermore, the E2-activated matrix mineralization was disrupted by PMA, a PKC activator, and SB202190, a p38 MAPK inhibitor, but not by wortmannin, a PI3K inhibitor. Matrix mineralization was also induced by the culture media from the E2-stimulated cell culture. This effect was hindered by PMA or heat treatment, but not by SB202190. These results indicate that E2 activates the p38 MAPK pathway via ERs independently from actions in the nucleus. Such activation may cause the secretion of certain signaling molecule(s), which inhibit the PKC pathway. Our study provides a novel pathway of E2 action that could be a therapeutic target to activate matrix mineralization under various diseases, including osteoporosis.

Effect of Curcuma longa on GPER-1 and Oxidative/Nitrosative Stress Biomarkers in Cardiac Ischemia-reperfusion Injury

Background: The death rate caused by cardiovascular diseases is increasing all over the world. To investigate the cardioprotective effects of Curcuma longa on cardiac ischemia-reperfusion (I/R) injury, which was further examined through G protein-coupled estrogen receptor 1 (GPER-1) and oxidative/nitrosative stress biomarkers. Methods: Twenty-four male Wistar-albino rats were divided into three groups (I/R group; n=8, sham group; n=8, treatment (Curcuma longa) group; n=8). 1 ml Curcuma longa 50 mg/kg was administered intraperitoneally in the treatment group. While catalase, superoxide dismutase and malondialdehyde values were measured by spectrophotometric method, the measurement of GPER-1 was performed by ELISA method. In the histopathological examination, the groups were evaluated in terms of hemorrhage, myocardial edema, myocytolysis and polymorphonuclear leukocyte (PMNL) infiltration. Result: Statistically significant differences were observed between the treatment and I/R groups in terms of oxidative stress (malondialdehyde) level and antioxidant enzyme activities (catalase, superoxide dismutase) (p less than 0.05). The GPER-1 level was higher in the treatment group compared to the I/R group (p less than 0.05). Histopathologically, PMNL infiltration, hemorrhage, edema and myocytolysis were observed in the I/R and sham groups, whereas only hemorrhage was detected in the treatment group. This study showed that treatment with Curcuma longa inhibited lipid peroxidation by restoring existing antioxidant enzymes against cardiac ischemia-reperfusion injuries.

Correlation between the RNA Expression and the DNA Methylation of Estrogen Receptor Genes in Normal and Malignant Human Tissues.

Estrogen plays a multifaceted function in humans via interacting with the estrogen receptors ERα, ERβ, and G protein-coupled estrogen receptor 1 (GPER1). Previous research has predominantly concentrated on elucidating the signaling route of estrogen. However, the comprehensive understanding of the expression profile and control of these estrogen receptors in various human tissues is not well known. In the present study, the RNA levels of estrogen receptors in various normal and malignant human tissues were retrieved from the human protein atlas, the cancer genome atlas (TCGA), and the genotype-tissue expression (GTEx) databases for analyzing the expression profile of estrogen receptors through gene expression profiling interactive analysis (GEPIA). The status of DNA methylation of estrogen receptor genes from TCGA were analyzed through the software Wanderer and cBioPortal. The MethSurv tool was utilized to estimate the relevance between specific cytosine-guanine (CG) methylation and tumor survival. The expression profile analysis revealed that ERα, ERβ, and GPER1 have unique expression patterns in diverse tissues and malignancies. The interesting results were the higher expression of ERβ RNA in the male testis than in females and the positive association between the RNA level of ERα and the androgen receptor in different human normal tissues. Especially, the significant changes in GPER1 expression in multiple malignancies showed a consistent decrease with no exception, which indicates the role of GPER1 in common tumor inhibition. The finding on the expression profile provides clues for exploring novel potential physiological and pathophysiological functions of estrogen. The DNA methylation analysis manifested that the expression of GPER1 and ERα showed a substantial correlation with the methylation of specific CG sites in the cis-regulating region of the gene. However, no such association was observed for ERβ. When comparing tumor tissues to normal tissues, the DNA methylation of certain CG sites of estrogen receptors showed a correlation with tumor survival but did not always correlate with the expression of that gene or with the expression of DNA methyltransferases. We proposed that the variation in DNA methylation at different CG sites in estrogen receptor genes had other functions beyond its regulatory role in its gene expression, and this might be associated with the progression and therapy efficiency of the tumor based on the modulation of the chromatin configuration.

Molecular characterization of GPR84 in domestic cats

G protein-coupled receptor 84 (GPR84) was cloned as an orphan receptor, and medium-chain fatty acids were then revealed as endogenous ligands. GPR84 is expressed in immune cells and is believed to protect liver function from lipotoxicity caused by overeating and high-fat diet intake. This study aimed to present the molecular characterization of GPR84 in domestic cats. The deduced amino acid sequence of the feline GPR84 shows high sequence homology (83–89 %) with the orthologues from other mammalians by cDNA cloning of feline GPR84. Remarkably high mRNA expression was observed in the bone marrow by Q-PCR analysis. The inhibition of intracellular cAMP concentration was observed in cells transfected with feline GPR84 and treated with medium-chain fatty acids. Immunostaining of GPR84 and free fatty acid receptor 2 (FFAR2)/GPR43 in the bone marrow, where high mRNA expression was observed, showed reactions in macrophages and myeloid cells. To clarify whether the receptor formed homo/hetero-merization, GPR84 and FFARs were analyzed using Nano-Luc binary technology and NanoLuc bioluminescence resonance energy transfer technologies, which revealed that GPR84 formed more heteromers with FFAR2 than homomers with each other. In addition, when GPR84 and FFAR2/GPR43 were cotransfected in the cell, their localization on the cell membrane was reduced compared with that when single receptors were transfected. These results indicated that GPR84 is a functional receptor protein that is expressed in cat tissues and may have a protein–protein interaction with FFAR2/GPR43 on the cell membrane.

Disordered GPR43/NLRP3 expression in peripheral leukocytes of patients with atrial fibrillation is associated with intestinal short chain fatty acids levels

BackgroundAtrial fibrillation (AF) is associated with circulating inflammation. Short-chain fatty acids (SCFAs) derived from gut microbiota (GM) regulate leukocyte function and inhibit the release of inflammatory cytokines, which are partly mediated by the G-protein-coupled receptor 43 (GPR43) signaling. This study aimed to investigate the expression of GPR43/NOD-like receptors family pyrin domain containing 3 (NLRP3) in leukocytes and the interaction with intestinal SCFAs levels in AF patients.MethodsExpressions of GPR43 and NLRP3 mRNA in peripheral blood leukocytes from 23 AF patients and 25 non-AF controls were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Expressions of leukocyte GPR43 and NLRP3 protein were evaluated by western blot analysis. The levels of plasma IL-1β were measured by enzyme-linked immunosorbent assay (ELISA). The fecal SCFAs levels based on GC/MS metabolome of corresponding 21 controls and 14 AF patients were acquired from our published dataset. To evaluate the expression of NLRP3 and GPR43 and the release of IL-1β, human THP-1 cells were stimulated with or without SCFAs (acetate, propionate, and butyrate), lipopolysaccharide (LPS), and nigericin in vitro, respectively.ResultsCompared to the controls, the mRNA expression in peripheral leukocytes was significantly reduced in AF patients (P = 0.011) coupled with the increase in downstream leukocyte NLRP3 mRNA expression (P = 0.007) and plasma IL-1β levels (P < 0.001), consistent with changes in GPR43 and NLRP3 protein expression. Furthermore, leukocyte GPR43 mRNA levels were positively correlated with fecal GM-derived acetic acid (P = 0.046) and negatively correlated with NLRP3 mRNA expression (P = 0.024). In contrast to the negative correlation between left atrial diameter (LAD) and GPR43 (P = 0.008), LAD was positively correlated with the leukocyte NLRP3 mRNA levels (P = 0.024). Subsequent mediation analysis showed that 68.88% of the total effect of intestinal acetic acid on AF might be mediated by leukocyte GPR43/NLRP3. The constructed GPR43–NLRP3 score might have a predictive potential for AF detection (AUC = 0.81, P < 0.001). Moreover, SCFAs treatment increased GPR43 expression and remarkably reduced LPS/nigericin-induced NLRP3 expression and IL-1β release in human THP-1 cells in vitro.ConclusionsDisrupted interactions between GPR43 and NLRP3 expression in peripheral blood leukocytes, associated with reduced intestinal GM-derived SCFAs, especially acetic acid, may be involved in AF development and left atrial enlargement by enhancing circulating inflammation.

GPR39-mediated ERK1/2 signaling reduces permethrin-induced proliferation of estrogen receptor α-negative cells

Certain insecticides are known to have estrogenic effects by activating estrogen receptors through genomic transcription. This has led researchers to associate specific insecticide use with an increased breast cancer risk. However, it is unclear if estrogen receptor-dependent pathways are the only way in which these compounds induce carcinogenic effects. The objective of this study was to determine the impact of the pyrethroid insecticide permethrin on the growth of estrogen receptor negative breast cancer cells MDA-MB-231. Using tandem mass spectrometric techniques, the effect of permethrin on cellular protein expression was investigated, and gene ontology and pathway function enrichment analyses were performed on the deregulated proteins. Finally, molecular docking simulations of permethrin with the candidate target protein was performed and the functionality of the protein was confirmed through gene knockdown experiments. Our findings demonstrate that exposure to 10–40 μM permethrin for 48 h enhanced cell proliferation and cell cycle progression in MDA-MB-231. We observed deregulated expression in 83 upregulated proteins and 34 downregulated proteins due to permethrin exposure. These deregulated proteins are primarily linked to transmembrane signaling and chemical carcinogenesis. Molecular docking simulations revealed that the overexpressed transmembrane signaling protein, G protein-coupled receptor 39 (GPR39), has the potential to bind to permethrin. Knockdown of GPR39 partially impeded permethrin-induced cellular proliferation and altered the expression of proliferation marker protein PCNA and cell cycle-associated protein cyclin D1 via the ERK1/2 signaling pathway. These findings offer novel evidence for permethrin as an environmental breast cancer risk factor, displaying its potential to impact breast cancer cell proliferation via an estrogen receptor-independent pathway.

Icariin attenuates vascular endothelial dysfunction by inhibiting inflammation through GPER/Sirt1/HMGB1 signaling pathway in type 1 diabetic rats

Icariin, a flavonoid glycoside, is extracted from Epimedium. This study aimed to investigate the vascular protective effects of icariin in type 1 diabetic rats by inhibiting high-mobility group box 1 (HMGB1)-related inflammation and exploring its potential mechanisms. The impact of icariin on vascular dysfunction was assessed in streptozotocin (STZ)-induced diabetic rats through vascular reactivity studies. Western blotting and immunofluorescence assays were performed to measure the expressions of target proteins. The release of HMGB1 and pro-inflammation cytokines were measured by enzyme-linked immunosorbent assay (ELISA). The results revealed that icariin administration enhanced acetylcholine-induced vasodilation in the aortas of diabetic rats. It also notably reduced the release of pro-inflammatory cytokines, including interleukin-8 (IL-8), IL-6, IL-1β, and tumor necrosis factor-alpha (TNF-α) in diabetic rats and high glucose (HG)-induced human umbilical vein endothelial cells (HUVECs). The results also unveiled that the pro-inflammatory cytokines in the culture medium of HUVECs could be increased by rHMGB1. The increased release of HMGB1 and upregulated expressions of HMGB1-related inflammatory factors, including advanced glycation end products (RAGE), Toll-like receptor 4 (TLR4), and phosphorylated p65 (p-p65) in diabetic rats and HG-induced HUVECs, were remarkably suppressed by icariin. Notably, HMGB1 translocation from the nucleus to the cytoplasm in HUVECs under HG was inhibited by icariin. Meanwhile, icariin could activate G protein-coupled estrogen receptor (GPER) and sirt1. To explore the role of GPER and Sirt1 in the inhibitory effect of icariin on HMGB1 release and HMGB-induced inflammation, GPER inhibitor and Sirt1 inhibitor were used in this study. These inhibitors diminished the effects of icariin on HMGB1 release and HMGB1-induced inflammation. Specifically, the GPER inhibitor also negated the activation of Sirt1 by icariin. These findings suggest that icariin activates GPER and increases the expression of Sirt1, which in turn reduces HMGB1 translocation and release, thereby improving vascular endothelial function in type 1 diabetic rats by inhibiting inflammation.

Effect of astrocyte GPER on the optic nerve inflammatory response following optic nerve injury in mice

Activated astrocytes are a primary source of inflammatory factors following traumatic optic neuropathy (TON). Accumulation of inflammatory factors in this context leads to increased axonal damage and loss of retinal ganglion cells (RGCs). Therefore, in the present study, we explored the role of the astrocyte G protein-coupled estrogen receptor (GPER) in regulating inflammatory factors following optic nerve crush (ONC), and analyzed its potential regulatory mechanisms. Overall, our results showed that GPER was abundantly expressed in the optic nerve, and co-localized with glial fibrillary acidic proteins (GFAP). Exogenous administration of G-1 led to a significant reduction in astrocyte activation and expression of inflammation-related factors (including IL-1β, TNF-α, NFκB, and p-NFκB). Additionally, it dramatically increased the survival of RGCs. In contrast, astrocytes were activated to a greater extent by exogenous G15 administration; however, RGCs survival was significantly reduced. In vitro, GPER activation significantly reduced astrocyte activation and the release of inflammation-related factors. In conclusion, activation of astrocyte GPER significantly reduced ONC inflammation levels, and should be explored as a potential target pathway for protecting the optic nerve and RGCs after TON.

Hemp Seed Oil Inhibits the Adipogenicity of the Differentiation-Induced Human Mesenchymal Stem Cells through Suppressing the Cannabinoid Type 1 (CB1).

Central and peripheral mechanisms of the endocannabinoid system (ECS) favor energy intake and storage. The ECS, especially cannabidiol (CBD) receptors, controls adipocyte differentiation (hyperplasia) and lipid accumulation (hypertrophy) in adipose tissue. In white adipose tissue, cannabidiol receptor 1 (CB1) stimulation increases lipogenesis and inhibits lipolysis; in brown adipose tissue, it decreases mitochondrial thermogenesis and biogenesis. This study compared the availability of phytocannabinoids [CBD and Δ9-tetrahydrocannabinol (THC)] and polyunsaturated fatty acids [omega 3 (ω3) and omega 6 (ω6)] in different hemp seed oils (HSO). The study also examined the effect of HSO on adipocyte lipid accumulation by suppressing cannabinoid receptors in adipogenesis-stimulated human mesenchymal stem cells (hMSCs). Most importantly, Oil-Red-O' and Nile red tests showed that HSO induced adipogenic hMSC differentiation without differentiation agents. Additionally, HSO-treated cells showed increased peroxisome proliferator-activated receptor gamma (PPARγ) mRNA expression compared to controls (hMSC). HSO reduced PPARγ mRNA expression after differentiation media (DM) treatment. After treatment with HSO, DM-hMSCs had significantly lower CB1 mRNA and protein expressions than normal hMSCs. HSO treatment also decreased transient receptor potential vanilloid 1 (TRPV1), fatty acid amide hydrolase (FAAH), and monoacylglycerol lipase (MGL) mRNAs in hMSC and DM-hMSCs. HSO treatment significantly decreased CB1, CB2, TRPV1, and G-protein-coupled receptor 55 (GPCR55) protein levels in DM-hMSC compared to hMSC in western blot analysis. In this study, HSO initiated adipogenic differentiation in hMSC without DM, but it suppressed CB1 gene and protein expression, potentially decreasing adipocyte lipid accumulation and lipogenic enzymes.

The Acetic Acid Produced by Lactobacillus Species Regulates Immune Function to Alleviate PEDV Infection in Piglets.

Porcine epidemic diarrhea virus (PEDV) infection results in significant mortality among newborn piglets, leading to substantial economic setbacks in the pig industry. Short-chain fatty acids (SCFA), the metabolites of intestinal probiotics, play pivotal roles in modulating intestinal function, enhancing the intestinal barrier, and bolstering immune responses through diverse mechanisms. The protective potential of Lactobacillus delbrueckii, Lactobacillus johnsonii, and Lactococcus lactis was first noted when administered to PEDV-infected piglets. Histological evaluations, combined with immunofluorescence studies, indicated that piglets receiving L. lactis displayed less intestinal damage, with diminished epithelial cell necrosis and milder injury levels. Differences in immunofluorescence intensity revealed a significant disparity in antigen content between the L. lactis and PEDV groups, suggesting that L. lactis might suppress PEDV replication, the intestine. We then assessed short-chain fatty acid content through targeted metabolomics, finding that acetate levels markedly varied from other groups. This protective impact was confirmed by administering acetate to PEDV-infected piglets. Data suggested that piglets receiving acetate exhibited resistance to PEDV. Flow cytometry analyses were conducted to evaluate the expression of innate and adaptive immune cells in piglets. Sodium acetate appeared to bolster innate immune defenses against PEDV, marked by elevated NK cell and macrophage counts in mesenteric lymph nodes, along with increased NK cells in the spleen and macrophages in the bloodstream. Acetic acid was also found to enhance the populations of CD8+ IFN-γ T cells in the blood, spleen, and mesenteric lymph, CD4+ IFN-γ T cells in mesenteric lymph nodes and spleen, and CD4+ IL-4+T cells in the bloodstream. Transcriptome analyses were carried out on the jejunal mucosa from piglets with PEDV-induced intestinal damage and from healthy counterparts with intact barriers. Through bioinformatics analysis, we pinpointed 189 significantly upregulated genes and 333 downregulated ones, with the PI3K-AKT, ECM-receptor interaction, and pancreatic secretion pathways being notably enriched. This transcriptomic evidence was further corroborated by western blot and qPCR. Short-chain fatty acids (SCFA) were found to modulate G protein-coupled receptor 41 (GPR41) and 43 (GPR43) in porcine intestinal epithelial cells (IPEC-J2). Post-acetic acid exposure, there was a notable upsurge in the ZO-1 barrier protein expression in IPEC-J2 compared to the unexposed control group (WT), while GPR43 knockdown inversely affected ZO-1 expression. Acetic acid amplified the concentrations of phosphorylated PI3K and AKT pivotal components of the PI3K/AKT pathway. Concurrently, the co-administration of AKT agonist SC79 and PI3K inhibitor LY294002 revealed acetic acid's role in augmenting ZO-1 expression via the P13K/AKT signaling pathway. This study demonstrates that acetic acid produced by Lactobacillus strains regulates intestinal barrier and immune functions to alleviate PEDV infection. These findings provide valuable insights for mitigating the impact of PEDV in the pig industry.

Far-Infrared Therapy Based on Graphene Ameliorates High-Fat Diet-Induced Anxiety-Like Behavior in Obese Mice via Alleviating Intestinal Barrier Damage and Neuroinflammation.

The consumption of a high-fat diet (HFD) has been implicated in the etiology of obesity and various neuropsychiatric disturbances, including anxiety and depression. Compelling evidence suggests that far-infrared ray (FIR) possesses beneficial effects on emotional disorders. However, the efficacy of FIR therapy in addressing HFD-induced anxiety and the underlying mechanisms remain to be elucidated. Here, we postulate that FIR emitted from a graphene-based therapeutic device may mitigate HFD-induced anxiety behaviors. The graphene-FIR modify the gut microbiota in HFD-mice, particularly by an enriched abundance of beneficial bacteria Clostridiaceae and Erysipelotrichaceae, coupled with a diminution of harmful bacteria Lachnospiraceae, Anaerovoracaceae, Holdemania and Marvinbryantia. Graphene-FIR also improved intestinal barrier function, as evidenced by the augmented expression of the tight junction protein occludin and G protein-coupled receptor 43 (GPR43). In serum level, we observed the decreased free fatty acids (FFA), lipopolysaccharides (LPS), diamine oxidase (DAO) and D-lactate, and increased the glucagon-like peptide-2 (GLP-2) levels in graphene-FIR mice. Simultaneously, inflammatory cytokines IL-6, IL-1β, and TNF-α manifested a decrease subsequent to graphene-FIR treatment in both peripheral and central system. Notably, graphene-FIR inhibited over expression of astrocytes and microglia. We further noticed that the elevated the BDNF and decreased TLR4 and NF-κB expression in graphene-FIR group. Overall, our study reveals that graphene-FIR rescued HFD-induced anxiety via improving the intestine permeability and the integrity of blood-brain barrier, and reduced inflammatory response by down regulating TLR4/NF-κB inflammatory pathway.

From Biological Activity to Stereoselectivity: A Portrait of Molecular and Mechanistic Profiles of the Therapeutic Potential of G-1 and LNS8801 as GPER-1 Activator in the Treatment of Waldenström’s Macroglobulinemia

Therapeutic activation of G protein-coupled estrogen receptor 1 (GPER-1) shows promise for treating Waldenström's Macroglobulinemia (WM). Recently, the selective small-molecule agonist G-1 or its enantiomer LNS8801 were reported as potent GPER-1 pharmacological activators, however, the molecular events surrounding their chirality towards GPER-1 is still unexplored. This study aimed to explore the molecular events surrounding the chirality of the selective small-molecule agonists G-1 and LNS8801 towards GPER-1. Molecular docking and dynamics simulations revealed strong binding affinities and key amino acid residues involved in the activation process. The results revealed favourable binding affinities of -29.87kcal/mol, and -28.09kcal/mol for G-1 and LNS8801 towards GPER-1, respectively. Per-residue energy decomposition and time-dependent analysis proved that Arg253 and Arg254 are key amino acid residues in binding these activators towards GPER-1. The activators affected stability, flexibility, and structure of GPER-1. G-1 exhibited greater rigidity when bound to GPER-1 compared to LNS880. This study's findings illuminate chirality and the potential for optimizing the enantiomer ratio to enhance inhibitory effects. These results could also reveal the molecular specifics and binding mechanism of the G-1 and its enantiomer LNS8801 with GPER-1, laying the basis for the rational design of enhanced chiral molecules against WM.

Abstract 568: Modulation of HER4 isoforms expression in estrogen-stimulated in vitro models

Abstract Introduction HER4, a tyrosine kinase receptor belonging to the Human Epidermal Growth Factor/HER/ErbB-family, has a complex biology that includes four isoforms (JMa-CYT1/2, and JMb-CYT1/2) and tissue specific oncogenic or tumour suppressor roles. In estrogen receptor positive (ER+) breast cancer (BC), HER4 isoforms can either act as a co-regulator of ESR1-mediated gene expression by localising to the nucleus, or activate apoptosis by localising to mitochondria. There is very little known about HER4 function in cutaneous melanoma, despite cutaneous melanoma showing the highest HER4 genomic alteration frequency (17.34%) across different cancers (cBioPortal). Due to the interplay between estrogen signalling and HER4 isoforms in ER+BC, this study investigates the RNA expression of HER4 isoforms along with hormone and nuclear receptors associated with 17β-estradiol stimulation in ER+BC and cutaneous melanoma in vitro models. Material and Methods CAMA1 (ER+BC) and SKMEL24 (cutaneous melanoma) cell lines were serum starved for 4h prior to stimulation with 17β-estradiol (0.5nM, 1nM, 5nM and 10nM). RNA was extracted from cells after 24h using TRIzol, followed by qRT-PCR to assess the expression of HER4 isoforms, the hormone receptors estrogen receptor 1 (ESR1/ER-alpha), estrogen receptor 2 (ESR2/ER-beta), progesterone receptor (PGR/PR), G protein-coupled estrogen receptor 1 (GPER1) and the nuclear receptors retinoic acid receptor alpha (RARA) and retinoid X receptor alpha (RXRA). Cell proliferation and caspase3/7 apoptosis analysis were performed at 24/48/72h in the 17β-estradiol stimulated cells by using the IncuCyte Live Cell Analysis Instrument (Sartorius). SN-38 (150nM) was used as positive control for apoptosis induction. Two-way ANOVA was used for the statistical analysis, p&amp;lt;0.05 was significant. Results The qRT-PCR analysis showed that upon stimulation of CAMA1 with 1nM of 17β-estradiol there is a numerical shift in the CYT-1/CYT-2 ratio towards CYT-2 (not statistically significant, p&amp;gt;0.05) and a significant increase in PGR (p= 0.0027) and RAR-A (p= 0.0248) expression compared to control. CAMA1 stimulated with any concentration of 17β-estradiol showed an increase in proliferation compared to the control. The stimulation of SKMEL24 with 5nM of 17β-estradiol showed an increase in JM-a (p=0.0075) and CYT-1 (p=0.0271) HER4 isoforms, GPER1 (p= 0.0286), and a dose-dependent increase in RARA (p=0.0011) compared to control, even in the absence of ESR1, ESR2 and PGR. SKMEL24 stimulated with 17β-estradiol showed a decrease in proliferation compared to control, but no apoptosis induction was found. Conclusion These findings suggest potential interplay between 17β-estradiol and HER4 isoforms leading to the activation of intracellular pathways that could influence tumour progression/suppression in melanoma. Further studies are needed to clarify the specific receptor interactions and pathways involved. Citation Format: Marta Valenti, Georg F. Bischof, John P. Crown, Denis M. Collins. Modulation of HER4 isoforms expression in estrogen-stimulated in vitro models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 568.

Qixian granule inhibits ferroptosis in vascular endothelial cells by modulating TRPML1 in the lysosome to prevent postmenopausal atherosclerosis

Ethnopharmacological relevanceQiXian Granule (QXG) is an integrated traditional Chinese medicine formula used to treat postmenopausal atherosclerotic (AS) cardiovascular diseases. The previous studies have found that QXG inhibited isoproterenol (ISO)-induced myocardial remodeling. And its active ingredient, Icraiin, can inhibit ferroptosis by promoting oxidized low-density lipoprotein (xo-LDL)-induced vascular endothelial cell injury and autophagy in atherosclerotic mice. Another active ingredient, Salvianolic Acid B, can suppress ferroptosis and apoptosis during myocardial ischemia/reperfusion injury by reducing ubiquitin-proteasome degradation of Glutathione Peroxidase 4 (GPX4) and down-regulating the reactive oxygen species (ROS)- c-Jun N-terminal kinases (JNK)/mitogen-activated protein kinase (MAPK) pathway. Aim of the studyThe objective of this research was to assess the possible impact of QXG on atherosclerosis in postmenopausal individuals and investigate its underlying mechanisms. Materials and methodsFemale ApoE−/− mice underwent ovariectomy and were subjected to a high-fat diet (HFD) to establish a postmenopausal atherosclerosis model. The therapeutic effects of QXG were observed in vivo and in vitro through intraperitoneal injection of erastin, G-protein Coupled Estrogen Receptor (GPER) inhibitor (G15), and silent Mucolipin Transient Receptor Potential Channel 1 (TRPML1) adenovirus injection via tail vein. UPLC-MS and molecular docking techniques identified and evaluated major QXG components, contributing to the investigation of QXG's anti-postmenopausal atherosclerotic effects. ResultsQXG increased serum Estradiol levels, decreased follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels, which indicated QXG had estrogen-like effects in Ovx/ApoE−/− mice. Furthermore, QXG demonstrated the potential to impede the progression of AS in Ovx/ApoE−/− mice, as evidenced by reductions in serum triglycerides (TG), total cholesterol (TC), and low-density lipoprotein-cholesterol (LDL-C) levels. Additionally, QXG inhibited ferroptosis in Ovx/ApoE−/− mice. Notably, UPLC-MS analysis identified a total of 106 active components in QXG. The results of molecular docking analysis demonstrated that Epmedin B, Astragaloside II, and Orientin exhibit strong binding affinity towards TRPML1. QXG alleviates the progression of atherosclerosis by activating TRPML1 through the GPER pathway or directly activating TRPML1, thereby inhibiting GPX4 and ferritin heavy chain (FTH1)-mediated iron pendant disease. In vitro, QXG-treated serum suppressed proliferation, migration, and ox-LDL-induced MMP and ROS elevation in HAECs. ConclusionQXG inhibited GPX4 and FTH1-mediated ferroptosis in vascular endothelial cells through up-regulating GPER/TRPML1 signaling, providing a potential therapeutic option for postmenopausal females seeking a safe and effective medication to prevent atherosclerosis. The study highlights QXG's estrogenic properties and its promising role in combating postmenopausal atherosclerosis.

GPR41 deficiency aggravates type 1 diabetes in streptozotocin-treated mice by promoting dendritic cell maturation.

Disturbances in intestinal immune homeostasis predispose susceptible individuals to type 1 diabetes (T1D). G-protein-coupled receptor 41 (GPR41) is a receptor for short-chain fatty acids (SCFAs) mainly produced by gut microbiota, which plays key roles in maintaining intestinal homeostasis. In this study, we investigated the role of GPR41 in the progression of T1D. In non-obese diabetic (NOD) mice, we found that aberrant reduction of GPR41 expression in the pancreas and colons was associated with the development of T1D. GPR41-deficient (Gpr41-/-) mice displayed significantly exacerbated streptozotocin (STZ)-induced T1D compared to wild-type mice. Furthermore, Gpr41-/- mice showed enhanced gut immune dysregulation and increased migration of gut-primed IFN-γ+ T cells to the pancreas. In bone marrow-derived dendritic cells from Gpr41-/- mice, the expression of suppressor of cytokine signaling 3 (SOCS) was significantly inhibited, while the phosphorylation of STAT3 was significantly increased, thus promoting dendritic cell (DC) maturation. Furthermore, adoptive transfer of bone marrow-derived dendritic cells (BMDC) from Gpr41-/- mice accelerated T1D in irradiated NOD mice. We conclude that GPR41 is essential for maintaining intestinal and pancreatic immune homeostasis and acts as a negative regulator of DC maturation in T1D. GPR41 may be a potential therapeutic target for T1D.

Role of GPR81 in regulating intramuscular triglyceride storage during aerobic exercise in rats.

Lactate, a metabolite of exercise, plays a crucial role in the body. In these studies, we aimed to investigate the role of G protein-coupled receptor 81 (GPR81), a specific receptor for lactate, in regulating lipid storage in the gastrocnemius muscle of rats. To achieve this, we measured the impact of sodium 3-hydroxybutyrate (3-OBA) concentration and time on the cAMP-PKA signaling pathway in the gastrocnemius muscles ofrats. Our investigation involved determining the effects of administering 3-OBA at a concentration of 3 mmol L-1 just 15 min before exercise. As expected, exercise led to a notable increase in intramuscular lactate concentration in rats. However, injecting 3-OBA prior to exercise yielded intriguing results. It not only further augmented the cAMP-PKA signaling pathway but also boosted the expression of lipolysis-related proteins such as hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL). Simultaneously, it decreased the expression of fat-synthesizing proteins, including acetyl CoA carboxylase (ACC) and fatty acid synthase (FAS), while increasing the protein expression of cytochrome c oxidase subunit Ⅳ(COX Ⅳ) and the activity of citrate synthetase (CS). Unfortunately, there was no significant change observed in intramuscular triglyceride (IMTG) content. In summary, our findings shed light on the role of lactate in partially regulating intramuscular triglycerides during exercise.