OBJECTIVES: To investigate the role of the calcium/calmodulin (CaM)-mediated activation of calcineurin (CN)-nuclear factor of activated T cells (NFAT) signaling pathway in mediating the regulatory effect of hyperforin (HY) on stress-induced depression-like disorder (DP) in mice. METHODS: C57BL/6J mice were randomly divided into control group, DP model group, and hyperforin treatment group (n=15). Behavioral changes of the mice were assessed using open field test (OFT), sucrose preference test (SPT), tail suspension test (TST), light/dark box test (LDB), and novel object suppression test (NSFT). Immunohistochemistry was used to detect tyrosine hydroxylase (TH) expression in the CA1 region of the hippocampus, and serum serotonin (5-HT) and norepinephrine (NA) levels were detected with ELISA. Western blotting was used to analyze the expressions of TNF-α, IL-1β, IL-2, and CN-NFAT pathway proteins. In cultured BV-2 microglial cells with lipopolysaccharide (LPS) stimulation, the effects of hyperforin and CN inhibitor (CNIS) on expressions of ionized calcium-binding adapter molecule 1 (IBA-1), 5-HT, NA, inflammatory cytokines and CN-NFAT pathway proteins were examined using immunofluorescence assay, ELISA or Western blotting. RESULTS: Compared with the control mice, the mice in DP group showed significantly reduced activity in OFT, decreased sucrose consumption in SPT, reduced shuttle crossing in LDB, and lowered food intake in NSFT with significantly increased immobility in TST. The mice with DP showed significantly decreased TH-positive neurons, lowered 5-HT and NA levels, and increased expressions of TNF-α, IL-1β, IL-2 and CaM-CN-NFAT pathway proteins. In cultured BV-2 cells, LPS stimulation strongly increased cellular IBA-1 expression, decreased the levels of neurotransmitters (5-HT and NA), and increased the levels of inflammatory cytokines and CN-NFAT signaling, and these changes were effectively reversed by treatment with hyperforin or CNIS. CONCLUSIONS Hyperforin improves stress-induced depression-like behaviors in mice and activated BV-2 cells by targeting the CN-NFAT signaling pathway.
Animals ; Mice, Inbred C57BL ; Mice ; Microglia/drug effects* ; Depression/etiology* ; Perylene/pharmacology* ; Calcineurin/metabolism* ; NFATC Transcription Factors/metabolism* ; Calcium Signaling/drug effects* ; Stress, Psychological ; Phloroglucinol/pharmacology* ; Signal Transduction ; Male ; Behavior, Animal/drug effects* ; Terpenes

OBJECTIVES: To investigate the effects of quercetin on cuproptosis and L-type calcium currents in the myocardium of diabetic rats. METHODS: Forty SD rats were randomized into control group and diabetic model groups. The rat models of diabetes mellitus (DM) induced by high-fat and high-sugar diet combined with streptozotocin (STZ) injection were further divided into DM model group, quercetin treatment group, and empagliflozin treatment group (n=10). Blood glucose and body weight were measured every other week, and cardiac function of the rats was evaluated using echocardiography. HE staining, Sirius red staining, and wheat germ agglutinin (WGA) analysis were used to observe the changes in myocardial histomorphology, and serum copper levels and myocardial FDX1 expression were detected. In cultured rat cardiomyocyte H9c2 cells with high-glucose exposure, the effects of quercetin and elesclomol, alone or in combination, on intracellular CK-MB and LDH levels and FDX1 expression were assessed, and the changes in L-type calcium currents were analyzed using patch-clamp technique. RESULTS: The diabetic rats exhibited elevated blood glucose, reduced body weight, impaired left ventricular function, increased serum copper levels and myocardial FDX1 expression, decreased L-type calcium currents, and prolonged action potential duration. Quercetin and empagliflozin treatment significantly lowered blood glucose, improved body weight, and restored cardiac function of the diabetic rats, and compared with empagliflozin, quercetin more effectively reduced serum copper levels, downregulated FDX1 expression, and enhanced myocardial L-type calcium currents in diabetic rats. In H9c2 cells, high glucose exposure significantly increased myocardial expressions of FDX1, CK-MB and LDH, which were effectively lowered by quercetin treatment; Elesclomol further elevated FDX1, CK-MB and LDH levels in the exposed cells, and these changes were not significantly affected by the application of quercetin. CONCLUSIONS Quercetin ameliorates myocardial injury in diabetic rats possibly by suppressing myocardial cuproptosis signaling and restoring L-type calcium channel activity.
Animals ; Quercetin/pharmacology* ; Calcium Channels, L-Type/metabolism* ; Diabetes Mellitus, Experimental/metabolism* ; Rats, Sprague-Dawley ; Rats ; Myocytes, Cardiac/drug effects* ; Myocardium/pathology* ; Male

OBJECTIVES: To investigate the changes in blood levels of calcium and bone metabolism biochemical markers in patients with primary osteoporosis receiving treatment with denosumab. METHODS: Seventy-three patients with primary osteoporosis treated in our Department between December, 2021 and December 2023 were enrolled. All the patients were treated with calcium supplements, vitamin D and calcitriol in addition to regular denosumab treatment every 6 months. Blood calcium, parathyroid hormone (PTH), osteocalcin (OC), type I procollagen amino-terminal propeptide (PINP), and type I collagen carboxy-terminal telopeptide β special sequence (β‑CTX) data before and at 3, 6, 9, and 12 months after the first treatment were collected from each patient. RESULTS: Three months after the first denosumab treatment, the bone turnover markers (BTMs) OC, PINP, and β-CTX were significantly decreased compared to their baseline levels by 39.5% (P<0.001), 56.2% (P<0.001), and 81.8% (P<0.001), respectively. At 6, 9, and 12 months of treatment, OC, PINP, and β-CTX remained significantly lower than their baseline levels (P<0.001). Blood calcium level was decreased (P<0.05) and PTH level increased (P<0.05) significantly in these patients at months of denosumab treatment, but their levels were comparable to the baseline levels at 6, 9, and 12 months of the treatment (P>0.05). CONCLUSIONS Denosumab can suppress BTMs and has a good therapeutic effect in patients with primary osteoporosis, but reduction of blood calcium and elevation of PTH levels can occur during the first 3 months in spite of calcium supplementation. Blood calcium and PTH levels can recover the baseline levels as the treatment extended, suggesting the importance of monitoring blood calcium and PTH levels during denosumab treatment.
Humans ; Denosumab/therapeutic use* ; Calcium/blood* ; Parathyroid Hormone/blood* ; Biomarkers/blood* ; Osteoporosis/blood* ; Osteocalcin/blood* ; Procollagen/blood* ; Female ; Collagen Type I/blood* ; Peptide Fragments/blood* ; Bone Density Conservation Agents/therapeutic use* ; Bone and Bones/metabolism* ; Male ; Middle Aged ; Vitamin D ; Peptides/blood* ; Aged

OBJECTIVES: To explore the toxic mechanism of Clostridium perfringens Beta1 toxin mediated by P2X7 receptor-induced calcium dyshomeostasis. METHODS: Ten-day-old BALB/c mice were randomly divided into control group, recombinant Beta1 toxin (rCPB1) group, PD151746 group, and PD151746+rCPB1 group, and all the treatment agents were administered by gavage. The changes in expressions of inflammatory factors in the jejunum of the mice were detected using antibody chip technology to explore the regulatory role of calcium dyshomeostasis in Beta1 toxin-induced inflammatory injury level. In the cell experiment, THP-1 cells were transfected with a si-RNA targeting P2X7 receptor and treated with rCPB1, and the changes in cell survival rate, levels of Ca²⁺, ROS and ATP, and expressions of pyroptosis and ferroptosis markers were determined. RESULTS: Oral administration of rCPB1 significantly increased the levels of inflammatory cytokines in the jejunal tissue of the neonatal mice, but their levels were significantly decreased after treatment with PD151746. In THP-1 cells, rCPB1 treatment significantly decreased cell survival and increased the levels of Ca²⁺, ROS, ATP and the expressions of pyroptosis and ferroptosis markers, and these changes were obviously attenuated by P2X7 receptor knockdown. CONCLUSIONS P2X7 receptor-mediated functional pore formation by Beta1 toxin can further lead to calcium dyshomeostasis, thereby triggering excessive accumulation of ROS to subsequently induce the co-occurrence of pyroptosis and ferroptosis.
Animals ; Pyroptosis/drug effects* ; Receptors, Purinergic P2X7/metabolism* ; Mice ; Mice, Inbred BALB C ; Ferroptosis/drug effects* ; Humans ; Calcium/metabolism* ; Macrophages/drug effects* ; Bacterial Toxins/toxicity*

Stromal interaction molecules (STIM)s are Ca²⁺ sensors in internal Ca²⁺ stores of the endoplasmic reticulum. They activate the store-operated Ca²⁺ channels, which are the main source of Ca²⁺ entry in non-excitable cells. Moreover, STIM proteins interact with other Ca²⁺ channel subunits and active transporters, making STIMs an important intermediate molecule in orchestrating a wide variety of Ca²⁺ influxes into excitable cells. Nevertheless, little is known about the role of STIM proteins in brain functioning. Being involved in many signaling pathways, STIMs replenish internal Ca²⁺ stores in neurons and mediate synaptic transmission and neuronal excitability. Ca²⁺ dyshomeostasis is a signature of many pathological conditions of the brain, including neurodegenerative diseases, injuries, stroke, and epilepsy. STIMs play a role in these disturbances not only by supporting abnormal store-operated Ca²⁺ entry but also by regulating Ca²⁺ influx through other channels. Here, we review the present knowledge of STIMs in neurons and their involvement in brain pathology.
Neurons/metabolism* ; Animals ; Humans ; Calcium/metabolism* ; Stromal Interaction Molecules/metabolism* ; Calcium Signaling/physiology* ; Calcium Channels/metabolism* ; Brain/metabolism*

Hemorrhagic shock is a common clinical emergency that can aggravate cell injury after resuscitation. Astrocytes are crucial for the survival of neurons because they regulate the surrounding ionic microenvironment of neurons. Although hemorrhagic shock and resuscitation (HSR) injury can impair cognition, it remains unclear how this insult directly affects astrocytes. In this study, we established an HSR model by bleeding and re-transfusion in mice. The social interaction test and new object recognition test were applied to evaluate post-operative cognitive changes, and the results suggest that mice experience cognitive impairment following exposure to HSR. In the HSR group, the power spectral density of β and γ oscillations decreased, and the coupling of the θ oscillation phase and γ oscillation amplitude was abnormal, which indicated abnormal neuronal oscillation and cognitive impairment after HSR exposure. In brief, cognitive impairment in mice is strongly correlated with Ca²⁺ signal strength in lateral septum astrocytes following HSR.
Animals ; Astrocytes/metabolism* ; Shock, Hemorrhagic/metabolism* ; Resuscitation/adverse effects* ; Male ; Mice ; Calcium Signaling/physiology* ; Mice, Inbred C57BL ; Septal Nuclei/metabolism* ; Cognitive Dysfunction/etiology* ; Disease Models, Animal ; Cognition Disorders/etiology*

Neuronal injury in glaucoma persists despite effective intraocular pressure (IOP) control, necessitating neuroprotective strategies for retinal ganglion cells (RGCs). In this study, we investigated the neuroprotective role of the γ-hydroxybutyrate analog HOCPCA in a glaucoma model, focusing on its effects on CaMKII signaling, oxidative stress, and neuroinflammatory responses. Retinal tissue from high IOP animal models was analyzed via proteomics. In vitro mouse retinal explants were subjected to elevated pressure and oxidative stress, followed by HOCPCA treatment. HOCPCA significantly mitigated the RGC loss induced by oxidative stress and elevated pressure, preserving neuronal function. It restored CaMKIIα and β levels, preserving RGC integrity, while also modulating oxidative stress and neuroinflammatory responses. These findings suggest that HOCPCA, through its interaction with CaMKII, holds promise as a neuroprotective therapy for glaucoma.
Animals ; Retinal Ganglion Cells/metabolism* ; Glaucoma/pathology* ; Oxidative Stress/drug effects* ; Neuroprotective Agents/pharmacology* ; Mice ; Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism* ; Mice, Inbred C57BL ; Disease Models, Animal ; Neuroinflammatory Diseases/drug therapy* ; Neuroprotection/drug effects* ; Male ; Intraocular Pressure/drug effects*

Microglial functions are linked to Ca²⁺ signaling, with endoplasmic reticulum (ER) calcium stores playing a crucial role. Microglial abnormality is a hallmark of Alzheimer's disease (AD), but how ER Ca²⁺ receptors regulate microglial functions under physiological and AD conditions remains unclear. We found reduced ryanodine receptor 2 (Ryr2) expression in microglia from an AD mouse model. Modulation of RyR2 using S107, a RyR-Calstabin stabilizer, blunted spontaneous Ca²⁺ transients in controls and normalized Ca²⁺ transients in AD mice. S107 enhanced ATP-induced migration and phagocytosis while reducing ramification in control microglia; however, these effects were absent in AD microglia. Our findings indicate that RyR2 stabilization promotes an activation state shift in control microglia, a mechanism impaired in AD. These results highlight the role of ER Ca²⁺ receptors in both homeostatic and AD microglia, providing insights into microglial Ca²⁺ malfunctions in AD.
Animals ; Microglia/pathology* ; Alzheimer Disease/pathology* ; Phagocytosis/drug effects* ; Ryanodine Receptor Calcium Release Channel/metabolism* ; Disease Models, Animal ; Mice ; Cell Movement/drug effects* ; Mice, Transgenic ; Calcium Signaling/physiology* ; Calcium/metabolism* ; Mice, Inbred C57BL ; Male ; Endoplasmic Reticulum/metabolism*

Diabetic cardiomyopathy (DCM) is a medical condition characterized by cardiac remodeling and dysfunction in individuals with diabetes mellitus. Sarcoplasmic reticulum (SR) and mitochondrial Ca²⁺ overload in cardiomyocytes have been recognized as biological hallmarks in DCM; however, the specific factors underlying these abnormalities remain largely unknown. In this study, we aimed to investigate the role of a cardiac-specific long noncoding RNA, D830005E20Rik (Trdn-as), in DCM. Our results revealed the remarkably upregulation of Trdn-as in the hearts of the DCM mice and cardiomyocytes treated with high glucose (HG). Knocking down Trdn-as in cardiac tissues significantly improved cardiac dysfunction and remodeling in the DCM mice. Conversely, Trdn-as overexpression resulted in cardiac damage resembling that observed in the DCM mice. At the cellular level, Trdn-as induced Ca²⁺ overload in the SR and mitochondria, leading to mitochondrial dysfunction. RNA-seq and bioinformatics analyses identified calsequestrin 2 (Casq2), a primary calcium-binding protein in the junctional SR, as a potential target of Trdn-as. Further investigations revealed that Trdn-as facilitated the recruitment of METTL14 to the Casq2 mRNA, thereby enhancing the m⁶A modification of Casq2. This modification increased the stability of Casq2 mRNA and subsequently led to increased protein expression. When Casq2 was knocked down, the promoting effects of Trdn-as on Ca²⁺ overload and mitochondrial damage were mitigated. These findings provide valuable insights into the pathogenesis of DCM and suggest Trdn-as as a potential therapeutic target for this condition.
Animals ; Diabetic Cardiomyopathies/pathology* ; RNA, Long Noncoding/genetics* ; Myocytes, Cardiac/metabolism* ; Mice ; Calsequestrin/genetics* ; Calcium/metabolism* ; Male ; Sarcoplasmic Reticulum/metabolism* ; Methyltransferases/metabolism* ; Mice, Inbred C57BL ; Mitochondria, Heart/metabolism* ; Disease Models, Animal ; Mitochondria/metabolism*

The 5-hydroxytryptamine 2A receptor (5-HT_2AR) is one of the key targets in the development of novel antidepressants. To develop new antidepressants targeting the 5-HT_2A receptor, this study established a high-throughput screening method for drugs targeting the 5-HT_2A receptor based on the principle of detecting calcium flux signals. The immunofluorescence assay and western blotting were employed to evaluate receptor expression levels in the 5-HT_2AR-CHO cell line. The reaction system parameters, including cell seeding density, DMSO concentration, and dye incubation time, were optimized with Z'-factor and signal window values as evaluation indicators. The specificity, precision, stability, and applicability of the method were assessed. Results indicated that the 5-HT_2AR-CHO cell line stably expressed high levels of the 5-HT_2A receptor. The optimized screening method involved a reaction system with 10 000 cells/well, 0.2% DMSO, and 2 h incubation with Calcium 6 dye. The method demonstrated excellent specificity, with inter-batch precision below 10% for the detection of 5-hydroxytryptamine (5-HT) at low, medium, and high concentrations. Testing four compounds that target the 5-HT_2A receptor- agonists 2,5-dimethoxy-4-iodoamphetamine (DOI), 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT), and lysergic acid diethylamide (LSD), along with the antagonist MDL100907-yielded Z'-factors (at EC₈₀) greater than 0.85 and signal window values over 0.91. The EC₅₀ values of these compounds were in the nanomolar range, and their potency rank order aligned with previously reported data, confirming the reliability of the established method. When being applied to the detection of 38 known active compounds, the method efficiently identified 5-HT_2A receptor agonists and antagonists while showing no response to non-target compounds. In conclusion, this study successfully constructs a high-throughput screening approach for 5-HT_2A receptor-targeting drugs based on calcium flux signals. The method possesses strong specificity, high sensitivity, and robust stability, being suitable for screening antidepressants targeting the 5-HT_2A receptor.
High-Throughput Screening Assays/methods* ; Receptor, Serotonin, 5-HT2A/metabolism* ; Animals ; CHO Cells ; Cricetulus ; Calcium Signaling/drug effects* ; Antidepressive Agents/pharmacology* ; Humans ; Serotonin 5-HT2 Receptor Antagonists/pharmacology* ; Calcium/metabolism*