EMPA is a high-affinity, reversible, and selective OX2 receptor antagonist. [3H]EMPA binds to human and rat OX2-HEK293 membranes with Kd values of 1.1 and 1.4 nM, respectively. EMPA competitively antagonizes orexin-A and orexin-B-induced [3H]inositol phosphate (IP) accumulation at hOX2 receptors with pA2 values of 8.6 and 8.8, respectively. EMPA displaces [3H]EMPA binding on cell membranes containing human and rat OX2 receptors with Ki values of 1.10±0.24 nM and 1.45±0.13 nM, respectively. In human and mouse V1a receptor binding assays, EMPA has IC50=5.75 µM, Ki=2.63 µM, IC50=12.8 µM, Ki=5.8 µM. In CHO (dHFr-) cells stably expressing hOX2 receptor, EMPA can inhibit the [Ca2+]i response induced by orexin-A or orexin-B, with IC50 values of 8.8±1.7 nM and 7.9±1.7 nM, respectively[1].
EMPA 细胞实验
Cell Line
Concentration
Treated Time
Description
References
Human coronary artery endothelial cells (HCAECs)
1 μM
24 h
EMPA ameliorates stretch induced oxidative stress and cell permeability via inhibiting PKC activity.
Redox Biol. 2024 Feb;69:102979
BV2 microglia
50 μM
24 hours
To evaluate the anti-inflammatory effects of EMPA on LPS-induced cell inflammation and NLRP3 inflammasome activation. Results showed that EMPA significantly suppressed the upregulation of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) mRNA levels and inhibited the expression of NLRP3 inflammasome-related proteins.
J Neuroinflammation. 2023 Dec 12;20(1):296
Human coronary artery endothelial cells (HCAECs)
1 μM
24 hours
EMPA reduces NOX activation and ROS generation induced by 10% stretch via inhibition of PKC activity
Redox Biol. 2024 Feb;69:102979
H9C2 cardiomyocytes
0.04-5 μM
2 or 24 hours
To evaluate the effect of EMPA on intracellular Na+ and Ca2+ concentrations, results showed that EMPA effectively suppressed the increase in intracellular Na+ and Ca2+ concentrations under high glucose conditions
Exp Mol Med. 2023 Jun;55(6):1174-1181
HL-1 cells
10, 50, 500 nM
24 hours
To evaluate the cytotoxic or cytoprotective effects of EMPA on cardiomyocytes. Co-incubation with EMPA increased cell viability, reduced intracellular Ca2+ content and oxidative stress.
Cardiovasc Diabetol. 2021 Jul 23;20(1):150
H9C2 cells
1 µM
72 hours
To investigate the effect of EMPA on IFN-γ-induced cell senescence, results showed that EMPA significantly inhibited IFN-γ-induced oxidative stress and senescence, and promoted cell proliferation.
Cardiovasc Diabetol. 2024 Jul 23;23(1):269
Porcine coronary artery endothelial cells
100 nM
24 hours
To evaluate the protective effect of EMPA on Ang II-induced endothelial senescence and dysfunction, results showed that EMPA inhibited Ang II-induced oxidative stress, increased senescence markers, and endothelial dysfunction.
Cardiovasc Diabetol. 2021 Mar 16;20(1):65
human ventricular cardiac myoblasts AC16
0.5 µM
7 days
To evaluate the effects of EMPA on high glucose-induced DNA methylation changes in the promoter regions of NF-κB and SOD2. Results showed that EMPA prevented HG-induced DNA demethylation by reducing TET2 binding to the promoter regions and restored normal gene expression levels.
Cardiovasc Diabetol. 2023 Feb 2;22(1):24
EMPA 动物实验
Species
Animal Model
Administration
Dosage
Frequency
Description
References
Mice
Diabetic and non-diabetic myocardial infarction models
Oral
30 mg/kg/day
1 week or 24 hours pretreatment
EMPA significantly reduced infarct size and myocardial fibrosis, improving cardiac function and survival rate.
Protein Cell. 2022 May;13(5):336-359
C57BL/6J male mice
Retinal ischemia and reperfusion (IR) injury model
Intravitreal injection
100 µg/ml
Single injection, observed for 7 days
To evaluate the therapeutic efficacy of EMPA in retinal IR injury. Results showed that EMPA significantly protected retinal ganglion cells (RGCs) from IR injury, attenuated local retinal inflammation, and restored the expression of mitochondrial dynamics-related genes Mfn1 and Opa1.
J Neuroinflammation. 2023 Dec 12;20(1):296
Rat
Langendorff perfused heart model
Perfusion
10 µM
10 min pretreatment followed by continuous perfusion
To investigate the effects of EMPA on cardiac function and metabolism during ischemia/reperfusion. Results showed that EMPA improved left ventricular-developed pressure, increased ATP and phosphocreatine levels, and enhanced Gibbs free energy for ATP hydrolysis. Additionally, EMPA shifted cardiac metabolism from glucose oxidation to increased ketone utilization.
Cardiovasc Res. 2023 Dec 19;119(16):2672-2680
Zebrafish
DM-HFrEF model
Immersion
0.2-5 μM
From 5 dpf, lasting more than 24 hours
To evaluate the effects of EMPA on survival, locomotion, and cardiac function in zebrafish, results showed that EMPA significantly improved survival, locomotion, and cardiac function
Exp Mol Med. 2023 Jun;55(6):1174-1181
C57Bl/6 mice
Non-diabetic mice
Oral gavage
10 mg/kg/day
Once daily for 10 days
To evaluate the protective effects of EMPA against DOXO-induced cardiotoxicity. EMPA improved cardiac function, reduced cardiac fibrosis and expression of inflammatory markers.
Cardiovasc Diabetol. 2021 Jul 23;20(1):150
C57BL/6J mice
HFpEF model
Oral gavage
10 mg/kg/day
Once daily for 28 days
To investigate the protective effect of EMPA on cardiac function in HFpEF mice, results showed that EMPA improved diastolic dysfunction, alleviated cardiac hypertrophy and fibrosis, and inhibited cardiac inflammation and aging processes.
Cardiovasc Diabetol. 2024 Jul 23;23(1):269
Rats
Aortic arch and thoracic aorta segments
In vitro incubation
100 nM
15 hours
To evaluate the inhibitory effect of EMPA on the upregulation of SGLT1 and SGLT2 protein expression in Ang II- and eNOS inhibitor-treated thoracic aorta segments, results showed that EMPA reduced oxidative stress and improved endothelial function.
Cardiovasc Diabetol. 2021 Mar 16;20(1):65
EMPA 动物研究
Animal study
Intraperitoneally injected at doses of 1-300 mg/kg, EMPA reversed [Ala11,D-Leu15]orexin-B-induced hypermobility in male NMRI mice in a dose-dependent manner, without significantly affecting the locomotor activity of the mice[1].Administered intraperitoneally at doses of 3-30 mg/kg, EMPA reduces baseline LMA in Wistar rats in a dose-dependent manner[1].
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