The nuclear factor kappaB (NF-kappaB) is a ubiquitously expressed transcription factor playing vital roles in innate immunity and other processes involving cellular survival, proliferation, and differentiation[2]. Neferine, is a major alkaloid present in the green embryos of Nelumbo nucifera Gaertn with anti-arrhythmia, anti-hypertensive and vaso-relaxant properties, and is also a NF-κB inhibitor. Neferine could inhibit LPS-ATP-induced oxidative stress and the activation of NLRP3 inflammasome signaling, and increased the endothelial cell viability and SOD production. siRNA which mediated the knockdown of NLRP3 promoted the neferine-induced inhibition effects of cell pyroptosis. Furthermore, these neferine-induced effects were reversed by the over-expression of NLRP3[3]. Neferine was distributed rapidly into different organ systems, with the highest concentrations found in the liver. In animal (rat) studies, neferine is known to be predominantly metabolized in the liver and undergoes initial bioconversion by CYP2D6 into liensinine, isoliensinine, dimethylliensinine, and dimethyl-isoliensinine[4]. The human lung adenocarcinoma A549 cells upon treatment with neferine induced cell cycle arrest in G1 phase and further induced apoptosis in a dose dependent manner along with events such as over production of reactive oxygen species, lipid peroxidation, depletion of the mitochondrial membrane potential, loss of cellular pool of anti-oxidants, MAPKs activation, DNA fragmentation and intracellular calcium accumulation[5]. Hep3B cells treated with neferine undergo cell cycle arrest, ER stress and apoptosis through activation of Bid, Bim, Puma, Bak,Bax, caspase-3, -6, -7, -8 and PARP. The upregulated proteins due to neferine include calcinexin,calpain-2, Bip, PDI and caspase-12. Formation of autophagosomes along with reduced migration ability in the Hep3B cells is also observed due to neferine treatment[6].
Neferine/甲基莲心碱 细胞实验
Cell Line
Concentration
Treated Time
Description
References
RBL-2H3 cells
1, 3, 10 µM
1 or 2 hours
Neferine inhibits PMA/A23187-induced phosphorylation of NF-κB pathway (IκBα and NF-κB).
Int J Mol Sci. 2021 Oct 12;22(20):10994.
MC3T3-E1 cells
1.56 µM ~ 50 µM
1, 3, 7 days
Nef at low concentrations showed no obvious toxicity to cells, while high concentrations (over 25 μM) significantly inhibited cell proliferation, but the inhibitory effect weakened by day 7. Nef promoted the alkaline phosphatase (ALP) activity of MC3T3-E1 cells, especially at day 7, where the ALP activity in the 25 μM and 50 μM groups was the highest.
J Biol Eng. 2023 Jul 17;17(1):45.
LNCaP cells
5, 10, 20 µM
18 hours
Neferine treatment significantly decreased the viability of LNCaP cells and enhanced TRAIL-induced apoptosis
Int J Oncol. 2020 May;56(5):1152-1161.
DU145 cells
5, 10, 20 µM
18 hours
Neferine treatment significantly decreased the viability of DU145 cells and enhanced TRAIL-induced apoptosis
Int J Oncol. 2020 May;56(5):1152-1161.
HaCaT cells
1, 3, 10 µM
20 min pretreatment, followed by 1 hour or 24 hours stimulation
Neferine inhibited the mRNA expression of pro-inflammatory cytokines and chemokines in TNF-α/IFN-γ-stimulated HaCaT cells.
Int J Mol Sci. 2021 Jul 30;22(15):8237.
RBL-2H3 cells
3 µM and 10 µM
20 minutes
Neferine inhibits PMA/A23187-induced elevation of intracellular calcium.
Int J Mol Sci. 2021 Oct 12;22(20):10994.
RBL-2H3 cells
10 µM
20 minutes
Neferine inhibits degranulation of mast cells induced by various activators (e.g., compound 48/80, A23187, anti-DNP/IgE + DNP/HSA).
Int J Mol Sci. 2021 Oct 12;22(20):10994.
BPH-1 cells
3 µM and 9 µM
24 and 48 hours
To investigate the effect of neferine (NF) in the regulation of oxidative stress and apoptosis. Results showed that NF significantly activated Nrf2 and its downstream proteins HO-1 and NQO1, reduced ROS and lipid peroxidation, upregulated SOD and GST, and significantly reduced cell proliferation.
Redox Rep. 2021 Dec;26(1):1-9.
Human umbilical vein smooth muscle cells (HUVSMCs)
0.1, 0.5, 1.0, 5.0 µM
24 hours
Neferine significantly inhibited Ang II-induced HUVSMC proliferation in a concentration-dependent manner. Neferine at 5.0 µmol/L increased HO-1 expression by 259%.
Acta Pharmacol Sin. 2010 Jun;31(6):679-86.
Primary cortical neurons
10 µM
24 hours
Nef dramatically reversed the decreased viability of primary cortical neurons induced by t-BHP and increased the expression of Nrf2 in nucleus.
Br J Pharmacol. 2019 Feb;176(3):400-415.
PC12 cells
1-10 µM
24 hours
Nef protected PC12 cells from t-BHP-induced cell injury, significantly increased the cell viability, decreased both the release of LDH and the generation of ROS.
Br J Pharmacol. 2019 Feb;176(3):400-415.
HaCaT cells
1, 3, 10, 30 µM
24 hours
To evaluate the effect of neferine on cell viability, results showed no cytotoxicity was observed at concentrations as high as 30 μM.
Int J Mol Sci. 2021 Jul 30;22(15):8237.
RBL-2H3 cells
1-30 µM
24 hours
Test the cytotoxicity of Neferine on mast cells, showing no effect on cell viability at 1-30 μM.
Int J Mol Sci. 2021 Oct 12;22(20):10994.
3T3-L1 preadipocytes
1, 2.5, 5, 10 µM
24, 48, 72 hours
To evaluate the effect of Neferine on the differentiation and lipid accumulation of 3T3-L1 preadipocytes. Results showed that Neferine inhibited lipid accumulation in a dose-dependent manner and downregulated the expression of PPARγ, C/EBPα, and SREBP-1c.
Nutrients. 2020 Jun 22;12(6):1858.
RBL-2H3 cells
1, 3, 10 µM
30 minutes
Neferine inhibits PMA/A23187-induced phosphorylation of MAPK pathway (p38, JNK, ERK).
Int J Mol Sci. 2021 Oct 12;22(20):10994.
HEK-293 cells
0 to 100 µM
48 hours
Neferine showed minimal cytotoxicity on normal kidney cells HEK-293.
Phytother Res. 2020 Sep;34(9):2366-2384.
SiHa cells
5, 10, 25, 50 µM
48 hours
Neferine significantly inhibited the proliferation of SiHa cells in a dose-dependent manner, showing 50% growth inhibition at 25μM.
Phytother Res. 2020 Sep;34(9):2366-2384.
HeLa cells
5, 10, 25, 50 µM
48 hours
Neferine significantly inhibited the proliferation of HeLa cells in a dose-dependent manner, showing 50% growth inhibition at 25μM.
Phytother Res. 2020 Sep;34(9):2366-2384.
VCaP cells
75 µM
48 hours
To evaluate the antiproliferative effect of Nef on VCaP cells, results showed that Nef significantly inhibited cell proliferation and induced cell death via ROS and autophagy
Int J Mol Sci. 2023 Sep 18;24(18):14242.
LNCaP cells
25 µM
48 hours
To evaluate the antiproliferative effect of Nef on LNCaP cells, results showed that Nef significantly inhibited cell proliferation and induced cell death via ROS and autophagy
Int J Mol Sci. 2023 Sep 18;24(18):14242.
HEK293 cells
0.1–100 µM
500 ms
To investigate the inhibitory effects of Neferine on Kv4.3 channels, results showed that Neferine dose-dependently inhibited Kv4.3 currents with an IC50 value of 8.437 μmol/L and a maximal inhibition of 44.12% at 100 μmol/L.
Acta Pharmacol Sin. 2015 Dec;36(12):1451-61.
RBL-2H3 cells
1, 3, 10 µM
6 hours
Neferine reduces mRNA expression of pro-inflammatory cytokines (IL-1β, IL-6, TSLP, TNF-α) in PMA/A23187-stimulated RBL-2H3 cells.
Int J Mol Sci. 2021 Oct 12;22(20):10994.
RAW264.7 cells
25 µM and 50 µM
7 days
Composites containing Nef inhibited RANKL-induced osteoclast formation and significantly reduced TRAP activity. Nef downregulated the expression of NFATC1 by inhibiting the NF-κB signaling pathway, thereby inhibiting osteoclast differentiation.
J Biol Eng. 2023 Jul 17;17(1):45.
CAL27 cells
0, 7.5, 15, 22.5, 30 µM
72 hours
To detect the inhibitory effect of neferine on cell proliferation, results showed that neferine significantly reduced the viability of CAL27 cells
Int J Mol Med. 2021 Jul;48(1):124.
HN30 cells
0, 7.5, 15, 22.5, 30 µM
72 hours
To detect the inhibitory effect of neferine on cell proliferation, results showed that neferine significantly reduced the viability of HN30 cells
Int J Mol Med. 2021 Jul;48(1):124.
HN6 cells
0, 7.5, 15, 22.5, 30 µM
72 hours
To detect the inhibitory effect of neferine on cell proliferation, results showed that neferine significantly reduced the viability of HN6 cells
Int J Mol Med. 2021 Jul;48(1):124.
HEK293 cells
10 µM
90 minutes (cannabinoid receptors) and 60 minutes (opioid receptors)
To evaluate the binding affinity of Neferine for δ- and μ-opioid receptors. Results showed that Neferine displayed affinities for δ- and μ-opioid receptors with Ki values of 0.7±0.1 and 1.8±0.2 µM, respectively, and was determined to be a weak δ agonist and weak μ partial agonist by GTPγS functional assay.
J Ethnopharmacol. 2015 Nov 4;174:57-65.
BPH-1 cells
100 µM
To evaluate the toxicity of Nef on benign prostatic hyperplasia cells, results showed that Nef had some effect on BPH-1 cells at higher concentrations
Int J Mol Sci. 2023 Sep 18;24(18):14242.
Primary white adipocytes
1, 2.5, 5, 10 µM
To evaluate the effect of Neferine on the differentiation of primary white adipocytes. Results showed that Neferine significantly reduced the protein expression of PPARγ, C/EBPα, and SREBP-1c.
Nutrients. 2020 Jun 22;12(6):1858.
Neferine/甲基莲心碱 动物实验
Species
Animal Model
Administration
Dosage
Frequency
Description
References
New Zealand white rabbits
Rabbit left ventricular wedge model
Arterial perfusion
1, 3, 10 μM
Not used
To investigate the effects of Neferine on cardiac electrophysiology in rabbits, results showed that Neferine dose-dependently prolonged QT intervals and action potential durations (APD), with a dramatic increase in APD10 at epicardial sites.
Acta Pharmacol Sin. 2015 Dec;36(12):1451-61.
Rats
KA-induced seizure model
Intraperitoneal injection
10 and 50 mg/kg
Single administration, observed for 72 hours
To evaluate the neuroprotective effects of Neferine on KA-induced seizures, results showed that Neferine significantly delayed seizure onset, reduced seizure severity, and decreased neuronal loss.
Int J Mol Sci. 2022 Apr 8;23(8):4130
BALB/c nude mice
CAL27 xenograft model
Intraperitoneal injection
10 mg/kg
Tumor volume was measured every 4 days until tumor volume reached 1000 mm³
To evaluate the inhibitory effect of neferine on tumor growth, results showed that neferine significantly inhibited tumor growth
Int J Mol Med. 2021 Jul;48(1):124.
Male Swiss Webster mice
Mouse tetrad assay model
Intraperitoneal injection
10, 30, 75 and 100 mg/kg
Single administration, behavioral tests performed 30 minutes post-injection
To evaluate the behavioral effects of Neferine in mice. Results showed that the acidic CHCl3 partition (enriched with Neferine) displayed decreased locomotion, increased catalepsy, antinociception, and hypothermia at doses of 75–100 mg/kg/ip, and also showed clonic-tonic seizures upon touch at 100 mg/kg.
J Ethnopharmacol. 2015 Nov 4;174:57-65.
Sprague-Dawley rats
Permanent middle cerebral artery occlusion (pMCAO) model
Intragastric administration
12.5, 25, 50 mg/kg
15 min prior to or 6 h after 24 h MCAO
Nef significantly decreased neurological scores and infarct volumes, increased the regional CBF, improved cerebral microstructure and regulated the activity of oxidative stress-related enzymes.
Br J Pharmacol. 2019 Feb;176(3):400-415.
BALB/c mice
DNCB-induced atopic dermatitis model
Intraperitoneal injection
3 mg/kg and 10 mg/kg
Once daily for 14 days
Neferine significantly decreased the skin barrier damage, scratching responses, and epidermal hyperplasia induced by DNCB.
Int J Mol Sci. 2021 Jul 30;22(15):8237.
BALB/c mice
DNCB-induced atopic dermatitis model
Intraperitoneal injection
3 mg/kg and 10 mg/kg
10 consecutive days
Neferine improves dermatitis appearance and mast cell infiltration caused by DNCB, and restores the expression of skin barrier proteins.
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