LPS (10 ng/mL)-stimulated cell migration was significantly inhibited by nociceptin (1 μg/mL), but addition on its own had no effect. Nociceptin (1 nM–10 μM) inhibits LPS-mediated cell migration in a concentration-dependent manner, with maximal effects occurring at 1 and 10 μM. Nociceptin has the ability to counteract the increase in IL-1β mRNA levels caused by LPS. In U87 cells, apoptosis is induced by NNC 55-0396 and nociceptin (1 μM). Through β-arrestin 2, nociceptin (1 μM) inhibits the rise in [Ca2+]i brought on by LPS in U87 cells. In U87 cells, nociceptin inhibits the phosphorylation of ERK and PKC brought on by LPS. The transcriptional activation of NF-kB and AP-1 reporter genes by LPS is inhibited by nociceptin [1].

LPS (10 ng/mL)-stimulated cell migration was significantly inhibited by nociceptin (1 μg/mL), but addition on its own had no effect. Nociceptin (1 nM–10 μM) inhibits LPS-mediated cell migration in a concentration-dependent manner, with maximal effects occurring at 1 and 10 μM. Nociceptin has the ability to counteract the increase in IL-1β mRNA levels caused by LPS. In U87 cells, apoptosis is induced by NNC 55-0396 and nociceptin (1 μM). Through β-arrestin 2, nociceptin (1 μM) inhibits the rise in [Ca2+]i brought on by LPS in U87 cells. In U87 cells, nociceptin inhibits the phosphorylation of ERK and PKC brought on by LPS. The transcriptional activation of NF-kB and AP-1 reporter genes by LPS is inhibited by nociceptin [1].

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N/OFQ (1 μM) antagonizes lipopolysaccharide (LPS, 10 ng/ml)-stimulated cell migration in human glioblastoma U87 cells in a wound healing assay. This inhibitory effect is prevented by the NOPr antagonist [NPhe¹]N/OFQ(1-13)NH₂ (10 μM). [1]
N/OFQ (1 μM) blocks LPS (10 ng/ml)-induced cell proliferation in U87 cells, as measured by [methyl-³H] thymidine incorporation. This effect is also prevented by the NOPr antagonist [NPhe¹]N/OFQ(1-13)NH₂ (10 μM). [1]
N/OFQ (1 μM) counteracts the LPS (10 ng/ml)-induced increase in IL-1β mRNA levels in U87 cells, as determined by real-time RT-PCR. [1]
N/OFQ (1 μM) induces apoptosis in U87 cells independently of LPS, as shown by annexin V/PI staining and increased caspase-3/7 activity. The pro-apoptotic effect is blocked by the NOPr antagonist [NPhe¹]N/OFQ(1-13)NH₂ (10 μM). [1]
N/OFQ (1 μM) antagonizes the LPS (10 ng/ml)-induced increase in intracellular free Ca²⁺ concentration ([Ca²⁺]i) in U87 cells, measured by rationetric microfluorometry using fura-2 AM. [1]
N/OFQ (1 μM) counteracts LPS (10 ng/ml)-induced phosphorylation of PKC and ERK 1/2 in U87 cells, as assessed by Western blot analysis. [1]
N/OFQ (1 μM) inhibits LPS (10 ng/ml)-mediated transcriptional activation of NF-κB and AP-1 reporter genes in U87 cells, as determined by a dual-luciferase reporter assay. [1]
Saturation binding assays using [¹²⁵I]-N/OFQ on U87 cell membranes determined a Bmax of 213 ± 12.5 fmol/mg protein and a Kd of 0.97 ± 0.25 nM for NOPr. [1]

Wound Healing Assay: U87 cells were grown to confluence in culture dishes. A sterile pipette tip was used to create scratches in the cell monolayer. After washing, serum-free medium containing the test compounds was added. Cell migration into the wound area was assessed after 18 hours by microscopy and quantified using analysis software. [1]
Cell Proliferation Assay: U87 cells were plated and treated for 24 hours. [methyl-³H] Thymidine was added for the final 5 hours. Cells were then harvested, and DNA was precipitated with trichloroacetic acid. Incorporated radioactivity was measured by liquid scintillation counting. [1]
Real-time RT-PCR: Total RNA was extracted from treated U87 cells, reverse transcribed, and amplified using gene-specific primers for IL-1β and NOPr. The L19 ribosomal protein gene served as an internal control. Relative mRNA levels were calculated using the ΔΔCT method. [1]
Cell Apoptosis Detection (Flow Cytometry): Treated U87 cells were stained with phycoerythrin-conjugated annexin V and 7-amino-actinomycin D (7-AAD) and analyzed by flow cytometry to distinguish viable, early apoptotic, and late apoptotic/necrotic cell populations. [1]
Caspase-3/7 Activation Assay: U87 cells were treated in 96-well plates. A caspase-3/7 substrate reagent was added, and after incubation, fluorescence was measured to determine caspase activity. [1]
Calcium Microfluorometry: U87 cells grown on coverslips were loaded with the calcium indicator fura-2 AM. Intracellular calcium concentration ([Ca²⁺]i) was monitored rationetrically in single cells using fluorescence microscopy upon stimulation with test compounds. [1]
Western Blotting: Treated U87 cells were lysed, and proteins were separated by SDS-PAGE, transferred to membranes, and probed with antibodies against phospho-PKC, phospho-ERK1/2, total ERK1/2, and β-actin. Signal was detected using enhanced chemiluminescence. [1]
Dual Luciferase Assay: U87 cells were co-transfected with firefly luciferase reporter plasmids containing NF-κB or AP-1 response elements and a Renilla luciferase control plasmid. After treatment, firefly and Renilla luciferase activities were measured sequentially. [1]
Saturation Binding Assay: Membranes prepared from U87 cells were incubated with increasing concentrations of [¹²⁵I]-N/OFQ with or without an unlabeled competitor. Bound radioligand was separated by filtration, and specific binding was analyzed to determine receptor density (Bmax) and equilibrium dissociation constant (Kd). [1]
Radioimmunoassay (RIA) for N/OFQ: Cell culture medium or cell homogenates from treated U87 cells were analyzed using a specific RIA kit to quantify immunoreactive N/OFQ (ir-N/OFQ) levels. [1]

Treatment with N/OFQ (1 μM) for 24 hours induced apoptosis in human glioblastoma U87 cells. [1]

[1]. Nociceptin/orphanin FQ antagonizes lipopolysaccharide-stimulated proliferation, migration and inflammatory signaling in human glioblastoma U87 cells. Biochem Pharmacol. 2017 Sep 15;140:89-104.

Nociceptin is a heptadecapeptide composed of phenylalanine (Phe), glycine (Gly), phenylalanine (Phe), threonine (Thr), glycine (Gly), alanine (Ala), arginine (Arg), lysine (Lys), serine (Ser), alanine (Ala), arginine (Arg), lysine (Lys), leucine (Leu), alanine (Ala), asparagine (Asn), and glutamine (Gln) residues linked in sequence. It is a metabolite found in both humans and rats. It is an organic molecular entity belonging to the polypeptide family. It is a pain-promoting peptide that can act as a specific endogenous agonist of the nociceptin receptor. N/OFQ is a heptadecapeptide that shares sequence homology with the opioid peptide dynorphin, but exhibits extremely low binding potency to classical opioid receptors. [1]
The binding of N/OFQ to NOPr affects glial cell function and may antagonize pro-inflammatory responses. [1]
Prolonged exposure of U87 cells to LPS (10 ng/ml) (72–96 h) downregulates NOPr mRNA expression and reduces the density of NOPr protein on the cell membrane. Therefore, under these conditions, N/OFQ loses its inhibitory effect on LPS-mediated cell migration, proliferation, and IL-1β mRNA increase. [1]
The inhibitory effect of N/OFQ on LPS-induced U87 cell responses is mediated by β-arrestin 2 and involves downstream signaling pathway components such as TRAF6, c-Src, PKC, and ERK 1/2. [1]
LPS treatment increases the content and release of N/OFQ in U87 cells, but this autocrine/paracrine effect does not lead to LPS-induced downregulation of NOPr mRNA. [1]

C79H129N27O22

1809.03727602959

1807.98

170713-75-4

16131448

White to off-white solid powder

1.5±0.1 g/cm3

1.657

-6.33

30

27

64

128

3720

15

C[C@H]([C@@H](C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)CNC(=O)CNC(=O)[C@H](CC2=CC=CC=C2)N)O

PULGYDLMFSFVBL-SMFNREODSA-N

InChI=1S/C79H129N27O22/c1-41(2)33-54(72(122)95-44(5)66(116)103-56(36-59(84)110)73(123)102-53(77(127)128)27-28-58(83)109)104-70(120)49(23-13-15-29-80)100-69(119)52(26-18-32-90-79(87)88)99-65(115)43(4)96-75(125)57(40-107)105-71(121)50(24-14-16-30-81)101-68(118)51(25-17-31-89-78(85)86)98-64(114)42(3)94-61(112)39-93-76(126)63(45(6)108)106-74(124)55(35-47-21-11-8-12-22-47)97-62(113)38-91-60(111)37-92-67(117)48(82)34-46-19-9-7-10-20-46/h7-12,19-22,41-45,48-57,63,107-108H,13-18,23-40,80-82H2,1-6H3,(H2,83,109)(H2,84,110)(H,91,111)(H,92,117)(H,93,126)(H,94,112)(H,95,122)(H,96,125)(H,97,113)(H,98,114)(H,99,115)(H,100,119)(H,101,118)(H,102,123)(H,103,116)(H,104,120)(H,105,121)(H,106,124)(H,127,128)(H4,85,86,89)(H4,87,88,90)/t42-,43-,44-,45+,48-,49-,50-,51-,52-,53-,54-,55-,56-,57-,63-/m0/s1

(2S)-5-amino-2-[[(2S)-4-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-6-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-6-amino-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S,3R)-2-[[(2S)-2-[[2-[[2-[[(2S)-2-amino-3-phenylpropanoyl]amino]acetyl]amino]acetyl]amino]-3-phenylpropanoyl]amino]-3-hydroxybutanoyl]amino]acetyl]amino]propanoyl]amino]-5-carbamimidamidopentanoyl]amino]hexanoyl]amino]-3-hydroxypropanoyl]amino]propanoyl]amino]-5-carbamimidamidopentanoyl]amino]hexanoyl]amino]-4-methylpentanoyl]amino]propanoyl]amino]-4-oxobutanoyl]amino]-5-oxopentanoic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~27.64 mM)
H2O : ≥ 50 mg/mL (~27.64 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (1.38 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (1.38 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (1.38 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 50 mg/mL (27.64 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)