COX-2; IL-1;βCaspase-3; Caspase-8; Caspase-9
Dopaminergic neurons - 6-OHDA is a neurotoxin that selectively destroys dopaminergic neurons by entering cells via dopamine transporters. It is widely used to induce Parkinson's disease (PD) symptoms in experimental models [1]
.
- Cyclooxygenase-2 (COX-2) - 6-OHDA induces COX-2 expression and nuclear translocation in neuronal cells, leading to PGE2 synthesis and pro-inflammatory cytokine production [1]
.
- EC50 values for cytotoxicity:
- Neuro-2a cells (mouse neuroblastoma): 111 μM (24 h incubation); 109 μM (48 h incubation) [1]
.
- SH-SY5Y cells (human neuroblastoma): 118 μM (24 h incubation); 107 μM (48 h incubation) [1]
.

Dopamine oxidation by hydrobromic acid (0-500 μM, 24 hours) decreases Neuro-2a cell survival and SH-SY5Y cell viability in a concentration-dependent way [1]. Dopamine oxidation with hydrobromic acid (75-150 μM, 0-24 h) stimulates the expression of COX-2. It also promotes the production of pro-inflammatory cytokines such IL-1β and PGE 2 biosynthesis [1]. Dopamine (0-150 μM, 12 h) and nuclear translocation are oxidized by hydrobromic acid [1]. Phosphorylation of p38 is induced by hydrobromic acid oxidation of dopamine (75 μM, 0–12 h) [3].

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Cytotoxicity in Neuronal Cell Lines: 6-OHDA decreased cell viability in both mouse Neuro-2a and human SH-SY5Y cells in a concentration-dependent manner, as measured by MTT reduction assay. After 24 h treatment, EC50 values were approximately 110-118 μM. Extending incubation to 48 h did not significantly increase cytotoxicity [1]
.
- COX-2 Induction: 6-OHDA rapidly and robustly induced COX-2 mRNA expression in a time-dependent manner. In Neuro-2a cells, 75 μM and 150 μM 6-OHDA significantly increased COX-2 mRNA at 24 h (P<0.05 and P<0.01, respectively). In SH-SY5Y cells, both concentrations significantly increased COX-2 mRNA at 24 h (P<0.05 and P<0.001, respectively) [1]
.
- COX-2 Nuclear Translocation: Immunochemistry revealed that 6-OHDA treatment caused translocation of COX-2 protein from the cytoplasm to the nucleus in both Neuro-2a and SH-SY5Y cells. The percentage of nuclear COX-2 significantly increased from approximately 25% in control cells to 40-45% in 6-OHDA-treated cells (P<0.001) [1]
.
- PGE2 Production: 6-OHDA stimulation substantially increased PGE2 levels in culture medium: nearly 5-fold in Neuro-2a cells (75 μM, 24 h, P<0.01) and 3-fold in SH-SY5Y cells (150 μM, 24 h, P<0.01). This increase was blocked by pretreatment with COX-2 inhibitor celecoxib (10 μM) or mPGES-1 inhibitor MPO-0057 (10 μM) [1]
.
- IL-1β Induction: 6-OHDA treatment significantly upregulated pro-inflammatory cytokine IL-1β mRNA levels. In Neuro-2a cells, 75 μM 6-OHDA increased IL-1β at 6 h (P<0.05) and 150 μM at 24 h (P<0.001). In SH-SY5Y cells, 150 μM 6-OHDA increased IL-1β at 6 h (P<0.05) [1]
.
- PGE2/EP2 Signaling: 6-OHDA-induced cytotoxicity was significantly reduced by EP2 receptor antagonists TG4-155 and TG6-10-1 in both cell lines (P<0.05 to P<0.001), but not by EP4 antagonist GW627368X. EP2 antagonists also reduced 6-OHDA-induced PGE2 secretion [1]
.
- Comparison with Celecoxib: COX-2 inhibitor celecoxib (10-20 μM) blocked up to 35% of 6-OHDA-induced cytotoxicity, similar to the protection afforded by EP2 antagonists, suggesting that COX-2-mediated injury is mostly attributed to EP2 receptor activation [1]
.

Degeneration of dopaminergic neurons in the substantia nigra is caused by hydrobromic acid oxidation of dopamine (5 μg/2 μL, injected unilaterally into the right striatum) [2].

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Cyclooxygenase-2 (COX-2) triggers pro-inflammatory processes that can aggravate neuronal degeneration and functional impairments in many neurological conditions, mainly via producing prostaglandin E2 (PGE2) that activates four membrane receptors, EP1-EP4. However, which EP receptor is the culprit of COX-2/PGE2-mediated neuronal inflammation and degeneration remains largely unclear and presumably depends on the insult types and responding components. Herein, we demonstrated that COX-2 was induced and showed nuclear translocation in two neuronal cell lines - mouse Neuro-2a and human SH-SY5Y - after treatment with neurotoxin 6-hydroxydopamine (6-OHDA), leading to the biosynthesis of PGE2 and upregulation of pro-inflammatory cytokine interleukin-1β. Inhibiting COX-2 or microsomal prostaglandin E synthase-1 suppressed the 6-OHDA-triggered PGE2 production in these cells. Treatment with PGE2 or EP2 selective agonist butaprost, but not EP4 agonist CAY10598, increased cAMP response in both cell lines. PGE2-initiated cAMP production in these cells was blocked by our recently developed novel selective EP2 antagonists - TG4-155 and TG6-10-1, but not by EP4 selective antagonist GW627368X. The 6-OHDA-promoted cytotoxicity was largely blocked by TG4-155, TG6-10-1 or COX-2 selective inhibitor celecoxib, but not by GW627368X. Our results suggest that PGE2 receptor EP2 is a key mediator of COX-2 activity-initiated cAMP signaling in Neuro-2a and SH-SY5Y cells following 6-OHDA treatment, and contributes to oxidopamine-mediated neurotoxicity [1].

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Cell Viability Assay[1]
Cell Types: Neuro-2a cells and SH-SY5Y cells
Tested Concentrations: 0-500 µM
Incubation Duration: 24 or 48 hrs (hours)
Experimental Results: Induction of neurotoxicity in Neuro-2a cells and SH-SY5Y cells in a concentration-dependent manner Causes cytotoxicity in SY5Y cells. In Neuro-2a cells, EC50=111 µM (incubation for 24 hrs (hours)) and 109 µM (incubation for 48 hrs (hours)); in SH-SY5Y cells, EC50=118 µM for 24 hrs (hours) and EC50=107 µM for 48 hrs (hours).

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RT-PCR[1]
Cell Types: Neuro-2a cells and SH-SY5Y cells
Tested Concentrations: 75 or 150 µM
Incubation Duration: 0, 6 or 24 hrs (hours)
Experimental Results: Rapid and robust induction of COX-2 in a time-dependent manner. Induces COX-2 activation, characterized by induction of expression and nuclear translocation. PGE2 in the culture medium increased Dramatically by nearly 5-fold in Neuro-2a cells (75 µM) and 3-fold in SH-SY5Y cells (150 µM). The pro-inflammatory cytokine interleukin 1β (IL-1β) was Dramatically upregulated in Neuro-2a cells and SH-SY5Y cells.

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Apoptosis analysis [3]
Cell Types: PC12 cells
Tested Concentrations: 0, 25, 50, 75, and 150 μM
Incubation Duration: 0, 2, 4, 6, 12, and 20 h
Experimental Results: Induced apoptosis of PC12 cells. Increased the activities of caspase-3, -8 and -9 in PC12 cells in a time- and concentration-dependent manner. Increased these caspase activities at 2-4 h and reached a maximum at 12 h. diminished cells with high mitochondrial membrane potential (JC-1 aggregate) in a time- and concentration-dependent manner.

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Western Blot Analysis[3]
Cell Types: PC12 cells
Tested Concentrations: 75 μM
Incubation Duration: 0, 3, 5, 6, 8, 10, and 12 h
Experimental Results: Increased the level of p-p38 in a time-dependent manner.

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Cell Culture: Neuro-2a cells were cultured in DMEM with 10% FBS, 1% MEM non-essential amino acids, penicillin/streptomycin. SH-SY5Y cells were cultured in 50% EMEM + 50% Ham's F-12 with 10% FBS, penicillin/streptomycin. Both were maintained at 37°C with 5% CO₂ [1]
.
- Cell Viability Assay (MTT): Cells were seeded in 96-well plates (5,000 cells/well). After treatment with various concentrations of 6-OHDA for 24 or 48 h, MTT (0.5 mg/mL) was added and incubated for 4 h at 37°C. Formazan was dissolved in DMSO and absorbance measured at 540 nm (reference 630 nm). Dose-response curves were generated and EC50 values calculated using OriginPro software [1]
.
- Immunocytochemistry: Cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, blocked with 10% horse serum, and incubated with primary antibodies against NeuN, tyrosine hydroxylase (TH), or COX-2 overnight, followed by Alexa Fluor-conjugated secondary antibodies and DAPI staining. Images were obtained using EVOS FL Auto Cell Imaging System. Fluorescence intensity was quantified using ImageJ software [1]
.
- Quantitative PCR (qPCR): Total RNA was isolated using Trizol with PureLink RNA mini kit. cDNA was synthesized from 1 μg total RNA using SuperScript III One-Step RT-PCR System. qPCR was performed using SYBR Green PCR Master Mix with primers for COX-2, IL-1β, and GAPDH. Cycling conditions: 95°C for 2 min, 40 cycles of 95°C for 15 s, 60°C for 1 min. Relative quantification was normalized to GAPDH [1]
.
- PGE2 Measurement: Cells were cultured in 24-well plates (90,000 cells/well for Neuro-2a; 150,000 cells/well for SH-SY5Y). After treatment, 50 μL medium was collected and PGE2 levels measured by ELISA according to manufacturer's protocol. Absorbance was measured at 450 nm [1]
.
- TR-FRET cAMP Assay: Cells were seeded in 384-well plates (4,000 cells/well) overnight. Medium was replaced with HBSS containing 20 μM rolipram. After 30 min incubation, cells were treated with compounds, then with EP agonists for 40 min. Cells were lysed with cAMP-d2 and anti-cAMP-cryptate. FRET signal was measured at 340 nm excitation with dual emissions at 665 nm and 590 nm. FRET signal was expressed as F665/F590 × 10⁴ [1]
.

The present study was undertaken to investigate the neuroprotective effects of resveratrol on 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease in rats. 6-OHDA-induced Parkinson's disease rat model involves chronic inflammation, mitochondrial dysfunction, and oxidative stress, and the loss of the dopaminergic neurons in the substantia nigra is the predominant lesion. Resveratrol has been shown to have anti-inflammatory actions, and thus was tested for its beneficial effects using 6-OHDA-induced Parkinson's disease rat model. Adult Sprague-Dawley (SD) rats were unilaterally injected with 6-OHDA (5 microg/2 microl) into the right striatum, and the striatum damage was assessed by rotational test, ultrahistopathology, and molecular alterations. Resveratrol (10, 20 and 40 mg/kg) was then given orally to Parkinson's disease rats, daily for 10 weeks to examine the protective effects. Rotational test (turns of rats) showed that resveratrol significantly attenuated apomorphine-induced turns of rats in 6-OHDA-injuried Parkinson's disease rat model as early as two weeks of administration. Ultrastructural analysis showed that resveratrol alleviated 6-OHDA-induced chromatin condensation, mitochondrial tumefaction and vacuolization of dopaminergic neurons in rat substantia nigra. Furthermore, resveratrol treatment also significantly decreased the levels of COX-2 and TNF-alpha mRNA in the substantia nigra as detected by real-time RT-PCR. COX-2 protein expression in the substantia nigra was also decreased as evidenced by Western blotting. These results demonstrate that resveratrol exerts a neuroprotective effect on 6-OHDA-induced Parkinson's disease rat model, and this protection is related to the reduced inflammatory reaction.[2].

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Cytotoxicity Profile: 6-OHDA causes concentration-dependent cell death in neuronal cell lines with EC50 values of 107-118 μM after 24-48 h exposure. Both Neuro-2a and SH-SY5Y cells showed similar sensitivity [1]
.
- Mechanism of Toxicity: 6-OHDA promotes neuronal inflammation through COX-2 induction, leading to PGE2 synthesis, IL-1β upregulation, and activation of EP2 receptor-mediated cAMP signaling. These inflammatory processes contribute to 6-OHDA-induced neurotoxicity [1]
.

[1]. Cyclooxygenase-2 contributes to oxidopamine-mediated neuronal inflammation and injury via the prostaglandin E2 receptor EP2 subtype. Sci Rep. 2017 Aug 25;7(1):9459.

[2]. Neuroprotective effect of resveratrol on 6-OHDA-induced Parkinson's disease in rats. Eur J Pharmacol. 2008 Dec 14;600(1-3):78-82.

[3]. Cell-permeable cAMP analog suppresses 6-hydroxydopamine-induced apoptosis in PC12 cells through the activation of the Akt pathway. Brain Res. 2006 Oct 3;1113(1):10-23.

[4]. Autoxidation and neurotoxicity of 6-hydroxydopamine in the presence of some antioxidants: potential implication in relation to the pathogenesis of Parkinson's disease. J Neurochem. 2000 Apr;74(4):1605-12.

neurotransmitter analogue that depletes norepinephrine reserves at nerve endings and leads to decreased dopamine levels in the brain. Its mechanism of action is related to the production of cellular lysogenic free radicals.

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Chemical Identity and Background: 6-OHDA (2,4,5-trihydroxyphenethylamine) is a synthetic analog of dopamine. It is a neurotoxin widely used to induce Parkinson's disease symptoms in experimental animals due to its ability to selectively destroy dopaminergic neurons via dopamine transporter-mediated uptake. The molecular mechanisms underlying its neurotoxicity remain largely unknown [1]
.
- Use in PD Research: 6-OHDA is commonly used to model Parkinson's disease in vitro and in vivo. The neuroblastoma cell lines Neuro-2a (mouse-derived) and SH-SY5Y (human-derived) preserve many aspects of substantia nigra pars compacta (SNpc) neurons and are widely used as in vitro models to study inflammation, oxidative stress, and apoptosis in dopaminergic neurons [1]
.
- Mechanism of Action in This Study: This study demonstrates that 6-OHDA rapidly induces COX-2 expression and nuclear translocation in neuronal cells, leading to PGE2 biosynthesis and pro-inflammatory cytokine (IL-1β) production. The neurotoxin activates cAMP signaling primarily through the PGE2 receptor EP2 subtype, and EP2 receptor inhibition provides neuroprotection against 6-OHDA-induced cytotoxicity [1]
.
- Dosing in Cell Culture: The study used 75 μM and 150 μM 6-OHDA for most experiments, corresponding approximately to EC25 and EC75 based on cytotoxicity curves. For Neuro-2a cells, 75 μM was used; for SH-SY5Y cells, 150 μM was used due to their higher COX-2 induction at this concentration [1]
.
- Time Course: 6-OHDA effects were examined at 6 h and 24 h time points. COX-2 induction, PGE2 production, and IL-1β upregulation were observed at both time points, with maximal effects typically at 24 h [1]
.
- Source: 6-OHDA used in this study was purchased from Sigma-Aldrich [1]
.

C8H12BRNO3

250.09

249

C, 38.42; H, 4.84; Br, 31.95; N, 5.60; O, 19.19

636-00-0

Oxidopamine hydrochloride;28094-15-7; 1199-18-4; 636-00-0 (HBr)

176170

Light brown to gray solid

406ºC at 760 mmHg

216-220 °C(lit.)

199.3ºC

1.963

5

4

2

13

142

0

OC1=CC(CCN)=C(O)C=C1O.[H]Br

MLACDGUOKDOLGC-UHFFFAOYSA-N

InChI=1S/C8H11NO3.BrH/c9-2-1-5-3-7(11)8(12)4-6(5)10;/h3-4,10-12H,1-2,9H2;1H

2,4,5-Trihydroxyphenethylamine hydrobromide

6-Hydroxydopamine hydrobromide; 6-OHDA hydrobromide; 6-OHDA HBr; 6 OHDA HBr; 6OHDA HBr; 6-OHDA Hydrobromide; 6 OHDA Hydrobromide; 6OHDA Hydrobromide; 6-Hydroxydopamine hydrobromide; 636-00-0; Oxidopamine hydrobromide; Oxidopamine (hydrobromide); 6-hydroxydopamine hbr; 6-OHDA; 2,4,5-Trihydroxyphenethylamine hydrobromide; 5-(2-aminoethyl)benzene-1,2,4-triol hydrobromide; 6-Hydroxydopamine Hydrobromide; 6Hydroxydopamine Hydrobromide; 6 Hydroxydopamine Hydrobromide; 6-Hydroxydopamine HBr; 6 Hydroxydopamine HBr

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.  (2). This product is not stable in solution, please use freshly prepared working solution for optimal results.

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 (~199.93 mM)
H2O : ~20 mg/mL (~79.97 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (10.00 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: ≥ 2.08 mg/mL (8.32 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 20.8 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: 50 mg/mL (199.93 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.)