mGluR/metabotropic glutamate receptor; phosphoserine phosphatase

DL-AP3 could attenuate OGD-induced injury by controlling neuronal viability and apoptosis. In addition, the protective effects of DL-AP3 on neurons might be associated with the expression of p-Akt1 and cytochrome C proteins.Study may provide a theoretical basis for the possible application of DL-AP3 in treating cerebral infarction. However, whether DL-AP3 can be used in clinical treatment of cerebral infarction still requires long-term investigations in in vivo models.[1]

---

Cerebral infarction is a type of ischemic stroke and is one of the main causes of irreversible brain damage. Although multiple neuroprotective agents have been investigated recently, the potential of DL-2-amino-3-phosphonopropionic acid (DL-AP3) in treating oxygen-glucose deprivation (OGD)-induced neuronal injury, has not been clarified yet. This study was aimed to explore the role of DL-AP3 in primary neuronal cell cultures. Primary neurons were divided into four groups: (1) a control group that was not treated; (2) DL-AP3 group treated with 10 μM of DL-AP3; (3) OGD group, in which neurons were cultured under OGD conditions; and (4) OGD + DL-AP3 group, in which OGD model was first established and then the cells were treated with 10 μM of DL-AP3. Neuronal viability and apoptosis were measured using Cell Counting Kit-8 and flow cytometry. Expressions of phospho-Akt1 (p-Akt1) and cytochrome c were detected using Western blot. The results showed that DL-AP3 did not affect neuronal viability and apoptosis in DL-AP3 group, nor it changed p-Akt1 and cytochrome c expression (p > 0.05). In OGD + DL-AP3 group, DL-AP3 significantly attenuated the inhibitory effects of OGD on neuronal viability (p < 0.001), and reduced OGD induced apoptosis (p < 0.01). Additionally, the down-regulation of p-Akt1 and up-regulation of cytochrome c, induced by OGD, were recovered to some extent after DL-AP3 treatment (p < 0.05 or p < 0.001). Overall, DL-AP3 could protect primary neurons from OGD-induced injury by affecting the viability and apoptosis of neurons, and by regulating the expressions of p-Akt1 and cytochrome c. [1]

---

DL-AP3 alleviated OGD-induced injury [1]
To explore the effects of DL-AP3 on neurons under hypoxic conditions, neurons were cultured under OGD conditions and treated with DL-AP3. DL-AP3 did not affect neuronal viability (p > 0.05) in DL-AP3 group. In OGD group, OGD significantly reduced neuronal viability after 24 and 72 hours (p < 0.001). However, DL-AP3 significantly attenuated the inhibitory effect of OGD on neuronal viability (p < 0.001), in OGD + DL-AP3 group. The results of cell viability assay in all groups are shown in Figure 1A. [1]
In DL-AP3 group, DL-AP3 had no significant effect on apoptosis (p > 0.05). However, OGD significantly increased the apoptotic cell rate in OGD group (p < 0.001), but this effect was significantly attenuated in OGD + DL-AP3 group (p < 0.01). The results of cell apoptosis in the four groups are presented in Figure 1B.

---

DL-AP3 protected neurons against OGD-induced injury by regulating p-Akt1 and cytochrome C expression [1]
To further investigate the molecular mechanism underlying the effect of DL-AP3 on OGD-induced injury, the protein expressions of p-Akt1 and cytochrome C in neurons were determined. In DL-AP3 group, DL-AP3 did not change the protein levels of p-Akt1 and cytochrome C (p > 0.05). However, OGD significantly down-regulated the level of p-Akt1 (p < 0.001) and up-regulated the level of cytochrome C (p < 0.001) in OGD group. Nevertheless, DL-AP3 significantly recovered the decreased levels of p-Akt1 and the increase of cytochrome C in OGD + DL-AP3 group (p < 0.001 and p < 0.05, respectively). The results of p-Akt1 and cytochrome C protein expression in the four groups are illustrated in Figure 2A and B.

Primary neurons were divided into four groups: (1) A control group that was not treated; (2) DL-AP3 group treated with 10 µM of DL-AP3 for 6 hours; (3) OGD group, in which neurons were cultured under OGD conditions for 12 hours; (4) OGD + DL-AP3 group, in which OGD model was first established and then the cells were treated with 10 µM of DL-AP3 for 6 hours.[1]
OGD model was established as previously described. Briefly, neurons were washed with glucose-free Earle’s Balanced Salt Solution and then were placed in a modular incubator chamber filled with gas mixture (95% N2 and 5% CO2) at 37°C. To terminate OGD, neurons were incubated under normal conditions.

---

Cell viability analysis [1]
Neuronal viability was determined using Cell Counting Kit-8, according to the manufacturer’s instructions. Briefly, primary neurons in the four groups were collected and seeded in 96-well plates with 2 × 103 cells/well. After 24-72-hour incubation, 20 µL of CCK-8 was added to each well and incubated for another 3 hours. Finally, the absorbance was measured by a microplate reader at a wavelength of 450 nm.

---

Apoptosis analysis [1]
Cell apoptosis was performed by Annexin V-FITC Apoptosis Detection Kit, according to the manufacturer’s instructions. Briefly, neurons in the four groups were collected and re-suspended in 200 µL of binding buffer containing 10 µL of Annexin-V-FITC. After 30 minutes of incubation in the dark at room temperature, 300 µL of phosphate buffered saline and 5 µL of propidium iodide solution were added into each sample and then the apoptotic cells were analyzed using flow cytometry.

---

Western blot analysis [1]
Cells in the four groups were collected and lysed in the lysis buffer. The protein concentration of the supernatant was determined using the BCA Protein Assay Kit, according to the manual. Equal amounts of proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. Then, the membranes were stained with primary antibodies: p-Akt1 (1:1000, ab66138), cytochrome C (1:5000; ab133504) or actin (1:1000; ab1801) overnight at 4°C. Subsequently, the blots were incubated with the horseradish peroxidase conjugated secondary antibodies for 1 hour at room temperature. The bands were visualized with the enhanced chemiluminescence detection kit, and data were analyzed using Image Lab software

[1]. DL-2-amino-3-phosphonopropionic acid protects primary neurons from oxygen-glucose deprivation induced injury. Bosn J Basic Med Sci. 2017 Feb 21;17(1):12-16.

2-amino-3-phosphonopropanoic acid is a non-proteinogenc alpha-amino acid that is alanine in which one of the hydrogens of the terminal methyl group has been replaced by a dihydroxy(oxido)-lambda(5)-phosphanyl group. It has a role as a metabotropic glutamate receptor antagonist and a human metabolite. It is a non-proteinogenic alpha-amino acid, a member of phosphonic acids and an alanine derivative.
1-Amino-propan-2-one-3-phosphate is a metabolite found in or produced by Escherichia coli (strain K12, MG1655).

---

In this study, we found that DL-AP3 could recover the down-regulation effects of OGD on the protein expression of p-Akt1 and could suppress the release of cytochrome C induced by OGD. These findings imply that the possible mechanism of DL-AP3 in protecting neurons from injury might be via up-regulating the expression of p-Akt1 and suppressing the release of cytochrome C.
Our results demonstrated that DL-AP3 could attenuate OGD-induced injury by controlling neuronal viability and apoptosis. In addition, the protective effects of DL-AP3 on neurons might be associated with the expression of p-Akt1 and cytochrome C proteins. Our study may provide a theoretical basis for the possible application of DL-AP3 in treating cerebral infarction. However, whether DL-AP3 can be used in clinical treatment of cerebral infarction still requires long-term investigations in in vivo models. [1]

C3H8NO5P

169.0728

169.014

C, 21.31; H, 4.77; N, 8.28; O, 47.31; P, 18.32

20263-06-3

3857

Solid powder

1.763 g/cm3

481.6ºC at 760 mmHg

227-229 °C(lit.)

245.1ºC

1.34E-10mmHg at 25°C

1.558

-5.2

4

6

3

10

174

0

P(CC(C(O)=O)N)(O)(O)=O

LBTABPSJONFLPO-UHFFFAOYSA-N

InChI=1S/C3H8NO5P/c4-2(3(5)6)1-10(7,8)9/h2H,1,4H2,(H,5,6)(H2,7,8,9)

2-amino-3-phosphonopropanoic acid

DL-AP-3; DL-AP3;DL-AP 3; AP3; 5652-28-8; 2-amino-3-phosphonopropanoic acid; 2-Amino-3-phosphonopropionic acid; 3-phosphonoalanine; Phosphonoalanine; DL-2-Amino-3-phosphonopropionic acid; 2-Amino-3-phosprop; ...; 20263-06-3; AP 3; AP-3

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

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