Perinatology.  2019 Sep;30(3):117-125. 10.14734/PN.2019.30.3.117.

SYM 2081 Exerts Neuroprotective Effect Modulating Anti-Apoptosis in the Hypoxic-Ischemic Brain Injury of Neonatal Rats

• Park JK
• Jang YY
• Jeong JE
• Lee EJ
• Choi EJ
• Chung HL
• Kim WT

• KMID: 2460024
• DOI: http://doi.org/10.14734/PN.2019.30.3.117

Abstract

OBJECTIVE
SYM 2081 ((2S,4R)-4-methylglutamic acid), an agonist and functional antagonist of the kainite receptor, has shown a neuroprotective effect in neurodegenerative diseases in adults but little is known concerning perinatal hypoxic-ischemic encephalopathy. This study is designed to evaluate whether SYM 2081 has preventive mechanisms via anti-apoptosis to gain further insight into neuroprotective roles of SYM 2081.
METHODS
In an in vivo animal model, the left carotid artery was ligated in 7-day-old Sprague-Dawley rat pups. The pups were divided into six groups: normoxia (N), hypoxia (8% Oâ‚‚, 92% Nâ‚‚) (H), H with shamoperation, H with operation (HO), HO treated with vehicle and HO treated with SYM 2081. In an in vitro model, the cultured embryonic cortical neuronal cells were divided into three groups: normoxia (95% air, 5% COâ‚‚), hypoxia (94% Nâ‚‚, 5% COâ‚‚) (Hc), and Hc treated with SYM 2081 before a hypoxic insult. Apoptosis was assessed using western blots and real-time polymerase chain reaction with Bcl-2, Bax, and caspase-3 antibodies and mRNAs.
RESULTS
In both in vitro and in vivo studies, Bcl-2 expression increased, whereas expressions of Bax, caspase-3, and the ratio of Bax/Bcl-2 were reduced with SYM 2081 treatment resulting in improved cell survival.
CONCLUSION
This study showed that SYM 2081 exerts a neuroprotective effect against hypoxic-ischemic injury through modulating apoptotic signaling pathways.

Keyword

4-methylglutamic acid; Hypoxia-ischemia; Brain; Apoptosis

MeSH Terms

Adult
Animals
Anoxia
Antibodies
Apoptosis
Blotting, Western
Brain Injuries*
Brain*
Carotid Arteries
Caspase 3
Cell Survival
Humans
Hypoxia-Ischemia, Brain
In Vitro Techniques
Models, Animal
Neurodegenerative Diseases
Neurons
Neuroprotective Agents*
Rats*
Rats, Sprague-Dawley
Real-Time Polymerase Chain Reaction
RNA, Messenger
Antibodies
Caspase 3
Neuroprotective Agents
RNA, Messenger

Figure

• 

  Fig. 1 Western blots of Bcl-2 (A; N, 100±2.2; H, 79.3±1.6; HS, 88.7±1.8; HO, 70.1±1.4; HV, 73.1± 1.5; HM, 77.8±1.6), Bax (B; N, 100±4.1; H, 88.1±3.5; HS, 94.4±3.8; HO, 144.7±5.9; HV, 127.8± 5.1; HM, 62.2±2.5), and caspase-3 (C; N, 100±3.2; H, 99.9±3.0; HS, 110.0±3.3; HO, 126.0±3.8; HV, 119.5±3.6; HM, 103.0±3.1) in the neonatal hypoxic-ischemic brain injury (in vivo) and the ratio of Bax/Bcl-2 expression (D). Data were shown as means±standard deviation (n=4). The (2S,4R)-4-Methylglutamic Acid (SYM 2081) was administered at 10 mg/kg. N, normoxia; H, hypoxia without operation; HS, hypoxia with Sham operation; HO, hypoxia with operation; HV, HO treated with vehicle; HM, HO treated with (2S,4R)-4-Methylglutamic Acid (SYM 2081). *P<0.05, statistically significant vs. HO.

• 

  Fig. 2 Real-time PCRs of Bcl-2 (A; N, 100±4.5; H, 85.3±3.4; HS, 99.3±3.9; HO, 72.2±2.8; HV, 51.1± 2.0; HM, 70.7±2.8), Bax (B; N, 100±4.1; H, 109.8±4.3; HS, 102.1±4.0; HO, 184.7±7.3; HV, 142.9± 5.7; HM, 102.1±4.1), and caspase-3 (C; N, 100±2.4; H, 116.1±2.3; HS, 118.1±2.5; HO, 224.2±4.4; HV, 215.8±4.3; HM, 78.2±1.6) mRNAs in the neonatal hypoxic-ischemic brain injury (in vivo) and the ratio of Bax/Bcl-2 expression (D). Data were shown as means±standard deviation (n=6). The (2S,4R)-4-Methylglutamic Acid (SYM 2081) was administered at 10 mg/kg. N, normoxia; H, hypoxia without operation; HS, hypoxia with Sham operation; HO, hypoxia with operation; HV, HO treated with vehicle; HM, HO treated with (2S,4R)-4-Methylglutamic Acid (SYM 2081). *P<0.05, statistically significant vs. HO; †P<0.05, statistically significant vs. HV.

• 

  Fig. 3 Western blots (A) of Bcl-2 (B; Nc, 100±2.1; Hc, 91.3±1.98; HMc, 103.5±2.1), Bax (C; Nc, 100±2.1; Hc, 106.1±2.1; HMc, 92.8±1.9), and caspase-3 (D; Nc, 100±3.3; Hc, 123.5±3.7; HMc, 108.6±3.3) in the embryonic cortical neuronal cell culture (in vitro) and the ratio of Bax/Bcl-2 expression (E). Data were shown as means±standard deviation (n=4). The (2S,4R)-4-Methylglutamic Acid (SYM 2081) was administered at 10 µg/mL. Nc, normoxia; Hc, hypoxia; HMc, hypoxia treated with (2S,4R)-4-Methylglutamic Acid (SYM 2081). *P<0.05, statistically significant vs. Hc.

• 

  Fig. 4 Real-time PCRs of Bcl-2 (A; Nc, 100±6.1; Hc, 43.5±2.6; HMc, 56.6±3.3), Bax (B; Nc, 100±6.4; Hc, 148.5±8.9; HMc, 61.9±3.7), and caspase-3 (C; Nc, 100±5.2; Hc, 131.9±6.6; HMc, 73.2±3.7) mRNAs in the embryonic cortical neuronal cell culture (in vitro) and the ratio of Bax/Bcl-2 expression (D). Data were shown as means±standard deviation (n=6). The (2S,4R)-4-Methylglutamic Acid (SYM 2081) was administered at 10 µg/mL. Nc, normoxia; Hc, hypoxia; HMc, hypoxia treated with (2S,4R)-4-Methylglutamic Acid (SYM 2081). *P<0.05, statistically significant vs. Hc.

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