301 - Tangeretin improves neuronal cell survival via phosphorylation of Akt after hypoxia-ischemia

Publication Number: 4295.301

Rajnish K. Yadav, University of Nebraska Medical Center, Omaha, NE, United States; Amanda O. Johnson, University of Nebraska College of Medicine, Omaha, NE, United States; Eric S. Peeples, University of Nebraska Medical Center, Omaha, NE, United States

Background: Neonatal hypoxic-ischemic brain injury (HIBI) is not only a serious threat to the lives of newborns but also one of the most significant causes of long-term neurological dysfunction in infants. Tangeretin is a lipophilic flavonoid, has antioxidant activity, anti-inflammatory potential, anti-cancer effects and potentially anti-ischemia. Currently, therapeutic hypothermia is the only treatment option available in high income countries, though outcomes remain poor despite optimal treatment. Additionally, a recent trial suggested negative outcomes after hypothermia in certain lower- and middle-income countries, emphasizing the need to explore other cost-effective and broadly deployable treatment options.

Objective: We hypothesize that treatment with Tangeretin improves neuronal cell viability and mitochondrial membrane potential (MMP) under oxygen and glucose deprivation with reperfusion (OGD/R) to mimic HIBI via phosphorylation of Akt.

Design/Methods: Neuro2a (N2a) cells underwent OGD/R (8-min flushing of hypoxia chamber with N2 at 20 psi) for 3 hours followed by 21 hours of reperfusion. Cells were treated during reperfusion after three hours of OGD/R and experimental groups included control cells maintained at normoxia with glucose-rich media, OGD/R, OGD/R+Tangeretin and OGD/R+Tangeretin+MK2206 (Allosteric Akt inhibitor). Cell viability was quantified by Alamar blue assay. Cell death after OGD/R was evaluated with TUNEL assay. MMP was determined by JC-1 fluorescence. Protein expression of pAkt (ser 473) was evaluated using western blot.

Results: Tangeretin (86.52±10.52) improves N2a cell viability against OGD/R (65.80±14.58). The co-treatment with MK2206 (54.71±8.95) significantly abolished the Tangeretin induced cell survival (Fig 1B). Consistent with these data, western blot analysis showed significant increased phosphorylation of Akt (Ser473) following Tangeretin treatment was inhibited by co-treatment with MK2206 (Fig 1C). Apoptotic cell death (Fig 2) and mitochondrial membrane depolarization (Fig 3) were increased in OGD/R compared with normoxia, and both increases were significantly attenuated in the presence of Tangeretin. These effects were also inhibited by MK2206 co-administration.

Conclusion(s): This study provides evidence that the Tangeretin exerts neuroprotection against OGD/R by enhancing neuronal survival in the N2a cell line, maintaining MMP and suppressing apoptosis. Future directions include assessing mitochondrial oxygen consumption rate, and glycolytic assay need to be evaluated to establish the role of mitochondrial metabolism mechanism in cell survival.

Fig. 1: Tangeretin reduces OGD/R-induced cytotoxicity via Akt in N2a cells.
Cells were treated under OGD-inducing conditions for 3 h and then reoxygenated in the presence of 10, 15, 20 μM tangeretin for 21 h.
(A) Cell viability after different concentration of tangeretin treatment was detected with Alamar blue assay.
(B) Role of Akt in cytotoxicity was determined with MK2206 combined with tangeretin using Alamar blue assay.
(C) Western blot analysis detecting the levels pAkt protein decreased MK2206 with combination of tangeretin in OGD/R-treated N2a cells. Data shown are means±SD. *p < 0.05 **p < 0.01 ***p < 0.001, ***p < 0.0001 vs OGD/R group.


Fig. 2: Tangeretin Inhibits OGD/R-induced apoptosis in N2a cells.
Tangeretin (10 μM) administered during reperfusion attenuated OGD/R-induced apoptosis using TUNEL apoptosis assay in N2a cells. TUNEL apoptosis assay was examined with a fluorescence microscope (magnification 20X objective field, 1280 X 960 pixels, scale bar =100 µm). Data shown are Mean±SD. Multiple comparisons were carried out using the one-way ANOVA analysis. Data shown are means±SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs OGD/R group.

Fig. 3: Tangeretin prevents OGD/R-induced cell mitochondrial depolarization in N2a cells.
Tangeretin (10 μM) reduced OGD/R-induced mitochondrial membrane depolarization in N2a cells. Images were examined with a fluorescence microscope (magnification 20X objective field, 1280X960 pixels, scale bar=100 µm). Data shown are means±SD, *p < 0.05 **p < 0.01 ***p < 0.001, ****p < 0.0001 vs OGD/R group.