Neuroprotective Effect of Fresh Gac Fruit Parts Against β-Amyloid-Induced Toxicity and Its Influence on Synaptic Gene Expression in HT-22 Cell Model

Abstract

Neurodegenerative diseases (NDs) have emerged as a significant global health crisis, disproportionately affecting the aging population. As longevity increases, the incidence, healthcare costs, and caregiver burden associated with NDs are escalating at an alarming rate. As of recent data, NDs such as Alzheimer's disease (AD) are not only significant health burdens but also reflect a complex interplay between socio-economic factors and healthcare systems worldwide. Gac fruit (Momordica cochinchinensis) is a rich source of bioactive compounds that has been used as food and traditional medicine. Gac fruit ameliorates memory deficits, enhances beta amyloid (Aβ)_1-42 clearance, and induces neurite outgrowth. In this study, we examined the anti-neurodegenerative and synaptic improvement effect of fresh gac fruit parts extracts (FGPEs) produced from different solvents. Results showed that the 80% ethanol extract of peel (PE-EtOH) and ethyl acetate extract of seed (SE-EtOAc) significantly protected HT-22 cells by attenuating Aβ-induced cell death, intracellular reactive oxygen species (ROS) production, mitochondrial dysfunction, and synaptic dysfunction. PE-EtOH protected synaptic functions by significantly increasing the postsynaptic density protein-95 (PSD-95) and reducing the neurexin 2 mRNA expression. In contrast, SE-EtOAc increased the PSD-95 and neurexin 3 and reduced the neurexin 2 expressions. These findings indicate that PE-EtOH and SE-EtOAc could have great potential in ameliorating Aβ-induced toxicity in an HT-22 cell model.

Keywords: Aβ toxicity; HT-22 cell; PSD95; gac fruit; neurexin; neurodegeneration; neuroligin; neuroprotection; synaptic function.

Figure 1

Neuroprotective effect of FGPE and memantine against Aβ-induced toxicity in HT-22. Protective effect of (A) peel extracts (PE-H₂O, PE-EtOH, PE-EtOAc, and PE-hexane) and memantine; (B) pulp extracts (PU-H₂O, PU-EtOH, PU-EtOAc, and PU-hexane) and memantine; (C) aril extracts (AR-H₂O, AR-EtOH, AR-EtOAc, and AR-hexane) and memantine; and (D) seed extracts (SE-H₂O, SE-EtOH, SE-EtOAc, and SE-hexane) and memantine. With (+) and without (−), gac fruit extracts and memantine concentration represented as (1, 5, 10, and 50 µg/mL). (E) Cell morphology after PE-EtOH (10 µg/mL), SE-EtOAc (10 µg/mL), or memantine (50 µg/mL) co-treated with Aβ (2.5 µM). Cell viability results were analyzed by comparing control (untreated) cells to Aβ control cells (treated with 2.5 µM Aβ) to assess the toxic effect of Aβ. Additionally, cells co-treated with FGPE or memantine and Aβ were compared to Aβ-treated cells to evaluate the protective effects of FGPE and memantine. PE-H₂O: water extract of peel; PE-EtOH: 80% ethanol extract of peel; PE-EtOAc: ethyl acetate extract of peel; PE-hexane: n-hexane extract of peel; PU-H₂O: water extract of pulp; PU-EtOH: 80% ethanol extract of pulp; PU-EtOAc: ethyl acetate extract of pulp; PU-hexane: n-hexane extract of pulp; AR-H₂O: water extract of aril; AR-EtOH: 80% ethanol extract of aril; AR-EtOAc: ethyl acetate extract of aril; AR-hexane: n-hexane extract of aril; SE-H₂O: water extract of seed; SE-EtOH: 80% ethanol extract of seed; SE-EtOAc: ethyl acetate extract of seed; and SE-hexane: n-hexane extract of seed. The data are expressed as mean ± standard deviation (SD) (n = 3). ^### p < 0.001 compared with control; * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with Aβ.

Figure 2

Protective effect of PE-EtOH and SE-EtOAc against Aβ-induced ROS production in HT-22 cells. (A) Immunofluorescence images of DHE stained cells after PE-EtOH treatment. Scale bar = 100 µm (B) Relative intensity of red fluorescence of PE-EtOH-treated cell. (C) Immunofluorescence images of DHE stained cells after SE-EtOAc treatment. Scale bar = 100 µm (D) Relative intensity of red fluorescence of SE-EtOAc-treated cell. With (+) and without (−), PE-EtOH and SE-EtOAc concentration represented as (1, 5, 10, and 50 µg/mL). Relative intensity of red fluorescence was calculated by image J software (version 1.54d; National Institutes of Health, Bethesda, MD, USA). DHE: dihydroethidium, PE-EtOH: 80% ethanol extract of peel, and SE-EtOAc: ethyl acetate extract of seed. The data are expressed as mean ± standard deviation (SD) (n = 3). ^### p < 0.001 compared with control, ** p < 0.01 and *** p < 0.001 compared with Aβ.

Figure 3

Protective effect of PE-EtOH and SE-EtOAc against Aβ-induced mitochondrial dysfunction in HT-22 cells. (A) JC-1-stained cell after PE-EtOH treatment. (B) Relative fluorescence intensity of PE-EtOH-treated cell, red fluorescence (healthy mitochondria), and green fluorescence (dysfunctional mitochondria). (C) Ratio of red and green fluorescence intensity of PE-EtOH-treated cells. (D) JC-1-stained cell after SE-EtOAc treatment. (E) Relative fluorescence intensity of red and green fluorescence of SE-EtOAc-treated cell against Aβ. (F) Ratio of red and green fluorescence intensity of SE-EtOAc-treated cells. With (+) and without (−), gac PE-EtOH and SE-EtOAc concentration represented as (1, 5, 10, and 50 µg/mL). PE-EtOH: 80% ethanol extract of peel, SE-EtOAc: ethyl acetate extract of seed. The data are expressed as mean ± standard deviation (SD) (n = 3). ^### p < 0.001 compared with control; * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with Aβ; and n.s not significant compared with Aβ.

Figure 4

Synaptic-related mRNA expression of PE-ETOH and SE-EtOAc treatment against Aβ-induced synaptic dysfunction. (A) PSD95 expression. (B) Neuroligin 2 expression. (C) Neurexin 2 expression. (D) Neurexin 3 expression. With (+) and without (−) PE-EtOH, SE-EtOAc, memantine, and Aβ. PE-EtOH: 80% ethanol extract of peel, SE-EtOAc: ethyl acetate extract of seed. The data are expressed as mean ± standard deviation (SD) (n = 3). ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001 compared with control; * p < 0.05, ** p < 0.01 compared with Aβ; and n.s no significance compared with Aβ.

Figure 5

HPLC chromatograms of phenolic compounds of PE-EtOH and SE-EtOAc. (A) Standards. (B) PE-EtOH. (C) SE-EtOAc. Detected at absorbance value 320 nm. (a) Gallic acid, (b) chlorogenic acid, (c) p-coumaric acid, (d) sinapic acid, (e) rutin, (f) myricetin, (g) quercetin, and (h) kaempferol. PE-EtOH: 80% ethanol extract of peel, SE-EtOAc: ethyl acetate extract of seed.