Alpha-Asaronol Alleviates Dysmyelination by Enhancing Glutamate Transport Through the Activation of PPARγ-GLT-1 Signaling in Hypoxia-Ischemia Neonatal Rats

Abstract

Preterm white matter injury (PWMI) is the most common form of brain damage in premature infants caused by hypoxia-ischemia (HI), inflammation, or excitotoxicity. It is characterized by oligodendrocyte precursor cell (OPC) differentiation disorder and dysmyelination. Our previous study confirmed that alpha-asarone (α-asaronol), a major compound isolated from the Chinese medicinal herb Acorus gramineus by our lab, could alleviate neuronal overexcitation and improve the cognitive function of aged rats. In the present study, we investigated the effect and mechanism of α-asaronol on myelination in a rat model of PWMI induced by HI. Notably, α-asaronol promoted OPC differentiation and myelination in the corpus callosum of PWMI rats. Meanwhile, the concentration of glutamate was significantly decreased, and the levels of PPARγ and glutamate transporter 1 (GLT-1) were increased by α-asaronol treatment. In vitro, it was also confirmed that α-asaronol increased GLT-1 expression and recruitment of the PPARγ coactivator PCG-1a in astrocytes under oxygen and glucose deprivation (OGD) conditions. The PPARγ inhibitor GW9662 significantly reversed the effect of α-asaronol on GLT-1 expression and PCG-1a recruitment. Interestingly, the conditioned medium from α-asaronol-treated astrocytes decreased the number of OPCs and increased the number of mature oligodendrocytes. These results suggest that α-asaronol can promote OPC differentiation and relieve dysmyelination by regulating glutamate levels via astrocyte PPARγ-GLT-1 signaling. Although whether α-asaronol binds to PPARγ directly or indirectly is not investigated here, this study still indicates that α-asaronol may be a promising small molecular drug for the treatment of myelin-related diseases.

Keywords: PPARγ; glutamic acid; oligodendrocyte precursor cells; white matter injury; α-asaronol.

FIGURE 1

Schematic diagram of the experimental design in vivo. (A) Schedule of animal experiments. (B) Molecular structure formula of α-asaronol.

FIGURE 2

α-Asaronol improves OPC differentiation and relieves dysmyelination in PWMI models. Representative immunofluorescence images for PDGFRα-labeled OPCs. (A,B) CC1-labeled mature oligodendrocyte and Olig2-labeled oligodendrocyte lineage (C,D) and MBP-labeled myelin sheath (E,F) in the corpus callosum at P3 + 4 d and P3 + 7 d after HI. Scale bar, 20 µm. (G,H) Quantification of the number of PDGFRα⁺ cells and the percentage of CC1+/Olig2+ cells. (I,K) Western blotting was performed to analyze PDGFRα and MBP protein levels. All data are expressed as the mean ± SEM. **p < 0.01, ***p < 0.001, ****p < 0.001 vs. sham group. ^# p < 0.05, ^## p < 0.01 vs. HI + NS group. N = 4 for each group.

FIGURE 3

α-Asaronol inhibits HI-induced activation of the ionotropic glutamate receptor AMPAR. The protein level of p-GluR2 (subunit 2 of AMPAR) was detected by western blot at P3 + 12 h, P3 + 4 d, and P3 + 7 d after HI. HI induced an obvious increase in p-GluR2 (Tyr876), and α-asaronol (50 mg/kg) treatment decreased the level of p-GluR2. All data are expressed as the mean ± SEM. ***p < 0.001, ****p < 0.0001 vs. sham. ^# p < 0.05, ^## p < 0.01 vs. HI + NS. N = 4 for each group.

FIGURE 4

α-Asaronol increases the expression of GLT-1 in the PWMI rat model. (A) GLT-1 protein expression was detected by western blotting at P3 + 12 h, P3 + 4 d, and P3 + 7 d. (B) GLT-1 was quantified and normalized against β-actin. (C) The mRNA level of GLT-1 was quantified by qPCR. All data are represented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 vs. sham group, ^# p < 0.05, ^## p < 0.01 vs. HI + NS group. N = 4 for each group.

FIGURE 5

α-Asaronol increases the expression of PPARγ in PWMI models. (A) Expression of PPARγ was detected at P3 + 12 h, P3 + 4 d, and P3 + 7 d after HI. (B) PPARγ was quantified and normalized against β-actin. (C) The mRNA level of PPARγ was quantified by qPCR. (D,E) Representative immunofluorescence images for GLT-1 and ATP1A1 double staining in the corpus callosum. Scale bar, 20 µm. (F,G) Molecular docking simulation of α-asaronol and PPARγ. All data are represented as the mean ± SEM. *p < 0.05, **p < 0.01 vs. sham group, ^# p < 0.05, ^### p < 0.001 vs. HI + NS group. N = 4 for each group.

FIGURE 6

α-Asaronol increases the expression of astrocytic GLT-1 under OGD/R conditions. (A) Gradient OGD/R conditions of astrocytes. (B) Cell viability of astrocytes. (C) Representative immunofluorescence images for GFAP and GLT-1 staining in the corpus callosum for all groups. Scale bar, 20 µm. (D) Statistical analysis of GLT-1 mean fluorescence intensity. (E) The expression of GLT-1 was detected and quantified. (F,G) Chromatographs of glutamate and quantitative analysis of glutamate concentration in the medium for all groups were performed. All data are represented as the mean ± SEM. **p < 0.01, ****p < 0.0001 vs. the control group, ^# p < 0.05, ^## p < 0.01 vs. the OGD/R group. N = 4 for each group.

FIGURE 7

α-Asaronol upregulated GLT-1 expression in astrocytes by activating PPARγ. (A) The morphology of astrocytes under the bright field. Scale bar, 50 µm. (B) The expression of PPARγ and GLT-1 was detected by western blotting. PPARγ and GLT-1 were quantified and normalized against β-actin. (C) Co-IP was used to examine the interaction of PPARγ and PGC1a or PPARγ and HDAC3. (D) Graphical image representing the ligand-independent repression and α-asaronol-dependent transactivation of PPARγ. All data are represented as the mean ± SEM. *p < 0.05, **p < 0.01 vs. control group, ^## p < 0.01 vs. OGD/R group, ^& p < 0.05, ^&&& p < 0.001 vs. OGD/R + ASA group. N = 4 for each group.

FIGURE 8

Conditioned medium from α-asaronol-treated astrocytes promotes OPC differentiation without influencing apoptosis. Representative immunofluorescence images for PDGFRα-labeled OPCs and CC1-labeled mature OLs (A) and MBP-labeled myelin sheath (B). Scale bar, 20 µm. (C–E) Statistical analysis of the numbers of PDGFRα⁺ cells, CC1⁺ cells, and MBP⁺ cells. (E,F) Representative flow cytometry images of OPCs and quantitative analysis of the percentage of apoptotic cells for all groups. Scale bar, 20 µm. All data are represented as the mean ± SEM. **p < 0.01, ****p < 0.0001 vs. the control group. ^# p < 0.05, ^## p < 0.01, ^#### p < 0.0001 vs. the OGD/R group. ^& p < 0.05, ^&&&& p < 0.001 vs. the OGD/R + ASA group. N = 4 per group.