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. 2024 May 16;14(5):589.
doi: 10.3390/biom14050589.

Gypenoside XVII Reduces Synaptic Glutamate Release and Protects against Excitotoxic Injury in Rats

Cheng-Wei Lu  1   2 , Tzu-Yu Lin  1   2 , Kuan-Ming Chiu  3   4 , Ming-Yi Lee  5 , Su-Jane Wang  6   7
Affiliations

Gypenoside XVII Reduces Synaptic Glutamate Release and Protects against Excitotoxic Injury in Rats

Cheng-Wei Lu et al. Biomolecules. .

Abstract

Excitotoxicity is a common pathological process in neurological diseases caused by excess glutamate. The purpose of this study was to evaluate the effect of gypenoside XVII (GP-17), a gypenoside monomer, on the glutamatergic system. In vitro, in rat cortical nerve terminals (synaptosomes), GP-17 dose-dependently decreased glutamate release with an IC50 value of 16 μM. The removal of extracellular Ca2+ or blockade of N-and P/Q-type Ca2+ channels and protein kinase A (PKA) abolished the inhibitory effect of GP-17 on glutamate release from cortical synaptosomes. GP-17 also significantly reduced the phosphorylation of PKA, SNAP-25, and synapsin I in cortical synaptosomes. In an in vivo rat model of glutamate excitotoxicity induced by kainic acid (KA), GP-17 pretreatment significantly prevented seizures and rescued neuronal cell injury and glutamate elevation in the cortex. GP-17 pretreatment decreased the expression levels of sodium-coupled neutral amino acid transporter 1, glutamate synthesis enzyme glutaminase and vesicular glutamate transporter 1 but increased the expression level of glutamate metabolism enzyme glutamate dehydrogenase in the cortex of KA-treated rats. In addition, the KA-induced alterations in the N-methyl-D-aspartate receptor subunits GluN2A and GluN2B in the cortex were prevented by GP-17 pretreatment. GP-17 also prevented the KA-induced decrease in cerebral blood flow and arginase II expression. These results suggest that (i) GP-17, through the suppression of N- and P/Q-type Ca2+ channels and consequent PKA-mediated SNAP-25 and synapsin I phosphorylation, reduces glutamate exocytosis from cortical synaptosomes; and (ii) GP-17 has a neuroprotective effect on KA-induced glutamate excitotoxicity in rats through regulating synaptic glutamate release and cerebral blood flow.

Keywords: cerebral blood flow; cortex; glutamate excitotoxicity; glutamate release; gypenoside XVII.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Effect of GP-17 on the 4-AP-evoked glutamate release from synaptosomes isolated from the cerebral cortex of adult rats. (A) Chemical structure of GP-17. (B) Glutamate release was measured under control conditions or in the presence of 10 μM GP-17. ***, p < 0.0001, two-tailed Student’s t test. (C) Dose-response curve for GP-17 inhibition of 1 mM 4-AP- and 15 mM KCl-evoked glutamate release. (D) Glutamate release by 1 mM 4-AP in the absence and presence of 10 μM GP-17 and absence and presence of omitting CaCl2 and adding 0.3 mM EGTA, 2 μM ω-CgTX GVIA, or 0.5 μM ω-AgTX IVA. ***, p < 0.0001 versus control group, One-way ANOVA with Tukey posthoc test. (E) FM1–43 release by 1 mM 4-AP in the absence (control) or presence of 10 μM GP-17. ***, p < 0.0001, two-tailed Student’s t test. GP-17 was added 10 min before depolarization and ω-CgTX GVIA or ω-AgTX IVA was added 20 min before this. n = 5 rats per group.
Figure 2
Figure 2
Involvement of PKA suppression in the inhibition caused by GP-17 on glutamate release from cerebrocortical synaptosomes. (A) Glutamate release by 1 mM 4-AP in the absence and presence of 10 μM GP-17 and absence and presence of 10 μM GF109203X, 10 μM KT5720, or 1 μM PKI 14–22. (B,C) Immunoblot results of PKA, PKA pThr197, synapsin I, synapsin I pSer9, SNAP-25, and SNAP-25 pThr138, and β-actin proteins in the synaptosomes from different groups. (C,D,F–I). The relative protein levels were quantified. ***, p < 0.001 versus the control group, #, p < 0.05 versus the 4-AP-treated group. One-way ANOVA with Tukey posthoc test. n = 5 rats per group. The details of the Western blot original images are in Supplementary Materials.
Figure 3
Figure 3
Effect of GP-17 on the neuronal degeneration in the entorhinal cortex of KA-treated rats. (A) FJB staining of 72 h after KA injection. FJB positive neurons are indicated by white arrows. Scale bars: 200 µm for 100×, 100 µm for 200×, 50 µm for 400×. (B,C). Statistical graph of FJB positive cells in the prefrontal cortex and entorhinal cortex in different groups. ***, p < 0.001 versus the control group, #, p < 0.05 versus the KA group. One-way ANOVA with Tukey posthoc test. n = 5 rats per group.
Figure 4
Figure 4
Effect of GP-17 on the concentration of glutamate (A) and GABA (B) and glutamate/GABA ratio (C) in the cortex from different groups. Glutamate and GABA levels were analyzed in the cortex of rats 72 h after KA injection. ***, p < 0.001 versus the control group, #, p < 0.05 versus the KA group. One-way ANOVA with Tukey posthoc test. n = 6 rats per group.
Figure 5
Figure 5
Effect of GP-17 on the protein expression levels of SNAT1, glutaminase, GDH, and VGLUT1 in the cortex from different groups. (A) Immunoblot results of SNAT1, glutaminase, GDH, and VGLUT1 in the cortex of rats 72 h after KA injection and the respective bar graphs. (B–E) The relative protein levels were quantified. ***, p < 0.001 versus the control group, #, p < 0.05 versus the KA group. One-way ANOVA with Tukey posthoc test. n = 5 rats per group. The details of the Western blot original images are in Supplementary Materials.
Figure 6
Figure 6
Effect of GP-17 on the protein expression levels of GluN2A and GluN2B in the cortex from different groups. (A) Immunoblot results of GluN2A, GluN2B, and GluN1 in the cortex of rats 72 h after KA injection. (B–D) The relative protein levels were quantified. ***, p < 0.001 versus the control group, #, p < 0.05 versus the KA group. One-way ANOVA with Tukey posthoc test. n = 5 rats per group. The details of the Western blot original images are in Supplementary Materials.
Figure 7
Figure 7
Effect of GP-17 on the cerebral blood flow in KA-treated rats. (A) The typical picture of cerebral blood flow in rats 72 h after KA injection. (B) The perfusion unit of cerebral blood flow in different groups. (C) Immunoblot results of GluN2A and GluN2B in the cortex of rats 72 h after KA injection. (D) The relative protein levels were quantified. **, p < 0.01 versus the control group, ***, p < 0.001 versus the control group. #, p < 0.05 versus the KA group. One-way ANOVA with Tukey posthoc test. n = 3–4 rats per group. The details of the Western blot original images are in Supplementary Materials.
Figure 8
Figure 8
Schematic representation of GP-17-mediated inhibition of glutamate release from synapsosomes (A) and the neuroprotective effects oof GP-17 on KA-induced glutamate excitotoxicity in rats (B). Graphs created with BioRender.com accessed on 20 February 2024.
See this image and copyright information in PMC

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