Abstract
Neuroprotective strategies in the treatment of stroke have been attracting a great deal of attentions. Our previous clinical and basic studies have demonstrated that protopanaxadiol ginsenoside-Rd (Rd), a monomer compound extracted from Panax ginseng or Panax notoginseng, has neuroprotective effects against ischemic stroke, probably due to its ability to block Ca2+ overload, an usual consequence of the overactivation of NMDA receptor (NMDAR). As an extending study, we explored here whether Rd exerted its neuroprotection as a novel NMDAR blocker. Our whole-cell patch-clamp results showed that Rd reduced NMDAR currents of cultured rat cortical neurons (EC50 = 7.7 μM) dose-dependently by acting on extrasynaptic NMDAR NR2b subunit. However, unexpectedly, cell transfection and radioligand binding assays revealed that Rd did not bind to the NMDAR channel directly. Alternatively, it inhibited the phosphorylation of NR2b at Ser-1303, a target of death associated protein kinase 1 (DAPK1). Moreover, cell-based and cell-free enzymatic assays showed that Rd did not inhibit the activity of DAPK1 directly, but blocked the activity of calcineurin, a key phosphatase for activating DAPK1. Importantly, other protopanaxadiol ginsenosides were also found to have potential inhibitory effects on calcineurin activity. Furthermore, as expected, calcineurin inhibition by cyclosporin A could mimic Rd’s effects and protect against NMDA-, oxygen glucose deprivation- or transient ischemic stroke-induced neuronal injury. Therefore, our present study provided the first evidence that Rd could exert an inhibitive effect on NMDAR-triggered currents and sequential excitotoxicity through mitigation of DAPK1-mediated NR2b phosphorylation by attenuating calcineurin activity.
Highlights
Ischemic stroke is the leading cause of brain injury, and it has a high morbidity, high mortality and high disability that is seriously threatening public health[1]
To explore whether NMDA receptor (NMDAR) is a potential target of Rd, we examined the effects of Rd on NMDAR currents of cultured rat cortical neurons (Fig. 1a)
We demonstrated that Rd, a kind of protopanaxadiol ginsenoside isolated from Panax ginseng or Panax notoginseng, protected against neuronal excitotoxic injury by inhibiting the activity of calcineurin phosphatase
Summary
Ischemic stroke is the leading cause of brain injury, and it has a high morbidity, high mortality and high disability that is seriously threatening public health[1]. A large number of potential neuroprotective agents, which have been showed to be obviously neuroprotective against cerebral ischemic injury in animal models, have proved not to be clinically efficacious[5,6]. A series of our previous clinical and basic studies have demonstrated that Rd could exert a remarkable neuroprotective effect against neuronal injury. Our recent evidence further showed that Rd protected cultured neurons against NMDA- or glutamate-induced excitotoxicity by inhibiting Ca2+ influx[21], indicating that Rd may act as an blocker of NMDA receptor (NMDAR), one of important receptor-operated Ca2+ channels (ROCC), whose overactivation-induced Ca2+ overload is the major cause for neuronal excitotoxicity[22,23,24,25]. The evidence above indicated that Rd may be a promising neuroprotectant with distinctive advantages for the treatment of ischemic stroke, and the mechanism may involve its potential capability of inhibiting of NMDAR. Here we explored whether Rd could serve as an NMDAR blocker to act against neuronal excitotoxicity
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