Neuroprotective Effect Of Ginsenoside Rd Against Ischemic Neuronal Damage: An In Vitro And In Vivo Study

Stroke ranks second as a cause of lost disability-adjusted life-years in the high-income countries and as a cause of death worldwide. In China, it is the second leading cause of death in women and the third in men. Ischemic stroke accounts for approximately 80% of acute stroke. Two major approaches have been developed to treat ischemic stroke: recanalization and neuroprotection. Thrombolysis with tissue plasminogen activator (tPA), limited by its narrow therapeutic time window and the concern of hemorrhagic complication, is still uncommon in use. The goal of neuroprotective strategies is to salvage ischemically threatened - but potentially viable– brain tissue suurounding the core of infarcted tissue and attenuate linical sequelae of stroke. Although a large number of neuroprotective interventions have failed to demonstrate the same efficacy as they showed in animal models, the development of neuroprotective therapy for acute ischemic stroke is still challenging.Ginsenoside Rd (GSRd) is a principal member of ginseng saponins and its content in the total notoginseng saponins is 4.07%, making it inexpensive for practical use. Recently, it has been demonstrated to produce a number of pharmacological actions such as inhibiting Ca2+ influx through receptor-and-store-operated Ca2+ channels, enhancing astrocyte differentiation from neural stem cells, attenuating oxidative damage and kainic acid-induced neurotoxicity, and diminishing 3-nitropropionic acid-induced motor impairment and cell loss in the striatum. We hypothesized that GSRd also possesses an ability to protect neuron from ischemic damage. Here we examined the effects of GSRd on neuron insult induced by oxygen glucose deprivation (OGD) in hippocampal neuron cultures and focal cerebral ischemia in rats.1. In vitro experimentsObjective To investigate the protective effect of GSRd against cell damage induced by OGD in cultured hippocampal neurons and its possible mechanicms. Methods Hippocampi were isolated from embryonic day 18 Spague-Dawley rat embryos. Cells were maintained in Neurobasal medium supplemented with 2% B27. OGD was carried out on 6-8 days in vitro (DIV) cultures. Hippocampal neurons were exposed to OGD for 2 h followed by a 24-h recovery (reoxygenation). For concurrent treatment, GSRd or vehicle was present in the culture medium during OGD/reoxygenation; for post-treatment, GSRd was added to the culture immediately after OGD. At 24 h post-OGD, cell survival was quantitatively assessed by LDH assay and double fluorescent staining with PI and Hoechst 33342. Formation of reactive oxygen species (ROS) was determined by use of fluorescent probe 2,7-dichlorofluorescein diacetate (DCFH-DA). Mitochondrial membrane potential (MMP) was measured using rhodamine 123. The fluorescence intensity was examined by flow cytometry. Cellular malondialdehyde (MDA) and superoxide dismutase (SOD) were determined by spectrophotometry using commercial kits. We also investigated the protective effect of GSRd against glutamate excitotoxicity. GSRd and subsequently L-glutamate were added to the medium. 1 h later, the glutamate-induced cell damage was assessed using LDH assay. Results Based on PI uptake method, 2-h OGD led to a marked increase of necrotic cells. GSRd (0.1-10μM) significantly decreased the number of necrotic neurons. Consistent results were obtained using LDH release assay. In contrast, neurons were not protected when GSRd treatment was started after OGD. 24 h after OGD, a 2.08-fold increase in the intracellular ROS was found by using DCFH-DA assay. However, GSRd (0.1-10μM) decreased the ROS production. OGD also significantly increased the level of MDA and decreased SOD in the cultures. GSRd (0.1-10μM) treatment resulted in a noticeable reduction in MDA content and elevation in SOD activity. After OGD the fluorescence of Rh 123 was decreased to 40.3% of basal level corresponding to depolarization. However, treatment with GSRd (1-10μM) significantly stabilized the MMP. Exposure to glutamate led to dramatic damage of cultured neurons as shown by LDH release assay right after the insult. The damage was almost abolished in the presence of GSRd 0.1-50μM (P<0.001). Conclusion We reported neuroprotective effects of GSRd in primary cultured rat hippocampal neurons against OGD. This protection was probably associated with inhibition of oxidative stress impairment, preservation of MMP and prevention of glutamate-induced excitotoxicity.2. In vivo experimentsObjective To conform the protective effect of GSRd against focal cerebral ischemia injury in rat. Methods Focal cerebral ischemia was induced by use of a suture occlusion model of the middle cerebral artery (2-h occlusion) in the rat. Immediately after reperfusion animals received bolus injections of GSRd or vehicle. Neurological deficits were scored and infarct volume was assessed by 2,3,5-triphenyltetrazolium chloride (TTC) staining 24 h after reperfusion respectively. Results At 24 h post-ischemia, a significant improvement in neurological score was observed in GSRd-treated animals (GSRd 10 mg/Kg, P=0.012; GSRd 30 mg/Kg, P=0.002) in comparison with vehicle-treated rats. The infarct volume in vehicle-treated animals was 232.7 mm3. Administration of GSRd at the dose of 10, 30 mg/Kg at the beginning of reperfusion significantly reduced infarct volume to 189.2 mm3 (P=0.018) and 178.7 mm3 (P=0.004), while the dose of 5 mg/Kg exhibited a borderline effect in the reduction of infarct size (200.3 mm3, P=0.074). Conclusion GSRd at 10 and 30 mg/Kg significantly reduced infarct volume and attenuated neurological deficits.