The Experimental Study Of Achyranthes Bidentata Ploypeptides On Neuroprotection

Objective:To observe the neuroprotective effect of Achyranthes Bidentata ploypeptides (ABPP) and its related mechanisms.Methods:(1) Middle cerebral artery occlusion (MCAO) was employed to establish focal cerebral ischemia-reperfusion model in rats, then the neurological behavior score and the percentage of cerebral infarction (with 2, 3, 5-triphenyltetrazolium chloride staining) were measured to observe the neuroprotective effect of ABPP in vivo.(2) N-methyl-D-aspartate (NMDA)-induced injury model was established in primary cultured rat embryonic hippocampal neurons, and methyl-thiazole-tetrazolium (MTT) assay was used to observe the neuroprotective effect of ABPP in vitro.(3) Hoechst33258 and propidium iodide (PI) double fluorescent staining, flow cytometry detection of PI, detection of lactate dehydrogenase and DNA ladder were applied to observe the effect of ABPP against NMDA-induced apoptotic cell death in primary cultured hippocampal neurons.(4) The primary cultured hippocampal neurons were loaded with fluo-3/AM (a fluorescent probe of calcium ion), and the dyed cells were used to observe the effect of ABPP on NMDA-induced rise in intracellular calcium ions with a confocal laser scanning microscope.(5) Western Blotting detection was performed to examine the effect of ABPP on the overexpression of Bax protein, which was induced by NMDA in cultured hippocampal neurons.(6) Caspase-3 activity assay was performed to examine the effect of ABPP on the protein activity levels of Caspase-3, which was enhanced by NMDA in cultured hippocampal neurons.(7) The primary cultured hippocampal neurons were loaded with Rhodamine 123 (a lipophilic cation fluorescent dye), and the dyed cells were to observe the effect of ABPP on NMDA-induced changes in the cellular mitochondrial transmembrane potential with a microplate fluorometer. In addition, the primary cultured hippocampal neurons were loaded with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), and the dyed cells were to observe the effect of ABPP on NMDA-induced changes in the intracellular radical oxygen species (ROS) with a microplate fluorometer.(8) With conventioanal whole-cell patch-clamp recording method, NMDA response was measured by amplitude of NMDA-induced peak current and steady-state current and its coefficient of desensitization. The influences of ABPP on these parameters were investigated in the same time.(9) By MTT assay, the effect of the two subunit-specific NMDA receptor antagonists, Ro-256981 and NVP-AAM007 on NMDA-induced cellular injury was observed in primary cultured hippocampal neurons in DIV8 and DIV13-14 respectively. Furthermore, the influence of ABPP on NMDA-induced neuronal injury was also investigated at the same time.(10) The primary cultured hippocampal neurons were loaded with fluo-3/AM, and the dyed neurons were used to observe the effect of Ro-256981 and NVP-AAM007 on NMDA or Bicuculline induced rise in intracellular calcium ions with a confocal laser scanning microscope. Furthermore, the influence of ABPP on the rise of intracellular calcium ion via NR2A-containing NMDA receptors or NR2B-containing NMDA receptors was also investigated respectively.(11) With whole-cell patch-clamp recording technique, the effect of Ro-256981 and NVP-AAM007 on NMDA induced current was measured by amplitude of NMDA-induced peak current and steady-state current and its coefficient of desensitization. Furthermore, the influence of ABPP on the NMDA induced current via NR2A-containing NMDA receptors or NR2B-containing NMDA receptors was also investigated by these parameters respectively.Results:(1) Focal cerebral ischemia-reperfusion, induced by MCAO, resulted in the mortality of 50%, and promoted the neurology deficit score of the survival rats and the percentage of cerebral infarction (p<0.01). ABPP (0.2mg/kg, iv) decreased the neurology deficit score and the percentage of cerebral infarction (p<0.01). (2) The results of MTT assay showed that NMDA could lower the activity of the primary cultured hippocampal neurons. Furthermore, with the increase of the concentration of NMDA or the incubation time the neuronal activity was fall-off. On the other hand, ABPP could inhibit the decrease of the neuonal activity in a concentration-dependent manner.(3) Hoechst33258 and propidium iodide (PI) double fluorescent staining, flow cytometry detection of PI, detection of lactate dehydrogenase and DNA ladder showed that NMDA could induce the apoptotic cell death in primary cultured hippocampal neurons, and ABPP (1μg/ml) could inhibit the apoptotic neuronal cell death.(4) Imaging of intracellular calcium ([Ca²⁺]_i) displayed that NMDA leaded to enhancement of intracellular fluorescence intensity in a concentration-dependent manner. Both AP-V and MK801 can inhibit the rise of the fluorescence intensity. Pre-, concurrent and post-treatment with ABPP could attenuate the enhanced fluorescence intensity induced by NMDA in concentration-dependent manner.(5) The Bax channel blocker can inhibit the NMDA-induced cell viability decrease by MTT assay. The results of RT-PCR and Western blot showed that NMDA could augment the expression of Bax in time dependence, and when the primary cultured hippocampal neurons incubated for 30min, the expression of Bax was significantly increased. Furthermore, ABPP could antagonize the up-regulation of Bax protein induced by NMDA (p<0.05).(6) Molecular probes DCFH-DA was used to monitor alterations of intracellular ROS levels. Exposure of the primary cultured hippocampal neurons to NMDA caused an elevation of ROS production in a concentration-dependent manner, and ABPP inhibited the elevation of ROS levels induced by NMDA, also showing a concentration-dependent pattern.(7) After exposure to NMDA, the mitochondrial membrane potential of cultured hippocampal neurons was lower than that in normal extracellular solution. ABPP inhibited the lowering of mitochondrial membrane potential induced by NMDA.(8) A three fold increase in caspase-3 activity was found after exposure of cultured hippocampal neurons to NMDA, but pretreatment with ABPP (1μg/ml) or MK-801 (10μM) protected neurons against the NMDA-induced increase in caspase-3 activity (p<0.05).(9) The results of MTT assay showed that either Ro-256981 or NVP-AAM007 could inhibit the acute lowering of cell viability induced by NMDA in primary cultured hippocampal neurons at DIV8 and DIV13-14 (p<0.05). However, ABPP could not inhibit the acute lowering of cell viability induced by NMDA. In primary cultured hippocampal neurons at DIV8, Ro-256981 and NVP-AAM007 could inhibit the delayed lowering of cell viability induced by NMDA (p<0.05). In addition, ABPP (0.1μg/ml) could inhibit the delayed lowering of cell viability induced by NMDA (p<0.01), but ABPP (10μg/ml) could not inhibit the delayed lowering of cell viability induced by NMDA, which was recovered by NVP-AAM007 (p<0.05). On the contrary, in primary cultured hippocampal neurons at DIV13-14, NVP-AAM007 could intensify the delayed lowering of cell viability induced by NMDA (p<0.01) and ABPP (10μg/ml) could inhibit the delayed lowering of cell viability induced by NMDA (p<0.01).(10) During the sustained exposure of hippocampal neurons to NMDA, the addition of NVP-AAM077 alone lowered the [Ca²⁺]_i, and the simultaneous addition of NVP-AAM077 and ABPP further lowered the [Ca²⁺]_i. On the contrary, during the sustained exposure of hippocampal neurons to NMDA, the addition of Ro25-6981 alone lowered the [Ca²⁺]_i, but the simultaneous addition of Ro25-6981 and ABPP inversely raised the [Ca²⁺]_i. In addition, during the sustained exposure of hippocampal neurons to bicuculline, the addition of NVP-AAM077 alone lowered the [Ca²⁺]_i, and the simultaneous addition of NVP-AAM077 and ABPP further lowered the [Ca²⁺]_i. On the contrary, during the sustained exposure of hippocampal neurons to bicuculline, the addition of Ro25-6981 alone lowered the [Ca²⁺]_i, and the simultaneous addition of Ro25-6981 and ABPP inversely raised the [Ca²⁺]_i, which can be blocked by NVP-AAM077.(11) ABPP could decrease the amplitude of NMDA-induced steady-state current and increase its coefficient of desensitization in cultured hippocampal neurons in DIV8. On the contrary, ABPP could enhance amplitude of NMDA-induced peak current and steady-state current and decrease its coefficient of desensitization in cultured hippocampal neurons in DIV13-14. In addition, treatment of Ro25-6981 alone can decrease amplitude of NMDA-induced peak current and steady-state current, and the simultaneous treatment of Ro25-6981 and ABPP could enhance amplitude of NMDA-induced peak current and steady-state current and decrease coefficient of desensitization of NMDA current. Furthermore, treatment of NVP-AAM007 alone can decrease amplitude of NMDA-induced peak current and steady-state current, and the simultaneous treatment of NVP-AAM007 and ABPP could reduce amplitude of NMDA-induced peak current and steady-state current and its coefficient of desensitization.Conclusions:(1) Achyranthes Bidentata ploypeptides (ABPP) have the neuroprotective effect in vivo and in vitro.(2) The protective effect of ABPP is probably related to its anti-apoptosis in primary cultured hippocampal neurons.(3) ABPP can inhibit the excess Ca²⁺ influx, intracellular ROS production, Bax protein over-expression, the activity of Caspase-3 and mitochondrial dysfunction, which were induced by over-stimulation of NMDA receptors.(4) ABPP can modulate NR2A- and NR2B-containing NMDA receptors differentially.