Ginsenoside Rb1 Improvs Beta-amyloid Peptide_25-35-induced Calcium Homeostasis By Inhibiting The Activity Of L-type Voltage-gated Calcium Channel In Hippocampal Neurons

Alzheimer's disease(AD) is a progressive neurodegenerative disease that is characterized by its typical clinical sympotoms of progressive cognitive impairment. The deposition of amyloidβprotein (Aβ), as insoluble fibrillar aggregates in neuritic plaque, is the dominant histological hallmark of Alzheimer's disease. Aβtends to form its aggregate state throughβ-sheet lamellar structure, and Aβ25-35 is its toxic fragment. Aβexerts its neurotoxicity through a variety of ways, elevation of intracellular calcium may play a key role of all.Ginsenoside Rb1, one protopanaxadiol type saponin, is one of the most important active compounds of ginseng. Our previous experiments confirmed that Rb1 has neuroprotective effects for AD . Most recently we found that Rb1 could decrease the elevation of intracellular calcium induced by aggregated Aβ25-35 so as to inhibit the activity of calpain and lower the level of phosphorylation of tau and help stabilize microtube.However, the underlie mechanism by which Rb1 decreases the concentration of Ca2+ in cell remains unknown.Because voltage-gated calcium channel and endoplasmic reticulum calcium stores plays an important role in calcium overload in cells induced by Aβ25-35 therefore in this paper we probed into the possible mechanism of Rb1's effect on calcium overload based on mechanisms of the two sides .Method: VGCC was recorded in primary cultured hippocampal neurons (7 days in vitro, DIV7)by using whole cell configuration of patch clamp technique.Drug was applied by extracellular bath or adding in the pipette solution or pretreatment,and its effect was determined by comparing the amplitude of IHVA before and after the drug application(or among different groups).A variety of calcium channel blockers or specific protein kinase inhibitors were involved in it to examine the effect of Aβ25-35 on VGCC and the possible mechanism of Rb1's effect.The hippocampal neurons were loaded with calcium-sensitive fluorescent indicator Fluo-3/AM. Intracellular calcium concentration ([Ca2+]i)changes were measured in different conditions of drug intervention by using laser scanning confocal microscopy so as to examine the effect of Aβ25-35 on endoplasmic reticulum calcium stores and the possible mechanism of Rb1's effect on calcium release of endoplasmic reticulum induced by Aβ25-35.Result: (1)There were multiple types of Ca2+ channel in primary cultured hippocampal neurons (L, N, P / Q, T, etc.), they could be distinguished on the basis of pharmacological and kinetic properties. Acute extracellular exposure of 10μmolL-1 aggregated Aβ25-35 has no effect on VGCC; However ICa of each group(aggregated Aβ25-35 preincubated for 3, 6, 12, 24h) all had augment compared to the control group with a pronounced effect in the preincubated-3h group. Preincubation with aggregated Aβdid not have an effect on whole-cell capacitance of hippocampal neurons compared to the control group.(2)Block of L-type Ca2+ channels by its specific inhibitor nifedipine completely prevent the current augmentation induced by preincubation with aggregated Aβ,however, block of N-type Ca2+ channels or P/Q-type Ca2+ channels by their specific inhibitorsω-conotoxin GVIA andω-agatoxin IVA,respectively,could not prevent the current augmentation induced by preincubation with aggregated Aβ. PKA specific inhibitor,H-89, partially inhibited calcium current in the preincubated group but MAPK specific inhibitor, PD98059, could not .(3) Groups of 2,10,20μM Aβ25-35 all increase [Ca2+]i in hippocampal neurons. 2-APB, an inhibitor of ER Ca2+ release through channels associated to IP3R, was shown to prevent the aggregated Aβ25-35-induced rise of [Ca2+]i, suggesting the involvement of Ca2+ release by ER. Treatment with aggregated Aβ25-35 for 1 h induce a significant decrease in the ER Ca2+ content (P<0.01), which was more pronounced 24 h after the addition of aggregated Aβ25-35. The increase in [Ca2+]i observed in aggregated Aβ25-35-treated cells was prevented by the PLC inhibitor U-73122 in the absence of extracellular Ca2+.(4)Ginsenoside Rb1 inhibited calcium channel current in hippocampal neurons in a dose-dependent manner and also had the inhibitory effect on VGCC induced by Aβ25-35. Nifedipine blocks Ginsenoside Rb1-sensitive VGCC induced by Aβ25-35 completely.However eitherω-conotoxin-GVIA(a selective blocker of N-type channels)orω-agatoxin IVA(a selective blocker of P/Q -type channels)could not dimininish Rb1-sensitive VGCC induced by Aβ25-35. Ginsenoside Rb1 induced a leftward shift of the steady-state inactivation curves of ICa to negative potential in the preincubated group without affecting its activation kinetics.The co-application of 10μmol/L Rb1 and forskolin did not cancel the reduction and the action of 10μmol/L Rb1 was not affected by staurosporine(a PKA / PKC inhibitor).Conclusion:Aggregated Aβ25-35 increased VGCC by regulating the phosphorylation of specific cell membrane calcium channel via PKA. IP3 pathway involved in the early release of Ca2+ from ER induced by Aβ25-35, the activation of PLC may involve in the process. Ginsenoside Rb1 high selectively targeted L-type calcium channel and inhibited channel activity by accelerating it access to the state of inactivation.Rb1 did not affect release of intracellular calcium induced by Aβ25-35. The above may be its molecular mechanisms in alleviating calcium overload induced by Aβ25-35 and the cell basis of ginseng to play pharmacological role in the Anti-Aging and the treatment of Alzheimer's disease.