Study On Functional Mechanisms Of Acid-sensing Ion Channels And Protection Of Puerarin During Acidotic-companied Ischemia And Reperfusion

Ischemic stroke is one of the major causes of human death and disabilities with main parthological mechanism of neuronal injury by ischemia. Acidosis is a common feature of ischemic brain. Accumulation of lactic acid as a byproduct of glycolysis and protons produced by ATP hydrolysis cause extracellular pH reduction during brain ischemia. Acidosis lasts for more than 4 hours with a pH below 6.0 under hyperglycemic conditions, which is expected to activate acid-sensing ion channels (ASICs). ASICs, which are activated by extracellular acidosis and mainly permeate Na+ currents, are members of Deg/ENaC super family. Up to now, six different ASIC isoforms, derived from four genes, have been identified. It has been reported that the wide spread expression of ASIC1a, which is the only acid sensing ion channel that conducts Ca2+, gives us a new clue to glutamate-independent intracellular Ca2+ accumulation and neuronal injury during ischemia. Although ASICs, especially ASIC1a, are considered to be new targets for brain ischemia, few drugs have been shown to effectively inhibit ASIC currents. The injury mechanisms of ASICs during ischemia and reperfusion also remain illuminated.In this study, we developed an in vitro acidotic model in rat hippocampal neurons. We investigated the effect of ASICs and ASIC1a during acidosis using techniques of patch clamp and laser scanning microscopy of confocal (LSMC).We also applied oxygen-glucose deprivation (OGD) and reperfusion during acidosis to simulate in vivo ischemia/reperfusion. The modulatory effect of ASICs on cell viability during OGD or reperfusion-companied acidosis was determined. The mechanism of ASICs modulated by OGD was also investigated. In the meantime, the modulatory effect of puerarin on ASICs and ASIC1a was also delineated, providing a mechanical insight into neuroprotective effect of puerarin during brain ischemia.The main results of this study are:1. Both amiloride, inbitor of ASICs and PcTxl,specific blocker of ASIC1a were able to protect hippocampal neurons from acidotic injuries.2. A home-made'Y'-tube perfusion system was employed to achieve a rapid extracellular solution change. The fast reduction extracellular pH induced a large inward current that was slow activated and slow inactivated, which could be dose-dependently inhibited by amiloride with IC50 of 13.8μM. This confirmed that the inwared current was ASICs.3. Extracellular acidosis was able to inhibit spontaneous current in 12-day cultured neurons. This inhibition was ASICs-independent.4. Puerarin could protect hippocampal neurons from acidotic injuries. Electrophysiological experiments showed that puerarin inhibit ASICs and ASIC1a current in a dose-dependent way with IC50 of 38.4μM and 9.31μM, indicating that puerarin prtected neurons by a mechanism of inhibing ASICs, especially ASIC1a.5. Acidosis induced neuronal damage during both OGD and reperfusion. However, it caused more detrimental injury during reperfusion. Application of neither amiloride nor PcTxl could protect neurons during OGD, while application of either amiloride or PcTxl improve neuronal viability during reperfusion. This result showed that the opening of ASICs, especially ASIC1a, induced cell injury during reperfusion other than ischemia. On the other hand, neither the amplitude nor desensitization of ASICs changed during OGD.Nevertheless, the recovery of ASICs from desensitization slowed down from 16.12±2.18s during normoxia to 69.54±21.10s during OGD, resulting to a reduced opening frequency of ASICs. This may be the protective mechanism of OGD from the acidotic injuries.In conclution, our results suggested that ASICs took part in the neuronal injuries during acidosis, but did not take part in the the acidosis-induced reduce of spontaneous currents. Futhermore, we also showed that OGD slowed down recovery of ASICs from desensitization, which may result the more detrimental effect of reperfusion during acidosis. On the other hand, puerarin protected neurons from acidotic injuries probably by inhibiting ASICs and ASIC1a. These results indicated that ASICs play an important role during ischemia/reperfusion, which gave us a new clue to drug development for stroke.