Research On Anti-acidosis And Mechanisms Of Acid-sensing Ion Channels 2a Subunit

Incidence,mutilation and mortality of ischemic cerebrovascular disease are high. Ischemic cerebrovascular disease,cardiac disease and malignant tumor become three major death causes in human beings.Exploring mechanisms of ischemic neuron injury will help prevention and treatment of ischemic brain injury.Although huge improvement on mechanisms of ischemic brain injury has been made from cytology molecular biology, but no effective neuro-protection drugs on stroke were found.It is well known that NMDAR activation and subsequent Ca²⁺ overload are critical for ischemia-induced cell death.However,clinical trials with agents targeting these mechanisms have been disappointing.Unfavorable experiences with mechanisms involving NMDA receptor channels have resulted in redirection of attention to other channels.Acidosis is a common feature of ischemia and is assumed to play a critical role in brain injury.Acid-sensing ion channels(ASICs) are H⁺- gated cation channels expressed throughout mammalian central and peripheral nervous system.ASICs are activated by acidic pH and sodium enters the cells through this channel causing cell depolarization and excitation.They belong to the DEG/ENaC(degenerin/epithelial sodium channel) superfamily.Until now,six subunits of ASICs encoded by 4 genes have been cloned: ASIC1a,ASIC1b,ASIC2a,ASIC2b,ASIC3 and ASIC4.It is associated with synaptic transmission and plasticity,spatial learning/memory,long-term potentiation(LTP),pain transduction and ischemia neuronal damage.It plays an important role in physiological and pathological conditions.ASIC1a and ASIC2a are widely expressed in central nervous system and closely associated with ischemia brain injury.ASIC1a played an important role on glutamate receptor-independent neuronal injury because of its high susceptibility for acid and high permeability for calcium.Recently Jonson et al presented evidence that transient global ischemia induces ASIC 2a protein expression in neurons that survive ischemia.Klenow fragmentmediated labeling of DNA strand breaks determined that ASIC2a induction did not occur in cells with detectable DNA damage.So we suggested a possible role for ASICs in mediating a cellular response to ischemia.Therefore our aim is to observe the possible effect and mechanism by depressing the expression of ASIC2a with RNA interference technique in acidosis environment.And we are attempt to build neuron model in vitro under OGD combined with acidosis to investigate ASIC2a expression change.First we amplified the different subunits of ASICs in C6 cells(one kind of rat neuroglioma) with semi-quantitative RT-PCR again.Cytotoxic edema was investigated by various pH values(6.5,6.0,5.5 or 5.0) for 2 hours with flow cytometry except that pH7.4 and pH7.0 had no influence on cellular volume.Replacement of extracellular Na⁺ by choline chloride and addition of 100μM amiloride led to obvious prevention of the acidosis-induced cell swelling.So we think that acidosis activated homomeric and heteromeric ASICs channel currents and that induced sodium inflow.We tested cell injury with relative LDH release.Cytotoxic edema increased relative LDH release and acute cell damage aggravated.Replacement of extracellular Na⁺ and addition of 100μM amiloride reduced acidosis-induced cell injury.Delayed cell injury was induced by various pH values for 2 hours and reperfusion 24 hours.Generally calcium participated in delayed cell injury.To elucidate whether calcium inflow played an important role on C6 injury by acidosis,we observed the effects on C6 under pH7.4 or pH6.0 acidic condition with[Ca²⁺]o rising from 0.2 mM to 1.3 mM.The results showed that the pH6.0 group with 1.3 mM[Ca²⁺]o had higher relative LDH release than 0.2 mM[Ca²⁺]o.And amiloride can reduce relative LDH release by 1.3 mM[Ca²⁺]o.Second we transfected specific siRNA into C6 cells and effective siRNA that was called C6-siRNA_2a was screened by Western blotting.Transfected with non-specific control was called C6-control.Compared to C6-control,C6-siRNA_2a induced more serious cytotoxic edema and relative LDH release under pH6.0 and pH5.0 extracellular solutions for 2 hours.But there was no difference between C6-control and C6-siRNA_2a about cell volume and injury after replacement of extracellular Na⁺ by choline chloride and addition of 100μM amiloride pretreatment.Delayed injury induced by reperfusion after 24 hours,MTT assay and relative LDH release were utilize to detect viability of C6 under various pH values.The same as before,C6-siRNA_2a produced more serious damage under pH6.0 and pH5.0 extracellular solutions after 24 hours.Under pH6.0 acidic condition with[Ca²⁺]_o rising from 0.2 mM to 1.3 mM,C6-siRNA_2a produced more relative LDH release implying reduced viability.That cytoplasm fluorescence intensity of calcium in C6-siRNA_2a dyed by fluo-3AM was higher than C6-control was direct experiment proof of calcium overloaded in pH6.0 and pH5.0 group.We recorded ion currents of ASIC1a,ASCI2a or ASIC1a/2a expressed in Xenopus laevis oocyte system.We found that pH₅₀ of ASIC1a,ASCI2a and ASIC1a/2a was 5.9,4.9 and 5.2 respectively.Only ASIC1a had permeability for calcium.So we concluded that ASIC1a proportionality increased in the all ASICs subunit because of down-regulation of ASIC2a and that raised susceptibility for acidosis,augmented sodium and calcium inflow, aggravated cytotoxic edema,acute and delayed injury.Third we build rat hippocampus neuron ODG combined with acidosis model in vitro to mimic in vivo circumstances.We observed that the expression of ASIC2a at pH6.0 and pH5.8 was significantly increased by 2-fold compared with pH 7.4 after prolonged 3h OGD/reperfusion at Oh.ASIC2a protein expression was also up-regulation at pH6.5 reperfusion at 7h.After that it gradually decreased to normal level at 24h. ASIC2a protein at pH 5.8 as well as ASICla was down-regulated dramatically because of a diminished capacity for protein synthesis induced by a big injury.Since acidosis is a central feature of brain ischemia,cell injury was assayed by the measurement of LDH release at various time points.Time-dependent injury was observed.In conclusion specific down-regulation ASCI2a in C6 induced more severious cytotoxic edema,acute cell injury and delayed cell injury implying that ASIC2a subunit could protect cell from acidosis in some degree.Besides we tested ASIC2a expression up-regulation in vitro model cell successfully.ASIC2a may be a potential protection targets for neuron.