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Acid sensing ion channel (acid-sensing ion channels, ASICs) H -gated cation channel, extracellular H concentration increased activation open extracellular Na through an open channel to enter the cells, resulting in cell depolarization excited. ASICs channel belongs to the epidermal sodium channel / degradation factor gene (ENaC / DEG, epithelial sodium channel / degenerin gene) in a superfamily, so far, six different subunits encoded by the four genes have been cloned, they are : ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3 and ASIC4. , ASIC1a is a very important subunit, in synaptic regulation passed its plasticity, spatial learning and memory, long-term potentiation (LTP), pain transmission and ischemic brain damage and other physiological and pathological activities play an important role. Tissue acidification is a very common pathological phenomena, such as stroke, seizures, inflammation, tumor tissue and metabolic abnormalities will cause local tissue acidification further tissue damage. Experimental results show that in the past ASIC1a involved in the acidification induced neuronal damage in the voltage-gated Ca 2 channels and glutamate receptor blocker existing circumstances, ASIC1a activation followed by intracellular Ca 2 overload can cause nerve injury time-dependent, and ASIC1a specific blocker (PcTX1) or the lowering extracellular Ca 2 inhibited. To further explore the relationship between inhibition of ASICs and acidification induced injury ASIC1a gene targeting RNAi (RNA interference) technology, designed to observe ASIC1a subunit expression was knocked down after the antacid possible neuroprotective role. First, ASICs subunits in rat glioma C6 cells was confirmed by RT-PCR, expression in rat hippocampal neurons and DRG neurons. Then built using RNAi technology the shRNA against of ASIC1a and ASIC2a subunit expression vector, effective interference plasmid was screened by western blotting. ASIC1a expression stably knocked down cell lines and stable negative control cell lines followed by the establishment of a stable strains ASIC1a knockdown effect was confirmed by semi-quantitative RT-PCR and western blotting. Colony formation rate was found by cell growth curve and measurable sets: to inhibit ASIC1a subunit expression did not change the growth and proliferation of cells in normal circumstances. By MTT assay and LDH assay and found to inhibit the ASIC1a subunit expression can significantly reduce the damage response induced by the acidification under acidic conditions (pH6.0, pH6.5, pH6.9) increased significantly: the number of cell survival, survival status improved. LDH release was found to inhibit ASIC1a subunit expression in rat hippocampal neurons transfected pshRNA1a2 interference plasmid, can significantly reduce the neuronal damage pH6.0 under acidic conditions, which is consistent with the experimental results obtained on C6 cells. In order to explore the ASIC1a subunit was knocked down cell antacid injury was a factor in the experiment, we compared the added amiloride (amiloride) to inhibit ASIC1a subunit expression differences of the pH6.0 injury protection role, and found : their protection role fairly, the difference was not statistically significant. Preliminary to inhibit the ASIC1a subunit expression antacid injury mechanism on C6 cells, we first prove a the permeable Ca 2 was acid-activated channels in C6 cells, then compared at pH6.0 under the conditions of the extracellular Ca 2 to rise from 0.2mM to 1.3mM while the shRNA1a2 (2) and shNEG cells. The results showed that the acidification can cause damage to the two groups of cells, but shRNA1a2 (2) compared with shNEG group, the degree of injury has been reduced, the number of cell death is significantly reduced. The above study shows that the knock-down the ASIC1a subunit expression can be significantly reduced acidification induced cell damage of ASIC1a subunit was knocked down may be the main reason of cells acid resistance, the ideas and experimental basis for the new brain protection strategy.
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