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Objective: Alzheimer's disease (Alzheimer's disease, AD), also known as Alzheimer's, based on memory, impaired cognitive function in neurodegenerative diseases as the main feature, typical pathological changes of senile plaques (senile plaque , SP), neurofibrillary tangles (neurofibrillary tangle, NFT) and a large number of neuron loss. β-amyloid protein (β-amyloid beta protein, Aβ) is the main component of senile plaques, composed of 39 to 43 amino acids. The study found that Aβ 25-35 , a short molecular fragment with complete peptide chain and Aβ neurotoxicity, and therefore is widely used in the experimental study of AD. AD etiology and pathogenesis of doctrine lot of that has been decided yet, but generally agreed that Aβ plays an important role in the pathogenesis of AD process. Therefore, in-depth study of its toxicity mechanism of action, has an important role in the understanding of AD pathological mechanism and prevention. The study confirmed that the voltage-dependent potassium channels are widely distributed in the central nervous system, mainly through the influence of the formation of the action potential, the shape of the action potential to adjust the frequency of the action potential, and adjust the resting membrane potential affect nerve function, including learning and memory. There are at least two kinds of voltage-gated potassium channels in hippocampal neurons, rapid activation and rapid inactivation of transient outward potassium current (I A ) and slowly to activate almost inactivating delayed rectifier potassium current (I K ). Studies have shown that potassium channels are involved in the damage and age-related cognitive function, and play an important role in learning and memory processes. I A current is the main component of the action potential repolarization early outward currents play an important role in regulating neuronal firing frequency, action potential generation and discharge mode. Large number of experiments prove that I A dynamics characteristics by phosphorylation regulation, I A channel hippocampal pyramidal cells can activate protein kinase A (protein kinase A, PKA) open probability is significantly reduced, resulting in a prolongation of the action potential, caused by the the Ca2 large influx, and trigger the release of neurotransmitter. Studies have confirmed that the cAMP / PKA-CREB pathway plays an important role in memory formation. PKA-CREB signaling pathway dysfunction exists in AD. Studies have shown, Aβ at low concentrations can cause dysfunction PKA-CREB signaling pathway, caused by closely related to learning and memory function in hippocampal CA1 region-term potentiation (long-term potentiation, LTP) is suppressed. Our previous study has observed that Aβ can significantly inhibit voltage-gated potassium channels (including I A and I K ), thus we speculated that Aβ 25 -35 in the I A current inhibition may be associated with the activation of the PKA pathway, or PKA may be mediated by the Aβ 25-35 I A current inhibition. Humanin (HN) is a recently discovered neuroprotective peptide, studies have shown, HN specific inhibition of familial Alzheimer's disease (familial Alzheimer's disease, FAD) gene mutations and Aβ deposition induced nerve cell death . And by the antagonistic of Aβ 25-35 induced PKC activation to antagonize Aβ 25-35 -induced inhibition of the current I K . HN whether through antagonizing Aβ 25-35 -induced PKA activation thereby antagonizing of Aβ 25-35 -induced I A inhibition of the current. ? speculate based on the above, we use the whole-cell patch clamp technique PKA potassium channel function to change the process and HN antagonism caused by Aβ. To clarify the Aβ caused the protective effect of potassium channel dysfunction mechanisms and HN. Method: 1, the separation of the hippocampal neurons extract selection 7-10 days SD rat hippocampal neurons isolated from the CA1 region of cell suspension, added to the petri dish. When the cells adherent 2ml clean the extracellular fluid under an inverted microscope for patch-clamp record. 2, the control group, Aβ Groups of experimental groups (5μmol / L), HN group (5μmol / L), 8-Brom-camp group (5μmol / L, 50 μmol / L, 500μmol / L), H-89 (10 μmol / L, ) Aβ group (5 μmol / L), electrophysiological measurement of whole-cell patch-clamp mode using voltage-clamp recording mode, the recording conditions given cells were recorded voltage-dependent depolarization pulses from -40 ~ 70mV (step 10mV) total potassium current and I K current, then the formula or rate I A . I K to whichever 158ms at the steady state current, while I the A whichever is the peak current. Compare different modes of administration in the same hippocampal neurons, caused by changes in potassium current. 4 x? S data are measured results of data analysis SPSS13.0 software for single-factor analysis of variance, and significant differences before and after dosing were analyzed by t-test. Inspection level α = 0.05, P lt; 0.05 indicated significant differences. Results: 1, Aβ 25-35 (5 μmol / L) of the voltage-dependent potassium current I A current was significantly inhibited. 70mV depolarizing pulses, Aβ 25-35 alone of the whole potassium current amplitude reduced to 2022.60 ± 3421.70 ± 409.98pA from the control group 442.68pA current amplitude compared with the control group decreased by 59.11 ± 12.94% (n = 5, p lt; 0.05); I, A Current at 2196.77 ± 170.56pA from the control group decreased to 1340.61 ± 259.71pA, compared with the control group, the current amplitude reduce 61.03 ± 11.82% (n = 5, p lt; 0.05). 2, PKA antagonist H-89 (10μmol / L) can block Aβ caused inhibition of I A . Joint use of the H-89 and Aβ canceled Aβ inhibition of potassium current, total potassium current I A current recovered to the level of the control group, relative to the normal current amplitude were 104.78 ± 10.19% (n = 5) and 119.68 ± 22.87% (n = 5); with a single application of Aβ 25-35 (5 μmol / L) current relative values ??were statistically significant (p lt; 0.05); different concentration of PKA agonist 8-brom-camp (5μmol / L, 50 μmol / L, 500μmol / L) dose-dependently inhibited the transient outward potassium current (I A ), relative to the current for the control group, the I A magnitude of the current were decreased to 87.18 ± 15.33% (n = 5, p gt; 0.05), and 59.91 ± 5.11% (n = 5, P LT; 0.05 ), 46.59 ± 2.56% (n = 5, p lt; 0.05). Delayed rectifier potassium current (I K ) rose slightly, but not significantly; 4, HN (5μmol / L) antagonize Aβ caused the whole potassium current and I A < / sub> current suppression; HN also can antagonize PKA agonist 8-brom-camp (50μmol / L) I A current suppression caused. Give HN (5μmol / L) after Aβ total potassium current and I to A current amplitude respectively from from 2022.60 ± 442.68pA and 1340.61 ± 259.71pA, to rise to from 2952.00 ± 272.01pA and 1928.63 ± 172.68pA, (n = 5, p lt; 0.05), 86.27 ± 7.95% of the control group and 87.79 ± 7.86%, respectively. Of Aβ 25-35 group, were statistically significant. Give HN (5 μmol / L), and after the 8-brom-camp I A relative current amplitude increased from 59.91 ± 5.11% for the control group, 78.68 ± 10.00% (n = 5). Conclusion: 1, acute administration of Aβ 25-35 inhibition of voltage-dependent potassium current, the I to A current. 2, PKA-mediated inhibition of Aβ on I A of I A inhibition may be one of the mechanisms of Aβ toxic effects through the activation of the PKA pathway. 3, HN antagonize Aβ 25-35 inhibition of I A , at the same time HN antagonistic PKA agonists caused of I A suppressed. Inhibition of Aβ activation of PKA and toxic effects may be caused by one of the mechanisms of HN antagonize the toxic effects of Aβ.
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