| Alzheimer’s disease(AD)is a neurological degenerative disease characterized by memory loss,cognitive impairment and behavioral and psychological symptoms of dementia.The most widely accepted pathological feature of AD is the senile plaques formed by abnormal deposition of neuropil β-amyloid peptides(Aβ)and neurofibril tangles(NFT)formed by abnormal phosphorylation of tau.There are also some neurophysiological features of AD such as increased network activity,epileptic activity,slowed neuro oscillation and decreased waveform complexity.Soluble oAβ is an upstream pathogenic factor of AD,which induces functional hyperexcitability by synapse drive mechanism,is toxic to primary neurons in rat and mouse models,inhibits long-term enhancement of hippocampus,and causes memory impairment.The main forms of oAβare oAβ1-40 and oAβ1-42 and the latter has the strongest toxicity.Therefore,inhibiting the toxicity of oAβ1-42 may be an important pathological mechanism to protect hippocampal neurons from damage.One of the earliest explanations of neuronal dysfunction and toxicity in AD was the channel hypothesis first proposed by Arispe et al.,which postulates that unregulated Aβion channels cause the loss of ion homeostasis(mainly by acquiring calcium ions)and ultimately trigger neuronal dysfunction and cell death.Anle138b,a diphenylpyrazole compound,is a low-polymer regulator has low toxicity,no detectable toxicity at therapeutic doses,high oral bioavailability,and good blood-brain barrier permeability.Anle 13 8b can block the Aβ channel and rescue the phenotype of amyloid-β-induced small mice model.Our previous work shows that either prophylactic or therapeutic administration of Anle138b can antagonize the toxic effect of oAβ1-42 on hippocampal neurons,but its mechanism is still unclear,especially whether Anle138b can protect rat hippocampal neurons against oAβ1-42-induced hippocampal neuronal hyperexcitation.In this experiment,SD rats born within 24 h were selected as experimental materials.On the basis of primary hippocampal neurons in vitro culture,hippocampal neurons were treated with oAβ1-42(100 nmol/L)continuously for 7 days to induce hippocampal hyper-excitation.Anle 13 8b was pre-cultured to establish an AD model induced by oAβ1-42.Lactate Dehydrogenase(LDH)kit and patch clamp technique were used to explore whether Anle138b can protect rat hippocampal neurons from oAβ1-42-induced rat hippocampal hyper-excitation and neurotoxicity.The results are as follows:1.Identification of primary hippocampal neurons in rats based on morphological and electrophysiological characteristics.The results showed that when a depolarizing current of 40 pA was applied,the average discharge frequency of action potentials in non-pyramidal neurons(11.74±0.76 Hz)was significantly faster than that in pyramidal neurons(5.45±0.65 Hz),with statistical significance(P<0.001).Pyramidal neurons were selected for this experiment.2.Primary hippocampal neurons from cultured rats were treated continuously with 100 nmol/L oAβ1-42 for 7 days.When hippocampal neurons were stimulated with a depolarizing current of 40 pA,compared with the control group,the oAβI-42 group showed a significant increase in the discharge frequency of action potentials(P<0.001),and the amount of LDH release also significantly increased(P<0.05),indicating that chronic oAβ1-42 treatment induced hyperexcitability and toxicity in primary cultured hippocampal neurons,which could serve as a cellular toxicity reaction model.3.When stimulated with a depolarizing current of 40 pA,treatment with different concentrations of Anle 13 8b(10,50,100 nmol/L)alone did not affect the discharge frequency of action potentials in hippocampal neurons(P>0.05),indicating that the above three concentrations of Anle138b themselves had no effect on hippocampal neurons and could be used in neuroprotection experiments.4.Single-cell patch clamp recording showed that when hippocampal neurons were stimulated with depolarizing currents of 10,20,30,40 pA,the discharge frequency of action potentials increased continuously,forming an input-output curve.Compared with the group treated with oAβ1-42 alone,the input-output curve of the oAβ1-42+Anle 138b group shifted significantly downwards.Statistical analysis of the slope of the input-output curve of the discharge frequency of action potentials showed that compared with the control group,chronic oAβ1-42 treatment could induce hyperexcitability in cultured hippocampal neurons(P<0.001),while Anle 13 8b could reduce oAβ1-42-induced hyperexcitability(P<0.05),with statistical significance.This indicates that Anle138b can prevent oAβ1-42-induced hyperexcitability in hippocampal neurons and has a neuroprotective effect.5.The analysis of action potential parameters shows that when giving hippocampal neurons 40 pA depolarizing current stimulation,compared with the oAβ1-42 group,the co-treatment group of Anle138b and oAβ1-42 prolongs the onset time of action potential(P<0.05)and enhances the post-superhypepolarization amplitude(P<0.001).6.Single-cell patch clamp recording shows that when giving hippocampal neurons 40 pA depolarizing current stimulation,compared with the group using oAβ1-42 alone,the discharge frequency of hippocampal neuron action potential in the 10 nmol/L Anle138b group had no significant change(P>0.05).However,the 50 nmol/L and 100 nmol/L Anle138b groups decreased the discharge frequency of hippocampal neuron action potential(P<0.001;P<0.01).7.When giving hippocampal neurons 40 pA depolarizing current stimulation,oAβ1-42 treatment for 5 days can induce hippocampal neuron overexcitation(P<0.001),and after 5 days of treatment,using hippocampal neuron culture solution alone for 2 days and 5 days,hippocampal neurons still maintain overexcitation(P<0.001).8.Compared with the group using oAβ1-42 alone for 5 days,the discharge frequency of hippocampal neurons in the 10 nmol/L Anle138b group had no significant change(P>0.05).However,the 50 nmol/L and 100 nmol/L Anle138b groups,co-treated with oAβ1-42 for 2 days,can reduce the already formed hippocampal neuron hyperexcitation induced by oAβ1-42(P<0.01;P<0.05),indicating that Anle138b above 50 nmol/L can reverse the formed hippocampal neuron hyperexcitation.9.When voltage-locked to-70 mV,Anle138b(100 nM)itself has no effect on the excitatory synaptic function of hippocampal neurons(P>0.05).Compared with the control group,the oAβ1-42 group had no significant change in amplitude and half-decay time(P>0.05),but frequency significantly increased(P<0.001).Compared with the oAβ1-42 group,the amplitude and half-decay time of the oAβ1-42+Anle138b group and the oAβ1-42+Anle138b 2 days group had no significant change(P>0.05),but frequency decreased significantly(P<0.0001),indicating that Anle138b can reduce the release of presynaptic glutamate.In summary,continuous administration of oAβ1-42 for 7 days can cause hyperexcitability and toxicity reactions in hippocampal neurons,making it a model for oAβ1-42 cytotoxicity.We used this model to evaluate the effect of Anle138b on oAβ1-42-induced hyperexcitability in hippocampal neurons and found that Anle138b not only prevented chronic oAβ1-42-induced hyperexcitability and synaptic toxicity,but also eliminated already formed hyperexcitability and synaptic toxicity induced by oAβ1-42.The mechanism of action of Anle 13 8b includes altering ion channel function in post-synaptic neurons and reducing the release of glutamate in pre-synaptic terminals.Therefore,small molecule inhibitors like Anlel38b may have great potential in the treatment of AD and are worth further investigation... |
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