The Effect Of Moxa Extract On The Apoptosis Of Primary Cultured Neurons And Its Mechanism

Glutamate(GLU)is a major excitatory neurotransmitter in the central nervous system of mammals.It plays an important role in many neurophysiological processes,such as neuron growth and development,synaptic stability and plasticity,learning and memory,and other neurotransmitters.However,the accumulation of glutamate in synapses can lead to neuronal toxic damage,aging and death,which is called "excitotoxicity" of neurons.The characteristic pathological changes of Alzheimer’s disease(AD)are extracellular senile plaques formed by amyloid beta deposition,intracellular neurofibrillary tangles formed by over-phosphorylation of Tau protein,loss of neurons and glial cell proliferation.Glutamate-mediated neurotoxic damage is one of the important mechanisms for the occurrence and development of Alzheimer’s disease.Therefore,how to effectively inhibit the apoptosis of neurotoxic cells induced by glutamate has become an important way to treat AD.Moxa smoke extract is an effective mixture extracted from moxibustion.It contains a variety of bioactive ingredients and has anti-free radical,bactericidal,anti-apoptotic and anti-oxidative effects.Previous experiments on the mechanism of moxibustion showed that moxibustion and moxibustion smoke could improve the learning and memory ability of AD model rats by antioxidant,regulating neurotrophic factors and reducing the toxicity of A β.These results suggest that moxa smoke and moxibustion therapy can be used to prevent and treat AD.Moxa smoke extract may be a promising drug for treating AD.On this basis,we used the Moxa smoke extract to interfere with the primary cultured normal neurons in the prefrontal cortex and hippocampus of one-day-old SD rats and the neurotoxic injury cell model induced by glutamate.We observed its anti-apoptotic effect on the normal cells and the injury cell models,and discussed the effect of moxa smoke extract on the normal neurons and glutamate-mediated neurons.Elucidating the protective effect and molecular mechanism of moxa smoke extract on normal nerve cell and AD cell models to provide theoretical basis for clinical application of moxibustion and moxa smoke extract to prevent and treat AD.OBJECTIVETo investigate the effects of moxa smoke extract on the prefrontal cortex and hippocampal neurons and glutamate-mediated neurotoxic injury models and its potential mechanism.METHODSNeurotoxicity injury models were established by primary culture of neurons in prefrontal cortex and hippocampus of neonatal SD rats in 24 hours after glutamate induction.The experiment was divided into two parts:1)To detect the effects of different concentrations of moxa smoke extract on the growth and apoptosis of normal nerve cells and its mechanism.The experimental groups were divided into blank control group,moxa smoke extract group and moxa smoke extract+ PI3K/Akt signaling pathway blocker group.2)To detect the effects of moxa smoke extract on the apoptosis of neurotoxic injury cells and its mechanism.The experimental groups were divided into blank control group,injury model group,moxa smoke extract + injury group and moxa smoke extract + injury + PI3K/Akt signaling pathway blocker group.MTT was used to detect the cell survival rate,TUNEL and DAPI immunofluorescence were used to detect the morphological changes of cells,and Western Blot was used to detect the expression of apoptotic protein.RESULT1.MTT and TUNEL assays showed that glutamate could decrease the survival rate and increase the apoptotic rate of nerve cells in a dose-dependent manner(P<0.001);moxa smoke extract in 0.1-1.0 μ g/mL could promote the growth of nerve cells and inhibit their apoptosis(P<0.01-0.001);moxa smoke extract in 0.2-1.2 μ g/mL could increase the survival rate of neurotoxic injury model cells induced by glutamate and decrease their apoptosis(P<0.01-0.001).2.Western Blot assay showed that glutamate could decrease the the expression of anti-apoptotic proteins of P-Akt and Bcl-2 and the expression of pro-apoptotic protein of active-Caspase-3 were up-regulated(P<0.01-0.001);moxa smoke extract chould reverse the effect of glutamate(P<0.01-0.001);the PI3K/Akt pathway blocker LY294002 could inhibit the apoptotic effect of moxa smoke extract(P<0.01-0.001)and decreased the expression of anti-P-Akt and Bcl-2 and increased the expression of active-Caspase-3(P<0.05-0.001).CONCLUSIONA certain concentration of moxa smoke extract can promote cell growth and delay neuronal apoptosis.Its mechanism may be to antagonize neuronal apoptosis induced by various factors by activating the PI3K/Akt signaling pathway and regulating the expression of downstream apoptosis-related proteins of Bcl-2 and active-Caspase-3.