Mechanism of suppressing astrocyte mitogen-activated protein kinase 14 to alleviate neuronal injury caused by glutamate excitatory toxicity / 中华创伤杂志

ABSTRACT

Objective:To explore the action mechanism of suppressing expression of mitogen- activated protein kinase 14（MAPK14）to alleviate glutamate excitatory toxicity and its neuronal protection effect.Methods:Lentivirus-mediated MAPK14 interference vector was synthetized by Shanghai Jikai Gene Chemical Technology Co.，Ltd. Astrocytes were obtained from SD rats 48 hours after birth，which were cultured in vitro and transfected by lentivirus-mediated transfection. According to the random number table，the cells were divided into three groups：（1）un-transfected group（normal group）with normal astrocytes and the cells were cultured in regular medium composed of Dulbecco's?modified Eagle's?medium（DMEM）；（2）negative control group with astrocytes transfected by MAPK14 no-loaded interference vector；（3）lentivirus transfected group with astrocytes transfected by MAPK14 interference vector. Seventy-two hours after transfection，astrocytes were co-cultured with neurons for 48 hours，and then they were cultured in a medium containing glutamate for 2 hours. The detection indexes included the optimal multiplicity of infection（MOI）value for astrocytes transfected by lentivirus vector，mRNA levels of MAPK14 and glial glutamate transporter 1（GLT-1）detected by rPCR 72 hours after transfection，protein levels of MAPK14 and GLT-1 detected by Western blot 72 hours after transfection，level of lactate dehydrogenase（LDH）and mortality of neurons measured by spectrophotometry and flow cytometry 2 hours after culturing in the medium with glutamate. Results:（1）The optimal MOI value for lentivirus transfecting astrocytes was 30，and astrocytes grew well after transfection.（2）Seventy-two after transfection，the mRNA level of MAPK14 in lentivirus transfected group（0.005 7±0.000 6）was significantly decreased as compared with un-transfected group（0.013 1±0.001 1）and negative control group（0.013 9±0.001 0）（ P<0.01），the mRNA level of GLT-1 in lentivirus transfected group（0.009 1±0.001 2）was not significantly changed as compared with un-transfected group（0.008 7±0.000 3）and negative control group（0.008 9±0.001 1）（ P>0.05）.（3）Seventy-two hours after transfection，the protein level of MAPK14 in lentivirus transfected group（0.29±0.04）was significantly decreased as compared with non-transfected group（0.61±0.05）and negative control group（0.63±0.01）（ P<0.01），the protein level of GLT-1 in lentivirus transfected group（0.73±0.06）was significantly increased as compared with un-transfected group（0.20±0.03）and negative control group（0.23±0.09）（ P<0.01）.（4）After astrocytes were co-cultured with neurons and subsequently cultured in the medium containing glutamate for 2 hours，the level of LDH in lentivirus transfected group［（109.67±2.40）U/L］was significantly lower than that in un-transfected group［（141.52±3.88）U/L］and negative control group［（141.29±3.61）U/L］（ P<0.01）. The mortality of neurons in lentivirus transfected group［（38.72±0.26）%］was significantly lower than that in un-transfected group［（52.94±1.36）%］and negative control group［（54.30±1.23）%］（ P<0.01）. Conclusions:The transfection with lentivirus-mediated MAPK14 interference vector can increase expression of GLT-1 in astrocytes to increase glutamate re-uptake and relieve the glutamate excitatory toxicity in neurons，which may provide a new experimental basis for future use of astrocyte gene regulation to alleviate neuronal injury caused by glutamate excitatory toxicity after traumatic brain injury.