Study On The Protective Effects And Mechanism Of 3-n-butylphthalide To AMPA Induced Primary Neuron Injury

Background: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease of unknown etiology, involving the upper and lower motor neurons of the spinal cord, brainstem, and cerebral cortex, and leading to severe disability and death within a few years. Riluzole is the only drug proven to have therapeutic effect to prolong survival times to several months. Mitochondria dysfunction is considered to play an important role in the mechanism of motor neuron death indicated by autopsy and transgenic model of ALS. NBP (3-n-butylphthalide) is a drug used to therapy cerebral infarction. It has been proven to have protective effect to mitochondria after ischemia. We tested whether NBP has therapeutic effect in ALS.Current researches manifest that glutamate excitotoxicity mediated by AMPA receptor play an important role in motor neuron death in ALS. AMPA receptor is a kind of Glutamate ion channel. It has been thought to be calcium-impermeable and the calcium influx caused by AMPA receptor owning to the voltage-gate channel activated by sodium influx through AMPA receptor. Now it is widely accepted that AMPA receptor can be divided into calcium-permeable and calcium-impermeable subtype and the calcium-permeable AMPA receptor is considered to have strong relationship with ALS.Objects The paper contains two parts. In the first chapter, we choose AMPA to cause neuron damage and investigate the effect of NBP to the injury. The second chapter is based on the theory that calcium influx through AMPA receptor play a major role in the mechanism of motor neuron lose in ALS. We investigate the intracellular calcium change caused by AMPA and the effect of NBP to this change.Methods In the first part, we choose lactate dehydrogenase (LDH) and mitochondrial transmembrane potential to investigate necrosis and apoptosis. Primary neuron were obtained from neonate rat and cultured in neurobasal A plus B27 to the 6～7 days. Then AMPA plus cyclothiazide were added to the culture medium and treated for 24 hours. The extra cellular fluid was collected and LDH were analysis. JC-1 was used to measure mitochondrial transmembrane potential difference in neuron to evaluate apoptosis. In the second part, we use evaluate the influence of NBP on calcium influx. The difference of LDH and mitochondrial transmembrane potential among blank, AMPA damage and NBP protect were analysis. In the second part, we investigate the intracellular calcium change caused by AMPA and the effect of NBP to this change. Then we excluded the sodium in extra cellular fluid. Under such condition, the calcium influx caused by calcium-impermeable AMPA receptor was removal and the effect of AMPA and NBP to the calcium influx through calcium-permeable receptor was evaluated.Result AMAP induced LDH rise in extra cellular fluid compared to contrast, suggesting the cultured neurons were undergoing necrosis. AMPA can also decrease mitochondrial transmembrane potential compared to vacuity group, suggesting apoptosis. NBP can release the change of LDH and mitochondrial transmembrane potential suggesting it may have therapeutic effect to both necrosis and apoptosis induced by AMPA receptor. The second part showed that NBP can release the rise of calcium concentration induced by AMPA and then we found the concentration of calcium can also be down regulation before AMPA stimulating. This suggests that NBP might change the calcium homeostasis. In the Na~+-free solution, NBP can also reduce the concentration of calcium before AMPA stimulating and during AMPA treatment; NBP can reduce the amplitude of calcium rise compare to blank suggesting AMPA can reduce the rise of calcium concentration caused by calcium-permeable AMPA receptor.Conclusion In this experiment, we find AMPA can damage the cultured cortex neuron, both necrosis and apoptosis may play a role in this duration. NBP showed protective effect to AMPA excitotoxicity to both mechanisms. Through investigating the effect to intracellular calcium, the result suggest NBP can down regulate the intracellular calcium concentration, this might have relationship with its effect to endocytoplasmic reticulum regulating calcium concentration. NBP can also down regulate the influx of calcium through calcium-permeable AMPA receptor.