The Experimental Study Of Valproic Acid Mediated Neuroprotective Function

Aim Valproic acid(VPA)is widely used as an anticonvulsant and mood-stabilizing drug.Accumulating evidence showed that VPA also has anti-cancer,anti-inflammatory and neuroprotective effects.Cerebral malaria(CM)is a severe neurological complication of infection with Plasmodium falciparum.However,there are still many challenges that need to be overcome before proper control or even elimination of malaria can be achieved.One of the most significant challenges is the increasing parasite resistance to virtually all drugs used for malaria therapy.Therefore,developing new drugs with increased efficacy is critical for controlling malaria.However,the role of VPA in experimental cerebral malaria(ECM)still needs further research.Therefore,the study aims to investigate the protection function of VPA in ECM model.Methods1.A suspension containing 1x10~5 parasitised red blood cells was injected via tail vein into C57BL/6 mouse to establish the ECM model,5 days later,serum was collected by heart puncture and centrifuged.2.Embryonic 13.5 mouse neural stem cell(NSCs)was successfully cultured in vitro,half medium was changed every three day,and they were passaged every 6 day.Then the third passage NSCs were passaged and plated on Poly-L-Lysine(PLL)-coated plates,3 days later,serum collected from ECM mouse and control mouse were added in the NSCs,1mM VPA was also added,then 5 days later,immunocytochemistry and RT-PCR was used to observe the effect of VPA on NSCs which cultured with serum collected from ECM.3.Embryonic mouse hippocampcal neuron was successfully cultured in vitro,half medium was changed every three day,and 7 days later,serum collected from ECM mouse and control mouse were added in the neuron,1mM VPA was also added,then 3 days later,DCFH-DA was used to detect the level of reactive oxygen species(ROS),CytoTox96~?non-radioactive cytotoxicity assay kit was used to detect the level of lactic dehydrogenase(LDH),Tunel apoptosis detection kit was used to detect the apotosis cell;4.A suspension containing 1x10~4 parasitised red blood cells was injected via tail vein into C57BL/6 mouse to establish the ECM model,300mg/kg VPA was injected intraperitoneally into ECM mouse for 5 days at day 1 post after infection.Survival rate and rapid murine coma and behavior scale(RMCBS)were recoded,haematoxylin-eosin staining was taken to observe the sequestered parasites and haemorrhage,evans blue was used to observe the blood brain barrier(BBB) permeability,IFN-γ,IL-1β,IL-6,IL-10 and TNF-αlevel were also detected;5.A suspension containing 1x10~4 parasitised red blood cells was injected via tail vein into C57BL/6 mouse to establish the ECM model,300mg/kg VPA was injected intraperitoneally into ECM mouse for 5 days at day 1 post after infection.Brain derived neurotrophic factor(BDNF),Glial cell line-derived neurotrophic factor(GDNF)and Nerve growth factor(NGF)level were deteceted,and patch clamp was used to measure the current of hippocampcal neuron.Results1.Immunocytochemistry results showed that theβ-Ⅲ-tubulin positive neurons decreased and GFAP positive astrocytes increased after addition of serum collected from ECM mouse,andβ-Ⅲ-tubulin positive neurons increased and GFAP positive astrocytes decreased after VPA addition;RT-PCR results also similar with the immunocytochemistry results.2.Light microscope,ROS,LDH and Tunel results showed that more neurons floated,ROS increased,LDH increased,Tunel increased after ddition of serum collected from ECM mouse,and less neuron floated,ROS decreased,LDH decreased,Tunel decreased after VPA addition;3.ECM model was successfully established,after VPA injection,the survival rate,weight,RMCBS decreased,and the haematoxylin-eosin staining result showed that less sequestered parasites and haemorrhage,evans blue showed the BBB was less disrupted,and the level of IFN-γ,IL-1β,IL-6,TNF-αdecreased,IL-10 increased;4.ECM model was successfully established,after VPA injection,the level of BDNF and GDNF increased,no change of the NGF level;and patch clamp recording showed the current increased of the hippocampcal neuron.Conclusion 1.Serum collected from ECM mouse could inhibit the NSCs differentiation into neurons,and VPA could rescue its effect on NSCs and the ratio of neuros increased;2.Serum collected from ECM mouse could induce the hippocampcal neurons apoptosis,and VPA could rescue its effect on hippocampcal neurons;3.VPA injection could improve the survival rate and the neurological symptoms,and decrease the sequestered parasites and haemorrhage,and BBB permeability;4.VPA injection could increase the current of hippocampcal neuron,and it could exert protection on ECM mouse.