Effects Of Folic Acid On Notch Signal Pathway In The Brain Tissues Of Rats With Focal Cerebral Infarction

Objective To study the effects of folic acid on Notch signal pathway related genes and proteins in the brain tissues of adult rats with focal cerebral infarction,and to explore the effects mechanisms of folic acid on prevention and treatment of cerebral infarction at the molecular level.Methods 48 adult male Sprague-Dawley rats were randomly divided into four groups according to body mass.They were sham operation(SO),middle cerebral artery occlusion(MCAO),MCAO+low dose folic acid(MCAO+LFA),and MCAO +high dose folic acid(MCAO+HFA).There were 12 rats in each group.SO and MCAO group were supplemented by saline for 10ml/(kg bw·d),MCAO+LFA and MCAO+HFA group were supplemented by folic acid for 4mg/(kg bw·d),12mg/(kg bw·d) respectively.The left cerebral infarction model was induced by occlusion of middle cerebral artery after 28-days folic acid supplementation.Serum folic acid concentration was measured with immune chemiluminescence before and after folic acid supplementation and after cerebral infarction.We observed the symptom and evaluated the neurological function on Zea longa five-point scale at 24h after the operation.And the brain infarction volume for the three rats of each group was detected by TTC staining method.Afler 3d,7d and 14d folic acid supplementation, the survival rats of each group were decapitated respectively and brains were rapidly removed and snap frozen in liquid nitrogen,frozen transverse sections were cut using a cryostat.The pathology change of brain was examined by hematoxylin-eosin(HE) staining at 3d.The expression levels of Notch1,Hes1,Hes5,Mash1,Neurogeninl and Neurogenin2 mRNA were detected by fluorescence in situ hybridization(FISH) method.The expression levels of Notch1,Hes1 and Hes5 proteins were detected by western blot method.Results The survival rats with focal cerebral infarction had the symptoms of Homer in left side,and the scores of neurological function were in 1 to 3,which showed that modeling was successful.The detection results showed that the concentration of serum folic acid in MCAO group was lower than that in SO group after cerebral infarction,and significantly lower than that in MCAO+LFA and MCAO+HFA group,and MCAO+LFA was lower than MCAO+HFA group before and after cerebral infarction(P＜0.05).There was significantly different between before and after cerebral infarction in MCAO,MCAO+LFA and MCAO+HFA groups respectively(P＜0.05).TTC staining showed that the ratio of infarction volume in MCAO was significantly higher than that in MCAO+LFA and MCAO+HFA group(P＜0.05).HE staining showed that neural cells were arranged orderly and intact in shape in SO group,the neural cells of infarction zone in MCAO group were necrosis,arranged asymmetrically,the cellular interspaces widened,the volume of some cells decreased with nuclear pyknosisand dyed darkly.While the pathology changes in the groups of folic acid supplementation were lighter than that in MCAO group.The results of fluorescence in situ hybridization showed that the expressions of related genes in notch signal pathway in rats with focal cerebral infarction were all detected at 3d.The expression levels of Notch1,and Hes5 mRNA were highest at 7d,and lowest at 14d.And that of Hes1 mRNA was highest at the time point of 14d.The fluorescence intensity of positive cells of the three genes in SO group were lower than that in MCAO group at each time point(P＜0.05),MCAO was lower than that in MCAO +LFA and MCAO+LFA group,and MCAO+LFA was lower than that in MCAO+LFA group at 7d and 14d(P＜0.05).The expression levels of Mash1,Neurogenin1 and Neurogenin2 mRNA in rats with focal cerebral infarction were lowest at 7d and highest at 14d.The fluorescence intensity of positive cells of Mash1 mRNA in MCAO group was significantly different from SO,MCAO+LFA and MCAO+HFA group(P＜0.05),and MCAO+LFA group was higher than that in MCAO+HFA group at 3d and 14d(P＜0.05).The fluorescence intensity of positive cells of Neurogenin1 mRNA in MCAO group were higher than that in MCAO+LFA group at 3d and 14d,and higher than that in MCAO+HFA group at each time point (P＜0.05),the MCAO+LFA group was higher than that in MCAO+HFA group at 7d and 14d(P＜0.05).The fluorescence intensity of positive cells of Neurogenin2 mRNA in SO group was lower than that in MCAO group at 3d and 14d,and MCAO group was higher than that in MCAO+LFA group(P＜0.05),and both of the two groups were all higher than that in MCAO+HFA group(P＜0.05).The results of western blot showed that the expression levels of Notch1,Hes1,Hes5 proteins were consistent with that of their genes respectively.And the expression levels of these three proteins in MCAO group were all lower than that in MCAO+LFA and MCAO+HFA group at 7d and 14d(P＜0.05),the expression levels of Notch1 and Hes1 proteins in MCAO+LFA group were all lower than that in MCAO+HFA group at the same time points(P＜0.05),the expression level of Hes5 protein in MCAO+LFA group was lower than that in MCAO+HFA group at 7d. Conclusion The focal cerebral infarction model of rats was established successfully by thread method.It showed that cerebral infarction can decrease the concentration of serum folic acid,and folic acid supplementation can increase the concentration of serum folic acid of rats,decrease the volume of cerebral infarction, and protect the neuron from necrosis,there was a dose-dependent relation. Supplementation of folic acid can promote the mRNA and protein expressions of Notch1,Hes1 and Hes5,decrease the mRNA expressions of Mash1,Neurogenin1 and Neurogenin2.Folic acid can enhance the therapy and recovery of cerebral infarction.