Study On The Mechanism For The Effect Of Folic Acid Deficiency On Neuronal Cell Injury By Mitochondrial STAT3 After Cerebral Ischemia-Reperfusion

ObjectiveTo observe the effects of folic acid deficency(FD)on damage of mitochondria after brain ischemia reperfusion injury by establishing middle cerebral artery artery occlusion(MCAO)model in SD rat,to investigate the role of mitochondrial STAT3 and its possible regulatory mechanisms in neuronal cell injury after the combinatorial intervention FD and different signaling pathway inhibitors in vivo and in vitro,and provide new ideas and experimental basis for prevention and treatment of cerebral infarction.Methods125 adult male SD rats were randomly divided into sham operation group(SHAM group),MCAO group,MCAO+FD group,MCAO+FD+AG490 group and MCAO+AG490 group.There were twenty-five rats in each group.The rats in SHAM and MCAO groups were fed with normal control diet.The rats in MCAO+FD and MCAO+FD+AG490 group were fed with folate deficient diet.Before the MCAO model,Folate deficiency feed was used in the MCAO+FD and MCAO+FD+AG490 group for preintervention of 25 d.The rats in MCAO+FD+AG490 and MCAO+AG490 group were injected with AG490 solution 1 h before surgery.The intraperitoneal injection concentration was 0.75 mg/mL of AG490 solution.The volume of cerebral infarction in rats was determined by triphenyltetrazolium chloride(TTC)staining.The apoptosis rate of neural cells were detected by Fluoro-Jade B(FJB)staining.The mitochondrial damage of brain was observed by transmission electron microscopy.The numbers of pS-STAT3 and pY-STAT3 positive cells in mitochondria of brain were detected by immunofluorescence staining.Western blot was used to detect the protein level of pY-STAT3,pS-STAT3 and JAK2/p-JAK2 in mitochondria of brain cells.In vitro model of N2 a cells subjected to OGD/R was established,and the combined intervention of AG490,LY294002,U0126 inhibitor and folic acid was used.The cell viability has been detected by CCK-8.Western blot was used to detect the protein levels of pY-STAT3,pS-STAT3,p-ERK1/2,p-AKT,and p-JNK in the cell mitochondria.ResultsCompared with MCAO group,the TTC staining after FD treatment showed that the volume of cerebral infraction increased,FJB staining showed that the apoptosis of neural cells was aggravated,the degree of mitochondrial damage was increased.Immunofluorescence staining showed that the numbers of pY-STAT3 and COX IV double-positive cells,pS-STAT3 and COX IV positive cells were significantly increased(P<0.05).Western blot showed that the protein level of pY-STAT3 and pS-STAT3 were significantly increased(P<0.05).Compared with MCAO+FD group,the numbers of pY-STAT3 and COX IV double-positive cells were significantly reduced,and the expression of protein level of pY-STAT3 were significantly decreased in MCAO+FD+AG490 group(P<0.05).The results of Western blot showed that the expression of pY-STAT3 protein in the mitochondria of N2 a cells reached a peak at 30 min and the pS-STAT3 protein reached its peak at 45 min after cell OGD/R.Compared with SHAM group,WB showed that the protein level of pY-STAT3,S-STAT3 and p-ERK1/2/p-AKT were increasedin OGD/R group(P<0.05).There was no significant change in p-JNK protein(P>0.05).CCK-8 assay showed that cell viability was decreased in OGD/R group(P<0.05).Compared with OGD/R group,WB showed that the protein level of pY-STAT3,pS-STAT3 and p-ERK1/2/p-AKT were significantly increased in OGD/R+FD group(P<0.05).CCK-8 assay showed that cell viability was decreased remarkably in OGD/R+FD group(P<0.05).Compared with OGD/R+FD group,WB showed that the protein level of pY-STAT3 were significantly decreased in OGD/R+FD+AG490 group(P<0.05).The protein level of pS-STAT3 were significantly decreased in OGD/R+FD+U0126 and in OGD/R+FD+LY294002 groups(P<0.05).CCK-8 assay showed that cell viability was increased remarkably in OGD/R+FD+AG490,OGD/R+FD+U0126,OGD/R+FD+LY294002 group,respectively(P<0.05).ConclusionsIt was found that FD could exacerbate the damage of brain tissue and mitochondria in rats,promote the expression of pY-STAT3 and pS-STAT3 in mitochondria,and these effects above were partly inhibited by AG490 except the protein of pS-STAT3.It is suggested that the folic acid deficiency may increase the pY-STAT3 expression in mitochondria via the JAK2-STAT3 pathway that aggravates brain tissue and mitochondrial damage after cerebral ischemia.In addition,the protein expression of pS-STAT3 may not be regulated by JAK2-STAT3 pathway.In vitro,it was found that FD could decrease the cell viability,promote the expression of pY-STAT3 and pS-STAT3 in mitochondria,and these effects above were partly inhibited by U0126 or LY294002 except pY-STAT3 expression.It is suggested that the FD may increase the pS-STAT3 expression in cell mitochondria via PI3K-AKT and MAPK-ERK1/2 pathway that aggravates neural cell damage after OCD/R.In addition,the protein of pY-STAT3 expression may not be regulated by p-JNK.In conclusion,FD may upregulate pY-STAT3 in mitochondria through JAK2-STAT3 pathway and upregulate pS-STAT3 in mitochondria through PI3K-AKT and MAPK/ERK1/2 pathway.Several pathways together contribute to the exacerbation of cerebral ischemia and the damage of nerve cells and mitochondria in FD-treated ischemic brains.