Protective Effects Of Folic Acid, Vitamin B₆ And Vitamin B₁₂ On Rats With Cerebral Ischemia

ObjectiveTo investigate the effects of folic acid, vitamin B₆ and vitamin B₁₂ on rats with focal cerebral ischemia, so as to explore the protective mechanisms.Methods 1. Developing a method for the determination of total homocysteine in plasma by high performance liquid chromatography. Plasma homocysteine was reduced by NaBH₄ and then derivatized by fluorogenic thoil-specific reagent SBD-F. The derivatives were separated isocratically by C18 reverses phase column and detected by fluorescent spectrophotometer. The results were calculated by the peak area. 2. Sprague-Dawley rats were randomly divided into four groups which were sham operation group (SO) supplemented by saline for 10 ml/(kg bw·d), ischemia model group (IM) supplemented by saline for 10 ml/(kg bwd), ischemia+folic acid group (IM+FA) supplemented by folic acid for 4 mg/(kg bw·d) and ischemia+ compound vitamins group (IM+CV) supplemented by folic acid for 4 mg/(kg bw·d), vitamin B₆ for 12 mg/(kg bw·d) and vitamin B12 for 24 1μg/(kg bw·d). There were 4 male and 4 female rats in each group respectively. The focal cerebral ischemia model was induced by occlusion of middle cerebral artery after 28-days intragastric administration. Plasma homocysteine and serum antioxidant indices of rats were measured before and after supplementation and after ischemia. Learning and remember abilities, brain antioxidant indices were measured after ischemia. Then all rats except SO group received magnetic resonance imaging (MRI) brain scan. Besides, the morphological changes of cerebral cortex in each group were observed by light microscope. 3. Chose P-actin as control gene. The expression of brain HO-1, HO-2 mRNA were detected by semi-quantity RT-PCR method after ischemia.Results 1. Calibration curve was linear in the range of 0-50 μmol/L for homocysteine. The recovery was 96.40-104.30%. Intra-sample coefficient ofvariation was 2.45-3.64%. Intra- and inter-day coefficients of variation were 2.56-3.91% and 3.19-3.96% respectively. 2. ?There was no significant difference in all indices in four groups before supplementation (P>0.05).(2)Levels of plasma homocysteine concentration in IM+FA and IM+CV groups were lower than those before supplementation and those in SO and IM groups (P<0.05 or P<0.0l) not only after 28-days supplementation but also after ischemia. Besides, homocysteine level in IM+CV group were also lower that in IM+FA group (PO.05). ?After supplementation, in IM+FA and IM+CV groups, serum SOD and GSH-Px activities were significantly higher, serum MDA contents were lower than those before supplementation and those in SO and IM groups (PO.05 or PO.01). There was no significant difference in serum LDH activities and NO contents in all groups CP>0.05). ?In IM+FA and IM+CV groups, SOD and GSH-Px activities in serum or brain were significantly higher, content of MDA and NO in serum or brain were lower (PO.01), serum LDH levels were lower than those in IM group (P<0.0l) after MCAO model was established. ?The neurological deficit scores and shock times in Y-type maze of IM+FA and IM+CV groups were lower than those in IM group (P<0.01). The correct times of IM+CV group in Y-type maze was higher than that in IM group (PO.05). ?6 hours after ischemia, ADC and DCavg in IM+FA and IM+CV groups were higher than those in IM group (PO.05 or P<0.01). ?On light microscopy, neurons in cerebral cortex of IM group shrunk. The cell's color was deeper. There were cytoplasm and nucleus pyknosis in IM group. Compared with IM group, the degree of pyknosis was lighten in IM+FA group. There was significant better recovery in cerebral tissue of IM+CV group. The cell morphology of IM+CV group was similar to that of SO group. 3. Levels of relative expression of brain HO-1 mRNA in IM+FA and IM+CV groups were lower than that in IM group (P<0.05 or .PO.01), levels of HO-2 mRNA expression were higher than SO group (P<0.05). There was no significant difference in levels of expression of HO-2 mRNA in IM+FA, IM+CV and IM groups (P>0.05).Conclusions 1. The HPLC method established for plasma homocysteinedetermination had high sensitivity and good reproducibility and may be applied for scientific research in homocysteine related diseases. 2. Supplementation of folic acid, vitamin B6 and vitamin B12 caused a significant decrease in plasma homocysteine concentration. The combined effect of folic acid, vitamin B6 and vitamin B12 was better than single effect of folic acid. 3. Supplement with folic acid could enhance the activities of SOD and GSH-Px, decreased the content of MDA, NO and activities of LDH, then improved the antioxidative defense of rats with cerebral ischemia. These effects could not be promoted by co-administration of vitamin B6 and vitamin B12. 4. Folic acid intake could not only enhance the learning and memory functions of rats with cerebral ischemia but also meliorate the neural function and pathological changes of cerebral cortex. Simultaneous administration of vitamin B6 and vitamin B12 showed additive effect. 5. Folic acid, vitamin B6 and vitamin B12 could down-regulate HO-1 mRNA expression but not influence the expression of HO-2 mRNA. Therefore, folic acid, vitamin B6 and vitamin B12 had protective effects on focal cerebral ischemia injury by decreasing the homocysteine contents, improving the antioxidative defense, meliorating the neural function and down-regulate HO-1 mRNA expression.