Interventional Effects Of 5-methyltetrahydrofolate On Alzheimer’s Disease Rats

ObjectiveAlzheimer’s disease(AD)is a degenerative disease of the nervous system characterized by impairment in learning and memory function.It is generally considered the result of the combined effects of genes and the environment.Studies have found that supplementing folic acid could inhibit AD development and improve patients’ memory.However,the ingested folic acid cannot be smoothly converted to the biologically active form,namely 5methyltetrahydrofolate(5-MTHF),required by the body without the methylenetetrahydrofolate reductase.That leads to the folate will unable to perform its normal physiological functions.The research has also illustrated that the bioavailability of 5-MTHF is about seven times that of folic acid.Therefore,direct 5-MTHF supplementation may be more beneficial to human health than folic acid,and 5-MTHF may be a potential substitute for folic acid.In terms of neurological diseases,data have shown the potential benefits of 5-MTHF on neuropathy in patients with depression.However,the research on the interventional impact of 5-MTHF on AD is still minimal.Hence,the purpose of this research was to evaluate the multitarget intervention effects of 5-MTHF on D-galactose(D-gal)and aluminum chloride(AlCl3)treated AD rats through the learning and memory function,the β-amyloid 1-42(Amyloid βprotein 1-42,Aβ1-42)and phosphorylated Tau protein(Phosphorylated Tau protein,p-Tau)contents,acetylcholinesterase(AChE)and nitric oxide synthase(NOS)activities,oxidative stress,endothelial cell function,hippocampal neuron morphology,amino acid neurotransmitters,apoptosis,and processing of amyloid precursor protein(APP)of rats,to provide new solutions for folic acid metabolism disordered population,and provide data support for researching and developing AD prevention and treatment drugs.Methods24 SPF male Wistar rats were divided into 2 groups(12 rats in per group):the control and model groups for model evaluation through the one-way ANOVA of body weight after adaptive feeding.In addition,72 SPF male Wistar rats were divided into 6 groups(12 rats in per group):the control,model,1 mg/kg 5-MTHF,5 mg/kg 5-MTHF,10 mg/kg 5-MTHF,and donepezil groups,to evaluate the interventional effects of 5-MTHF on AD rats.The model group was administered 60 mg/(kg·d)D-gal and 10 mg/(kg·d)AlCl3 solution intraperitoneally for 6 weeks to establish the AD rat model.The 1 mg/kg 5-MTHF group,5 mg/kg 5-MTHF group,10 mg/kg 5-MTHF group,and donepezil group were gavaged 1 mg/(kg·d)5-MTHF,5 mg/(kg·d)5MTHF,10 mg/(kg·d)5-MTHF and 1 mg/(kg·d)donepezil for 6 weeks based on intraperitoneal injection 60 mg/(kg·d)D-gal and 10 mg/(kg·d)AlCl3 for six weeks.The control group and model group was given a volume-matched normal saline every day.After the intervention,the Morris water maze was utilized to assess rats’ learning and memory function.After the behavioral experiment,two rats were randomly selected from each group for cardiac perfusion.Brain tissue was taken for H-E staining to observe the neuronal morphology in the CA1 region of the rat hippocampus.For the other rats,blood was collected from the abdominal aorta and serum was isolated.The brain tissue was quickly removed and hippocampus isolated on ice.Then the serum and tissue were stored in a-80℃ refrigerator until subsequent analysis.Kits were utilized to determine the Aβ1-42 and p-Tau contents,the AChE and NOS activities in the brain of rats,and the superoxide dismutase(SOD)activity,the malondialdehyde(MDA),tumor necrosis factor-alpha(TNF-α),endothelin-1(ET-1),interleukin-6(IL-6),nitric oxide(NO),and vascular endothelium Growth factor(VEGF)levels in the serum.HPLC was utilized to determine the aspartic acid(Asp),glycine(Gly),glutamic acid(Glu),and y-aminobutyric acid(y-GABA)concentrations in the brain of rats.The qRT-PCR was utilized to determine the mRNA expressions of a disintegrin and metallopeptidase domain 10(ADAM 10),β-site amyloid precursor protein cleaving enzyme 1(BACE1),and cysteinyl aspartate specific proteinase 3(caspase3)in rat hippocampus.Western blot was utilized to determine the protein expressions of AD AM 10 and caspase3 in rat hippocampus.Results1.Model evaluationThe water maze represented that the latency of all groups of rats decreased gradually with training time(P<0.05).On days 3-4 of the spatial navigation experiment,the model rats spent more time locating the platform than the controls(P<0.05).In the probe task,the model rats had decreased number of crossing the platform and time spent in the target quadrant than the controls(P<0.05).H-E staining of the hippocampus showed no apparent neuronal abnormality,and the pyramidal cells were neatly arranged,compact,and with clear boundaries in the control rats’CA1 region.The model rats had reduced numbers of pyramidal cells,dim outline,and neuronal atrophy in the CA1 area.2.Effects of 5-MTHF on the growth and development of AD ratsThe rats in the control group maintained an average growth rate,stable mental state,and regular diet and drinking during the experiment.Rats in the model group had sparse and dull hair,sluggishness,decreased exercise,and growth retardation.The body weight of rats in each group increased gradually with experimental time.The body weight of the model rats showed a downward tendency in the 5-6 weeks than the controls.but no statistical significance was discovered.There was no apparent statistical difference between the 5-MTHF intervention group,the donepezil group,and the model groups’ body weight.3.Effects of 5-MTHF on learning and memory functions of AD ratsThe water maze results represented that the latency of all groups of rats gradually decreased with increasing training time(P<0.001).On days 2-4 of the spatial navigation task,the model rats had a longer latency than the controls(P<0.05).On days 3-4,the 5-MTHF intervention and donepezil groups had a shorter time to find the platform than the model group(P<0.05).In the probe task,the model rats had decreased number of crossing the platform and time spent in the target quadrant than the controls(P<0.01).Compared to the model group,the 5 mg/kg 5-MTHF,10 mg/kg 5-MTHF,and donepezil groups showed different improvement degrees in these two indicators(P<0.05).4.Effects of 5-MTHF on Aβ1-42,p-Tau,AChE,and NOS in brain tissue of AD ratsThere were apparent statistical differences in the Aβ1-42 and p-Tau levels in the brain tissue of rats in each group(P<0.001).The model rats had elevated Aβ1-42 and p-Tau concentrations than the controls(P<0.001).The 5-MTHF intervention group and the donepezil group had increased Aβ1-42 concentration than the model group(P<0.001).The p-Tau concentration in the 5 mg/kg 5-MTHF,10 mg/kg 5-MTHF,and donepezil groups was lower than that in the model group(P<0.05).The model rats had the raised AChE and NOS activities in the brain than the controls(P<0.001).After supplementation with 5-MTHF,the AChE and NOS activities decreased in the 10 mg/kg 5-MTHF group compared with the model group(P<0.05).5.Effects of 5-MTHF on the neuronal morphology in the hippocampal CA1 region of AD ratsH-E staining of the hippocampus showed no significant neuronal abnormalities in the CA1 region of the control rats,and the pyramidal cells were neatly arranged,dense,and well-defined.Rats in the model group had reduced pyramidal cell numbers,were poorly defined,and showed neuronal atrophy in the CA1 area.The 5-MTHF and donepezil intervention improved the phenomenon of pyknosis and irregular structure of AD rats’ CA1 region neurons.6.Effects of 5-MTHF on serum antioxidant and cytokine indexes of AD ratsThe model rats had decreased serum SOD activity than the controls(P<0.001).The SOD activity of 5-MTHF intervention and donepezil groups was elevated than the model rats(P<0.05).The model rats had raised serum MDA content than the controls(P<0.001).The 5 mg/kg 5-MTHF group,10 mg/kg 5-MTHF group,and donepezil group had lower MDA levels than the model group(P<0.01).The model rats had increased serum ET-1,NO,TNF-α、and IL-6 levels than the controls(P<0.001),The ET-1 level of 5 mg/kg 5-MTHF group and the 10 mg/kg 5-MTHF group rats decreased more than the model group(P<0.01).The 5-MTHF intervention and donepezil group rats had reduced TNF-α,NO,and IL-6 concentrations than the model group(P<0.05).The control rats had increased serum VEGF content than the model group(P<0.001).The 10mg/kg 5-MTHF and donepezil group rats had more VEGF than the model group(P<0.05).7.Effects of 5-MTHF on the levels of amino acid neurotransmitters of AD ratsThe Asp,Glu,Gly,and γ-GAB A contents in the brain of rats in each group were statistical difference(P<0.001).The Asp level in the model group was higher than that in the control group(P<0.001).and the 5mg/kg 5-MTHF group and Donepezil group had lower Asp content than the model group(P<0.05).The control group’s Glu,Gly.and γ-GABA levels were higher than those in the model group(P<0.05).After supplementing 5-MTHF,the 10 mg/kg 5-MTHF group had raised Glu level than the model group(P=0.002).8.Effects of 5-MTHF on ADAM10,BACE1,and caspase3 mRNA expression and ADAM10 and caspase3 protein expression of AD rat hippocampusThe model rats had reduced hippocampal ADAM 10 mRNA and protein expression than the controls(P<0.01).The 10 mg/kg 5-MTHF and donepezil groups increased the ADAM 10 mRNA and protein expression than the model group(P<0.05).The model rats had higher hippocampal BACE1 mRNA expression more than the controls(P=0.007).After supplementation with 5-MTHF,the expression of BACE1 mRNA was reduced in the 5 mg/kg 5-MTHF and the 10 mg/kg 5-MTHF groups compared to the model group(P<0.05).No statistical discrepancy was observed in the hippocampal caspase3 mRNA and protein expression among groups.Conclusion5-MTHF can alleviate D-gal and AICl3-induced cognitive dysfunction in AD rats.The underlying mechanism may be accomplished by inhibiting the increase of Aβ1-42 and p-Tau,alleviating endothelial cell dysfunction and cholinergic damage,and regulating APP processing,oxidative stress,and excitatory amino acids release.