Effect And Its Mechanism Of Ketogenic Diet And D-β-hydroxybutyrate On Parkinson's Disease

Parkinson's disease(PD),also named by paralysis agitans,first described by James Parkinson of British doctor in 1817.The cardinal symptoms of PD included resting tremor,rigidity,gait disturbance,postural instability and bradykinesia.Life quality were influenced seriously.A major pathological hallmark of PD was the degeneration of nigrostriatal dopaminergic neurons,resulting in the reduced release of dopamine(DA)into the striatum.The etiology and mechanism of PD were incompletely understood up to now.Researchers proposed that apoptosis induced by oxidative stress and mitochondrial dysfunction was probably one of mechanisms.Postmortem shows that the number of dopaminergic neurons in SN of PD patients decreased significantly and apoptotic cells were observed.Reactive oxygen species(ROS)induced by oxidative stress,lipid peroxidation and malondialdehyde(MDA)increased.Glutathione(GSH)decreased and mitochondrial dysfunction appeared.Using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP),6-hydroxydopamine (6-OHDA)and rotenone for PD rat or mouse model,the number of dopaminergic neurons in SN decreased significantly and apoptotic cells were observed.ROS and MDA increased.GSH level,mitochondrial membrane potential and adenosine triphosphate(ATP)production decreased.Using neurotoxins above for dopaminergic neurons in vitro,rat pheochromocytoma cell line(PC12)and neuroblastoma cell line(SH-SY5Y),cell viability decreased and apoptotic cells appeared.The apoptotic rates increased,the expression of some anti-apoptotic genes down-regulated and some pro-apoptotic genes up-regulated.Caspase-3 activity,oxidative stress,intracellular ROS,lipid peroxidation and MDA increased. Intracellular GSH,mitochondrial membrane potential and ATP production decreased.Therefore apoptosis of dopaminergic neurons induced by oxidative stress and mitochondrial dysfunction was probably one of mechanisms.The current treatment option available for PD primarily was pharmacological dopamine(DA)replacement-Levodopa(L-dopa,a precursor of dopamine)and its compounds.However,replacement therapy did not impede the progress of the disease so far clinically.In vivo and vitro,L-dopa and its compounds are toxic to dopaminergic neurons.Therefore replacement of L-dopa could not stop the loss of dopaminergic neurons and cure PD.People try their best to explore some therapeutic methods for impeding the progress of PD and protecting dopaminergic neurons.It was reported that ketogenic diet(KD)or D-β-hydroxybutyrate(DβHB)of ketone bodies(KB)were neuroprotective.The ketogenic diet(KD)was a high-fat, low-carbohydrate,low-protein diet that has been used for more than 80 years for the treatment of medically children or adult intractable epilepsy from 1921 clinically.The contol rate of KD for epilepsy was 66.7%.After high-fat diet,the level of ketone bodies(KB)increases and ketosis is formed chronically in body. Recent clinical evidence to substantiate the value of D-β-hydroxybutyrate(DβHB) in the treatment of PD has come from Van Itallie and co-workers.These researchers placed 5 patients with PD onto a very strict ketogenic diet to raise their plasma KB levels to 1-7 mM.After one month,their Unified Parkinson's Disease Rating Scale(UPDRS)disability scores showed a mean 43%reduction and the patients claimed a "moderate" to "very good" improvement in their disorder.The main components of KB were DβHB and acetoacetate(AcAc).There were some evidences of experiments in vivo and in vitro that KB induced by KD was neuroprotective.KD and DβHB were neuroprotective for acute and chronic neuronal damage.DβHB Na was neuroprotective against hypoxia in serum-free hippocampal primary cultures.DβHB inhibited the degeneration of dopaminergic neurons induced by MPTP,movements of mice improved and ATP production of SN increased.DβHB inhibited dopominergic neurons damage by MPP+ in vitro and SH-SY5Y cells damage by rotenone.Based on above experiments,the neuroprotective mechanisms of KD and KB's main compotent DβHB were to be explored in detail.In this study,the models of PD made by 6-hydroxybutyrate(6-OHDA)in vitro and in vivo were utilized.In vivo,Wistar rats were pretreated with KD;in vitro, PC12 cells were pretreated with the main compound DβHB of KB.Nissl staining, immunohistochemistry,high performance liquid chromatography(HPLC),cell viability measurement,inverted microscope observation,acridine orange(AO) staining,flow cytometry,reverse transcriptase polymerase chain reaction(RT-PCR) and microplate reader were used.The neuroprotection of KD and DβHB for dopaminergic neurons was explored through inhibition of apoptosis and oxidative stress and keeping mitochondrial function.The proofs were provided that the therapeutic function of KD for PD and neuroprotection for PD.All the experiments were divided into 5 parts as abstracts following:Part 1:The therapeutic effect of KD on neuroprotection of dopaminergic neuronsObjective:To study the therapeutic effect of KD on neuroprotection of dopaminergic neurons.Methods:After rats were treatmented with normal diet(ND)and KD for 2 weeks,6-OHDA was injected in the right striatum.The four groups(ND,KD, 6-OHDA+ND and 6-OHDA+KD)were used for this experiment and there were 5 rats in each group.After 6-OHDA injection,ND or KD were continued for 2 weeks.The number of neurons in SN were counted using Nissl staining and TH immunohistochemistry.The fiber density was measured using tyrosine hydroxylase(TH)immunohistochemistry.DA and its metabolites dihydroxy-phenylaceticacid(DOPAC)and homovanillic acid(HVA)were measured by HPLC.The neuroprotection of KD was analyzed for dopaminergic neurons.Results:1.Using Nissl staining and TH immunohistochemistry:The number of dopaminergic neurons of SN decreased in rats with ND right striatal injecting 6-OHDA for 2 weeks compared to rats with ND without striatal injecting 6-OHDA, P＜0.01;the number of dopaminergic neurons of SN increased in rats with KD right striatal injecting 6-OHDA compared to rats with ND striatal injecting 6-OHDA for 2 weeks,P＜0.05;the number of dopaminergic neurons of SN had no difference between KD and ND without right striatal injecting 6-OHDA,P＞0.05.Using TH immunohistochemistry,the fiber density of striatum decreased in rats with ND striatal injecting 6-OHDA for 2 weeks compared to rats with ND without right striatal injecting 6-OHDA,P＜0.01;the fiber density of striatum increased in rats with KD right striatal injecting 6-OHDA compared to rats with ND right striatal injecting 6-OHDA for 2 weeks,P＜0.05;the fiber density of striatum had no difference between KD and ND without right striatal injecting 6-OHDA,P＞0.05.2.Using HPLC,DA and its metabolites DOPAC and HVA decreased significantly decreased in rats with ND right striatal injecting 6-OHDA for 2 weeks compared to rats with ND without right striatal injecting 6-OHDA,P＜0.01;DA and its metabolites DOPAC increased significantly in rats with KD right striatal injecting 6-OHDA compared to rats with ND right striatal injecting 6-OHDA for 2 weeks,P＜0.05,HVA increased but had no difference significantly,P＞0.05;DA and its metabolites DOPAC and HVA had no difference between KD and ND without right striatal injecting 6-OHDA,P＞0.05.Conclusions:KD was neuroprotective on dopaminergic neurons Part 2:The effect of DβHB on apoptosis induced by 6-OHDA in PC12 cellsObjective:To study the effect of DβHB on apoptosis induced by 6-OHDA in PC12 cells.Methods:PC12 cells were pretreated with DβHB(final concentration 4 mM) for 1 h prior to 6-OHDA(final concentration 50 and 100μM)and then cultured for 24 h.The five groups(control,50μM 6-OHDA,100μM 6-OHDA,4 mM DβHB+ 50μM 6-OHDA and 4 mM DβHB+100μM 6-OHDA)were used for this experiment using measurements of MTT cell viability,inverted microscope, acridine orange(AO)staining and flow cytometry for morphological changes and apoptotic rates of apoptosis.Results:The cell viability decreased in PC12 cells treated with 6-OHDA (final concentration 50 and 100μM)and the survival rates were 75.77±0.84%and 47.16±2.37%of control,P＜0.05.Using inverted microscope and AO staining,cell death increased,cell shrank and process shortened.All these changes were apoptotic morphology.Using flow cytometry,apoptotic rates were 27.17±2.64% and 37.89±2.39%(control,1.94±0.52%)and increased compared to the control, P＜0.05.PC12 cells were pretreated with DβHB(final concentration 4 mM)and then co-cultured with 6-OHDA for 24 h.The cell viability increased compared to corresponding 6-OHDA alone and the survival rates were 86.62±1.66%and 65.74±1.49%of control,P＜0.05.Cell death decreased,cell shrinkage inhibited and apoptotic rates decreased(19.38±3.20%and 33.24±2.32%)compared to corresponding 6-OHDA alone,P＜0.05.Conclusions:DβHB inhibited the apoptosis induced by 6-OHDA in PC12 cells.Part 3:The effect of DβHB on expression of bcl-2/bax mRNA ratio and caspase-3 activity in PC12 cells Objective:To study the effect of DβHB on expression of bcl-2/bax mRNA ratio and caspase-3 activity.Methods:Cell culture,drug treatments and groups of this study were the same as described Part 2.Bcl-2 mRNA,bax mRNA and the ratio of bcl-2/bax mRNA were measured using reverse transcription polymerase chain reaction (RT-PCR)and caspase-3 activity was measured by multi-detection microreader plate.Results:The expression of bcl-2 mRNA decreased,bax mRNA increased and then the ratio of bcl-2/bax mRNA decreased in PC12 cells treated with 6-OHDA (final concentration 50 and 100μM)compared to the control,P＜0.05.Caspase-3 activity increased in PC12 cells treated with 6-OHDA and caspase-3 activity were 125.83±1.54%and 146.27±2.41%of control compared to the control,P＜0.05. PC12 cells were pretreated with DβHB(final concentration 4 mM)and then co-cultured with 6-OHDA for 24 h.The expression of bcl-2 mRNA increased,bax mRNA decreased and then the ratio of bcl-2/bax mRNA increased in PC12 cells compared to corresponding 6-OHDA alone respectively,P＜0.05.Caspase-3 activity decreased in PC12 cells and caspase-3 activity were 115.61±2.75%and 132.75±3.11%of control compared to corresponding 6-OHDA alone respectively, P＜0.05.Conclusions:DβHB inhibited the apoptosis induced by 6-OHDA in PC12 cells.The expression of bcl-2/bax mRNA ratio decreased in PC12 cells induced by 6-OHDA.Caspase-3 activity increased in PC12 cells induced by 6-OHDA.DβHB inhibited the changes as above.Part 4:The effect of DβHB on oxidative stress induced by 6-OHDA in PC12 cellsObjective:To study the effect of DβHB on oxidative stress induced by 6-OHDA in PC12 cells Methods:Cell culture,drug treatments and groups of this study were the same as described Part 2.Intracellular ROS,MDA and intracellular GSH were measured using multi-detection microreader plate.Results:Intracellular ROS increased in PC12 cells induced by 6-OHDA(final concentration 50 and 100μM)and the ROS levels were 167.69±9.40%and 289.8±9.42%of control compared to control,P＜0.05;MDA increased in PC12 cells induced by 6-OHDA and the MDA levels were 201.24±9.68%and 291.18±17.59%of control compared to control;P＜0.05.However,intracellular GSH decreased in PC12 cells induced by 6-OHDA and the GSH levels were 75.45±4.97%and 53.98±3.20%of control compared to control,P＜0.05.PC12 cells were pretreated with DβHB(final concentration 4 mM)and then co-cultured with 6-OHDA for 24 h.Intracellular ROS decreased and the ROS levels were 155.57.69±6.32%and 275.53±8.28%of control compared to 6-OHDA alone respectively,P＜0.05.MDA decreased and the MDA levels were 156.33±9.07% and 220.37±19.81%of control compared to 6-OHDA alone respectively,P＜0.05. Intracellular GSH increased and the GSH levels were 82.29±0.78%and 61.01±4.36%of control compared to 6-OHDA alone respectively,P＜0.05.Conclusions:DβHB inhibited oxidative stress induced by 6-OHDA in PC12 cells.Part 5:The effect of DβHB on mitochondrial dysfunction induced by 6-OHDA in PC12 cellsObjective:To study the effect of DβHB on mitochondrial dysfunction induced by 6-OHDA in PC12 cellsMethods:Cell culture,drug treatments and groups of this study were the same as described Part 2.Mitochondrial membrane potential and ATP production were measured multi-detection microreader plate.Results:Mitochondrial membrane potential decreased in PC12 cells induced by 6-OHDA(final concentration 50 and 100μM)and mitochondrial membrane potential were 78.81±2.96%and 42.01±2.95%of control compared to control, P＜0.05;ATP production decreased in PC12 cells induced by 6-OHDA and ATP levels were 62.23±4.46%and 32.2±4.37%of control compared to control, P＜0.05.PC12 cells were pretreated with DβHB(final concentration 4 mM)and then co-cultured with 6-OHDA for 24 h.Mitochondrial membrane potential increased and the mitochondrial membrane potential were 88.79±3.36%and 59.29±4.72%of control compared to 6-OHDA alone respectively,P＜0.05.ATP increased and the ATP levels were 85.25±4.65%and 58.94±4.84%of control compared to 6-OHDA alone respectively,P＜0.05.Conclusions:DβHB inhibited loss of mitochondrial membrane potential and decrease of ATP production induced by 6-OHDA in PC12 cells.