Neuroprotective Effects Of Citicoline On Dopaminergic Neurons And Its Mechanism

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the death ofmesencephalic nigrostriatal dopaminergic neurons. It is not clear about etiology of PD. It isknown as hypothesis that the pathogenesis and mechanisms in the disease may includemitochondrial dysfunction, glutamate– mediated excitotoxicity, oxidative stress, apoptosis,inflammation and so on. The incidence of PD is increase with advancing age. At present, PDcan not be cured radically. Only it is symptomatic treatment for PD with prescriptionlevodopa which is a dopamine precursor and replacement of the brain's dopamine.Levodopa is administered for a long time, which is harmful to patients. So it is veryimportant to find some new agent and medicine to prevent and treat PD. Citicoline isconsidered as an optimal agent in promising neuroprotective medicine. It is safety and itsside effects are few. Its price is relative lower. It could have potential therapeutic efficacy ina variety of diseases in membrane disorder, dysfunction or degeneration results in cellulardeath and tissue ischemia and necrosis. It is used to treat cerebral ischemia, stroke, cognitiveimpairment, glaucoma and so on in clinical trials. Citicoline also be used in the treatment ofPD. To study on some neuroprotective mechanism of citicoline, we established a PD modelof mesencephalic dopaminergic neurons cultured in vitro in embryonic 15-day (E15) rat.The toxicities of 6-hydroxydopamine and glutamate were used to induce PD model.The neuroprotective effects of citicoline were investigated and evaluated from PDpathogenesis. Mitochondrial dysfunction, glutamate–mediated excitotoxicity, oxidativestress, apoptosis, immune factors and inflammation were detected for the neuroprotectiveeffects of citicoline. The results showed that citicoline could protect mitochondrial functionby increasing mitochondrial membrane potential, attenuate glutamate–mediatedexcitotoxicity by decreasing intracellular free calcium concentration, reduce oxidative stressby reducing ROS and elevating anti-oxidative enzyme action, inhibit neuron apoptosis byup-regulate Bcl-2 and down-regulate Bax pathway, and increase neuron percentage of Sphase in cell cycle by increasing DNA synthesis and viability.1 Cell model of PD and neuroprotective effects of citicoline in vitroThe neuron viability was evaluated by MTT assay.E15 rat mesencephalic dopaminergicneurons cultured primarily in vitro. In day 12, 50mmol/L 6-OHDA was added into theculture medium and incubated for 30 minutes. The viability of dopaminergic neurons wasdecreased to 40% – 50%. In day 6, 8 and 10, various concentrations of citicoline (2, 1, 0.1,0.01 and 0.001 mmol/L) were added, respectively, into the culture medium and incubatedfor 6 days, in day 12, 6-OHDA was added. This is neuroprotective effects of citicolinemodel in vitro.Tyrosine hydroxylase (TH) immunocytochemistry and TH protein expression weremeasured in order to identify dopaminergic neurons. Dopaminergic neurons showed brownstain that is TH positive cells (TH+). In citicoline group, TH+ significantly increased andneuron processes were more and longer than those in 6-OHDA group. The percentage of THprotein expression in citicoline group was significantly higher than that in 6-OHDA group(P < 0.01). The percentage of TH protein expression was 89.87% in 2 mmol/L citicolinegroup, 9.91% in 6-OHDA group and 81.53% in control, respectively.2 The viability of neurons evaluated by MTT assay2.1 The dose-and time-effects of 6-OHDA on the loss of dopaminergic neurons andtheir viability evaluated by MTT assayVarious final concentrations(25, 50, 100, 200 and 500 μmmol/L)of 6-OHDA wereadded, respectively, into the culture medium in day 12 and incubated for 0.5, 2, 6, 24 and 48hours, respectively. The result showed that the viability decreased significantly for 0.5 and 2hours in 50 μmmol/L 6-OHDA.2.2 Effect of various concentration citicoline on primary culture of mesencephalicdopaminergic neuronsThe mesencephalic dopaminergic neurons in culture were prepared from day-15embryonic Wistar rat. The neurons were plated at concentrations of 2 × 105/ml in 96-wellCostar multi-well plastic plates previously coated with poly-D-lysine. In day 6, 8 and 10,various concentrations of citicoline were added, respectively, into the culture for 6 days. Inday 12, the neurons were collected for MTT assay. The data showed that, as compared withthat in the control, the viability increased in 2, 1 and 0.1 mmol/L citicoline group (P < 0.05).The neuroprotective effect of citicoline was dose-dependent.2.3 Neuroprotective effect of various concentration citicoline for different days on6-OHDA-induced PD modelThe results demonstrated that the neuron viability increased with the number of timesof citicoline (0.1, 0.01and 0.001mmol/L) added into the primary culture. After citicoline wasadded into the culture medium with 3 times, it could attenuate the viability decreased by6-OHDA neurotoxin for 6 days.2.4 Neuroprotective effect of various concentration citicoline on 6-OHDA-induced PDmodelThe neuron viability in 1, 0.1, 0.01 and 0.001 mmol/L citicoline group wassignificantly higher than that in 6-OHDA group (P <0.01);as compared with that in thecontrol, the neuron viability increased significantly in 0.01 and 0.001 mmol/L citicolinegroup (P < 0.01).2.5 Glutamate-mediated excitotoxicityWe observed the effect of various concentration glutamate (250, 500 and 750 μmol/L)on the viability of mesencephalic neurons in primary culture for 0.5, 2, 6 and 24 hours,respectively. The results showed that after the culture was treated with 500 μmol/Lglutamate for 0.5 hours in day 12, the neuron viability was decreased (P < 0.01). There wasthe difference in the neuron viability between 500 and 750 μmol/L glutamate (P < 0.01).2.6 Neuroprotective effect of various concentration citicoline on glutamate-mediatedexcitotoxicityCiticoline was added into mesencephalic primary culture in day 6, 8 and 10 for 6 days.After 500 μmol/L glutamate was added into the culture medium for 0.5 hours, the neuronviability in the medium was significantly lower than that in the control;while that inciticoline group was higher that in glutamate group,even it was significantly higher thanthat in the control (P < 0.01). It showed that citicoline could attenuate glutamate-mediatedexcitotoxicity.3 Effect of citicoline on mitochondrial membrane potential of dopaminergic neuronsIt was one of the earliest changes of cell apoptosis for the level of mitochondrialmembrane potential to decrease. So we can measure MMP to evaluate neuron apoptosischange. The final concentration of 10g/L Rh123 was added in the culture for 0.5 hours. Thefluorescence intensity was detected with flow cytometry. The results showed that MMP in6-OHDA group was significantly lower than that in the control and citicoline groups (P <0.01). While MMP in citicoline group increased significantly. It was proved that citicolinecould attenuate the decrease of MMP induced by 6-OHDA.4 Effect of citicoline on the transudatory amount of lactate dehydrogenase (LDH) indopaminergic neurons induced by 6-OHDALDH is released into the culture medium from dead cell. We can evaluate the injureddegree of neurons by detecting the activity of LDH in the culture medium. The resultsshowed that, as compared with that in the control, the transudatory amount of LDH in6-OHDA group increased (448.1 U/ml), while the transudatory amount of LDH in thecontrol was 329.2 U/ml. In addition, as compared with that in 6-OHDA group, thetransudatory amount of LDH in citicoline group (except 0.001 mmol/L group) decreased,while the transudatory amount of LDH in citicoline group was higher than that in the control.The results demonstrate that citicoline could reduce the transudatory amount of LDHinduced by 6-OHDA, but it couldn't make the LDH level of injured neurons recover tonormal level.5 Effect of citicoline on the percentage of [Ca2+]i of neurons treated by 6-OHDAIntracellular Ca2+ ions play vital roles in regulating neuronal signaling and apoptosis.Using highly sensitive Ca2+ fluorescent dye, Fluo-3/AM, the changes of intracelluar freecalcium ions ([Ca2+]i) in dopaminergic neurons treated by 6-OHDA measured with flowcytometry. The results showed that after the culture incubated for 45 minutes at 37℃ with 5μmol/L Fluo-3/AM and 0.0625% Pluronic F-127, the percentage of [Ca2+]i ofmesencephalic dopaminergic neurons in the rats treated by 6-OHDA was significantly(49.30%), higher than that in the control (42.40%);while compared with that in 6-OHDAgroup, that in citicoline group decreased significantly. The percentage of [Ca2+]i of theneurons treated by both 6-OHDA and 0.1 mmol/L citicoline was (17.09%) lower than that ofonly treated by 6-OHDA without citicoline.The results indicate that citicoline has protectiveeffect on reducing neurons apoptosis caused by increasing [Ca2+]i of neurons induced by6-OHDA neurotoxin.6 Effect of citicoline on neuron apoptosis, protein related to apoptosis and cell cycleprogression6.1 Effect of citicoline on neuron apoptosis induced by 6-OHDAUsing PI dye, the percentage of neuron apoptosis and cell cycle progression weredetected by flow cytometry. The results showed that the percentage of apoptosis of neuronsin 6-OHDA group was significantly (22.63%) higher than that in the control (10.77%);while that in citicoline group(except 2 mmol/L group)decreased, as compared with that in6-OHDA group (P <0.01). The data suggest that citicoline could reduce neurons apoptosisinduced by 6-OHDA.6.2 Effect of citicoline on cell cycle progression of neurons treated by 6-OHDAThe results showed that the percentage of neurons treated by 1 and 0.1 mmol/Lciticoline in G2/M phase decreased, while that in S phase increased. That is to say, thepercentage of neurons entered into DNA synthesis increased and the ability of neuronproliferation enhanced. So citicoline can exert its neuroprotective effect by promotingneuron proliferation.6.3 Effect of citicoline on Bcl-2, Bax protein expression in neuron induced by 6-OHDABcl-2 and Bax protein expression were detected with flow cytometry usingimmunocytochemistry indirectly labeled antibody. The result showed that Bax expressionwas 49.54% in 6-OHDA group and 35.20% in control. After the neuron in the culture wastreated with citicoline, as compared with those in 6-OHDA group without citicoline, Baxexpression decreased significantly, Bcl-2 expression increased, and the ratio of Bax/Bcl-2descend. The ratio of Bax/Bcl-2 was in 0.1 mmol/L citicoline group, even lower than that inthe control (P < 0.01). So neuron apoptosis in citicoline group decreased and viabilityincreased.7 Effect of citicoline on oxidative stress of neurons in cell model of PDPD is one of the commonest neurodegenerative diseases, and oxidative stress has beenevidenced to play a vital role in its causation.7.1 Effect of citicoline on reactive oxygen species (ROS) of neurons in cell model of PDFluorescent probe for the detection of ROS is a promising tool with which enhance ourunderstanding of the physiological roles of ROS. Using 2',7'-dichlorodihydrofluorescein(DCFH), ROS was detected with flow cytometry. Mesencephalic neurons in primary culturewere plated at a density of 5 × 105 neurons/ml in 24-well plates, 1 ml/well, 3 parallel wells.Citicoline was added into the culture in day 6, 8 and 10 for 6 days. In day 12, 50μmol/L6-OHDA was added into the culture for 2 hours. Then, 10 μmol/L DCFH was added into theculture medium at 37℃ for 45 minutes, digested and collected the neurons. The resultshowed that the accumulation of intracellular ROS in 6-OHDA group increased (39.96%).As compared with that in 6-OHDA group, ROS in citicoline group (citicoline + 6-OHDA)decreased, but it was higher than that in the control. The data suggest that citicoline couldattenuate the increase of ROS induced by 6-OHDA, but it couldn't recover to normal level.7.2 Effects of citicoline on anti-oxidative enzyme and MDA of neurons in PD model7.2.1 Neuron sample treatment: Rat mesencephalon neurons treated by citicoline for 6days. Then in day 12, 50μmol/L 6-OHDA was added into culture for 2 hours. So, the cellmodel of PD was set up. The neurons were digested, collected and broke up by ultrasonic(10 Amperage, 10 s/time, 3 times), then centrifugated and collected for experiment.7.2.2 Malondialdehyde (MDA): The content of MDA in neurons can reflect indirectly theinjured degree of neurons. The results showed that the content of MDA in neurons descendin 2, 1, 0.1 and 0.01 mmol/L citicoline group, as compared with that in 6-OHDA group (P <0.01). The content of MDA in 2 mmol/L citicoline group was lower than that in the control.7.2.3 Catalase (CAT): The results indicated that the activity of CAT in neurons was lowerin 6-OHDA group. As compared with that in 6-OHDA group, the level of CAT in citicolinegroup (except 2 mmol/L) increased significantly (P < 0.01), and the activity of CAT washigher than that in the control (P < 0.05).7.2.4 Superoxide dismutase (SOD): The results showed that the enzymatic activities oftotal SOD in neurons were 71.8 U/mgprot in the control, 58.44 U/mgprot in 6-OHDA group,and 63.77 U/mgprot in 0.01 mmol/L citicoline group, and 63.91 U/mgprot in 0.001 mmol/Lciticoline group. The activities of total SOD in neurons in 0.01 and 0.001 mmol/L citicolinegroup was significantly higher than that in 6-OHDA group (P < 0.01).7.2.5 Glutathione peroxidase (GSH-Px): The results showed that GSH-Px activity ofneurons in 0.1 mmol/L citicoline group increased significantly (52.99 U/mgprot), ascompared with that in 6-OHDA group, and it was higher than that in the control (23.71U/mgprot), P < 0.01.8 Nitric oxide (NO)100μl supernatant culture medium was collected and the production of NO in themedium was determined. The result showed that the amount of NO in 6-OHDA group was145.71μmol/L. Only the amount of NO in 0.001 mmol/L citicoline group was lower thanthat in 6-OHDA group and also significantly lower than that in the control (P < 0.01), but,that in 1 mmol/L citicoline group was higher than that in the control.9 Nitric oxide synthase (NOS)The result showed that the content of cNOS in supernatant culture medium of neuronswas very much, while iNOS was few. The contents of both cNOS and iNOS in 6-OHDAgroup decreased. On the other hand, the contents of both tNOS and iNOS in citicoline group(except 2 mmol/L group) and cNOS in citicoline group (except 1 mmol/L group) weresignificantly higher than those in 6-OHDA group (P < 0.01). The results showed the changeof NOS was similar to NO result. The contents of tNOS and cNOS in the control (exceptiNOS) were higher that those in 6-OHDA group. The result was different from the otherreports about NO and NOS.