Neuroprotective Effects Of Glabridin On MPTP-induced Parkinson’s Disease And Its Mechanism

BackgroudsParkinson’s disease(PD)is a nervous system disease characterized by degenerative disorder of the substantia nigra and striatum neural pathway,which can be the second disease impacting on human health,the clinical feature is static tremor,muscle rigidity,bradykinesia and equilibrium disorder.According to the current situation,we have no effective management to restore the degenerative neurons,drug therapy is the major managemengt and just symptomatic treatment.These drugs can remission symptoms,and the rational of these drugs is based on the chief target or signal path which is the mechanism of PD.It is the vital subjects and hotspot to develop the drugs which can preventPDin the field of medicine,and there are several bio-active Chinese preparations using for this along with the improvement of the purification level for traditional Chinese medicine.Glabridin(GLA)has the neuroprotective effect through the anti-inflammatory effects and increasing the expression of antioxidant effect to downgrade the oxidative stress reaction between cells.Therefore GLA can protect the neurocyte from apoptosis(DNA fragmentation,protein oxidation,mitochondrial peroxidation).Extracellular regulated protein kinases(ERK)is a popular protein kinase-level link recent year,extracellular signal-regulated kinase(ERK1/2)belong to Ser/Thr protein kinase family,which can participate in the regulation of neuroplasticity in physiological and pathological.The sustainable excitation ERK1/2 pathway plays direct action in the damagement mediated by dopamine neurotoxin in vitro.In addition,in the substantia nigra neuron of Parkinsons’autopsy brains,phosphorylation ERK1/2 is accumulated,this prompt the ERK1/2 is relecanted with dopaminegic nerve damage.1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)is a neurotoxin that has a targeted effects on the neurons involved in PD,and has been widely used in the establishment of animal models of PD.The study ofPDetiology and neurobiology helps to identify new targets and new drugs for the treatment of such diseases and is important for improving human quality of life and health.Objective(1)Study on the improvement of behavior and cognitive ability of traditional Chinese herbal medicine and GLA in MPTP-inducedPDin Mice,as well as the protection of thesubstantia nigra,striatum and cortex,hippocampus and its mechanism.(2)To analyze the effect of GLA on the Neuroplasticity regulation participate in ERK signaling pathway,and to explore the role of ERK signaling pathway in the pathogenesis of PD,and to further confirm the mechanism of GLA in the treatment of MPTP-inducedPDin mice.Methods(1)Animal grouping and model processing:140 cases of C57BL/6N mice were randomly divided into blank control group,model(MPTP)group,GLA control(GLA)group,model +GLA(MPTP + GLA)low dose group,model +GLA(MPTP + GLA)medium dose group,model +GLA(MPTP + GLA)in the high dose group,model + levodopa group(MPTP + LD),20 rats in each group.MicePDmodel was prepared by intraperitoneal injection of MPTP(20 mg/kg),Blank control group:intraperitoneal injection the equal volume of physiological salinewith with model group;Model group:MPTP 20mg/kg intraperitoneal injection once a day for 7 days;GLA(GLAB)control group:GLAB,50mg/kg,gavage,once a day for 7 days;GLA low dose group:1 hour after MPTP administration of GLA set 20mg/kg gavage,once a day for 7 days;GLA medium dose group:1 hour after MPTP administration GLA set 30mg/kg gavage,once a day for 7 days;GLA high dose group:1 hour after MPTP administration GLA set 50mg/kg gavage,once a day for 7 days;levodopa group:lh after MPTP administration levodopa 40mg/kg gavage,once a day for 7 days.The behavior and cognitive changes of mice were observed 3 hours after the completion of each administration.(2)Behavior and cognitive ability test1)Movement coordination ability detection:after the end of the administration of fatigue rotarod test,before the formal test,the mice first received 2 days of training,5 minutes a day,the speed of 40rpm.On the third day,the test was started and the speed was adjusted to 60 rpm.The test was started at the same time for 3 days.Avoid circadian rhythm effects on mice behavior.Take 5 mice each time placed in the fatigue rotarod bar(diameter:3 cm),to 20s of adaptation time,after the mice stand firm,start the tester,so that the rod speed slowly accelerated to set Speed,record each mouse from the bar on the drop time respectively,set the maximum time of 5 minutes,up to 5 minutes,the bar automatically stop the ball to avoid the high value to the results caused by greater error.Each mouse was measured three times and averaged as the average score for the mouse experiment.2)Mice learning and memory ability test:after the end of the administration of each group of mice Y-type electric maze test was performed,the voltage is 40 V,using a fixed number of random method(ie,safe area in irregular order).Learning ability:shocl the mice which placed in the starting area after 3 min,the mice fled to the safe area,the lights for 15s after the extinguished rest,the next operation is resumed after 45s,10 consecutive trials 9 times to achieve the correct standard.Memory capacity:24 hours after the same method to test the memory preservation of rats.(3)HE staining of brain tissue:pathological changes of striatum and hippocampus were observed by hematoxylin-eosin staining.After treatment with 4%paraformaldehyde solution,the sections were stained with hematoxylin-eosin.The pathological changes of striatum and hippocampus were observed under light microscope(x 400).(4)Correlation factor determination:the levels of TNF-a and IL-18 in hippocampus,striatum and cortex and hippocampus were detected in strict accordance with the instructions step by ELISA.The content of MDA in midbrain ventral,striatum and cortex,hippocampus tissue were measured by thiobarbituric acid.The content of SOD in midbrain ventral,striatum and cortex,hippocampus tissue were measured by xanthine oxidase method.(5)Immunohistochemistry was used to detect the expression of TH and ERK:Immunohistochemistry was used to detect the expression of TH and ERK in the striatum,cortex and hippocampus:the mice were anesthetized and the brain specimens were fixed,using freezing microtome cut slices having a thickness of 14 microns.3%hydrogen peroxide blocked the endogenous peroxidase,PBS rinse,bovine serum albumin solution(30g/L)was incubated,each group were added mouse anti-TH monoclonal antibody(1:1000)or ERK protein kinase Clone antibody(1:100).After washing with PBS,the secondary antibody was added dropwise and stained with DAB.Light microscope striatum,cortex and hippocampus brown particles increased positive.(6)Western blot was used to detect the expression of TH and ERK protein:The expression of TH and ERK protein in the striatum,cortex and hippocampus were detected by Western-blot:after the specimens were cut,the samples were homogenized and the total protein was extracted and protein quantification,20 ul of the lysate was dissolved and subjected to SDS-PAGE.After electrophoresis,the protein was transferred to the NC membrane and blocked,add the first resistance.(GAPDH,ERK,TH,respectively,according to 1:1000,1:2000,1:1000,diluted with fresh clinker),and incubated overnight at 4℃.The next day,used TBST to wash away unbound primary antibodies.Add a certain percentage of diluted horseradish peroxidase(HRP)labeled secondary antibody(1:1000),and after incubated at room temperature,used TBST to separate the unbound secondary antibody.Developed by ECL method,the developed photos were scanned with a scanner,using ScanImage software to take the gray value,got the data after the analysis.The changes of TH and ERK protein expression in striatum,cortex and hippocampus were detected.Results(1)The typical behavioral performance,behavioral ability,learning and memory ability ofPDmice in MPTP group were significantly lower than those in control group.There was no significant difference between the GLA control group and the blank control group.Compared the MPTP + GLA group and the MPTP +LD group with MPTP group the behavioral ability,learning and memory ability was substantial improvement.MPTP +LD group was superior to MPTP + GLA group,the difference was statistically significant.(2)HE staining shows:compared with the control group,MPTP group striatum,hippocampal neuron reduction,neurodegeneration,glial cell proliferation;there was no significant difference between the GLAB control group and the blank control group;Compared with MPTP group,MPTP + GLA group and MPTP + LD group,neuronal degeneration,neurodegenerative improvement,glial cell hyperplasia reduced.MPTP + GLA group and MPTP + LD group between the difference was not statistically significant.(3)Compared with the control group,the levels of TNF-α,IL-18 and MDA in the ventral,striatum,cortex and hippocampus of MPTP group were significantly increased,while the content of SOD was significantly decreased.Compared with MPTP group,the levels of TNF-α,IL-18 and MDA in the substantia nigra and cortical hippocampus of the MPTP +GLA and MPTP + LD group were significantly lower and positively correlated with the GLA dose,and the content of SOD was negatively correlated with the GLA dose.There was no significant difference between MPTP + GLA group and MPTP + LD group(P>0.05),while the levels of TNF-α and IL-18 in medium dose high dose MPTP + GLA group were lower than those in MPTP + LD group.The content of MDA in MPTP + GLA high dose group had lower than MPTP + LD group.The content of SOD in MPTP + GLA high dose group was higher than MPTP + LD group,but there was no significant difference between MPTP + GLA low and medium dose group and MPTP + LD group.(4)Immunohistochemical results showed that:TH-positive neurons were observed in the striatum,cortex and hippocampus of control group,arranged in a striped manner,occasionally with p-ERK positive cells.Compared with control group,the number of TH-positive neurons in striatum,cortex and hippocampus of MPTP group decreased,and p-ERK positive cells increased.The number of TH-positive neurons in striatum,cortex and hippocampus of MPTP + GLA group was lower than that of MPTP group,and the number of p-ERK positive cells was also lower than that of model group.Compared with MPTP + LD group,the number of TH-positive neurons in striatum,cortex and hippocampus of MPTP + GLA group was increased,and the number of p-ERK positive cells was also increased,but the difference was not statistically significant.(5)Western blot analysis showed that:Compared with the control group,the expression of TH protein in the hippocampus and substantia nigra of MPTP group was decreased and the expression of p-ERK protein was significantly increased,in MPTP + GLA group and MPTP + LD group compared with MPTP group,the expression of TH protein was increased,the expression of p-ERK was suppressed.In the MPTP + LD group the expression of TH protein was higher in striatum,cortex and hippocampus than that in the MPTP + GLA group,and the expression of p-ERK protein was increased but the difference was not statistically significant.Conclusions(1)Traditional Chinese medicine GLA can improve MPTP-induced behavior and learning and memory ability of rats,and can prevent MPTP-induced dopaminergic neuronal injury and glial cell proliferation.(2)GLA play a role in protecting neurons through anti-inflammatory,anti-oxidation,and anti-inflammatory,antioxidant effect is better than levodopa.(3)ERK signaling pathway is in association with the nerve injury in brain tissue of PD.(4)GLA can protect the behavior and learning and memory ability ofPDmice induced by MPTP through inhibition of ERK signaling pathway.