The Neuroprotective Effect Of Cholecystokinin-8 In MPTP-induced PD Mouse Model

Objective:1.Observe the effect of cholecystokinin-8(CCK-8)on the motor function of MPTP-induced Parkinson’s mouse model using behavioral tests,and explore the neuroprotective effect of cholecystokinin-8 on MPTP-induced Parkinson’s mouse model;2.To explore the mechanism of the neuroprotective effect of CCK-8 by observing the effects of it on dopaminergic neurons,α-synuclein,glial fibrillary acidic protein,inflammation-related indicators and apoptosis in MPTP-induced Parkinson’s mouse modelMethods:1.Grouping:36 8-week-old male C57 mice were divided into:(1)Control group;(2)MPTP model group;(3)CCK-8+MPTP intervention group2.Parkinson’s modeling method:intraperitoneal injection of MPTP(30 mg/Kg)for one week.3.(1)Control group:Inject simple 0.9%sodium chloride solution intraperitoneally for 7 days(0.2 ml/d);(2)MPTP model group:intraperitoneal injection of simple MPTP solution for 7 days(30 mg/kg/d);(3)CCK-8+MPTP group:MPTP solution(30 mg/kg/d)was intraperitoneally injected,and CCK-8 solution(25 nmol/kg/d)was injected 30 minutes later for 7 days.4.Treatment:All mice were monitored for body weight and blood glucose(days 1 and 7),and behavioral tests were performed on the 7th day,including the rotarod test,the open field test,and the gait test.All mice were sacrificed at the same time on the 2nd day after the end of the drug injection,their heads were decapitated,and brain tissue specimens were collected.Then all mice were used for immunohistochemistry and Western-blot assay respectively,to determine of tyrosine hydroxylase(TH),α-synuclein(α-syn),glial fibrillary acidic protein(GFAP),ion-calcium junction receptor molecule-1(IBA-1),nuclear transcription factor(NF-κB),glial cell-derived neurotrophic factor(GDNF),and apoptosis-related proteins(Bax,Bcl-2)of substantia nigra and striatum.Results:1.Blood glucose and body weight levels:there was no significant difference in blood glucose and body weight levels of mice at the same time on the 1 st and 7th day between the groups(p<0.05).2.Behavioral test results:2.1.General observation:the MPTP group had less activities,limbs were stiff,dorsal arching and limb tremor appeared.These were all improved in CCK-8+MPTP group.2.2.Rotarod test:compared with control group,the sustainable rotating rod movement time of mice in MPTP group was shortened,it was longer in CCK-8+MPTP group than that in MPTP group(p<0.05).2.3.Open field test:the walking distance of MPTP group was decreased compared with control group,it was significantly improved in CCK-8+MPTP compared with MPTP group(p<0.05).2.4.Gait test:the swing speed and stride length of the MPTP group mice are lower than those of the control group,and the stance time and step cycle are larger than those of the control group.After the intervention of CCK-8,the speed and stride length were larger than those in the MPTP model group,and the stance time and step cycle were lower than those in the control group(p<0.05).3.Results of immunohistochemical staining and Western-blot:3.1.The expression level of TH:Western blot:in the substantia nigra striatum,the TH level was significantly decreased in MPTP group,it was higher in the CCK-8+MPTP group than that in MPTP group(p<0.05).3.2.The expression level of α-syn:Western blot:in the substantia nigra,the expression of α-syn in MPTP group was increased compared with the control group,this of CCK-8+MPTP group was lower than that in MPTP model group(p<0.05).3.3.The expression level of neuroinflammatory response related factors(GFAP,Iba-1,NF-κB):Immunohistochemistry:in the striatum,the number of GFAP positive was significantly increased in the MPTP group,this in the CCK-8+MPTP group was less than that in MPTP group(p<0.05).Compared with the control group,the number of Iba-1 positive cells of MPTP model group was significantly increased;It was less in the CCK-8+MPTP group(p<0.05).Compared with the control group,the number of NF-κB positive cells of the MPTP model group increased,this in the CCK-8+MPTP group was less than that in MPTP group(p<0.05).Western blot:(1)GFAP expression level:in substantia nigra,the level of GFAP in MPTP group was increased compared with the control group,it was significantly decreased in CCK-8+MPTP group(p<0.05).(2)The NF-κB level in MPTP model group was increased compared with the control group,it was lower in CCK-8+MPTP group(p<0.05).3.4.The expression level of GDNF:Immunohistochemistry:in striatum region,the positive GDNF cells was significantly decreased in MPTP group compared with control group,this in CCK-8+MPTP group was higher than in MPTP model group(p<0.05).Western blot:in striatum region.the GDNF level was significantly decreased in MPTP group compared with control group,this was higher in CCK-8+MPTP group than that in MPTP model group(p<0.05).3.5.The expression level of apoptosis-related protein(Bax,Bcl-2):Western blot:(1)In striatum region,the Bax level was significantly increased in MPTP group compared with control group,this was lower in CCK-8+MPTP group than that in MPTP model group(p<0.05).(2)In striatum region,the Bcl-2 level was significantly decreased in MPTP group compared with control group,this was higher in CCK-8+MPTP group than that in MPTP model group(p<0.05).Conclusion:1.Through observing behavioral tests that CCK-8 can improve MPTP-induced motor dysfunction in a mouse model of Parkinson,it has a neuroprotective effect on MPTP-induced Parkinson’s mouse model;2.CCK-8 can reduce the loss of dopaminergic neurons and the accumulation of pathologicalα-syn,inhibit the neuroinflammatory response and cell apoptosis,and promote the expression of GDNF in the substantia nigra and striatum in the MPTP-induced Parkinson’s mouse model.