Neuroprotective Effects Of D-Ala2-GIP-glu-PAL In The Acute And Chronic Parkinson’s Disease Mouse Models Induced By MPTP

Objective:1.To study whether D-Ala2-GIP-glu-PAL can reverse motor symptoms and pathological lesions,including the loss of dopaminergic neuron and fibers,reduction of synapse,and increased expression of α-synuclein in substantial nigra pars compacta(SNpc)and striatum,in the acute and chronic Parkinson’s disease(PD)mouse model induced by 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP).And further to determine whether D-Ala2-GIP-glu-PAL has a neuroprotective effect in the acute and chronic PD mouse model.2.To study whether D-Ala2-GIP-glu-PAL can inhibit astrocyte and microglia activation,and regulate the expression of apoptosis gene Bax and Bcl-2,and modulate the expression of p-CREBS133 in the SNpc induced by MPTP.And further to explore the action mechanism of D-Ala2-GIP-glu-PAL in the treatment of acute PD mice model.3.To study whether D-Ala2-GIP-glu-PAL can reduce astrocyte and microglia activation,inhibit lipid peroxidation,and modulate the expression of BDNF in the SNpc and striatum induced by MPTP and probenecid.And further to explore the pharmacological mechanism of D-Ala2-GIP-glu-PAL in the treatment of chronic PD mice model.Methods:1.The acute PD mouse model1.1 Animals and drug treatments: 32 male C57BL/6 mice(25 ±1 g)were divided into four different groups(n=8 per group)consisting of(i)a control group treated with saline alone;(ii)a group treated with D-Ala2-GIP-glu-PAL;(iii)a group treated with MPTP alone;and(iv)a group treated with D-Ala2-GIP-Glu-PAL in combination with MPTP.Mice received four intraperitoneal injections of MPTP(20 mg/kg body weight)or saline(0.9%)control at 2-h intervals.D-Ala2-GIP-glu-PAL(25nmol/kg body weight)was intraperitoneal injected for 7 days after MPTP injection.Control mice received equal volumes of the vehicle solution.1.2 Behavioral assessment: The pole test was conducted on the 5th day after MPTP treatment.The open-field test was conducted on the 6th day after MPTP treatment.1.3 Sample preparation: All animals were sacrificed on the 7th day after MPTP injection.Brains of 4 mice per group were removed and fixed in the 4% paraformaldehyde/PBS(pH7.4),and further were embedded in paraffin.And sections encompassing the SNpc and the striatum were cut for immunohistochemistry.And whole brains of another 4 mice in every group were removed immediately and the SNpc was dissected out for Western Blot.1.4 The expression of tyrosine hydroxylase(TH)of the SNpc and the striatum,glial fibrillary acidic protein(GFAP)and ionized calcium-binding adaptor molecule 1(IBA1)in the SNpc of mice were detected by immunohistochemistry.1.5 The expression of synaptophysin(SYN),Bax,Bcl-2 and p-CREBS133 in the SNpc were detected by Western Blot.2.The chronic PD mouse model2.1 Animals and drug treatments: 48 male C57BL/6 mice(25 ±1 g)were divided into four different groups(n=12 per group)consisting of(i)a control group treated with vehicle solution;(ii)a group treated with D-Ala2-GIP-glu-PAL;(iii)a group treated with MPTP and probenecid;and(iv)a group treated with D-Ala2-GIP-glu-PAL in combination with MPTP and probenecid.Mice were intraperitoneal injected with 10 doses of MPTP(25 mg/kg in saline)and probenecid(250 mg/kg in dimethyl sulfoxide)for 5 weeks with an interval of 3.5 days between consecutive doses.D-Ala2-GIP-glu-PAL(25nmol/kg body weight,)intraperitoneal was treated one time per day during MPTP and probenecid injection and continued for 1 week after MPTP and probenecid injection.Control mice received equal volumes of the vehicle solution.2.2 Behavioral assessment: The open-field test,the pole test,and rotarod test were conducted on the 6th weeks.2.3 Sample preparation: All animals were sacrificed on the end of the 6th weeks.Whole brains of half of mice per group were removed immediately and ventral midbrain was dissected out for Western Blot.And Brains of another half of mice per group were removed and fixed in the 4% paraformaldehyde/PBS(pH7.4),and further were embedded in paraffin.And sections encompassing the SNpc and the striatum were cut for immunohistochemistry.2.4 The expression of TH,α-synuclein,GFAP,IBA1,4-Hydroxynonenal(4-HNE),and BDNF in the SNpc and striatum were detected by immunohistochemistry.2.5 The expression of α-synuclein and BDNF in the SNpc were detected by Western Blot.Results:1.The acute PD mouse model1.1 Behavioral assessment: The results of open-field test showed that the number of crossing line and rearing in MPTP group mice exhibited a significant reduction versus the control group mice(P<0.01).However,D-Ala2-GIP-glu-PAL treatment reversed these effects on crossing line and rearing(P<0.05).The results of pole test showed that MPTP injection induced the bradykinesia and movement unbalance of mice,with more T-turn and T-LA(P < 0.001),compared with the control.However,treatment with D-Ala2-GIP-glu-PAL significantly reversed these effects induced by MPTP(P<0.01).1.2 Immunohistochemistry: On the 7th day after MPTP injection,the number of TH-positive cells in the SNpc was decreased to 27.00% of control(P<0.001),and the average optical density of TH staining in the striatum was decreased to 56.86% of control(P<0.001).However,D-Ala2-GIP-glu-PAL treatment restored numbers of dopaminergic neurons to 59.4% in SNpc(P<0.05)and the expression of TH to 83.64% in the striatum(P<0.01)in MPTP-treated mice.The areas of GFAP positive cells and the areas of IBA1 positive cells in the SNpc of MPTP group mice were significantly higher than those in control group(P<0.001).The areas of GFAP positive cells and the areas of IBA1 positive cells in the SNpc of MPTP + D-Ala2-GIP-glu-PAL group mice were significantly lower than those in group MPTP(P<0.001).1.3 Western Blot: The expression of SYN was much reduced in mice treated with MPTP(P<0.001)compared to control.D-Ala2-GIP-glu-PAL treatment partially reversed the reduction(P<0.01)compared to the MPTP group.Expression of the anti-apoptotic signaling molecule Bcl-2 in SNpc was reduced by MPTP treatment,expression of the pro-apoptotic signaling molecule Bax in SNpc was increase by MPTP treatment,and ratio of Bax/Bcl-2 was finally increase(P < 0.01),compared with control group.While D-Ala2-GIP-glu-PAL partly decreased the ratio of Bax/Bcl-2 by promoting Bcl-2 expression(P<0.01).There was a significant decrease in levels of p-CREBS133 of the SNpc in MPTP group(P<0.001),compared with control group.However,D-Ala2-GIP-glu-PAL treatment moderate reversed the decrease(P<0.05).2.The chronic PD mouse model2.1 Behavioral assessment: The results of open-field test showed that the number of crossing line and rearing in MPTP and probenecid group mice exhibited a significant reduction versus the control group mice(P < 0.01).However,D-Ala2-GIP-glu-PAL treatment reversed these effects on crossing line and rearing(P<0.05).The results of pole test showed that MPTP and probenecid injection induced more T-turn and T-LA of mice,compared with the control(P<0.001).However,treatment with D-Ala2-GIP-glu-PAL significantly reversed these effects(P<0.05).The results of rotarod test showed that drop latency in MPTP and probenecid group mice was less than control group(P<0.001).However,treatment with D-Ala2-GIP-glu-PAL significantly reversed these effects induced by MPTP and probenecid(P<0.05).2.2 Immunohistochemistry: After MPTP and probenecid injection for 5 weeks,the numbers of TH positive cells in the SNpc was decreased to 33.99% of control group(P<0.001),and the average optical density of TH staining in the striatum was decreased to 56.75% of control group(P<0.001).However,D-Ala2-GIP-glu-PAL treatment restored numbers of TH positive cells to 59.21% in SNpc(P<0.05)and the overall levels of TH to 73.33% in the striatum(P<0.05)in MPTP and probenecid-treated mice.The average optical density of α-synuclein positive staining in the SNpc and striatum was significantly increased by MPTP and probenecid injection for 5 weeks(P<0.001)compared with control mice.However,D-Ala2-GIP-glu-PAL treatment decreased the effects induced by MPTP and probenecid(P<0.05).The areas of GFAP positive cells and the areas of IBA1 positive cells in the SNpc and striatum of MPTP and probenecid group mice were significantly higher than those in control group(P<0.001).The areas of GFAP positive cells and the areas of IBA1 positive cells in the SNpc and striatum of MPTP and probenecid + D-Ala2-GIP-glu-PAL group mice were significantly lower than those in MPTP and probenecid group(P<0.001).The average optical density of 4-HNE staining in the SNpc and striatum was significantly increased by MPTP and probenecid injection for 5 weeks(P<0.001),compared with the control group.However,D-Ala2-GIP-glu-PAL treatment decreased the levels of 4-HNE induced by MPTP and probenecid(P<0.05).The areas of BDNF positive staining in SNpc was much decreased in mice treated with MPTP and probenecid(P<0.001),compared to control.While D-Ala2-GIP-glu-PAL treatment partially reversed the decrease(P<0.05)compared to the MPTP and probenecid group.2.3 Western Blot: The expression of α-synuclein in SNpc was increased in mice treated with MPTP and probenecid(P < 0.001),compared to control.While D-Ala2-GIP-glu-PAL treatment partially reversed the increase(P<0.05)compared to the MPTP and probenecid group.The expression of BDNF in SNpc was much decreased in mice treated with MPTP and probenecid(P < 0.001),compared to control.While D-Ala2-GIP-glu-PAL treatment partially reversed the decrease(P<0.05)compared to the MPTP and probenecid group.Conclusions:1.Our findings demonstrated that in the acute and chronic PD mouse model induced by MPTP,D-Ala2-GIP-glu-PAL can significantly reverse PD-like motor symptoms and pathological lesions,including the loss of dopaminergic neuron and increased expression of α-synuclein in the SNpc and striatum.D-Ala2-GIP-glu-PAL has shown a promising neuroprotective effect in the acute and chronic mouse PD model.2.The neuroprotective activity of D-Ala2-GIP-glu-PAL may be related to the activation of GIP-R signal transduction pathway of dopaminergic neurons in the SNpc,further regulating gene transcription mediated by CREB,promoting the expression of Bcl-2 and inhibiting cell apoptosis,inhibiting lipid peroxidation,promoting the production of BDNF,inhibiting the glial activation and neuroinflammation.