| The complexity of Parkinson's disease is accompanied by clinical problems,including an inability to make a definitive diagnosis at the earliest stages of the disease and difficulties in the management of symptoms at later stages.Furthermore,there are no treatments that slow the neurodegenerative process.To explore drugs with neuroprotective or disease-modifying properties has become one of the key points of PD clinical treatment.A large number of reports have reported that Glucagon-like peptide 1(GLP-1)receptor agonists can show the advantage of neuroprotective effects in neurodegenerative diseases and enhance cell biological functions in the central nervous system.However,its specific mechanism is still unclear.Fasciculation and elongation protein zeta-1(FEZ1)is specifically expressed in the nervous system,involved in axon growth,microtubule transport,and neurodevelopment.At the same time,Previous studies in our laboratory have shown that the expression of FEZ1 in a PD model is significantly higher than that of neurons,and participate in the differentiation of dopamine neurons and the regulation of dopamine release.Therefore,this study investigated whether GLP-1/GIP dual-receptor agonists DA-JC1 achieves effective protection by regulating the expression of astrocyte FEZ1 in the PD model,as well as provides a new,better reference for the treatment and prevention of Parkinson's disease.Objective:By establishing a PD model,the effects of GLP-1/GIP dual-receptor agonists DA-JC1 on the PD model and the expression change of FEZ1 before and after treatment were studied,and the significance and possible mechanism of FEZ1 expression change in it were discussed.Methods:(1)Firstly,ensure the optimal concentration value of GLP-1/GIP dual-receptor agonist DA-JC1 through using CCK-8 assay and then establish a PD cell model by inducing SH-SY5Y cells in vitro with 6-OHDA.The prevention group(DA-JC1+6-OHDA),model group(6-OHDA),treatment group(6-OHDA+DA-JC1),and normal group(Control)was set up.The prevention group was treated with 6-OHDA in advance 24 h,and the treatment group was received with 6-OHDA after 24 h.The SH-SY5Y cells were observed with morphological changes,the cell viability was detected by CCK-8 assay and the expression of FEZ1/BCL-2/BAX protein in each group was detected by western blot after DA-CJ1 injection is processed separately.(2)PD rats were established by stereotactic injection of 6-OHDA(2 ?g/?l)in the medial forebrain bundle(MFB),and the prevention,treatment,model and sham operation groups were set up respectively.The sham group only operated on the dura mater.DA-JC1(25 nmol/kg)was administered intraperitoneally insistent for 6 days,and the diet and body weight of each group were recorded daily.(3)On the 1st,3rd,and 7th day,the behavioral changes were recorded by the mining experiment,and the change in the number of rotations before and after treatment was recorded by apomorphine(APO).(4)Western blot was used to detect the expression of FEZ1/TH/GFAP/CREB(cAMP response element-binding protein)in substantia nigra and striatum.The expression changes of TH/GFAP in substantia nigra and striatum were observed by immunohistochemistry.The interaction relationship of FEZ 1/CREB was observed by the Co-IP experimentResults:(1)The best use concentration of DA-JC1 was 1 ?mol/L and the optimal concentration of 6-OHDA was 150 ?mol/L by CCK-8 assay.Microscopical observation showed that the cells in the prevention group were complete and the suspension cells were small.The apoptosis rate of CCK-8 in the prevention group(13.32%)was significantly lower than that in the treatment group(31.50%)and the model group(32.48%)(P<0.01)Western blot analysis also found that FEZ1 was expression highest in the prevention group,and the same results found the expression of Bcl-2 was increased.(2)Rats showed obvious rotational behavior 2 weeks after 6-OHDA injury.By recording the body weight and diet,it was found that the bodyweight of PD rats after DA-JC1 injection decreased significantly and the growth rate was slow.There was no significant difference in the daily diet weight statistics of each group.(3)The results of the behavioral level experiment showed that the level of exercise ability in the treatment group was significantly improved(P<0.01),and the rats in the treatment group showed more emotional relief ability(P<0.05).The data of apomorphine induction within 30 min showed The number of rotations in the treatment group(p<0.001)and the prevention group(p<0.05)was significantly lower than that in the model group,and the behavior score of the rats was improved.(4)The results of western blot analysis of FEZ 1 were staying the same with the cell model.The expression of TH and GFAP protein also had a similar trend with FEZ1.Immunohistochemical results showed that DA-JC1 treatment had obvious protective effects on the morphology and quantity of TH neurons in the substantia nigra.Co-immunoprecipitation experiments showed that CREB interacts with FEZ1,indicating that the expression of FEZ1 is regulated by the cAMP-PKA pathway.Conclusion:(1)DA-JC1 has a proliferative effect on SH-SY5Y cells;(2)DA-JC1 can improve the motor symptoms of PD rats;(3)DA-JC1 can regulate the expression of FEZ1 through cAMP-PKA pathway,and then play a neuroprotective role in PD models;(4)FEZ1 can be used as a potential target for the treatment of PD diseases. |
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