Study On The Mechanism Of Ghrelin Reducing Dopaminergic Neuron Apoptosis By Regulating Autophagy And Endoplasmic Reticulum Stress

Background:Parkinson’s disease(PD)is a type of neurodegenerative disease characterized by progressive loss of dopaminergic neurons and formation of Lewy bodies in the substantia nigra pars compacta(SNpc)of the midbrain.The exact mechanism of PD neuronal degeneration is not clear.Evidence has demonstrated that endoplasmic reticulum stress(ERS),autophagy dysfunction,and other mechanisms are involved in PD neuronal death.The current treatment of PD is mainly dopamine replacement therapy,which will be accompanied by certain side effects and cannot delay the progression of the disease,while alternative strategies such as stem cell therapy,neuro-nucleus surgery and deep brain stimulation have strict indications.Drug therapy is still the cornerstone of PD treatment.Therefore,it is of great significance to find a new drug for the treatment of PD.Ghrelin is a brain-gut peptide,which can cross the blood-brain barrier.Studies have shown that the Ghrelin/GHS-R system exerts neuroprotective effects through anti-inflammatory,anti-apoptotic,etc.The specific mechanism of Ghrelin still needs to be further explored.Objective:In this study,1-methyl-4-phenyl-1,2,3,6-tetrahydropyran(MPTP)was used to treat C57BL/6 mice to construct an animal model of PD.To explore whether Ghrelin exerted neuroprotective effects via regulating autophagy and ERS,which can provide new targets for PD drug treatment in the future.Methods:All mice were housed in an SPF-grade room and adapted to the surrounding environment for 1 week,the mice were divided into 4 groups:Control group,Ghrelin group,MPTP group,and MPTP+Ghrelin group according to the random number table.MPTP was injected intraperitoneally for 5 days,and Ghrelin was pre-administered for 6 days.The fatigue rota-rod test and the pole test are explored to detect the motor coordination of mice.The pathological changes of SNpc and STR were measured by immunohistochemical experiments to detect the number of TH+neurons and the density of TH+nerve fibers.Western blot assay to detect the expression of GHS-R1a and TH in the SNpc and STR,as well as the expression of α-synuclein,GRP78,CHOP,p-IRE1α,p-ASK1,p-JNK,Cleaved caspase-12,Cleaved caspase-3,LC3B-II,p62 in the SNpc.The expression of TH co-localized with GHS-R1a,α-synuclein,GRP78,and CHOP in the dopaminergic neurons was determined by double fluorescence immunostaining.Results:1.In the MPTP mice model of PD,the latency time on the rota-rod apparatus wassignificantly shortened,while the total spending time of climbing rods was significantly extended.Besides,the number of TH+ neurons,the density of TH+nerve fibers,and the expression of TH were all decreased.All the above indicated that the animal model of PD was constructed successfully.Pretreatment with Ghrelin significantly antagonized the neurotoxicity,ameliorated the behavioral results,and increased the expression of TH induced by MPTP.2.The protein expression of GHS-R1a was decreased induced by MPTP in the SNpc and STR of the mice and the fluorescence intensity in dopaminergic neurons was also reduced indicating that MPTP inhibited the expression of the receptor,but pretreatment with Ghrelin increased the expression of GHS-R1a,which was contributed to the binding of Ghrelin and better to exerted protective effects.3.The results of western blot assay and immunofluorescence double staining both indicated that the expression of α-synuclein and p-α-synuclein were significantly increased in the MPTP-treated mice,while administration of Ghrelin effectively reduced the aggregation and phosphorylation of α-synuclein.4.The expression of ERS marker proteins GRP78 and CHOP were increased in MPTP-lesioned mice,indicating that the activation of ERS.Administration of Ghrelin attenuated the expression of GRP78 and CHOP.Besides,Ghrelin also inhibited the activation of IRE1α-ASK1-p-JNK and caspase-12/caspase-3 signaling pathway induced by MPTP,which is beneficial to reduce the apoptotic response of dopaminergic neurons.5.In the MPTP mice model of PD,our results showed that autophagy was significantly inhibited in the MPTP group of the SNpc indicated by the down-regulation of LC3B-Ⅱ and up-regulation of p62.Administration of Ghrelin to MPTP-treated mice significantly reversed the phenomenon,indicating that Ghrelin promoted the autophagic process.Conclusions:Ghrelin promoted autophagy,reduced the accumulation and phosphorylation of α-synuclein,further inhibited the apoptosis caused by ERS,and exerted neuroprotective effects in dopaminergic neurons.