The Effect And Underlying Mechanism Of Hydrogen Sulfide Slow-releasing Donor GYY4137 On MPTP-induced Mouse Model Of Parkinson's Disease

Objective:To study the possible role and underlying mechanism of hydrogen sulfide(H2S)slow-releasing donor GYY4137(GYY)in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-induced mouse model of Parkinson's disease(PD).Methods:Male C57BL/6J mice(8-10 weeks old;23-28 g weight)were used in this study.The mice were randomly divided into six groups and subjected to different treatments:control,PD mouse model group,three different dosages of GYY+ MPTP,and GYY only group.PD mouse model was established by intraperitoneally(i.p.)injecting the mice with 14 mg/kg MPTP four times at 2-h intervals.Saline was given as control.GYY+MPTP group:GYY,at the dosage of 12.5 mg/kg,25 mg/kg or 50 mg/kg,was administered(i.p.)three days before MPTP injection and lasted for two weeks after MPTP treatment.GYY only group:the mice received GYY treatment without MPTP injection.Behavioral tests including rotarod,grid walking,balance beam walking and crack melon seeds were performed to assess the motor function.Immunohistochemistry in combination with DAB staining was applied to examine the number of tyrosine hydroxylase(TH)positive(dopaminergic)neurons in the substantia nigra pars compacta(SNpc).High-performance liquid chromatography(HPLC)was used to measure the striatal dopamine(DA)level.The protein levels of TH and neuronal nitric oxide synthease(nNOS)in the striatum and the nNOS expression in opaminergic neurons in vitro were detected by western blotting.The striatal nitrite content was evaluated by Griess reagent.The level of nitrated alpha-synuclein(Tyr125 and Tyr133)in the striatum was detected by the specific antibody against nitrated a-syn with western blot.Results:(1)Behavioral assessments:Rotarod,grid walking and balance beam walking testes demonstrated that compared to saline-treated group,the motor function in MPTP-injected mice was significantly impaired at one and two weeks after MPTP injection.Co-treatment with different doses of GYY alleviated the MPTP-induced motor dysfunction to a different extent,and the effect of 50 mg/kg GYY was significant.Crack melon seeds test revealed no significant difference among all tested groups.(2)Immunohistochemistry study,in combination with western blotting and HPLC assay,showed that MPTP injection caused an approximate 40%decrease in the number of dopaminergic neurons in the SNpc,and an obvious reduction of TH protein expression and dopamine content in the striatum compared to control group,and this effect was significantly attenuated in the mice co-treated with 50 mg/kg GYY.(3)Acute MPTP injection caused a marked increase of nNOS expression and nitrite level in the mouse striatum compared to saline treatment.The increase was alleviated in the mice with GYY co-treatment,and the effect of 50 mg/kg GYY was significant.(4)MPP+ caused an obvious increase of nNOS expression in dopaminergic neurons,and 100 ?M GYY can alleviate the increase of nNOS.(5)The striatal nitrated a-syn level was markedly elevated in MPTP-treated mice,and this elevation was abolished by 50 mg/kg GYY co-treatment.(6)Treatment with 50 mg/kg GYY alone or saline did not affect the motor behavior or other tested indexes.Conclusion:The study demonstrates a protective action of GYY against dopaminergic neurondegeneration induced by MPTP in the nigrostriatal pathway via its inhibition on nitrative stress and a-syn nitration.