Hydrogen Sulfide Prevents 6-ohda-induced Dopaminergic Neurons Apoptosis And Loss In Substantia Nigra Via Upregulation Of Leptin

Background and ObjectiveThere are two pathological changes in Parkinson’s disease:the loss of dopamine neurons and the accumulation of Lewy bodies composed ofα-syncline.Hydrogen sulfide（H₂S）has a definite neuroprotective effect on Parkinson’s disease,but its mechanisms are not clear.Leptin,an adipose-derived hormone,plays a neuroprotective effect.Therefore,we used the single injection of 6-OHDA into left striatum as the Parkinson’s disease model to explore whether the protection of H₂S against6-OHDA-induced dopaminergic neurons apoptosis and loss in substantia nigra is via upregulation of Leptin.MethodsMale SD rats were injected with 6-hydroxydopamine 6-OHDA（AP:+1.0 mm;ML:+3.0 mm;DV:-4.5 mm）into the striatum to establish the pathologic model of Parkinson’s disease.The number of survival dopaminergic neuron was assessed by TH+cells,and the percentage of apoptosis of neuron was evaluated by tunel staining in substantia nigra.Multi-mode microplate reader was used to detect the activity of Caspase-3 in the substantia nigra.The expression of Bcl-2 and Bax protein in the substantia nigra was detected by Western blotting.Results:1.H₂S suppresses the loss of dopaminergic neurons in the substantia nigra（SN）of 6-OHDA-treated rats.After pretreated with NaHS（30 or 100μmol/kg,i.p.）for 1 w,rats were injected 8μg of6-OHDA into the left striatum and continued to 4-w treatment with NaHS（30 or 100μmol/kg,i.p.）.TH-positive cells were detected to assess the survival of dopaminergic neuron.Compared with control group,the number of TH positive cells in the SN of 6-OHDA-treated group was decrease.However,NaHS（100μmol/kg/day,i.p.）significantly increased the number of TH positive cells in the SN of 6-OHDA-treated group,which indicated that H₂S suppressed the loss of dopaminergic neuron induced by 6-OHDA.2.H₂S inhibits the apoptosis of neurons in the SN of6-OHDA-treated rats.In the SN of 6-OHDA-treated group,the percentage of tunnel positive cells and the activity of Caspase-3 were increased,the expression of anti-apoptotic protein Bcl-2 was downregulation,and the expression of pro-apoptotic protein Bax was upregulation.However,Na HS（100μmol/kg/day,i.p.）significantly declined the percentage of tunnel positive cells and the activity of Caspase-3,upregulated the expression of bcl-2,and downregulated the expression of Bax in the SN of 6-OHDA-treated group.These data suggested that H₂S protects the SN against 6-OHDA-caused apoptosis.3.H₂S upregulates the expression of Leptin in the SN of6-OHDA-treated rats.To determine whether H₂S regulates the Leptin pathway,the level of Leptin protein in the SN was examined by western blotting.Compared with control group,the expression of Leptin was decreased in the SN of 6-OHDA-treated group.However,the expression of Leptin in the SN of 6-OHDA-treated group was increased following the intraperitoneal injection of 100μmol/kg/day NaHS.4.Blockade of Leptin reverses the inhibitory role of H₂S in6-OHDA-exerted loss of dopaminergic neurons in the substantia nigra（SN）of rats.To determine the mediatory role of Leptin in the protective effect of H₂S,we firstly investigated whether the Leptin receptor antibody（OBR）reverses the inhibitory role of H₂S in6-OHDA-exerted loss of dopaminergic neurons in the substantia nigra（SN）of rats.The results showed that Leptin-OBR（2μg,i.c.v.）abolished the protective effect of H₂S on 6-OHDA-induced loss of rat substantia nigra dopamine neurons,indicating that Leptin mediates the protection of H₂S against 6-OHDA-exerted loss of dopaminergic neurons in the substantia nigra（SN）of rats.5.Blockade of Leptin reverses the protection of H₂S against6-OHDA-exerted apoptosis in the SN of rats.To determine the mediatory role of Leptin in the protective effect of H₂S,we also investigated whether the Leptin receptor antibody（OBR）reverses the protective action of H₂S in 6-OHDA-exerted apoptosis in the SN of rats.In the SN of 6-OHDA-treated rats,NaHS（100μmol/kg/day,i.p.）significantly reduced the percentage of tunel positive cells and the activity of Caspase-3,upregulated the expression of bcl-2,and downregulated the expression of Bax in the SN of 6-OHDA-treated group.However,in Leptin-OBR（2μg,i.c.v.）-cotreated group,the percentage of tunel positive cells and the activity of Caspase-3 were increased,the expression of anti-apoptotic protein Bcl-2 was downregulation,and the expression of pro-apoptotic protein Bax was upregulation.Leptin mediates the protection of H₂S against 6-OHDA-exerted apoptosis in the substantia nigra（SN）of rats.ConclusionH₂S attenuates 6-OHDA-induced loss of dopaminergic neurons and apoptosis in the SN via up-regulating of Leptin.