Action Of Excessive Induced By 6-OHDA Via P66SHC Pathway On Neuron Cell Injury And The Protective Effect Of Hydrogen Sulfide

Objective:Parkinson’s disease（PD）is a common neurodegenerative disease.Neuronal cell death is a well-recognized pathological mechanism of PD.Studies have shown that H₂S can inhibit neuronal apoptosis and exert anti-PD effect,but the underlying mechanisms need to be further explored.We previously reported that H₂S can inhibit the production of mitochondrial ROS（Mitochondria ROS,MtROS）and reduce the level of oxidative stress in the body.So we used 6-OHDA（a drug that directly acts on dopaminergic nerve energy）to construct a PD model of SH-SY5Y cells,whether Hydrogen sulfide can reduce the synthesis of MtROS in the P66SHC pathway and ameliorate the autophagy caused by6-OHDA,thereby protect neuronal cells against 6-OHDA-induced apotosis.Method:MTT was used to detect the viability of SH-SY5Y cells.Western blot was used to detect endogenous CBS protein,P-p66shc,T-p66shc and autophagy marker index LC3II/I.Protein expression of Beclin1;generation of mitochondrial oxygen free radicals by Mito-sox;qualitative detection of apoptotic cells by Hoechst 33258 staining;quantitative detection of apoptotic cells by flow cytometry。Result:1.Treatment of SH-SY5Y cells with different concentrations of 6-OHDA（0,12.5,25,50,100,150μmol/L）for 24 hours can reduce cell viability in a concentration-dependent manner,while giving different concentrations of NaHS（exogenous H₂S for Body,10,30,100,300μmol/L）Pretreatment for 30 min or overexpression of CBS（the main synthetase of endogenous H₂S in nerve cells）can combat6-OHDA-induced SH-SY5Y cell damage to varying degrees.2.NaHS（100μmol/L）pretreatment for 30min or overexpression of CBS can significantly inhibit the increase of P66SHC phosphorylation and mitochondrial oxygen free radical enhancement in SH-SY5Y cells induced by 6-OHDA.3.In the SH-SY5Y cell line of C59S mutation（ie,mutation of cysteine at position 59 in the CH₂ domain of P66SHC protein）,NaHS pretreatment for 30 min could not counteract the increase of P66SHC phosphorylation and mitochondrial oxygen free radical enhancement induced by 6-OHDA.It is also unable to counteract the damage of SH-SY5Y cells induced by 6-OHDA,and the use of NAC（scavenger of ROS）can also counteract the enhancement and damage of mitochondrial oxygen free radicals in SH-SY5Y cells induced by6-OHDA.4.6-OHDA treatment for 24h significantly up-regulated the ratio of autophagy marker protein LC3II/LC3I,Beclin1 protein level,and promoted autophagy.However,after H₂S,NAC pretreatment or CBS overexpression,the ratio of LC3II/LC3I and Beclin1 protein expression were down-regulated,and 6-OHDA-induced autophagy was improved,which was reversed by C59S mutation.5.Administration of H₂S can significantly inhibit the apoptosis of SH-SY5Y cells induced by 6-OHDA;administration of autophagy promoter RA（rapamycin）or C59S mutation can counteract the effect of H₂S and further aggravate apoptosis.Conclusion:6-OHDA up-regulates P66SHC phosphorylation to increase mitochondrial oxygen free radical-increased autophagy to induce neuronal cell damage;H₂S down-regulates 6-OHDA to induce P66SHC phosphorylation to reduce mitochondrial oxygen free radical-induced autophagy to reduce neuronal cell damage.