The Study On The Regulation Of Exogenous Hydrogen Sulfide To APP Alpha Metabolic Pathway In Pheochromocytoma Cells

ObjectiveTo observe the effects of NaHS, the precursor of hydrogen sulfide（H2S）, on cell survival and growth of pheochromocytoma （PC12） cells,and acquire the optimum experimental concentration for cell survivaland growth. To observe the effects of NaHS on APP alpha metabolicpathway and alpha secretase in PC12cells, then to explore therelationship of APP alpha metabolic pathway and alpha secretaseinfluenced by Exogenous H2S.MethodsWe took PC12cells in vitro as the research object, with NaHS asthe donor of exogenous hydrogen sulfide. PC12cells were divided into8groups: control group, NaHS10mol/L group, NaHS25mol/Lgroup, NaHS50mol/L group, NaHS100mol/L group, NaHS200mol/L group, NaHS500mol/L group, NaHS1000mol/L group.The eight groups were treated with0,10,25,50,100,200,500, and 1000mol/L NaHS, respectively. MTT method was used to test the cellsurvival and growth. We observed the influence of the differentconcentrations of NaHS on cell survival rate. Then we selected thesuitable NaHS concentration for experiment. After PC12cells weretreated with experimental NaHS concentration, Western blot assay wasused to detect the proteins levels of APP, ADAM10, ADAM17. ELISAmethod was used to analyze the levels of sAPP干,A平40,A平42, andsAPP平in cellular culture medium. We observed the influence of thedifferent concentrations of NaHS on APP alpha metabolic pathway andalpha secretase activity.Results1.The results of the cell survival and growth of PC12cells showedas follows: the higher NaHS concentrations (10200mol/L were, thebetter cell survival and growth were（P0.05）;among theseconcentrations of NaHS, the appropriate concentrations of NaHS onculturing neuron were100mol/L and200mol/L. With the increaseof NaHS concentrations5001000mol/L, the cell growth andsurvival decreased.500mol/L and1000mol/L of NaHS had toxiceffect on culturing neuron.2. The results of the expressions of APP showed as follows:compared with the control group, the expressions of APP in differentNaHS concentrations groups had no significant difference P0.05. 3. The results of the expressions of sAPP α, Aβ40, Aβ42, andsAPP β protein showed as follows: compared with control group, withthe increase of NaHS concentrations10200mol/L,the expression ofsAPP α protein increased, and the expressions of Aβ40,Aβ42andsAPP β protein decreased, which showed an concentration dependence.The results showed that differences have statistical significance（P0.05）.4. The results of the expressions of ADAM10and ADAM17protein showed as follows: the expressions of ADAM10, ADAM17protein in different NaHS levels groups more increased than the controlgroup, which showed an increasing sequence in10100mol/L NaHSgroups P0.05.It showed that NaHS enhanced alpha secretaseactivity in the PC12cells. The expressions of ADAM17protein in100mol/L and200mol/L NaHS groups had no significant differenceP0.05. But the expressions of ADAM10protein in100mol/LNaHS group was obviously higher than that in other groups P0.01.Conclusion1.100mol/L and200mol/L of the concentrations of NaHS aremore beneficial to the survival and growth of PC12cell in vitro culture.500mol/L and1000mol/L of the concentrations of NaHS havetoxic effect.2. Exogenous H₂S might up regulate the expressions of sAPP α, and decrease pathological products including Aβ40, Aβ42, sAPP β inPC12cell, which means exogenous H₂S could enhance APPnon amyloid metabolic pathway （alpha metabolic pathway）.Exogenous H₂S could increase the expressions of ADAM10andADAM17protein, which means exogenous H₂S could enhancealpha secretase activity. The reinforcement of APP non amyloidmetabolic pathway （alpha metabolic pathway） might be related to theincrease of alpha secretase activity influenced by exogenous H₂S.