| PART1:PROTECTIVE EFFECT OF EXOGENOUS HYDROGEN SULFIDE AND DONEPEZIL IN PC12CELLS DAMAGED BY A B25TO35Objective:To observe the protective effect of exogenous hydrogen sulfide and donepezil in PC12cells pheochromocytomas (pheochromocytoma cells, PC12) damaged by Aβ25to35(beta amyloid peptide25to35, Aβ25-35). Methods:PC12cells were cultured in vitro and sulfur hydrogenated sodium (sodium hydro sulfide, NaHS) were provided as exogenous H2S donor.The study were devided into six groups: control group, Aβ25to35group, NaHS group, NaHS+Aβ25to35group, donepezil group, donepezil+Aβ25to35group.the concentration of NaHS were10,25,50,100,200,500,1000μmol/L respectively, Aβ25-35donepezil were20μmol/L. After treatment of PC12with the different group for24h, the cell survival rates were measured by (3-(4,5)-dimethylthiahiazo(z-y1)-3,5-diphenytetrazoliumromide, MTT). Results: (1) Aβ25to35significantly reduces the PC12cell survival rate (P<0.05);(2) After treatment with different concentrations of NaHS,10μmol/L of NaHS did not affect the PC12cell survival rate (P>0.05),25μmol/L～200μmol/L of NaHS improves the PC12cell survival rate (P<0.05), moreover, the toxicity of PC12cell induced by Aβ25-35were concentration-dependently inhibited by NaHS from10μmol/L～200μmol/L and significantly improve the survival rate (P<0.05) of PC12. However,500and1000μmol/L NaHS is toxic to PC12cells (P>0.05);(3) Donepezil reduces the toxic effect of Aβ25to35in PC12cells and increases the survival rate of PC12cells (P<0.05). Conclusion:H2S and donepezil have protctive effect on cell damage induced by PC12cells. PART2:THE INFLUENCE OF EXOGENOUS HYDROGEN SULFIDE AND DONEPEZIL ON APP/Ap METABOLIC PATHWAY IN PC12CELLSObjective:To investigate the influence of exogenous hydrogen sulfide and donepezil on APP/Ap (amyloid precursor protein/beta amyloid peptide, APP/Aβ) metabolic pathway in PC12cells. Methods:PC12cells were cultured in vitro and sulfur hydrogenated sodium (sodium hydrosulfide, NaHS) were provided as exogenous H2S donor. The study were devided into seven groups:control group, NaHS10μmol/L,25μmol/L,50μ mol/L,100μmol/L,200μmol/L group, donepezil20μmol/L. After treatment of PC12with the different group for24h, the Aβ40、Aβ42、sAPPα、sAPPβ level were measured by ELISA,the protein expression of APP metabolism were measure by Western blot. Results:(1) There have no difference of APP with seven groups(P>0.05);(2) The Aβ40, Aβ42and sAPP-β protein expression level were decreased, and meanwhile the sAPPa protein level were increased by NaHS with different concentrations and donepezil group. Conclusion:Exogenous H2S and donepezil can induce the APP/Ap metabolic pathway to the amyloid pathway way (a way). PART3:THE INFLUENCE OF EXOGENOUS HYDROGEN SULFIDE AND DONEPEZIL ON A-SECRETASE ENZYME (ADAM10/ADAM17) IN PC12CELLSObjective:To observe the influence of exogenous H2S and donepezil the H2S and investigate the association of H2S and donepezil with the tranform of APP metabolic pathway. Methods:PC12cell were cultured in vitro and sulfur hydrogenated sodium (sodium hydrosulfide, NaHS) were provided as exogenous H2S donor. The study were devided into seven groups:control group, NaHS10μmol/L,25μmol/L,50μmol/L,100μmol/L,200μmol/Lgroup, donepezil20μmol/L. After treatment of PC12with the different concentration for24h, the ADAM10and ADAM17were measured by Western blot. Result:Compared with control group, the ADAM10and ADAM17protein level were increased in NaHS concentration and donepezil group. along with the increasing the concentration of NaHS, the ADAM10and ADAM17protein level were decreased (P<0.05). Whereras, the ADAM17and ADAM10were not significantly changed in NaHS100μmol/L and NaHS200μmol/L group(P>0.05), but the highest protein level ADAM17and was in NaHS100μmol/L group (P<0.01). Conclusion:(1) Exogenous H2S and donepezil increase the a-secretase (ADAM10/ADAM17) protein expression level;(2) The exogenous H2S and donepezil transform APP/Aβ metabolic pathway to the amyloid pathway and this tranformation probablely related to the increasing level of ADAM10/ADAM17. PART4:THE RESEARCH ON THREE CELL SIGNALLING OF EXOGENOUS HYDROGEN SULFIDE AND DONEPEZIL SHIFT APP ALPHA METABOLIC PATHWAYSObjective:To observe the effects of PI3-K, MAPKs and JNK three signaling pathways on APP a metabolic pathway resulted by exogenous hydrogen sulfide and donepezil in PC12cell. Methods:PC12cell were cultured in vitro and NaHS were exogenous H2S donor. The study were devided into two parts:(1)The first step includs four groups:control group, LY group (LY29400220μmol/L), SB group (SB20358030μmol/L), SP group (SP60012520μmol/L).(2)The second parts includes nine group (control, NaHS100μmol/L, NaHS100μmol/L+SP, NaHS100μmol/L+LY, NaHS100μmol/L+SB, donepezil20μmol/L group, donepezil20μmol+SP, donepezil20μmol/L+LY, donepezil20μmol/L+SB). Before addition of NaHS100μmol/L or donepezil20μmol/L to PC12cells, the SP600125, LY294002and SB203580were first added.After24hours intervention, Aβ40, Aβ42, sAPPa, sAPPβ protein level were measured by ELISA. the APP, ADAM10, ADAM17protein were measured by Western blot. Results:(1) The APP protein level is not inhibited by (P>0.05) SP600125,LY294002and SB203580, however, SP600125and LY294002inhibitor ADAM10and ADAM17protein expression.(2) SB203580have no no obvious effect (P>0.05) on Aβ40, Aβ42, sAPPβ, sAPPα protein expression. Whereas, LY294002, SP600125enhance Aβ40, Aβ42, sAPPβ protein level and reduce sAPPa protein expression (P<0.01).(3) Compared with control group,SP600125and LY294002reduce the the effect of H2S of increasing level of ADAM10;SB203580, SP600125and LY294002reduce the effect of donepezil of increasing level of ADAM10and ADAM17.(4) Compared with control group, SP600125and PI3-K LY294002inhibit the effect of H2S of reducing Aβ40, Aβ42, sAPPβ protein expression(P<0.01); SB203580, SP600125and LY294002reduce the effect of donepezil of reducing Aβ40, Aβ42, sAPPβ protein expression (P<0.01).(5) Compared with control group,SP600125and PI3-K LY294002inhibit the effect of H2S of increasing sAPPa protein expression (P<0.01); SB203580, SP600125and LY294002reduce the effect of donepezil of increasing sAPPa protein expression (P<0.01).Conclusion:Exogenous H2S can uprise the ADAM10and ADAM17and significantly improve the expression of sAPPa and effectively reduce the production of Aβ40、Aβ42and sAPPβ in vitro culture PC12. The PI3-K or JNK signal pathway may be involved in this procedure. Donepezil has similar effects with H2S, its mechanism may be related to PI3-K, JNK or p38MAPK pathway. |
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