| Part1: The protein expression and activity of CBS enzyme and levelof endogenous hydrogen sulfide and in the cortex and hippocampus ofAPP/PS1transgenic miceObjective: To measure the protein expression and activity of CBSenzymes and the level of endogenous hydrogen sulfide in the cortex andhippocampus of3-,6-,9-,12-month-old APP/PS1transgenic mice.Methods: To measure the CBS enzyme expression byimmunohistochemistry analysis; To measure the activity of CBS enzymesand the level of endogenous hydrogen sulfide in the cortex andhippocampus of3-,6-,9-,12-month-old APP/PS1transgenic mice bysulf-electrode method.Results: The protein expression and activity of CBS enzyme were notreduced because of orderly neurons in the brain of6-month-old micecompared with3-month-old mice. There are no significant difference in thelevel of endogenous hydrogen sulfide in the cortex and hippocampusbetween3and6-month-old APP/PS1transgenic mice. The protein expression and activity of CBS enzyme were reduced caused by deceasedand unstuck CBS neurons, especially in the brain of12-month-old mice;The level of endogenous hydrogen sulfide in the cortex and hippocampusof9and12-month-old mice were decreased significantly.Conclusion: The expression and activity of CBS enzyme werereduced with unstuck and deceased CBS neurons; The level of endogenoushydrogen sulfide in the cortex and hippocampus of9and12-month-oldAPP/PS1mice were decreased, especially in12-month-old mice. PART2: Effects of exogenous hydrogen sulfide on the metabolism ofamyloid peptide precursor in APP/PS1transgenic miceObjective: To explore if supplementing exogenous sources of H2Swould improve AD-derived memory impairments and/or reduce theproduction of Aβ; we sought to determine if this same exogenousapplication of H2S could shift APP processing away from the pathogenicbeta-secretase pathway and toward the non-pathogenic alpha-secretasepathway. Then we will determine the optimizing dose of NaHS effect onthe metabolism of APP.Methods:10,20,50μmol/kg NaHS was then administeredintraperitoneally (i.p.) to4-and10-month old AD transgenic mice every other day for two months，the control group treated with equal dose ofphysiologic saline. When the mice were6months old and12months old.①The Morris and Water Maze were used to measure the improvedmemory impairment of mice.②The expression of capase3weremeasured by immunohistochemistry analysis to ensure the toxicity ofNaHS to mice.③Enzyme linked immunosorbent assay (ELISA) wereused to measure the level of Aβ1-40/Aβ1-42of NaHS treatment mice andcontrol group.④The protein expression of BACE-1, PS1, C99,ADAM10, ADAM17, C83were measured by Western blot.Results:①All NaHS treatment groups showed the same swimmingspeed in the Morris water maze when compared with age-matched controls.However, in the place navigation test (PNT),50μmol/kg NaHS treatmentsignificantly reduced both the escape latencies and swimming distance ofsix-month-old transgenic mice when compared to age-matched salinecontrols on the2ed,3rd,4thday in90s (p<0.05). There was no difference inescape latencies and swimming distance among the10and20μmol/kgNaHS treatment groups and saline controls (p>0.05). Compared toage-matched controls, twelve-month-old transgenic mice, that receivedtreatments of NaHS (50μmol/kg) had both decreased escape latencies andswimming distance on the2ed,3rd,4thday in90s. However,10and20μmol/kg NaHS treatment didn,t reduced the escape latencies of mice(p<0.05). On the fifth day (probe trial with no platform present), mice treated with50μmol/kg of NaHS showed a higher percentage of time spentin the target quadrant when compared to age-matched controls at both six-and twelve-months of mice. Moreover, mice treated with50μmol/kg ofNaHS had more crossings over the platform location when compared toage-matched controls at both six-and twelve-months of mice.②50μmol/kg NaHS treatment reduced the expression of capase-3inboth six-and twelve-month-old transgenic mice.③50μmol/kg of NaHS resulted in a decrease in Aβ-40and Aβ-42levels in both six-and twelve-month-old transgenic mice when comparedto age-matched controls (p<0.05). Similarly, treatment with50μmol/kg ofNaHS reduced both the number and size of Aβ-42in six-and twelve-month-old transgenic mice when compared to age-matched controls.Interestingly, both10and20μmol/kg of NaHS treatment did not changelevels of eitherAβ-40orAβ-42(p>0.05).④At six months of age, transgenic mice treated with50μmol/kg ofNaHS had a reduced expression of BACE1(p<0.05). Both20and50μmol/kg of NaHS treatment significantly decreased expression levels ofBACE1in twelve-month-old mice. In accordingly,20and50μmol/kg ofNaHS treatment mice also showed reduced levels of APP-CTFβ (C99) atsix and twelve months of age when compared to age-matched treatmentgroups.⑤Compared to age-matched treated mice, we observed a significant decrease in protein levels of PS1in the50μmol/kg NaHS treatment groupat both six-and twelve-months.20μmol/kg of NaHS treatment alsoreduced expression of PS1in six-and twelve-month-old mice, but10μmol/kg of NaHS had no effect on PS1protein levels at any time point.⑥NaHS treatment did not change the expression levels of ADAM17in six-month-old mice (p>0.05); however, there was a dose-dependenceincrease in expression levels of ADAM17in the cortex of twelve-month-old months NaHS treated mice when compared to age-matchedcontrols, especially in50umol/kg NaHS treatmed mice. NaHS treatmenthad no effect on the expression levels of ADAM10in both six-andtwelve-month-old transgenic mice (p>0.05). A parallel increase in levels ofits corresponding CTFα (C83) in50μmol/kg NaHS treated mice at twelvemonths of age, but we did not observe the a change in C83levels insix-month-old mice.Conclusion:50μmol/kg NaHS application were no toxical fortransgenic mice, resulted in a shift from the plaque-forming beta pathwayto the non-plaque forming alpha pathway of APP cleavage and reduced theproduction of Aβ with decrease in the expression of BACE1and PS1aswell as increase in the expression of ADAM17, improved the cognitionimpairment in six and twelve month APP/PS1mice. PART3: Effect of PI3K/AKT, MAPK/ERK path way on exogenoushydrogen sulfide affecting APP metabolismObjective: The aim of the present study is to investigate the effect ofPI3K/AKT, MAPK/ERK path way on exogenous hydrogen sulfide affectingAPP metabolism.Methods: Firstly, the expression of capase3were measured byimmunohistochemistry analysis to ensure the toxicity of NaHS to mice in12-month-old nomal mice, APP/PS1transgenic mice and50μmol/KgNaHS-treament transgenic mice; Then we examined the protein expressonof BACE1, PS1, ADAM17, pAKT, pP38MAPK, pERK, pJNK by Westonblot. At the same as application of exogenous H2S, the administration ofPI3K/AKT inhibitor5and10mg/kg LY294002by lateral cerebral ventricleor intraperitoneal injection MAPK/ERK inhibitor2.5and5mg/kgPD98059to11-month-old transgenic mice for4weeks. Memory functionwas measured through the Morris water maze in transgenic mice,50umol/kg NaHS treated transgenic mice, NaHS+5.0mg/kg and NaHS+10mg/kg LY294002, NaHS+2.5mg/kg and NaHS+5mg/kg PD98059transgenic mice. Aβ level was determined via ELISA andimmunohistochemical analyses. BACE1, PS1and ADAM17secretaseprotein expression was measured by Western blot analysis to ensure which pathway correlating with the change of secretase caused by NaHS.Results:①The normal twelve-month-old mice show us littleexpression of caspase-3, but there are large expression of caspase-3intwelve-month-old mice APP/PS1transgenic mice, NaHS treatment reducedthe expression of capase-3in transgenic mice.②In twelve-month-old AD transgenic mice, the expression ofBACE1, PS1evidently were increased, but the expression of ADAM17was reduced compared with normal mice (p<0.05). After APP/PS1transgenic mice were treated by50umol/kg NaHS for4weeks, theexpression of BACE1, PS1were decreased and the expression of ADAM17increased according compared with control mice (p<0.05).③pP38MAPKs was increased in twelve-month-old APP/PS1transgenic mice compared with normal mice (p<0.05), there were nodifference in the expression of pAkt, pERK, pJNK between AD transgenicmice and normal mice (p>0.05).50umol/kg NaHS for4weeks activatedpAkt, pERK, and attenuated the expression of pP38MAPK, but notchanged the expression of pJNK in twelve-month-old APP/PS1transgenicmice compared with normal mice.④All50μmol/kg NaHS treatment transgenic mice, NaHS+5.0mg/kg and NaHS+10.0mg/kg LY294002, NaHS+2.5mg/kg and5.0mg/kg PD98059treament transgenic mice showed the same swimmingspeed in the Morris water maze compared with12-month-old transgenic mice. In the place navigation test (PNT),50μmol/kg NaHS treatmentsignificantly reduced the escape latencies on3rd,4thin twelve-month-oldtransgenic mice when compared to age-matched transgenic mice in90s.However, there were no diference in the escape latencies between NaHS+5mg/kg and NaHS+10mg/kg LY294002transgenic mice and transgenicmice in90s (p>0.05).Compared with12-month-old transgenic mice, the escape latencies ofNaHS+2.5mg/kg PD98059and NaHS+5.0mg/kg PD98059transgenicmice were reduced in90s (p<0.05). There were no diference in the escapelatencies between NaHS+2.5mg/kg PD98059and NaHS+5.0mg/kgPD98059(p>0.05). On the fifth day (probe trial with no platform present),the percentage of time spent in the target quadrant and the numbers ofcrossing the platform location in NaHS treated transgenic mice were muchmore than of transgenic mice (p<0.05). There were no difference in thepercentage of time spent in the target quadrant and the numbers of crossingthe platform location between NaHS+5mg/kg and NaHS+10mg/kgY294002and transgenic mice (p>0.05). But the numbers of crossing theplatform location in NaHS+2.5mg/kg and NaHS+5.0mg/kg PD98059were more much than that of transgenic mice (p<0.05).⑤50μmol/kg of NaHS resulted in a decrease in Aβ-40and Aβ-42levels in twelve-month-old transgenic mice when compared to transgenicmice (p<0.01). There were no difference in the level of Aβ-40and Aβ-42in NaHS+5mg/kg and NaHS+10mg/kg LY294002transgenic micecompared with12-month-old transgenic mice (p>0.05). Interestingly,compared with12-month-old transgenic mice both NaHS+2.5mg/kg andNaHS+5mg/kg PD98059treatment reduced the levels of either Aβ-40orAβ-42. The number and size of Aβ-42were same as the levels of eitherAβ-40orAβ-42in evey group mice (p>0.05).⑥In NaHS+5mg/kg and NaHS+10mg/kg LY294002treated mice,the expression of BACE1and PS1were increased compared with NaHStreatment group. However, the expression of ADAM17was not changed inthe brains of mice. There were no difference in the expression of BACE1,PS1between NaHS+5mg/kg and NaHS+10mg/kg LY29004treamentmice (p>0.05).⑦There were no difference in the expression of BACE1, PS1andADAM17between NaHS+2.5mg/kg and NaHS+5mg/kg PD98059andNaHS treated mice (p>0.05).Conclusions:50μmol/kg NaHS reduced the expression of BACE1and PS1in transgenic mice and increased the expression of ADAM17.50μmol/kg NaHS reduced the expression of pP38MAPKs as well as increasedthe expression of pAKT, pMAPKs/ERK. The inhibitor LY294002was usedto inhabit the PI3K/Akt. The expression of BACE1and PS1were increased,but the expression of ADAM17was similar with the NaHS treated mice.The inhibitor PD98059was used to inhibit MAPKs/ERK. The expression of BACE1, PS1and ADAM17were similar with that of NaHS treamedmice. Exogenous H2S may exerts its reducing effects of BACE-1and PS1protein expression through PI3K/Akt but not MAPK/ERK signalingpathway. |
PDF Full Text Request