| Objective:1By stimulating C6cell with Aβ25-35of different concentrations, observe the viabilityand morphology of the cell and test the change of protein expression of thioredoxinbinding protein(TBP-2), thioredoxin (TRX), receptor for advanced glycation endproducts(RAGE)，low-density lipoprotein receptor-related protein(LRP1)，thereforeoffering evidence for the forming of AD cell model.2By stimulating C6cell with Liuweidihuang serum(LWDHS)of differentconcentrations, observe the viability and morphology of the cell and test the change ofprotein expression of TBP-2、 TRX、 RAGE、 LRP1， screen the appropriateconcentration of LWDHS, providing basis for the study of the effect of LWDHS onC6cell model.3By intervening AD cell model with the screened appropriate concentration ofLWDHS, observe the expression effect of LWDHS on AD cell model’s viability,morphology and TBP-2、TRX、RAGE、LRP1and discuss the molecular mechanismof its effect.Methods：1Prepare Aβ25-35protein solutions: make up1000μmol/L’s mother liquor from1mg’sAβ25-35with sterile super-pure water, place it in a37℃incubator and make it aged for5d, then separate them to-80℃environment and reserve. Dilute them to workingconcentration before using.2By dividing Aβ25-35into five concentration group as0.1μmol/L、1μmol/L、10μmol/L、20μmol/L、40μmol/L and intervene C6cell for24h, test the effect ofAβ25-35on C6cell viability and observe the cell morphological changes by MTTmethod. To test the protein expression changes of TBP-2、TRX、RAGE、LRP1in C6 cells caused by Aβ25-35using Western Blot method.3Make drug serum: SD male rat (250±20g）, Liuweidihuangwan concentration pill(Wanxi Pharmaceutical),1.08g/kg orally,2/day, make LWDHS from abdominal aorticblood2hours after gavage on the fourth day’s morning.4Divide LWDHS into five concentration level as2.5%,5%,10%,20%,30%and effectC6cell for24h, screen the best concentration for increasing PC6cells’ vitality, andobserve the changes in cell morphology and apply the Western Blot method to detectthe protein expression changes of related proteins TBP-2、TRX、RAGE、LRP1.5Intervene Aβ25-35effected AD cell model with the best LWDHS concentration,observe cell morphology, vitality in microscope, and apply the Western Blot methodto detect the protein expression changes of proteins TBP-2、TRX、RAGE、LRP.Results:1After stimulation of C6cell by different concentration level of Aβ25-35, cell activityand vitality rate are remarkably decreased. Cell morphology was observed under anmicroscope: compared with normal C6cells, as the dose of the Aβ25-35increased,thenumber of the C6cells stimulated by Aβ25-35reduced, cell size became smaller andcell protuberance became shorter, and cell debris and apoptotic/dead cells is graduallyincreased, showing changed dependency on dose. It showed an increased proteinexpression of TBP-2&RAGE and an reduced expression of TRX and LRP1.2After stimulating C6cell by different LWDHS, cell activity and vitality rate areremarkably increased. Cell morphology was observed under an inverted microscope,as the concentration of LWDHS increased,the number of the C6cells increased, cellaggregation fused to single layer, cytoplasmic process became larger and number ofcell protuberance became larger, cell protuberance became thicker and longer. Atthe same time, by detecting the20%,30%concentration level of LWDHS, cell bodyincreased, cell number stoped rising, showing a differentiation tendency. Western Blottesting results showed that as concentration increased, protein expression of TBP-2'RAGE reduced a lot, while protein expression of TRX and LRP1increased much andboth depend on dose.3Stimulation of LWDHS inhibit the reduced cell viability caused by Aβ25-35.Cellmorphology was observed under an microscope, compared to normal control group,the number of the C6cells reduced, cell protuberance became shorter, and apoptoticcells showed in Aβ25-35group; there was an reduced apoptotic cells in LWDHSintervention group and cell size became larger, cell protuberance became longer. LWDHS inhibit the increase of protein expression of TBP-2and RAGE induced byAβ25-35, restored the decreased TRX and LRP1expression.Conclusion:1The toxic damage of Aβ25-35on C6cell was positively correlated with its dose, andAβ25-35inhibit cell proliferation activity significantly; Aβ25-35participate in ADforming by adjusting its expression on TBP-2、TRX、RAGE、LRP1. Insulin signalingand PI3K/AKT may participate in the pathogenesis of AD during this process, whichindicated that oxidative stress and inflammatory reaction plays an important role inleading to Alzheimer’s disease.2LWDHS played a protection role for cell apoptosis caused by Aβ25-35by adjustingthe expression of TBP-2、TRX、RAGE、LRP; TBP-2/RAGE is probably an importantconnector in AD treatment.Methods:... |
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