The Influence Of Decreased Intracellular PH Of SH-SY5Y Cells On Presenilin-1Expression

ObjectiveTo study the expression and activity changes of presenilin-1whenintracellular pH decreased. Further explore the relationship betweenintracellular pH(pHi) and activity of γ-secretase.MethodsSH-SY5Y cells were cultured routinely. One part of cells were transfectedby adenovirus interference plasmids pAd-miR-NHE1and adenovirus vectorpAd-miR-NC. According to the optimum MOI to inhibit the expression of NHE1,so as to decrease the intracellular pH. The other parts of cells were cultured inacidic mediums. Based on the pH of6.8,6kinds of mediums of which pHdecreased0.5in turns were prepared. The optimum acidic medium weredetermined through observing the cells growth curves. Acidulated mediumgroup was established through activating the acid sensing ion channel topromoting the ion exchange. There were four groups of cells including normalcells group, negative control group (transfected by empty vector), NHE1inhibited group (transfected by SiRNA) and acidulated medium group. Theintracellular pH of different groups were evaluated through the pHi standardcurve detected by the fluorescence indicator BCECF-AM influorospectrophotometer. The presenilin1activity of different groups weremeasured by ELISA and the mRNA expressions of presenilin1were detected byReal-Time PCR. Western blotting was used to analyze the different presenilin1 expression levels among different groups.ResultsSH-SY5Y cells were transfected by adenovirus interference plasmidstargeting human NHE-1mRNA to inhibit NHE-1expression. After culturing thenormal cells in acidic medium with different pH for72hours, the optimum pH forcells growing was determined to be6.3according to the growth status andgrowth curve of cells. The pHi of NHE1inhibited group and acidulated mediumgroup were6.62±0.02and6.99±0.02respectively, which were significantlylower than the normal cells group（pHi=7.27±0.02） and negative control group（pHi=7.24±0.03）(P<0.05). There were no obvious differences of pHi betweenthe normal cells group and negative control group (P>0.05). That suggests theacidic cell model have been constructed. ELISA results showed that theactivities of PS1in NHE1inhibited group and acidulated medium group were26.3±4.2ng/L and23.5±4.0ng/L respectively, more than that in normal cellsgroup（9.1±0.6ng/L） and negative control group（7.8±1.1ng/L）(P<0.05). PCRresults showed that the mRNA expression of PS1in NHE1inhibited group andacidulated medium group were1.75±0.04and1.39±0.03respectively, morethan that in normal cells group （1.23±0.01）and negative control group（1.00）(P<0.05). Western Blotting results showed that the expression of PS1protein inNHE1inhibited group and acidulated medium group had an obvious increasingcompared with the normal cells group and negative control group(0.70±0.01and0.68±0.08vs0.54±0.01and0.55±0.05)(P<0.05), while there was no obviousdifference of the protein expression between NHE1inhibited group andacidulated medium group and no obvious difference between the normal cellsgroup and negative control group(P>0.05).ConclusionDecreased pHi could cause presenilin1expression and activity increased inSH-SY5Y cells. Presenilin1was considered to be the major component and activity center of γ-secretase at present, which suggests that decreased pHicould stimulate the activity of γ-secretase, and producing more Aβ as a result.