The Study On The Relationship Between The Intracelluar PH And β-secrete Enzymes

ObjectiveTo investigate the changes of β-secrete enzyme-1expression and activityunder the condition of intracellular acidification. Further explore the relationshipbetween the intracellular pH and β-secretases.MethodsHuman neuroblastoma (SH-SY5Y) cells were cultured. To build NHE-1gene knocking down cell model and negative control cell group, SH-SY5Y cellswere transfected successfully by recombinant adenovirus interfering plasmidpAd-miR-NHE1and negative control plasmid. SH-SY5Y cells were also culturedwith the serum-free culture medium for five days to build the serum-free cellmodel. There were4groups of cells including normal control group, NHE-1gene knocking down group, negative control group and serum-free group. Thechanges of intracellular pH were detected by fluorescence spectrophotometerrespectively. The expression of β-secrete enzyme-1was detected by Real-timePCR and Western Blotting, and the activity of β-secrete enzyme-1was detectedby ELISA. Single-factor analysis of variance and LSD-t testing were used toanalyzing whether there were statistical significants among the4groups.ResultsUnder the condition of serum-free, the cells were in the best state at thefouth day. The intracellular pH of NHE-1gene knocking down group was6.91±0.055.The intracellular pH of serum-free group was7.03±0.062. The intracellular pH of negative control group was7.22±0.026, and the intracellularpH of normal cell group was7.19±0.066. Compared with the normal cell and thenegative control group, the intracellular pH of NHE-1gene knocking down groupand serum-free group was lower than the normal cell and the negative controlgroup, the difference has significance (P<0.05). The diffrence of intracellular pHbetween the normal cell group and the negative control group has nosignificance(p>0.05). In the NHE-1gene knocking down group, the β-secreteenzyme-1mRNA and protein expression were5.65±0.33and0.37±0.030respectively. The β-secrete enzyme-1mRNA and protein expression of theserum-free group were2.40±0.55and0.32±0.031respectively. Compared withthe normal cell group (0.92±0.34;0.12±0.020) and the negative control group(1.00±0.00;0.14±0.021), the β-secrete enzyme-1mRNA expression, proteinlevel were significantly higher in NHE-1gene knock down group and serum-freegroup than that in normal control group and negative control group(p<0.05). Thedifference of β-secrete enzyme-1mRNA expression, protein level between thenormal cell group and the negative control group has no significance(p>0.05). Inthe NHE-1gene knocking down group, the β-secrete enzyme-1activity was449.45±65.95ng/L. The β-secrete enzyme-1activity of serum-free group was468.60±58.59ng/L. Compared with the normal cell group(290.65±82.33ng/L)andthe negative control group (327.11±82.32ng/L), the β-secrete enzyme-1activitywere significantly higher in NHE-1gene knock down group and serum-freegroup than that in normal control group(p<0.05). The difference of β-secreteenzyme-1activity between the normal cell group and the negative control grouphas no significance(p>0.05).ConclusionsThe NHE-1gene knocking down group and serum-free group weresuccessfully constructed to make the intracellular environment acidification.Demonstrated that intracellular pH decreased can cause β-secretases expression increasing and activity improving, which may contribute to theabnormal processing of APP and Aβ producing.