Amiloride Is Protective In PC12 Cells Via Autophagy Pathway

Objective1 To observed the influence of acid-(pH6.0) on PC12 cells survival acidosis,lactate dehydrogenase (LDH) leakage rate,apoptosis rate and intracellular free calcium concentration, and view the phenomenon of Ami protected PC12 cells against acid-induced injury;2 To observed the influence of MPP~+ on PC12 cells survival acidosis,LDH leakage rate,apoptosis rate and intracellular free calcium concentration, and view the phenomenon of Ami protected PC12 cells against MPP~+-induced injury;3 To analysis the influence of acid-/MPP~+ on autophagy-lysosome pathway (ALP) of PC12 cells, and view the antagonistic action of Ami.MethodsPC12 cells were treated with either MPP~+ or acidic (pH6.0) medium for 24 h, while giving Ami as intervention: The cells viability was analyzed by MTT assay; The cells injury was assessed by LDH assay; Flow cytometry was used to study the apoptotic and calcium influx; Western blot analysis were used to study the autophagic mechanisms.Results1 After 24 h, acidic medium (pH6.0) treatment resulted in significantly lower survival rate (P<0.001), the supematant of LDH leakage was significantly higher (P<0.001), the apoptosis rate increased (P<0.001), cells intracellular calcium concentration was significantly higher (P<0.05); 100μM of Ami effectively improve the cell survival rate (P<0.001), reduced LDH leakage rate (P<0.001), and inhibited apoptosis (P<0.001) and calcium influx (P<0.01);2 After 24 h, MPP~+ medium treatment resulted in significantly lower survival rate (P<0.001), the supematant of LDH leakage was significantly higher (P<0.001), the apoptosis rate increased (P<0.05), cells intracellular calcium concentration was significantly higher (P<0.01); 100μM of Ami effectively improve the cell survival rate (P<0.001), reduced LDH leakage rate (P<0.001), and inhibited apoptosis (P<0.01) and calcium influx (P<0.05);3 After 24 h, acid-/MPP~+ inhibited the expression of LC3-II (P<0.001), while induce more expression of LAMP2a (P<0.05); but Ami was upregulation of LC3-II protein (P<0.01), which is associated decrease expression of Lamp2a (P<0.05).Conclusions1 Ami can protected PC12 cells against acid-induced injury.2 Ami can protected PC12 cells against MPP~+-induced injury.3 Acid-/MPP~+ inhibited the activity of Macroautophagy, while increased the activity of chaperone-mediated autophagy (CMA); but Ami can up-regulation the activity of Macroautophagy and decrease the activity of CMA.