ABSTRACT
AIM:To investigate the neuroprotective effect of progesterone against adenosine triphosphate (ATP)-injured human neuroblastoma SH-SY5Y cells.METHODS:The SH-SY5Y cells in the logarithmic phase were divided into different groups according to the progesterone and ATP concentrations.The cell viability was measured by CCK-8 assay.The membrane permeability was detected using fluorescent dye YO-PRO-1.Cytosolic Ca2+ concentration was measured with fluorescent dye Fluo-3/AM.The expression of purinergic P2X7 receptor was assessed by Western blot.RESULTS:The viability of the SH-SY5Y cells was significantly decreased (P ＜ 0.05) and YO-PRO-1 uptake was obviously increased (P ＜ 0.05) in a concentration-dependent manner compared with control group when SH-SY5Y cells were treated with ATP at 1,3,5 and 7 mmol/L for 2 h.The viability reduction of the SH-SY5Y cells induced by ATP was obviously counteracted by treatment with progesterone at 3,10 and 30 nmol/L for 30 min (P ＜ 0.05) as compared with ATP group.YO-PRO-1 fluorescence enhancement induced by ATP in SH-SY5Y cells was significantly reduced (P ＜ 0.05) by progesterone (30 nmol/L) or P2X7 receptor antagonist KN-62 (500 nmol/L) pretreatment for 30 min,and no obvious difference between treatments with progesterone and KN-62 was observed.Cytosolic Ca2+ fluorescence intensity in normal group was a little,but that in ATP group was increased (P ＜ 0.05).Progesterone or KN-62 pretreatment significantly decreased the cytosolic fluorescence intensity of Ca2+ induced by ATP (P ＜ 0.05).However,no obvious difference between treatments with progesterone and KN-62 was found.The expression of P2X7 receptor in ATP group was significantly higher than that in control group (P ＜ 0.05),and progesterone inhibited ATP-induced P2X7 receptor expression (P ＜ 0.05).CONCLUSION:Progesterone inhibits P2X7 receptor expression,membrane pore formation,intracellular Ca2+ increase and cell death induced by ATP,so progesterone may protect SH-SY5Y cells against ATP-induced injuries.
ABSTRACT
AIM:To investigate the neuroprotective effect of progesterone against adenosine triphosphate (ATP)-injured human neuroblastoma SH-SY5Y cells.METHODS:The SH-SY5Y cells in the logarithmic phase were divided into different groups according to the progesterone and ATP concentrations.The cell viability was measured by CCK-8 assay.The membrane permeability was detected using fluorescent dye YO-PRO-1.Cytosolic Ca2+ concentration was measured with fluorescent dye Fluo-3/AM.The expression of purinergic P2X7 receptor was assessed by Western blot.RESULTS:The viability of the SH-SY5Y cells was significantly decreased (P ＜ 0.05) and YO-PRO-1 uptake was obviously increased (P ＜ 0.05) in a concentration-dependent manner compared with control group when SH-SY5Y cells were treated with ATP at 1,3,5 and 7 mmol/L for 2 h.The viability reduction of the SH-SY5Y cells induced by ATP was obviously counteracted by treatment with progesterone at 3,10 and 30 nmol/L for 30 min (P ＜ 0.05) as compared with ATP group.YO-PRO-1 fluorescence enhancement induced by ATP in SH-SY5Y cells was significantly reduced (P ＜ 0.05) by progesterone (30 nmol/L) or P2X7 receptor antagonist KN-62 (500 nmol/L) pretreatment for 30 min,and no obvious difference between treatments with progesterone and KN-62 was observed.Cytosolic Ca2+ fluorescence intensity in normal group was a little,but that in ATP group was increased (P ＜ 0.05).Progesterone or KN-62 pretreatment significantly decreased the cytosolic fluorescence intensity of Ca2+ induced by ATP (P ＜ 0.05).However,no obvious difference between treatments with progesterone and KN-62 was found.The expression of P2X7 receptor in ATP group was significantly higher than that in control group (P ＜ 0.05),and progesterone inhibited ATP-induced P2X7 receptor expression (P ＜ 0.05).CONCLUSION:Progesterone inhibits P2X7 receptor expression,membrane pore formation,intracellular Ca2+ increase and cell death induced by ATP,so progesterone may protect SH-SY5Y cells against ATP-induced injuries.