The Membrane Pore Mechanism Of Progesterone Intervening On SH-SY5Y Cell Injury Induced By ATP

Background At present,the ATP?Adenosine-5'-triphosphate,ATP?is one of the important signaling molecule in the nervous system,the research showed that the P2X7 receptor?P2X7R?is a major receptor mediated ATP toxicity.ATP at physiological concentration?micromolar?,may activate P2X7 R and form ion channel which can selectively mediate Ca²⁺ and Na+ cations flow.then participate in a series of physiological effects,such as the regulation of transmitter release,synaptic modification and neurotrophic effect and so on.However,extracellular high concentration of ATP can produce cytotoxicity which can induce membrane pores information and allow various cations and organic substances less than 900 D such as NMDG+ and YO-PRO-1 throwing into cells.Then the apoptosis and necrosis may take place.Moreover excessive activation of P2X7 R may aggravate the damage of brain tissue because of Ca²⁺ influx increasing and overloading of Ca²⁺ forming which is related to apoptosis or necrosis of neurons.At present progesterone has been used in many animal experiments such as acute toxic injury,traumatic injury in the central nervous system,spinal cord injury,stroke and others.It can delay cell necrosis and apoptosis,reduce the inflammatory response and ischemic cerebral edema,promote the myelin synthesis and blood-brain barrier reconstruction,and can improve the age-related dementia?Alzheimer's disease?,decrease epilepsy and other neurodegenerative lesions occurrence and development.But So far the brain injury treatment is still a great problem for man,clinical intervention methodsis a little.progesterone may be able to bring a glimmer of hope for patients.Since the increase in ATP concentration is a common phenomenon in multiple central or peripheral neuronal secondary injuries,the protective effect of progesterone on neuronal secondary injury has also been demonstrated.In this study,we tried to observe the effect of progesterone on ATP-induced SH-SY5 Y cell injury and explore the mechanism of progesterone neuron protection,and provide experimental basis for its clinical application.Objectives This study attempts to clarify the effect of progesterone on ATP-induced SH-SY5 Y cell injury and its mechanism.Methods1 The determination of cell viability1.1 SH-SY5 Y cell were cultured in high glucose DMEM containing 1 0%fetal bovine serum.SH-SY5 Y cell in the logarithmic phase with a density of 5 × 104 cells / L were inoculated in 96-well plates and were cultured in 37 ?,5 % CO2 thermostat box for 24 hour.And then were divided into to 4 groups:?1?blank group:There was only DMEM culture medium but no cells in the plate.?2?control group:the cells were treated without ATP?3?ATP group:the cells treated with 1,3,5,7 m M ATP respectively.Two hours later,the cell vitality was detected by Cell Counting Kit-8?CCK-8?.1.2 Cells detection method was the same as 1.1,then six groups were randomly divided into control group,ATP group,Progesterone?10 ? 30 ? 100 ? 300 n M?+ATP group.Cells were incubaed with different density of Progesterone for 30 min.The next step was to treated with 5 m M ATP for 2 h.And Progesterone was always remained in the sphere of reaction.The cell vitality were detected by Cell Counting Kit-8.The cell vitality of 0 m M ATP group was considered 100% as control group.2 The detection of SH-SY5 Y cell membrane formation2.1 SH-SY5 Y cell in the logarithmic phase with a density of 5 × 104 / L were inoculated in 96-well plates,which were randomly assigned to the following groups :control group,ATP group?1,3,5,7 m M ATP?.ATP action time of each group was 2h,then YO-PRO-1?2?M?were added to the system for 1 h,and the fluorescence intensity of each group was detected by microplate reader. 2.2 SH-SY5 Y cells with a denfity of 5 × l04 cells / L in the logarithmic phase were cultured in 96-well plates,which were randomly assigned to 4 groups:control group,ATP group,Progesterone + ATP group,KN-6²⁺ATP group.ATP was 5 m M,Progesterone was30 n M and KN-62 was 500 n M.Progesterone or KN-62 were incubated for 30 min,and then incubated with ATP for 2 hour.Finally,YO-PRO-1?2?M?were added to the system for 1 h.The fluorescence intensity of each group was detected by microplate reader.3 The effect of Progesterone or KN-62 intracellular free calcium concentration by furo-3/AM3.1 SH-SY5 Y cell in the logarithmic phase with a density of 5 × 104 / L were inoculated in 96-well plates.They were randomly assigned to the following groups:control group,ATP group?1,3,5 m M ATP?.Cells of each group were treated by ATP for15 min,30 min,60 min respectively.Followed by washing 3 times with Hank'S solution.Then incubated in 5?M Fluo-3/AM and 2?M Hoechst 33342 for 30 minutes at 37 ?.Fluorescence intensity of intracellular fluorescence was observed under fluorescence microscope.3.2 SH-SY5 Y cell in the logarithmic phase with a density of 5 × 104 / L were inoculated in 96-well plates.They were randomly assigned to the following groups:control group,ATP group,Progesterone + ATP group,KN-6²⁺ATP group.ATP was 5 m M,Progesterone was 30 n M and KN-62 was 500 n M.First,Cells were incubated in Progesterone or KN-62 for 30 min,and then incubated with ATP for another 1 hour,followed by washing 3 times with Hank'S solution.Finally,incubated in 5?M Fluo-3/AM and 2?M Hoechst 33342 for 30 minutes at 37 ?.Fluorescence intensity of intracellular fluorescence was observed under fluorescence microscope.4 The effect of Progesterone on P2X7 receptor expression in ATP-induced SH-SY5 Y cells The SH-SY5 Y cells in the logarithmic growth phase were randomly divided into 3groups: control group,ATP?5 m M?group,Progesterone?30 n M?+ATP group.The cell membrane proteins were extracted measured by BCA.The expression levels of P2X7 receptor in SH-SY5 Y cells were detected by Western blottingResults1 The determination of cell viability1.1 The effect of ATP on the vitality of SH-SY5 Y cells.If the vitality of control group considered as 100%,that of?l,3,5,7 m M?ATP group were respectively 80.4%,71.7%,56.1%,36.2%.All had significant difference?P < 0.01?compared with the control group.1.2 Effect of Progesterone on the vitality of SH-SY5 Y cells induced by ATP.Cells were treated with ATP?5 m M?for 2 h after pretreated with?10,30,100,300 n M?Progesterone.The vitality of?3,10,30,100,300 n M?Progesterone were respectively were 62.8%,64.6%,74.4%,59.7% which obviously decreased that of the control group?100%?.While the vitality of?30 n M?Progesterone +ATP group was obviously higher than that of ATP group?P < 0.01?.It made clear that the protection of Progesterone on the SH-SY5 Y cells injured by ATP was in a dose-dependant manner.But 100 n M and 300 n M of Progesterone had no futher beneficial?P >0.05?.2 The detection of SH-SY5 Y cell membrane formation2.1 The membrane pore formation of SH-SY5 Y cells induced by ATP.The YO-PRO-1?molecular weight 629D?dyes was made use to representative of the membrane formation.The SH-SY5 Y cells were separately treated with different concentration of ATP for 2 h.,The intracellular fluorescence intensity of the of 1,3,5,7 m M ATP groups were respectively 103.5%,108.6%,120.5%,140.6%Considering that of the control group?0 m M ATP group?as 100 %.The fluorescence intensity of YO-PRO-1 in 5 m M,7m M ATP group were obviously increased in comparison with that of control group?P <0.01?.The research find that ATP could promote the membrane pore formation.2.2 The effect of Progesterone or KN-62 on the membrane pore formation induced by ATP in SH-SY5 Y cells.The intracellular fluorescence intensity of the 2 groups respectively were 103.9%?98.23% after the cells pretreated by Progesterone or KN-62 for 30 min followed by ATP and YO-PRO-1 for 2 h.The intracellular fluorescence intensity of both 2 groups were increased lower than that of 5 m M ATP group?P <0.01?.It showed that both Progesterone or KN-62 could inhibit the membrane pore formation induced by ATP in SH-SY5 Y cells.3 The changes of Ca²⁺concentration induced by ATP in SH-SY5 Y cells The result of Fluo-3 showed that Progesterone or KN-62 pretreatment significantly decreased the fluorescence intensity in the cells induced by 5 m M ATP?P <0.05?.Compared with the control group,the expression of P2X7 receptor in ATP group was significantly higher than that of contal group?P <0.05?,and the expression in Progesterone group was lower than that of in ATP group?P <0.05?.4 The effect of Progesterone on P2X7 receptor expression in ATP-induced SH-SY5 Y cells The result of Western blotting showed that the expression of P2X7 receptor protein was significantly increased after treating with 5 m M ATP for 2 h.While the expression up-regulation of P2X7 R protein induced by ATP was obviously decreased by pretreating with 30 n M Progesterone.It showed that Progesterone could inhibit the effect of ATP on P2X7 receptor expression in SH-SY5 Y cellsConclusion KN-62 can decrease the intracellular free calcium concentration and the fluorescence intensity of YO-PRO-1 in SH-SY5 Y cells induced by ATP and improve the survival rate of cells.those indicate that the P2X7 receptor plays a key role in the cells injury induced by ATP.Progesterone is similar to the role of P2X7 receptor antagonist KN-62,can significantly inhibit the membrane pore formation and reduce the intracellular Ca²⁺concentration induced by ATP,and improve the survival rate of cells.