| BackgroundATP (Adenosine triphosphate, ATP) is an important energy molecule and purinergic neurotransmitter in the body. It can activate the P2 (especially P2X7) receptor, modulating the brain function and may cause rapid depolarization of the cells, which resulting in an influx of extracellular calcium and affecting the intracellular Ca2+ concentration ([Ca2+]1). While with repeated or prolonged activation by ATP at high concentration, the receptor formed "large pore", allowing the permeation of large molecular weight organic cation up to 900Da and ATP itself. ATP can stimulate the maturation and secretion of interleukin-ip (IL-1β) in microglia after the accumulation of pro-IL-1β caused by bacterial lipopolysaccharide (LPS), and then the occurrence and development of the inflammation could be promoted.Hydrogen sulfide (H2S) has been postulated to be the third gaseous signaling molecule after nitric oxide (NO) and carbon monoxide (CO). The protection of H2S on the injured cells have been confirmed by many researches in the latest years. However the relationship between H2S and the ATP-P2X purinergic signaling mechanisms is still rarely reported. Our experiment is based on the study of ATP-P2X purinergic signaling pathway to explore the effect and role of H2S and to uncover the molecular mechanism and important target.ObjectivesThe rat microglia injury model were established by treating with ATP and the effects and mechanisms of H2S protecting rat microglia against injured were explored.Methods1 The effect of NaHS on the rat microglia vitality induced by ATP1.1 Rat microglia were cultured in high glucose DMEM containing 10% fetal bovine serum. Rat microglia with a density of 1x105cells/L in the logarithmic phase were inoculated in 96-well plates and were cultured in 37 ℃,5% CO2 culture box for 24 hour. Then were randomly divided into four 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 0.3 mmol/L,1 mmol/L,3mmol/L,5mmol/L, 1 Ommol/L ATP respectively. Three hours later, the cell vitality was assayed by MTT assay. 1.2 Cells processing method was as same as 1.1, then five groups were randomly divided into control group, ATP group, NaHS (100、200、400μmol/L)+ATP group. Cells were pretreated with different concentration of NaHS for 30 min, followed by treatment with 3mmol/L ATP for 3h, and NaHS was always in the reaction system. The cell vitality were assayed by MTT assay, the cell vitality of Ommol/L ATP group was considered 100% as control group.2 The effect of NaHS on membrane pore formation induced by ATP in rat microglia2.1 Rat microglia in the logarithmic phase inoculating in 96-well plates with a density of 1x105cells/L were randomly divided into following groups:control group, ATP group (0.3 mmol/L,1 mmol/L,3mmol/L,5mmol/L,10mmol/L ATP). Fluorescence intensity (485 nm excitation,516 nm emission) of YO-PRO-1 was determined by Reader after exposed to different concentration of ATP and 2μmol/L YO-PRO-1 for 1h.2.2 Rat microglia in the logarithmic phase were inoculating in 96-well plates with a density of 1x105cells/L were randomly divided into four groups:control group, ATP group, NaHS+ATP group, KN-62+ATP group. ATP was 3mmol/L, NaHS was 200μmol/L, KN-62 was 500nmol/L. The fluorescence intensity was determined by the Reader when the cells were treated by YO-PRO-1 for 1 h following 30 min by NaHS or KN-62.3 The effect of NaHS on P2X7 receptor expression in ATP-induced Rat microgliaRat microglia in the logarithmic phase inoculating in 60mm petri dishes were randomly divided into three groups:control group, ATP (3mmol/L) group, NaHS (200μmol/L)+ATP group. The expression of P2X7 receptor protein were analyzed by Western blotting.4 The effect of NaHS on IL-1β release from activated rat microglia-induced by ATP-LPS.Rat microglia in the logarithmic phase inoculating in 60mm petri dishes were randomly divided into three groups:control group, ATP alone group, LPS group, ATP+LPS group, NaHS+ATP+LPS group. ATP was 3mmol/L, LPS was 1μg/mL, NaHS was 200μmol/L.Cell culture supernatants were collected and centrifuged when rat microglial cells were stimulated with LPS for 12 h and ATP for 3h after pretreated with NaHS for 30 min. IL-1β in cell culture supernatants were measured using a commercially available ELISA kits according to the manufacturer’s instruction.Results1 The effect of NaHS on the rat microglia vitality induced by ATP1.1 The effect of ATP with different concentration on the vitality of rat microglia The vitality of 0.3mmol/L ATP group was 95.17% considering that of control group as 100%. There was no significant difference in vitality between control group and 0.3mmol/L ATP group (P>0.05). The vitality of lmmol/L,3mmol/L,5mmol/L, 10mmol/L ATP group were respectively 83.03%,72.13%,68.40%,52.50%. All had significant difference (P<0.01) compared with the control group.1.2 NaHS influence to the vitality of rat microglia induced by ATP Cells were treated with ATP (3mmol/L) for 3h after pretreated with 100μmol/L,200pmol/L,400umol/L NaHS. The vitality of 100μmol/L,200μmol/L,400μmol/L NaHS were respectively 78.41%, 91.56%,82.97% considering the control group vitality as 100%. While the vitality of 200μmol/LNaHS+ATP group was apparently higher than that of ATP alone group (P< 0.01). It indicated that the protection of NaHS on the rat microglia injured by ATP was in a dose-dependant manner.200μmol/L of NaHS had reached the bestand 400μmol/L had no futherbeneficial (P>0.05).2 The effect of NaHS on membrane pore formation induced by ATP in rat microglia2.1 The membrane pore formation of rat microglia induced by ATP The YO-PRO-1 (molecular weight 629D) dyes was used to represent the membrane formation. The rat microglia were separately treated with different concentration of ATP for 1 h. Considering the intracellular fluorescence intensity of control group (Ommol/L ATP group) as 100%, those of 0.3mmol/L, lmmol/L,3mmol/L,5mmol/L,10mmol/L ATP were respectively 100.31%,107.86%,112.96%,143.17%,151.82%. The fluorescence intensity of YO-PRO-1 in 3mmol/L,5mmol/L, and 10mmol/L ATP group were significant increased compared with that of control group (P<0.01). The results demonstrated that ATP could accelerate the membrane pore formation and YO-PRO-1 uptake in a dose-dependent manners.2.2 The effect of NaHS or KN-62 on the membrane pore formation induced by ATP in rat microglia. The intracellular fluorescence intensity of the two groups were respectively 101.17%,102.23% after the cells pretreated by NaHS or KN-62 for 30 min followed by ATP and YO-PRO-1. The intracellular fluorescence intensity of both two groups were obviously weaker than that of 3mmol/L ATP group (P< 0.01). It showed that both NaHS and KN-62 could antagonize the membrane pore formation induced by ATP in rat microglia.3 The effect of NaHS on P2X7 receptor expression in ATP-induced Rat microglia The result of Western blotting showed that the expression of P2X7 receptor protein was significantly increased after 3mmol/L ATP for 3h. While the expression upregulation of P2X7 receptor protein mediated by ATP was significantly reduced by pretreating with 200μmol/L NaHS (P<0.05). It showed that NaHS could block the effect of ATP on P2X7 receptor expression in Rat microglia.4 The effect of NaHS on IL-1β release from activated rat microglia-induced by ATP-LPS.Extracellular IL-1β was detected to reflect the activation of rat microglia. The results showed that extracellular IL-1β of ATP alone group or LPS group had no significant difference compared with that of control group (P>0.05). While the extracellular IL-1β of ATP+LPS group was obviously increased compared to ATP alone group and LPS group (P <0.05). However applicating with NaHS 30min before ATP+LPS, the extracellular IL-1β was obviously decreased than that of ATP+LPS group(P<0.05). Also It demonstrated that both ATP and LPS could not evoke the release of IL-1β, but the combined effect of ATP and LPS could activated the rat microglia and facilitate the release of IL-1β.ConclusionsThe high concentration of ATP can decrease cell vitality, upregulate the expression of P2X7 receptor protein, promote membrane pore formationin rat microglia. The combining effect of ATP and LPS can activate the rat microglia and facilitate the IL-1β release more. But NaHS could against the above effects of ATP, so we suppose that there is an important role of purinergic signal pathway in neuroprotection of hydrogen sulfide. |
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