NaHS对ATP诱导大鼠小胶质细胞P2X受体表达的影响

目的：观察硫化氢（H₂S）供体硫氢化钠（NaHS）对ATP致伤的大鼠小胶质细胞细胞活力、细胞膜通透性及P2X7受体表达的影响。方法：实验取对数期形态结构及生长分化良好的大鼠小胶质细胞，随机分4组，每组设3个复孔。①正常对照组：常规培养，不进行ATP处理。②ATP组：接种细胞24 h后ATP处理。③NaHS+ATP组：NaHS预先孵育30 min后再用ATP处理，并且NaHS始终存在于反应体系中。④KN-62（P2X₇受体阻断剂）+ATP组：KN-62预先孵育30 min，其余同NaHS+ATP组。MTT检测各组细胞活力，荧光染料YO-PRO-1检测各组相对荧光单位（RFU）反映膜的通透性，Western blot检测各组P2X₇受体表达水平。结果：①与对照组相比，不同浓度的ATP （1、3、5、10 mmol/L）作用3 h均可明显降低大鼠小胶质细胞活力，NaHS （200 μmol/L）干预后大鼠小胶质细胞活力较ATP组明显增加（P<0.01），但NaHS达400 μmol/L浓度时，其保护作用未进一步增加。②随着ATP浓度的增加，大鼠小胶质细胞内YO-PRO-1的荧光强度显著增加，NaHS预处理可明显减少细胞对YO-PRO-1的摄取（P<0.01）。③ATP （3 mmol/L）能上调P2X₇受体蛋白表达水平，而NaHS （200 μmol/L）预孵育则可明显抑制ATP引起的P2X₇受体蛋白表达的上调（P<0.01）。结论：NaHS可减少ATP致伤的大鼠小胶质细胞的P2X₇受体表达、降低通透性、增加细胞活力，提示调控P2X₇受体的表达和功能可能是H₂S神经保护作用的重要环节。

Effect of NaHS on ATP-induced P2X receptor expression in rat microglia

Objective: To observed the effect of sodium hydrosulphide (NaHS), a donor of H₂S on the cell viability,the membrane permeability and the expression of P2X₇ receptor induced by adenosine triphosphate(ATP) in rat microglia.Methods: Rat microglia in logarithmic growth phase was randomly divided into 4 groups. In control group, the cells were cultured without ATP treatment. In ATP group, the cells were treatment with ATP after cultured for 24 hours. In NaHS+ATP group, the cells were incubated with NaHS for 30 min before ATP, and NaHS always existed in the reaction system. In KN-62+ATP group, the cells were pretreated with KN-62 for 30 min, the others were as the same as NaHS+ATP group. The cell viability was detected by MTT. Fluorescent dyes YO-PRO-1 was used to observe the membrane permeability. The expression of P2X7 receptor was examined by immunofluorescence staining.Results: ① Compared with control group, the cell viability dropped after treatment with ATP (1、3、5、10 mmol/L) for 3 hours. When pre-incubation with NaHS(200 μmol/L), the cell viability was apparently higher than that of ATP alone group(P<0.01), while 400 μmol/L had no further beneficial.②The YO-PRO-1 fluorescence intensity was obviously elevated by ATP in rat microglia, but this effect was counteracted by NaHS pretreatment (P<0.01). ③ The expression of P2X7 receptor protein was significantly increased after ATP(3 mmol/L) for 3 h. While the expression upregulation of P2X7 receptor protein induced by ATP was significantly counteracted by pretreating with NaHS(200 μmol/L) (P<0.01).Conclusion: NaHS could reduce the expression of P2X7 receptor, decrease membrane permeability, and increase the cell viability in rat microglia injured by ATP. So the cytoprotection of hydrogen sulfide may be related to the expression and function of P2X7 receptor.