摘要
目的:观察硫化氢(H_2S)供体硫氢化钠(Na HS)对ATP致伤的大鼠小胶质细胞细胞活力、细胞膜通透性及P2X_7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。(2)ATP组:接种细胞24 h后ATP处理。(3)NaHS+ATP组:Na HS预先孵育30 min后再用ATP处理,并且Na HS始终存在于反应体系中。(4)KN-62(P2X_7受体阻断剂)+ATP组:KN-62预先孵育30 min,其余同Na HS+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P2X_7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,Na HS(200μmol/L)干预后大鼠小胶质细胞活力较ATP组明显增加(P<0.01),但Na HS达400μmol/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,Na HS预处理可明显减少细胞对YO-PRO-1的摄取(P<0.01)。(3)ATP(3 mmol/L)能上调P2X_7受体蛋白表达水平,而Na HS(200μmol/L)预孵育则可明显抑制ATP引起的P2X_7受体蛋白表达的上调(P<0.01)。结论:Na HS可减少ATP致伤的大鼠小胶质细胞的P2X_7受体表达、降低通透性、增加细胞活力,提示调控P2X_7受体的表达和功能可能是H_2S神经保护作用的重要环节。
Objective： To observed the effect of sodium hydrosulphide （NariS）, a donor of It2 S 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 ceils were cultured without ATP treatment. In ATP group, the cells were treatment with ATP after cultured for 24 hours. In NariS ＋ ATP group, the cells were incubated with NariS for 30 min before ATP, and NariS always existed in the reaction system. In KN-62 ＋ ATP group, the cells were pretreated with KN-62 for 30 rain, the others were as the same as NariS ＋ ATP group. The cell viability was detected by MTF. Fluorescent dyes YO-PRO-I 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 NariS（200 μmol/L）, the cell viability was apparently higher than that of ATP alone group（ P 〈 0.01 ） , while 400μmol/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 NariS 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 NariS（200 μmol/L） （ P 〈 0.01 ）. Conclusion： NariS could reduce the expression of P2X7 receptor, decrease membrane permeability, and increase the cell viability in rat microglia injured by ATP. So the cyto- protection of hydrogen sulfide may be related to the expression and function of P2X7 receptor.
出处
《中国应用生理学杂志》
CAS
CSCD
2017年第6期519-523,共5页
Chinese Journal of Applied Physiology
基金
国家自然科学基金资助项目(81271376
81371346)
河南省高等学校重点科研项目计划(16A310011
16A310013)
