H_2S对ATP诱导小胶质细胞活化的影响

The effects of H_2S on ATP-induced microglial activation

目的:研究硫化氢(hydrogen sulfide,H_2S)对三磷酸腺苷(adenosine triphosphate,ATP)诱导大鼠小胶质细胞炎性因子释放的调节,并探讨小胶质细胞条件培养基对神经元样细胞凋亡的影响。方法:选定的大鼠小胶质细胞随机分4组:(1)正常对照组:常规培养;(2)ATP组:细胞接种24 h后用ATP处理;(3)Na HS(sodium hydrosulfide,H_2S供体)+ATP组:ATP处理前用Na HS预孵育30 min,且Na HS始终存在于反应体系中;(4)KN-62(异喹啉衍生物,嘌呤受体拮抗剂)+ATP组:ATP处理前用KN-62预孵育30 min。用ELISA检测各组细胞上清液中TNF-α、IL-6的变化,用Western Blot检测各组丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)P38和JNK表达水平。用人神经母细胞瘤细胞(SH-SY5Y)研究条件培养基对神经元的影响,其方法与上述大鼠小胶质细胞处理相同。用倒置显微镜观察SH-SY5Y细胞形态变化及凋亡情况,用MTT法检测各组细胞活力。结果:当ATP浓度3 mmol/L时,大鼠小胶质细胞释放TNF-α和IL-6水平增加(P<0.05),同时上调大鼠小胶质细胞p-P38、p-JNK蛋白表达水平(P<0.05)。当ATP浓度5 mmol/L时,小胶质细胞活化后的条件培养基可以使SH-SY5Y细胞形态发生改变,其细胞活力降低(P<0.05)。ATP引起的上述变化可以通过Na HS预处理而逆转(P<0.05),其作用与P2X7R阻断剂KN-62相类似。结论:H_2S可下调被ATP诱导的p-P38和pJNK MAPK蛋白表达,减少TNF-α和IL-6等炎性因子的释放,从而对神经元产生保护作用。

Objective： To investigate the effect of hydrogen sulfide （H2S） on the release of inflammatory cytokines in microglias induced by adenosine triphosphate, and to investigate the effect of conditioned medium of microglias on the apoptosis of neuron like cells. Methods： The mieroglia selected was divided into 4 groups randomly： （ 1 ） normal con- trol group： routine culture; （2） ATP group： the cells were treated with ATP after cultured for 24 hours; （3） Naris （ the donor compound of sodium hydrosulfide） ＋ ATP group ： the cells were treated with ATP after treated with NariS for 30 minutes, and NariS was constantly present in the reaction system; （4） KN-62 （isoquinoline derivative, purine receptor antagonist） ＋ ATP group： the cells were treated with ATP after treated with KN-62 for 30 minutes. The changes of TNF-ot and iL-6 in the supernatant of each group were detected by ELISA, and the levels of mitogen-activated protein kinase （MAPK）, P38 and JNK were detected by Western Blot. Human neuroblastoma cells （SH-SY5Y cells） were used for investigation into the effects of conditioned medium on neurons, similar to those treated with microglia. The morphological changes and apoptosis of SH-SY5Y cells were observed by inverted microscope, and the cell viability of each group was detected by MTT. Results ： At ATP concentration of 3 mmol/L, the concentrations of TNF-α and IL-6 released by microglia in rats were increased （P 〈 0.05）, the expression of p-P38 and p-JNK protein was up-regulated in microglia （P 〈 0.05）. At the concentration of 5mmol/L ATP, the conditioned medium with activated microglia could change the morphology of SH-SY5Y cells and decrease their cell viability （P 〈 0.05）. These changes induced by ATP could be reversed by pretreatment with NariS （P 〈 0.05 ）, which was similar to the P2X7R blocker KN-62. Conclu- sion： H2S can down regulate the expression of p-P38 and p-JNK MAPK proteins induced by ATP and reduce the release of inflammatory factors such as TNF-α, IL-6 and so on, thus protecting neurons.