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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。
AIM: To investigate the regulation of hydrogen sulfide (H2S) on the release of inflammatory cytokines in rat microglia cells induced by adenosine triphosphate (ATP) and to explore the effect of microglia conditioned media on neuronal apoptosis influences. Methods: The selected rat microglia cells were randomly divided into 4 groups: (1) normal control group: conventional culture; (2) ATP group: treated with ATP 24 h after cell inoculation; (3) sodium hydrosulfide H 2 S donor) + ATP group: Pretreatment with Na HS for 30 min before ATP treatment, and Na HS was always present in the reaction system; (4) KN-62 (isoquinoline derivatives, purinergic receptor antagonist) + ATP Group: Preincubated with KN-62 for 30 min prior to ATP treatment. The changes of TNF-α and IL-6 in the supernatant of each group were detected by ELISA. The expression of P38 and JNK in mitogen-activated protein kinase (MAPK) of each group were detected by Western Blot. The effect of conditioned media on neurons was studied using human neuroblastoma cells (SH-SY5Y) in the same manner as the above microglia treatment in rats. Morphological changes and apoptosis of SH-SY5Y cells were observed by inverted microscope. Cell viability was measured by MTT assay. Results: When the concentration of ATP was 3 mmol / L, the levels of TNF-α and IL-6 released from rat microglia increased (P <0.05), and the protein expressions of p-P38 and p-JNK in microglia were increased Level (P <0.05). When ATP concentration was 5 mmol / L, the conditioned medium of activated microglia could change the morphology of SH-SY5Y cells and decrease the cell viability (P <0.05). The above ATP-induced changes were reversed by Na HS pretreatment (P <0.05), similar to P2X7R blocker KN-62. CONCLUSION: H 2 S can down-regulate the expression of p-P38 and pJNK MAPK induced by ATP and decrease the release of inflammatory cytokines such as TNF-α and IL-6, thereby protecting neurons.