褪黑素通过抑制NLRP3减轻MPP+诱导的HT22细胞焦亡/炎性损害

Melatonin alleviates MPP+-induced pyroptosis/inflammatory damage of HT22 cells by inhibiting NOD-like receptor protein 3

摘要:

目的 研究褪黑素(MT)预处理抑制NOD样受体热蛋白结构域相关蛋白3(NLRP3)水平，减轻神经细胞焦亡，改善帕金森病症状的作用及分子机制。方法 正常培养的HT22细胞记为空白对照组（Sham组）；细胞进行10 mmol/L 1-甲基-4-苯基-吡啶离子(MPP⁺)处理24 h，制成帕金森病细胞模型后，记为MPP⁺组；先用5 μmmol/L MT预处理6 h后，再造模后，记为MPP⁺+MT组；各组细胞处理后经培养24 h后取材。采用CCK8检测细胞死亡变化；采用荧光免疫组化法和Western-blotting方法检测NLRP3分布和表达变化；使用标准ELISA试剂盒检测IL-1β和 IL-18表达变化。结果 与Sham组相比，MPP⁺组神经细胞大量死亡，NLRP3在存活细胞的胞膜和胞浆中高表达，相应的炎症因子IL-1β和IL-18也大量释放，两组间差异有统计学意义(P<0.05)。相较于MPP⁺组，MPP⁺+MT组细胞存活率升高，细胞中NLRP3染色阳性率降低，表达量也降低，相应的IL-1β和IL-18表达量也降低，两组间差异有统计学意义(P<0.05)。结论 MT可有效抑制NLRP3表达和炎症因子的释放，减轻神经细胞焦亡，从而减轻MPP⁺造成的HT22细胞损伤。

Abstract:

Objective To investigate the role and molecular mechanism of melatonin （MT） pretreatment in inhibiting the level of NOD-like receptor protein 3 （NLRP3）， reducing neuronal pyroptosis， and improving Parkinson’s disease.Methods Normally cultured HT22 cells were established as blank control group （sham group）； the cells were treated with 10 mmol/L MPP⁺ for 24 hours to establish a cell model of Parkinson’s disease （MPP⁺ group）； the cells pretreated with 5 μmmol/L MT for 6 hours before modeling were established as MPP⁺+MT group； samples were collected after the cells were cultured for 24 hours after treatment. CCK8 assay was used to measure the change in cell death； fluorescence immunohistochemistry and Western blotting were used to measure the changes in the distribution and expression of NLRP3； a standard ELISA kit was used to measure the changes in the expression of interleukin-1β （IL-1β） and interleukin-18 （IL-18）.Results Compared with the sham group， the MPP⁺ group had the death of a large number of nerve cells， a high expression level of NLRP3 in the membrane and cytoplasm of the surviving cells， and the secretion of large amounts of the corresponding inflammatory factors IL-1β and IL-18 （P<0.05）. Compared with the MPP⁺ group， the MPP⁺+MT group had a significant increase in cell viability and significant reductions in the positive rate of NLRP3 staining and the expression levels of NLRP3， IL-1β， and IL-18 （P<0.05）.Conclusions MT can effectively inhibit the expression of NLRP3 and the release of inflammatory factors， reduce nerve cell pyroptosis， and thus improve the damage of HT22 cells caused by MPP⁺.