同型半胱氨酸通过促进细胞焦亡和上调琥珀酸脱氢酶亚基A诱导神经炎症反应

Homocysteine induces neuroinflammation by improving pyroptosis and upregulating SDHA

目的探索同型半胱氨酸(Hcy)是否通过促进细胞焦亡和上调琥珀酸脱氢酶亚基A(SDHA)蛋白表达诱导神经炎症反应。方法使用不同浓度Hcy(1.25、2.50、5.00 mmol/L)处理小鼠海马神经元细胞系HT22细胞。CCK-8检测细胞活力;免疫荧光法观察细胞形态和焦亡相关蛋白消皮素(GSDMD)与细胞膜的共定位情况;Western blotting检测细胞核苷酸结合寡聚化结构样受体蛋白3(NLRP3)、天冬氨酸蛋白水解酶-1前体(pro-Caspase-1)和Caspase-1的活性片段(cleaved-Caspase-1)、凋亡相关斑点样蛋白(ASC)、GSDMD和GSDMD的N端结构域(GSDMD-N)等焦亡相关蛋白及SDHA蛋白表达水平。酶联免疫吸附测定法检测炎症因子白细胞介素-1β(IL-1β)和白细胞介素-18(IL-18)水平。结果Hcy可显著降低HT22细胞活力,损伤HT22细胞突触,增加GSDMD与细胞膜的共定位和细胞膜破裂程度,上调HT22细胞NLRP3、pro-Caspase-1、cleaved-Caspase-1、ASC、GSDMD和GSDMD-N等焦亡相关蛋白及SDHA蛋白表达水平,并增加IL-1β和IL-18水平。结论Hcy可诱导神经炎症反应,其机制与其促进细胞焦亡和上调SDHA蛋白表达密切相关。

Aim To demonstrate whether homocysteine(Hcy)induces neuroinflammation by improving pyroptosis and upregulating the expression of succinate dehydrogenase subunit A(SDHA).Methods HT22 cells were treated with different concentrations of homocysteine(Hcy,1.25,2.50,5.00 mmol/L).The cell viability was tested by CCK-8.The cell morphology as well as the co-localization of gasdermin D(GSDMD)and cell membrane were observed by immunofluorescence.The expressions of pyroptosis-associated proteins,nucleotide-binding oligomerization domain-like receptorprotein 3(NLRP3),pro-Caspase-1,cleaved-Caspase-1,apoptosis-associated speck-like protein containing a caspase recruitment domain(ASC),GSDMD,and N-terminal fragment of GSDMD(GSDMD-N),as well as SDHA were detected by Western blotting.The levels of interleukin-1β(IL-1β)and interleukin-18(IL-18)were measured by Enzyme-linked immunosorbent assay(ELISA).Results Hcy decreased the cell viability,damaged the synapse,increased the co-localization of GSDMD and cell membrane,enhanced the rupture of cell membrane,increased the expressions of pyroptosis-related proteins,NLRP3,pro-Caspase-1,cleaved-Caspase-1,ASC,GSDMD and GSDMD-N,upregulated the expression of SDHA,elevated the levels of IL-1βand IL-18.Conclusion Hcy induces neuroinflammation,which is corelated with improving pyroptosis and upregulating the expression of SDHA.