叶酸通过NLRP3/ASC/Caspase-1信号通路对HT22细胞拟帕金森损伤作用的研究

doi:10.3877/cma.j.issn.2095-9141.2022.04.002

目的

探讨叶酸对小鼠HT22细胞拟帕金森（PD）损伤产生的影响。

方法

将小鼠HT22细胞作为研究对象，随机分为对照组、模型组、叶酸组及治疗组4组。采用CCK-8、LDH、TUNEL以及免疫荧光试验检测叶酸对MPP⁺诱导的细胞损伤的活性影响以及NOD样受体蛋白3（NLRP3）荧光表达的影响；采用Western免疫印迹和qPCR实验检测叶酸对MPP⁺诱导细胞损伤后NLRP3、凋亡相关微粒蛋白（ASC）、Caspase-1表达的影响；利用ELISA检测叶酸对MPP⁺诱导细胞损伤后白细胞介素（IL）-18和IL-1β表达的影响；最后采用Western免疫印迹检测NLRP3抑制剂CY-09对MPP⁺诱导细胞损伤后ASC、Caspase-1、IL-18和IL-1β表达的影响。

结果

MPP⁺诱导HT22细胞引起拟PD损伤，CCK-8、LDH、TUNEL实验检测结果显示叶酸可显著缓解损伤引起的细胞活性的降低和凋亡细胞的增加；免疫荧光试验检测结果显示叶酸可降低损伤后细胞中NLRP3荧光强度的升高；Western免疫印迹和qPCR试验检测结果显示叶酸可降低NLRP3、ASC、Caspase-1在蛋白水平和mRNA水平的升高；ELISA检测结果显示，叶酸治疗可显著缓解损伤后细胞内IL-18和IL-1β的分泌；最后采用Western免疫印迹检测显示CY-09可以阻断损伤后细胞内ASC、Caspase-1、IL-18和IL-1β蛋白水平的升高。

结论

叶酸通过降低IL-18和IL-1β表达保护MPP⁺致HT22细胞拟PD损伤，潜在的保护机制可能与NLRP3/ASC/Caspase-1信号通路密切相关。

关键词: 帕金森病, 叶酸, NOD样受体蛋白3, 白细胞介素-1β, 白细胞介素-18

Objective

To investigate the effect of folic acid on Parkinson's like injury in mouse HT22 cells.

Methods

Mouse HT22 cells were used as the study subjects, and were randomly divided into four groups: Control group, MPP⁺ group (Model group), Folic acid group and MPP⁺ + Folic acid (Treatment group). CCK-8, LDH, TUNEL and immunofluorescence assay were used to detect the effect of folic acid on cell viability and expression of NLRP3 induced by MPP⁺. The effects of folic acid on the expression of NLRP3, ASC and Caspase-1 after MPP⁺-induced cell injury were detected by Western blot and qPCR. ELISA was used to detect the effect of folic acid on the expression of interleukin (IL)-18 and IL-1β after MPP⁺-induced cell injury. Finally, Western blot was used to examine the expression of ASC, Caspase-1, IL-18 and IL-1β after CY-09 treatment.

Results

MPP⁺ can cause Parkinson’s like injury to HT22 cells. The results of CCK-8, LDH and TUNEL experiments showed folic acid can significantly alleviate the decrease of cell viability and the increase of apoptotic cells caused by injury. Immunofluorescence test showed that folic acid could reduce the increase of NLRP3 fluorescence intensity in injured cells. Western blot and qPCR test showed that folic acid decreased the increase of NLRP3, ASC and Caspase-1 in protein level and mRNA level. ELISA results showed that folic acid treatment significantly alleviated the secretion of intracellular IL-18 and IL-1β after injury. Finally, Western blot showed that CY-09 could block the increase of ASC, Caspase-1 and IL-18 and IL-1β protein levels in cells after injury.

Conclusion

Folic acid can cause Parkinson's like injury to HT22 cells by decreasing IL-18 and IL-1β expression and protecting MPP⁺, and the potential protective mechanism may be closely related to the NLRP3/ASC/Caspase-1 signaling pathway.

Key words: Parkinson’s disease, Folic acid, NOD-like receptor protein 3, Interleukin-1β, Interleukin-18

图1 叶酸对MPP⁺诱导的细胞损伤的活性影响A：CCK-8实验检测；与对照组比较，^aP<0.05；与模型组比较，^bP<0.05；B：LDH实验检测；与对照组比较，^aP<0.05；与模型组比较，^bP<0.05；C：TUNEL阳性细胞率，与对照组比较，^aP<0.05；与模型组比较，^bP<0.05；D：TUNEL实验检测

图2 叶酸对MPP⁺诱导细胞损伤后NLRP3荧光表达的影响A：NLRP3免疫荧光染色代表性图片；B：NLRP3荧光密度量化；与对照组比较，^aP<0.05；与模型组比较，^bP<0.05；NLRP3：NOD样受体蛋白3；MPP⁺：1-甲基-4-苯基吡啶

图3 Western blot检测叶酸对MPP⁺诱导细胞损伤后NLRP3、ASC、Caspase-1蛋白的表达NLRP3：NOD样受体蛋白3；MPP⁺：1-甲基-4-苯基吡啶；ASC：凋亡相关微粒蛋白

图4 叶酸对MPP⁺诱导细胞损伤后NLRP3、ASC、Caspase-1表达的影响A~C：Western blot检测结果量化（A：NLRP3蛋白；B：ASC蛋白；C：Caspase-1蛋白）；D~F：qPCR检测（D：NLRP3 mRNA；E：ASC mRNA；F：Caspase-1 mRNA）；与对照组比较，^aP<0.05；与模型组比较，^bP<0.05

图5 叶酸对MPP⁺诱导细胞损伤后炎症因子表达的影响A：叶酸对MPP⁺诱导细胞损伤后IL-18表达的影响；B：叶酸对MPP⁺诱导细胞损伤后IL-1β表达的影响；与对照组比较，^aP<0.05；与模型组比较，^bP<0.05

图6 CY-09对MPP⁺诱导细胞损伤后ASC、Caspase-1、IL-18和IL-1β表达的影响A：Western blot检测；B~E：Western blot结果的量化（B：ASC；C：Caspase-1；D：IL-18；E：IL-1β)；与对照组比较，^aP<0.05；与模型组比较，^bP<0.05

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Effect of folic acid on Parkinson's like injury of HT22 cells through the NLRP3/ASC/Caspase-1 signaling pathway

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Effect of folic acid on Parkinson's like injury of HT22 cells through the NLRP3/ASC/Caspase-1 signaling pathway