目的:观察二氢山柰酚（dihydrokaempferol，DHK）对IGF2BP2介导的N6‑甲基腺苷（N6‑methyladenosine，m6A）甲基化修饰的影响，阐明其抑制细胞焦亡发挥神经保护效应的分子机制。 方法:小鼠原代海马神经元细胞随机分为空白组、模型组、DHK低剂量组、DHK高剂量组及IGF2BP2抑制剂CWI1‑2组，采用Aβ_1‑42诱导构建阿尔茨海默病（Alzheimer′s disease，AD）体外细胞焦亡模型。CCK‑8法检测细胞活力，流式细胞术检测细胞焦亡比例，生化试剂盒检测超氧化物歧化酶（superoxide dismutase，SOD）活性与丙二醛（malondialdehyde，MDA）含量，免疫荧光法检测活性氧（reactive oxygen species，ROS）水平，Western blot检测NLRP3、cleaved caspase‑1、GSDMD‑NT、mature IL‑1β、pro‑caspase‑1及IGF2BP2蛋白表达，RT‑qPCR检测IGF2BP2 mRNA表达，斑点杂交检测RNA m6A甲基化水平。 结果:与空白组比较，模型组细胞活力显著降低，焦亡细胞比例显著升高，SOD活性显著降低，MDA含量与ROS水平显著升高，NLRP3、cleaved caspase‑1、GSDMD‑NT、mature IL‑1β及IGF2BP2蛋白表达显著上调，pro‑caspase‑1蛋白表达显著下调，m6A修饰显著增加，差异均有统计学意义（P<0.01）。相较模型组，DHK可剂量依赖性提升细胞活力、降低焦亡比例、改善氧化应激指标、抑制焦亡相关蛋白表达、下调IGF2BP2表达并降低总RNAm6A甲基化水平，差异均有统计学意义（P<0.01）。 结论:DHK可能通过抑制IGF2BP2表达、降低NLRP3 mRNA的m6A甲基化修饰水平，阻断NLRP3炎症小体活化介导的细胞焦亡通路，进而发挥抗AD神经元损伤的神经保护效应。

Objective:To investigate the effect of dihydrokaempferol （DHK） on IGF2BP2‑mediated N6‑methyladenosine （m6A） methylation modification， and clarify its molecular mechanism of exerting neuroprotective effects by inhibiting neuronal pyroptosis. Methods:Primary mouse hippocampal neurons were randomly divided into control group， model group， low‑dose DHK group， high‑dose DHK group and IGF2BP2 inhibitor CWI1‑2 intervention group. The in vitro Alzheimer′s disease （AD） cell pyroptosis model was established by Aβ_1‑42 induction. Cell viability was detected using a CCK‑8 assay， and the ratio of pyroptotic cells was determined using flow cytometry； Superoxide dismutase （SOD） activity and malondialdehyde （MDA） content were measured using biochemical detection kits； Reactive oxygen species （ROS） level was observed by immunofluorescence staining. Western blot was applied to detect the protein expressions of NLRP3， cleaved caspase‑1， GSDMD‑NT， mature IL‑1β， pro‑caspase‑1 and IGF2BP2. RT‑qPCR was used to test IGF2BP2 mRNA expression， and dot blot assay was performed to determine total RNA m6A methylation level. Results:Compared to the control group， the model group exhibited significantly decreased cell viability， increased proportion of pyroptotic cells， reduced SOD activity， elevated MDA content and ROS level. The protein levels of NLRP3， cleaved caspase‑1， GSDMD‑NT， mature IL‑1β and IGF2BP2 were markedly upregulated， while pro‑caspase‑1 protein expression was downregulated， accompanied by a notable increase in m6A methylation level （P<0.01）. Compared to the model group， DHK treatment dose‑dependently improved cell viability， reduced pyroptosis rate， ameliorated oxidative stress indicators， suppressed the expression of pyroptosis‑related proteins， downregulated IGF2BP2 expression and decreased total RNA m6A methylation level with statistically significant differences （P<0.01）. Conclusion:DHK may inhibit IGF2BP2 expression and reduce m6A methylation modification level of NLRP3 mRNA， thereby blocking NLRP3 inflammasome‑mediated pyroptosis pathway and exerting neuroprotective effects against neuronal injury in AD.

R749.16;R741

黑龙江省中医药科研项目（ZHY19?003）；黑龙江省自然科学基金面上项目（H2018063）