目的 探讨白藜芦醇（resveratrol， Res）通过调控Toll样受体4（toll-like receptor 4， TLR4）基因及其甲基化水平缓解同型半胱氨酸（homocysteine， Hcy）诱导小鼠胰岛β细胞氧化损伤的机制。 方法 采用CCK-8法检测不同浓度的Hcy和Res对小鼠胰岛β细胞活力的影响；设置分组：对照（Control）组、Hcy组、Hcy + Res共处理组，丙二醛（MDA）试剂盒、谷胱甘肽过氧化物酶（GSH-Px）活性试剂盒、酶联免疫吸附法（ELISA）检测各组细胞MDA水平、GSH-Px活力、胰岛素水平；荧光染色法检测各组细胞氧化应激状态；Western blot和qRT-PCR检测各组细胞TLR4蛋白和mRNA的表达水平；巢式甲基化检测各组细胞TLR4 DNA甲基化表达水平，Western blot检测各组细胞DNA甲基转移酶1（DNMT1）蛋白水平；构建DNMT1小干扰RNA（siRNA）并将其转入胰岛β细胞，设置分组：Hcy组、Hcy + Res组、Hcy + si-NC组、Hcy + si-DNMT1组、Hcy + Res + si-DNMT1组；Western blot检测各组TLR4蛋白水平；采用上述试剂盒检测敲低DNMT1各组MDA水平、GSH-Px活力以及胰岛素水平；构建TLR4 siRNA并将其转入胰岛β细胞的基础上，设置分组：Hcy组、Hcy + si-NC组、Hcy + si-TLR4组、Hcy + Res + si-NC组、Hcy + Res + si-TLR4组，为进一步研究Res自身对TLR4的独立调控作用，增设分组Res + si-NC组、Res + si-TLR4组，采用上述试剂盒检测MDA水平、GSH-Px活力以及胰岛素水平。 结果 用100 μmol/L的Hcy干预后，细胞活力降低，用10 μmol/L的Res干预后，能够有效缓解Hcy对胰岛β细胞的损伤；MDA试剂盒、荧光染色结果显示，与Control组相比，Hcy组MDA和活性氧（ROS）水平升高（P 0.05），Hcy + Res组则下降（P 0.05），GSH-Px活性试剂盒、ELISA法结果显示，与Control组相比，Hcy组GSH-Px活性和胰岛素水平降低（P 0.05），Hcy + Res组则升高（P 0.05）；Western blot和qRT-PCR结果显示，与Control组相比，Hcy组细胞TLR4蛋白水平、mRNA水平显著升高（P 0.05），Hcy + Res组则显著下降（P 0.05），而巢式甲基化结果显示，TLR4甲基化水平与mRNA表达呈负相关；相较于Control组，DNMT1的蛋白水平在Hcy组中低表达（P 0.05），在Hcy + Res组中高表达（P 0.05）；与Hcy + si-NC组相比，Hcy + si-DNMT1组TLR4蛋白表达显著升高（P 0.05），与Hcy + Res组相比，Hcy + Res+si-DNMT1组TLR4蛋白表达进一步升高（P 0.05）；与Hcy + si-NC组相比，Hcy + si-DNMT1组MDA水平显著升高（P 0.05），与Hcy + Res组相比，Hcy + Res + si-DNMT1组MDA水平进一步升高（P 0.05）；而GSH-Px活力、胰岛素水平变化方向与MDA变化相反；进一步敲低TLR4后MDA水平下降（P 0.05），Res干预后则更低（P 0.05）；而GSH-PX活性和胰岛素水平升高（P 0.05），Res干预后则更高（P 0.05）。 结论 Res能够减轻Hcy诱导的胰岛β细胞氧化损伤，这一过程与TLR4表达及其甲基化水平改变有关。

Objective This study aims to explore the mechanism through which resveratrol （Res） mitigates homocysteine （Hcy）-induced oxidative damage in mouse pancreatic β-cells by regulating the expression and methylation level of the Toll-like receptor 4 （TLR4） gene. Methods The effects of different concentrations of homocysteine and resveratrol on the viability of mouse pancreatic β-cells were evaluated using the CCK-8 assay. In each group， the levels of malondialdehyde （MDA）， the activity of glutathione peroxidase （GSH-Px）， and the levels of insulin were measured using an MDA kit， a GSH-Px activity kit， and enzyme-linked immunosorbent assay （ELISA）， respectively. The oxidative stress status was assessed through fluorescence staining. The protein and mRNA expression levels of TLR4 in the control group， the Hcy group， and the homocysteine + resveratrol （Hcy + Res） co-treatment group were detected via Western blot and qRT-PCR. The DNA methylation level of TLR4 was determined using nested methylation-specific PCR， and the protein expression of DNMT1 （DNA methyltransferase 1） was measured by Western blot. Small interfering RNA fragments targeting DNMT1 were constructed and transfected into pancreatic β-cells. Subsequently， the TLR4 protein expression was detected by Western blot. The MDA levels， GSH-Px activity， and insulin levels in the DNMT1 knockdown groups were measured using the aforementioned kits. Likewise， small interfering RNA fragments targeting TLR4 were constructed and transfected into pancreatic β-cells， and then the MDA levels， GSH-Px activity， and insulin levels were measured. Results Cell viability decreased after treatment with 100 μmol/L Hcy. In contrast， intervention with 10 μmol/L Res effectively mitigated the Hcy-induced damage to pancreatic β-cells. Results from the MDA assay and fluorescence staining indicated that， when compared with the Control group， the Hcy group had elevated MDA and reactive oxygen species （ROS） levels （P 0.05）， while these levels declined in the Hcy+Res group （P 0.05）. Findings from the GSH-Px activity assay and ELISA demonstrated that， relative to the Control group， the Hcy group had reduced GSH-Px activity and insulin levels （P 0.05）， whereas the Hcy + Res group showed increased levels （P 0.05）. Western blot and qRT-PCR results showed that the expression levels of TLR4 protein and mRNA were significantly higher in the Hcy group than in the Control group （P 0.05）， while they were significantly lower in the Hcy + Res group （P 0.05）. Conversely， results from nested methylation-specific PCR showed that the TLR4 DNA methylation levels displayed an opposite trend. The expression of DNMT1 protein was lower in the Hcy group （P 0.05） and higher in the Hcy + Res group （P 0.05） compared with the Control group. The expression of TLR4 protein was significantly higher in the Hcy + si-DNMT1 group than in the Hcy + si-NC group （P 0.05）， and it was further increased in the Hcy + Res + si-DNMT1 group compared with the Hcy + Res group （P 0.05）. Compared with the Hcy + si-NC group， the MDA levels were significantly elevated in the Hcy + si-DNMT1 group （P 0.05）. Moreover， when compared with the Hcy + Res group， the MDA levels were further increased in the Hcy + Res + si-DNMT1 group （P 0.05）. The changes in GSH-Px activity and insulin levels were opposite to those of MDA. Further knockdown of TLR4 led to a decrease in MDA levels （P 0.05）， and there was an even greater reduction after Res intervention （P 0.05）. Conversely， GSH-Px activity and insulin levels increased （P 0.05）， and there was a further elevation after Res intervention （P 0.05）. Conclusion Resveratrol alleviates the oxidative damage in pancreatic β-cells induced by homocysteine， a process that is associated with alterations in TLR4 expression and its methylation level.