目的 探讨β-羟基丁酸（β-hydroxybutyrate,BHB）对高浓度胰岛素诱导的SH-SY5Y细胞能量代谢障碍的改善作用,并分析其与SIRT2信号通路的关系。方法 采用150 nmol/L胰岛素作用48 h建立SH-SY5Y细胞胰岛素抵抗模型,随后给予10 mmol/L BHB干预24 h;采用CCK-8法检测细胞活力,2-NBDG荧光探针检测葡萄糖摄取能力,ELISA法检测炎症因子及氧化应激指标,DCFH-DA和JC-1荧光染色分别检测细胞活性氧（reactive oxygen species,ROS）水平及线粒体膜电位,生化试剂盒检测ATP含量、NAD⁺/NADH比值及线粒体呼吸链复合物活性,Western blot检测磷酸化胰岛素受体底物-1（丝氨酸307位点）[phosphorylated insulin receptor substrate-1 at serine 307,p-IRS-1（Ser307）]、沉默信息调节因子同源蛋白2（sirtuin 2,SIRT2）、乙酰化α-微管蛋白（赖氨酸40位点）[acetylated α-tubulin at lysine 40,Ac-α-tubulin（Lys40）]、过氧化物酶体增殖物激活受体γ共激活因子1α（peroxisome proliferator-activated receptor gamma coactivator 1-alpha,PGC-1α）及线粒体转录因子A（mitochondrial transcription factor A,TFAM）蛋白表达。结果 与正常对照组相比,模型组细胞活力及葡萄糖摄取能力显著下降,p-IRS-1（Ser307）表达升高,提示胰岛素抵抗模型构建成功;模型组白细胞介素（interleukin,IL）-6、IL-17及肿瘤坏死因子α（tumor necrosis factor-alpha,TNF-α）水平升高,白细胞介素-10（interleukin-10,IL-10）水平降低,ROS及丙二醛（malondialdehyde,MDA）增加,超氧化物歧化酶（superoxide dismutase,SOD）活性下降;同时线粒体膜电位降低,ATP含量、NAD⁺/NADH比值及呼吸链复合物Ⅰ、Ⅱ、Ⅲ、Ⅳ活性均明显下降。BHB干预后,细胞活力显著提高,炎症因子失衡得到改善,ROS生成减少,SOD活性升高,MDA含量下降,线粒体膜电位恢复,ATP生成及呼吸链功能增强;进一步机制研究显示,BHB显著上调SIRT2蛋白表达,降低Ac-α-tubulin（Lys40）/α-tubulin比值,并促进PGC-1α和TFAM表达。结论 BHB可显著改善高浓度胰岛素诱导的SH-SY5Y细胞能量代谢障碍,其作用机制可能与激活SIRT2、增强去乙酰化功能并促进线粒体生物发生有关。
Abstract
Objective To investigate the ameliorative effects of β-hydroxybutyrate (BHB) on energy metabolism disorder in SH-SY5Y cells induced by high-concentration insulin and analyze its relationship with the SIRT2 signaling pathway. Methods An insulin resistance model was established in SH-SY5Y cells by treatment with 150 nmol/L insulin for 48 h, followed by intervention with 10 mmol/L BHB for 24 h. Cell viability was assessed using the CCK-8 assay, and glucose uptake capacity was measured with the 2-NBDG fluorescent probe. Inflammatory cytokines and oxidative stress markers were determined by ELISA. Cellular reactive oxygen species (ROS) levels and mitochondrial membrane potentials were evaluated using DCFH-DA and JC-1 fluorescent staining, respectively. ATP content, NAD⁺/NADH ratio, and the activities of mitochondrial respiratory chain complexes were measured using biochemical assay kits. Western blot was employed to detect the protein expression of phosphorylated insulin receptor substrate-1 at serine 307 [p-IRS-1 (Ser307)], sirtuin 2 (SIRT2), acetylated α-tubulin at lysine 40 [Ac-α-tubulin (Lys40)], peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), and mitochondrial transcription factor A (TFAM). Results Compared with the normal control group, the model group exhibited significantly reduced cell viability and glucose uptake capacity, along with increased p-IRS-1(Ser307) expression, indicating successful construction of the insulin-resistant model. The model group also showed elevated levels of interleukin-6 (IL-6), interleukin-17 (IL-17), and tumor necrosis factor-α (TNF-α), decreased interleukin-10 (IL-10), accumulation of ROS and malondialdehyde (MDA), decreased superoxide dismutase (SOD) activity, reduced mitochondrial membrane potential, and markedly lower ATP content, NAD⁺/NADH ratio, and activities of respiratory chain complexes I, II, III, and IV. Following BHB intervention, cell viability was significantly improved, inflammatory imbalance corrected, ROS production reduced, SOD activity elevated, MDA content declined, mitochondrial membrane potential was restored, and ATP generation and respiratory chain function enhanced. Further mechanistic studies revealed that BHB significantly upregulated SIRT2 protein expression, decreased the Ac-α-tubulin (Lys40)/α-tubulin ratio, and promoted the expression of PGC-1α and TFAM. Conclusion BHB can significantly ameliorate high-concentration insulin-induced energy metabolism disorder in SH-SY5Y cells, and its mechanism is associated with the activation of SIRT2, enhancement of deacetylase function, and promotion of mitochondrial biogenesis.
关键词
β-羟基丁酸 /
胰岛素抵抗 /
SIRT2 /
线粒体功能 /
SH-SY5Y细胞
Key words
β-hydroxybutyrate /
insulin resistance /
SIRT2 /
mitochondrial function /
SH-SY5Y cells
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基金
国家自然科学基金（No.82374547）; 海南省自然科学基金（No.326MS0442）; 中山市中医药传承创新发展科研专项重点项目（2024B3024）; 广东省教育厅生物医药重点专项（No.2025ZDZX2012,2025ZDZX2106）; 广东食品药品职业学院校级课题（No.2025ZR18）
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