胰高血糖素调控巩膜组蛋白乳酸化及Notch1通路延缓近视的初步研究

A preliminary study of glucagon in delaying myopia progression by regulating scleral histone lactylation and the Notch1 signaling pathway

摘要:

目的 探讨胰高血糖素对缺氧下人眼巩膜成纤维细胞（HSFs）糖酵解代谢重编程的调控作用，并利用镜片诱导型近视（LIM）豚鼠模型验证其通过“糖酵解-乳酸化-Notch1”通路延缓近视进展的机制。方法 将HSFs分为以下4组：常氧对照组、缺氧模型组、缺氧+胰高血糖素组、缺氧+2-DG组。采用CCK-8法检测不同浓度（10～5 000 nmol/L）胰高血糖素对细胞增殖活力的影响。在缺氧条件下，设置不同浓度胰高血糖素梯度，检测乳酸生成量，以确定最适干预浓度。以最适浓度进行药物干预，采用实时荧光定量聚合酶链反应检测缺氧诱导因子-1α及Notch1的mRNA表达水平。体内实验选用33只3周龄豚鼠，随机分为对照组、LIM组及LIM+胰高血糖素组，每组11只。LIM组及LIM+胰高血糖素组豚鼠右眼均佩戴-6.0 D负透镜。LIM+胰高血糖素组每4 d向右眼玻璃体腔内注射胰高血糖素溶液（1 μmol/L）15 μL。诱导4周后，检测屈光度及眼轴长度。通过眼底后极部照相观察眼底形态学。采用Western blotting检测巩膜组蛋白H3第18位赖氨酸乳酸化（H3K18la）、Notch1及α-平滑肌肌动蛋白（α-SMA）的表达。结果 CCK-8法检测结果显示，在10～5 000 nmol/L浓度范围内分别处理HSFs 24、48及72 h后，各组细胞增殖活力均>95%。浓度梯度实验结果显示，胰高血糖素对缺氧诱导的乳酸生成具有剂量依赖性抑制作用，在1 000 nmol/L时趋于平台。缺氧处理12 h后，缺氧模型组乳酸生成量升至（3.16±0.04）mmol/gprot，较常氧对照组（2.28±0.03）mmol/gprot升高（P <0.05）；1 000 nmol/L胰高血糖素干预后，缺氧细胞乳酸含量降至（2.45±0.05）mmol/gprot，HIF-1α及Notch1的基因表达亦同步下调（P <0.05）。体内实验中，与LIM组相比，LIM+胰高血糖素组豚鼠右眼的近视性屈光漂移及眼轴增长均受到抑制（P <0.05），眼底豹纹状改变减轻，巩膜组织中升高的H3K18la、Notch1及α-SMA蛋白表达同步下调（P <0.05）。结论 胰高血糖素通过抑制巩膜糖酵解，减少乳酸积聚，下调H3K18la修饰，并阻断Notch1介导的巩膜成纤维细胞向肌成纤维细胞转化，从而延缓近视进展。

Abstract:

Objective To investigate the regulatory effect of glucagon on glycolytic metabolic reprogramming in human scleral fibroblasts (HSFs) under hypoxia and to verify, using a guinea pig model of lens-induced myopia (LIM), its mechanism of delaying myopia progression through the "glycolysis-lactylation-Notch1" pathway.Methods HSFs were divided into four groups: normoxic control, hypoxia model, hypoxia + glucagon, and hypoxia + 2-DG. The effects of different concentrations of glucagon (10–5 000 nmol/L) on cell viability were assessed using the CCK-8 assay. Under hypoxia, a glucagon concentration gradient was established, and lactate production was measured to determine the optimal intervention concentration. At the optimal concentration, quantitative real-time polymerase chain reaction was used to determine the mRNA expression levels of hypoxia-inducible factor-1α (HIF-1α) and Notch1. For the in vivo experiment, 33 three-week-old guinea pigs were randomly divided into a control group, an LIM group, and an LIM + glucagon group. The right eyes of guinea pigs in the LIM and LIM + glucagon groups were fitted with -6.0 D lenses. The LIM + glucagon group received an intravitreal injection of 15 μL of glucagon solution (1 μmol/L) into the right eye every 4 days. After 4 weeks of induction, refraction and axial length were measured, fundus morphology was observed by posterior pole photography, and Western blotting was used to determine the expression of histone H3 lysine 18 lactylation (H3K18la), Notch1, and α-smooth muscle actin (α-SMA) in the sclera.Results After treatment with 10–5 000 nmol/L glucagon for 24, 48, and 72 h, cell viability remained > 95% in all groups. The concentration-gradient experiment showed that glucagon dose-dependently inhibited hypoxia-induced lactate production, with the effect reaching a plateau at 1 000 nmol/L. After 12 h of hypoxia, lactate production in the hypoxia model group increased to (3.16 ± 0.04) mmol/gprot, which was significantly higher than that in the normoxic control group [(2.28 ± 0.03) mmol/gprot] (P < 0.05). Treatment with 1 000 nmol/L glucagon reduced the lactate content of hypoxic cells to (2.45 ± 0.05) mmol/gprot and simultaneously downregulated HIF-1α and Notch1 mRNA expression (P < 0.05). In vivo, compared with the LIM group, the myopic refractive shift and axial elongation of the right eye were inhibited in the LIM + glucagon group (P < 0.05), tessellated fundus changes were alleviated, and the elevated levels of H3K18la modification and Notch1 and α-SMA protein expression in the sclera were simultaneously downregulated (P < 0.05).Conclusion Glucagon inhibits scleral glycolysis, reduces lactate accumulation, downregulates H3K18la modification, and blocks Notch1-mediated transdifferentiation of scleral fibroblasts into myofibroblasts, thereby delaying myopia progression.