Aloin mitigates hypoxic injury in rat cardiomyocytes: inhibiting oxidative stress and ferroptosis

doi:10.12307/2025.525

Abstract

Abstract: BACKGROUND: Myocardial cell hypoxic injury is closely associated with oxidative stress and ferroptosis. Previous studies have shown that aloin has various effects such as antioxidant, anti-inflammatory, and anti-tumor activities.
OBJECTIVE: To investigate the effects of aloin on oxidative stress and ferroptosis in hypoxia-induced H9C2 cells.
METHODS: A hypoxia model was established using H9C2 myocardial cells. Firstly, cell viability was determined to confirm the lack of cytotoxicity of aloin and to determine its optimal therapeutic concentration. Subsequently, the effects of aloin on hypoxia-induced lactate dehydrogenase release, reactive oxygen species production, and mitochondrial oxidative stress in H9C2 cells were evaluated using assay kits, dihydroethidium fluorescent probes, and MitoSOX™ Red fluorescent probes, respectively. To verify the effect of aloin on ferroptosis, intracellular Fe2+ content and lipid peroxidation level were detected using fluorescence staining and flow cytometry, respectively. Then, the expression levels of ferroptosis regulatory factors glutathione peroxidase 4, acyl-CoA synthetase long-chain family member 4, and nuclear factor E2-related factor 2 were detected using western blot assay and real-time fluorescence quantitative PCR techniques. Finally, the role of ferroptosis in aloin-mediated myocardial protection was further confirmed by using the ferroptosis inducer Erastin.
RESULTS AND CONCLUSION: (1) Compared with the control group, the viability of H9C2 cells in the hypoxia group was significantly decreased, lactate dehydrogenase release, reactive oxygen species level, mitochondrial oxidative stress degree, Fe2+ content, and lipid peroxidation degree were significantly increased, while glutathione peroxidase 4 and nuclear factor E2-related factor 2 mRNA and protein expression levels were significantly decreased, and acyl-CoA synthetase long-chain family member 4 mRNA and protein expression were significantly increased (all P < 0.05). (2) Compared with the hypoxia group, both low and high doses of aloin reversed the changes in above indicators (all P < 0.05). (3) Compared with the hypoxia+aloin group, the hypoxia+aloin+Erastin group showed a significant decrease in H9C2 cell viability and a significant increase in lactate dehydrogenase release (both P < 0.01). The results indicate that aloin has a protective effect on hypoxia-treated H9C2 cells in a dose-dependent manner, mainly achieved by inhibiting oxidative stress and ferroptosis.

Key words: aloin, myocardial cell hypoxic injury, oxidative stress, ferroptosis, mitochondria

CLC Number:

Tan Mingyue, Jin Yifeng, Zhang Jun, Li Hongxia. Aloin mitigates hypoxic injury in rat cardiomyocytes: inhibiting oxidative stress and ferroptosis[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(25): 5335-5344.

Figures/Tables 2

2.1 芦荟素对H9C2细胞活力的影响芦荟素是从芦荟植物中分离出的蒽醌化合物(分子结构式见图1A)，它是食品中常见的“天然香料”[24]。为明确芦荟素的安全性，使用不同浓度(0，5，10，20，50，100，150，200，250，300 μmol/L)芦荟素对H9C2细胞进行24 h干预。然后，使用CCK-8法检测细胞活力。结果显示，与未接受处理的对照组(0 μmol/L)相比，当芦荟素浓度在0-200 μmol/L 范围内时，细胞活力并未出现显著变化(P > 0.05)，见图1B。然而，芦荟素浓度达到250，300 μmol/L时，细胞活力显著下降(P < 0.05，P < 0.001)，见图1B。由此可见，在0-200 μmol/L的浓度范围内，芦荟素对H9C2细胞的毒性作用极小。基于这一结果，后续的实验将集中在这一浓度范围内，以探寻芦荟素对H9C2细胞的最佳治疗浓度。 2.2 不同浓度芦荟素对缺氧条件下H9C2细胞活力及乳酸脱氢酶释放的影响 2.2.1 确定药物干预浓度将缺氧诱导的H9C2细胞与不同浓度的芦荟素(0，5，10，20，50，100，150，200 μmol/L) 共培养24 h，使用CCK-8法检测细胞活力。缺氧导致H9C2细胞活力显著下降(P < 0.001)。与未经芦荟素处理的H9C2细胞相比，当芦荟素浓度处于5 μmol/L时，细胞活力并未出现明显变化(P > 0.05)；当芦荟素浓度升至10，20，50，100，150，200 μmol/L时，H9C2细胞活力逐渐恢复(P < 0.05，P < 0.001)，见图2A。值得注意的是，在50，100，150，200 μmol/L芦荟素处理组之间，细胞活力未观察到明显差异(P > 0.05)。50 μmol/L处理组的细胞活力优于20 μmol/L处理组(P < 0.05)。因此，选择20，50 μmol/L作为后续实验的芦荟素低、高浓度。 2.2.2 乳酸脱氢酶释放情况与对照组相比，缺氧组H9C2细胞乳酸脱氢酶释放显著升高(P < 0.001)；与缺氧组相比，使用20，50 μmol/L芦荟素处理后乳酸脱氢酶释放明显减少(P < 0.01，P < 0.001)，并且芦荟素高剂量组对乳酸脱氢酶释放的抑制作用强于芦荟素低剂量组(P < 0.05)，见图2B。 2.3 芦荟素对缺氧条件下H9C2细胞内活性氧生成及线粒体氧化应激的影响 2.3.1 活性氧生成超氧化物阴离子(DHE)荧光探针检测H9C2心肌细胞活性氧水平，与对照组相比，缺氧组H9C2细胞内荧光强度显著增加(P < 0.001)，这说明细胞中活性氧生成显著增加；与缺氧组相比，芦荟素组细胞内荧光强度均明显降低(P < 0.001)，且芦荟素高剂量组的荧光强度低于芦荟素低剂量组(P < 0.001)，见图3A，B。这一结果说明，20，50 μmol/L芦荟素均能抑制细胞内活性氧生成，且这种抑制作用呈剂量依赖性。 2.3.2 线粒体氧化应激采用MitoSOX Red荧光探针检测H9C2细胞线粒体中氧化应激情况。MitoSOX染色结果显示，缺氧可诱导H9C2细胞内线粒体氧化应激程度明显升高(P < 0.001)，使用芦荟素干预后细胞内线粒体氧化应激相较于缺氧组均降低(P < 0.001)；芦荟素高剂量组对线粒体氧化应激的抑制作用强于芦荟素低剂量组(P < 0.01)，见图3C，D。 2.4 芦荟素对缺氧条件下H9C2细胞内亚铁含量的影响细胞内铁超载是铁死亡的驱动因子，也是铁死亡的主要特征之一。利用对Fe2+敏感的荧光染料FerroOrange来检测H9C2细胞内Fe2+的水平。FerroOrange染色结果表明，与对照组相比，缺氧组的H9C2细胞内荧光强度显著增强(P < 0.001)，这说明缺氧损伤会导致H9C2细胞内Fe2+含量增加。然而，经20，50 μmol/L芦荟素处理后，H9C2细胞内的荧光强度均明显降低(P < 0.001)；芦荟素高剂量组的荧光强度低于芦荟素低剂量组(P < 0.01)，见图4。这一结果说明，芦荟素可以有效降低细胞内亚铁累积，而且50 μmol/L芦荟素降低效果更好。"

2.5 芦荟素对缺氧处理的H9C2细胞脂质过氧化程度的影响脂质过氧化累积是铁死亡的核心特征之一。通过脂质过氧化荧光探针C11 BODIPY染色并使用流式细胞术检测荧光强度。结果显示，与对照组相比，缺氧组H9C2细胞内荧光强度显著增强(P < 0.001)，这表明缺氧损伤会明显加剧H9C2细胞内的脂质过氧化程度。与缺氧组相比，芦荟素干预明显逆转了脂质过氧化的上升(P < 0.001)，其中芦荟素高剂量组的效果优于芦荟素低剂量组(P < 0.001)，见图5。 2.6 芦荟素对缺氧条件下H9C2细胞中铁死亡相关mRNA表达的影响实时荧光定量PCR(RT-qPCR)检测结果显示，与对照组相比，缺氧组细胞中Nrf2、GPX4 mRNA表达水平均显著下调(P < 0.001)，而ACSL4 mRNA表达水平明显上调(P < 0.001)；芦荟素低剂量组的Nrf2 mRNA表达与缺氧组相比无明显变化(P > 0.05)，但芦荟素高剂量组Nrf2 mRNA表达水平显著上升(P < 0.001)，并且与低剂量组相比也显示出统计学差异(P < 0.05)；与缺氧组相比，芦荟素干预组的GPX4 mRNA表达水平均升高(P < 0.05，P < 0.001)，ACSL4 mRNA表达水平均降低(P < 0.001)，且芦荟素高剂量组对GPX4和ACSL4的调控效果强于芦荟素低剂量组(P < 0.01)，见图6。 2.7 芦荟素对缺氧H9C2细胞中铁死亡相关蛋白表达的影响 Western blot检测结果显示，与对照组相比，缺氧组Nrf2与GPX4的蛋白表达明显降低(P < 0.01，P < 0.001)，ACSL4的蛋白表达显著升高(P < 0.01)；与缺氧组相比，经芦荟素处理后，GPX4的蛋白表达均升高(P < 0.05，P < 0.001)，且这种促进作用呈剂量依赖性(P < 0.05)；芦荟素低剂量组的Nrf2与ACSL4蛋白表达相较于缺氧组差异无显著性意义(P > 0.05)，芦荟素高剂量组的Nrf2蛋白表达显著升高(P < 0.01)，ACSL4蛋白表达明显下降(P < 0.01)，见图7。 2.8 芦荟素通过抑制铁死亡改善H9C2细胞缺氧损伤 2.8.1 确定Erastin干预浓度上述实验明确了芦荟素能够有效抑制由缺氧引发的H9C2细胞铁死亡。为了深入探究芦荟素是否通过抑制铁死亡来保护H9C2细胞，使用铁死亡诱导剂Erastin进行了研究。在实验中，选用了不同浓度的Erastin(0，1，2.5，5，10 μmol/L)对H9C2细胞进行24 h的干预处理。通过CCK-8法检测细胞活力。实验结果显示，与未接受Erastin处理的对照组(0 μmol/L)相比，当Erastin浓度为1 μmol/L时，对细胞活力并无显著影响(P > 0.05)；然而，当浓度提高到2.5，5，10 μmol/L时，细胞活力出现明显下降(P < 0.001)，见图8A。基于上述结果，选择2.5 μmol/L Erastin进行后续实验。 2.8.2 Erastin对芦荟素干预的H9C2细胞活力及乳酸脱氢酶释放的影响为了验证铁死亡诱导剂Erastin是否确实能够逆转芦荟素对缺氧细胞的保护作用，在芦荟素(50 μmol/L)治疗的基础上，加入了铁死亡诱导剂Erastin(2.5 μmol/L) 进行干预。与缺氧+芦荟素组对比，缺氧+芦荟素+Erastin组的细胞活力显著下降(P < 0.001)，乳酸脱氢酶释放明显升高(P < 0.01)，见图8B，C。这一结果表明芦荟素是通过抑制铁死亡改善H9C2细胞的缺氧损伤。"

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