The Role and Mechanism of Mitochondrial Ferroptosis in Uremic Toxin-Associated Myocardial Injury
-
摘要:
目的 探讨尿毒症毒素硫酸吲哚酚（indoxyl sulfate，IS）在慢性肾病（chronic kidney disease，CKD）大鼠心肌损伤中的作用及其通过线粒体途径介导铁死亡的机制。 方法 采用5/6肾切除法建立CKD模型，将大鼠随机分为假手术组、CKD组、IS组、CKD+IS组及CKD+IS+铁死亡抑制剂Ferrostatin-1（Fer-1）组。通过比色法检测心肌组织超氧化物歧化酶（superoxide dismutase，SOD）活性、丙二醛（malonaldehyde，MDA）、还原型谷胱甘肽（reduced glutathione hormone，GSH）及Fe2+含量；采用二氢乙啶（dihydropyridine，DHE）荧光法观察活性氧（reactive oxygen，ROS）水平；RT-qPCR与Western blot检测铁死亡相关基因长链脂肪酸-CoA合成酶4（acyl-coA synthetase long-chain family member 4，ACSL4）、胱氨酸/谷氨酸逆转运蛋白（solute carrier family 7 member 11，SLC7A11）和GPX4的mRNA及蛋白表达；TUNEL法评估心肌细胞凋亡；同时检测线粒体ATP含量并通过透射电镜观察其超微结构变化。 结果 与假手术组相比，CKD组大鼠体重下降，心脏质量增加（P < 0.05），SOD与GSH水平下降，MDA、Fe2+及ROS显著升高（P < 0.05）；ACSL4表达上调，而SLC7A11与GPX4显著下调（P < 0.05），并伴随大量心肌细胞凋亡及ATP含量减少（P < 0.05）。慢性IS暴露进一步加剧上述变化（P < 0.05），表现为线粒体肿胀、嵴断裂及膜密度增加等典型铁死亡超微结构特征；而Fer-1干预可逆转这些损伤，使ATP水平恢复、线粒体形态改善。 结论 IS可通过激活线粒体途径诱导铁死亡，加重CKD大鼠心肌氧化应激与能量代谢障碍，从而促进心肌细胞凋亡；Fer-1干预可显著缓解IS诱导的线粒体损伤与心肌铁死亡。 Abstract: Objective To investigate the role of the uremic toxin indoxyl sulfate （IS） in myocardial injury in chronic kidney disease （CKD） rats and to elucidate the mechanism of mediating ferroptosis through the mitochondrial pathway. Methods A CKD model was established using the 5/6 nephrectomy method. Rats were randomly divided into five groups: sham-operated, CKD, IS, CKD+IS, and CKD+IS+ferroptosis inhibitor Ferrostatin-1 （Fer-1）. Myocardial tissue levels of superoxide dismutase （SOD） activity, malondialdehyde （MDA）, reduced glutathione （GSH）, and Fe2+ were determined using colorimetric assays. Reactive oxygen species （ROS） levels were assessed by dihydroethidium （DHE） fluorescence staining. The mRNA and protein expression of ferroptosis-related genes （ACSL4, SLC7A11, and GPX4） were measured by RT-qPCR and Western blotting. Myocardial apoptosis was evaluated using TUNEL staining. Mitochondrial ATP content was determined, and ultrastructural changes were observed using transmission electron microscopy （TEM）. Results Compared with the sham group, CKD rats showed decreased body weight, increased heart mass （P < 0.05）, reduced SOD, GSH levels, and significantly elevated MDA, Fe2+, and ROS levels （P < 0.05）. ACSL4 expression was markedly upregulated, while SLC7A11 and GPX4 were downregulated （P < 0.05）, accompanied by extensive myocardial apoptosis and decreased ATP content （P < 0.05）. Chronic IS exposure further exacerbated these changes （P < 0.05）, manifesting typical ultrastructural features of ferroptosis, including mitochondrial swelling, cristae rupture, and increased membrane density （P < 0.05）. Fer-1 intervention effectively reversed these alterations, restoring ATP levels, and improving mitochondrial morphology. Conclusion: IS can induce ferroptosis via activation of the mitochondrial pathway, aggravating myocardial oxidative stress and energy metabolism disorders in CKD rats, thereby promoting cardiomyocyte apoptosis. Fer-1 intervention significantly alleviated IS-induced mitochondrial damage and myocardial ferroptosis. -
图 2 各组大鼠体质量、心脏质量比较 （n = 15，$\bar x \pm s $）
A：各组大鼠体质量；B：各组大鼠心脏质量。*P < 0.05；**P < 0.01；***P < 0.001。
Figure 2. Comparison of body weight and heart mass among groups （n = 15，$\bar x \pm s $）
图 3 各组大鼠心肌线粒体SOD活性、GSH含量、MDA、Fe2+及ROS水平比较（×200）
A：各组大鼠心肌线粒体SOD活性；B：各组大鼠心肌线粒体GSH含量；C：各组大鼠心肌线粒体MDA含量；D：各组大鼠心肌线粒体Fe2+含量；E-F：各组大鼠心肌线粒体ROS水平。*P < 0.05；**P < 0.01；***P < 0.001。
Figure 3. Comparison of myocardial mitochondrial SOD activity，GSH content，MDA ，Fe2+ ，and ROS levels among groups （×200）
图 4 各组大鼠心肌线粒体途径铁死亡相关指标mRNA和蛋白表达量比较（$ \bar x \pm s$，n = 6）
A：各组大鼠心肌线粒体ACSL4 mRNA含量；B：各组大鼠心肌线粒体SLC7A11 mRNA含量；C：各组大鼠心肌线粒体GPX4 mRNA含量；D：各组大鼠心肌线粒体ACSL4、SLC7A11、GPX4蛋白印迹图；E：各组大鼠心肌线粒体ACSL4蛋白定量分析；F：各组大鼠心肌线粒体SLC7A11蛋白定量分析；G：各组大鼠心肌线粒体GPX4蛋白定量分析。*P < 0.05；**P < 0.01；***P < 0.001。
Figure 4. Comparison of mRNA and protein expression levels of mitochondrial pathway ferroptosis-related markers in myocardial tissue among groups（$\bar x \pm s $，n = 6）
图 5 各组大鼠心肌组织细胞凋亡情况比较和心肌线粒体凋亡相关指标蛋白表达情况（×200，$ \bar x \pm s$，n = 6）
A：各组大鼠心肌组织细胞凋亡免疫组化图；B：各组大鼠心肌组织细胞凋亡定量分析；C：各组大鼠心肌线粒体Bax、Bcl-2蛋白印迹图；D：各组大鼠心肌线粒体心肌线粒体Bax蛋白定量分析；E：各组大鼠心肌线粒体心肌线粒体Bcl-2蛋白定量分析。*P < 0.05；**P < 0.01；***P < 0.001。
Figure 5. Comparison of myocardial apoptosis and protein expression of mitochondrial apoptosis-related indicators among groups （×200，$\bar x \pm s $，n = 6）
-
[1] Jankowski J, Floege J, Fliser D, et al. Cardiovascular disease in chronic kidney disease: Pathophysiological insights and therapeutic options[J]. Circulation, 2021, 143(11): 1157-1172. doi: 10.1161/CIRCULATIONAHA.120.050686 [2] Lim Y J, Sidor N A, Tonial N C, et al. Uremic toxins in the progression of chronic kidney disease and cardiovascular disease: Mechanisms and therapeutic targets[J]. Toxins (Basel), 2021, 13(2): 142. doi: 10.3390/toxins13020142 [3] Lu P H, Yu M C, Wei M J, et al. The therapeutic strategies for uremic toxins control in chronic kidney disease[J]. Toxins (Basel), 2021, 13(8): 573. doi: 10.3390/toxins13080573 [4] Karbowska M, Hermanowicz J M, Tankiewicz-Kwedlo A, et al. Neurobehavioral effects of uremic toxin-indoxyl sulfate in the rat model[J]. Sci Rep, 2020, 10(1): 9483. [5] Lai Y R, Cheng B C, Lin C N, et al. The effects of indoxyl sulfate and oxidative stress on the severity of peripheral nerve dysfunction in patients with chronic kidney diseases[J]. Antioxidants (Basel), 2022, 11(12): 2350. doi: 10.3390/antiox11122350 [6] Cui Y, Zhang Y, Zhao X, et al. ACSL4 exacerbates ischemic stroke by promoting ferroptosis-induced brain injury and neuroinflammation[J]. Brain Behav Immun, 2021, 93: 312-321. doi: 10.1016/j.bbi.2021.01.003 [7] Jiang P, Zhou L, Zhao L, et al. Puerarin attenuates valproate-induced features of ASD in male mice via regulating Slc7a11-dependent ferroptosis[J]. Neuropsychopharmacology, 2024, 49(3): 497-507. doi: 10.1038/s41386-023-01659-4 [8] Ma T, Du J, Zhang Y, et al. GPX4-independent ferroptosis-a new strategy in disease’s therapy[J]. Cell Death Discov, 2022, 8(1): 434. [9] Li W, Xiang Z, Xing Y, et al. Mitochondria bridge HIF signaling and ferroptosis blockage in acute kidney injury[J]. Cell Death Dis, 2022, 13(4): 308. doi: 10.1038/s41419-022-04770-4 [10] Li H, Ding J, Liu W, et al. Plasma exosomes from patients with acute myocardial infarction alleviate myocardial injury by inhibiting ferroptosis through miR-26b-5p/SLC7A11 axis[J]. Life Sci, 2023, 322: 121649. doi: 10.1016/j.lfs.2023.121649 [11] Tsai L T, Weng T I, Chang T Y, et al. Inhibition of indoxyl sulfate-induced reactive oxygen species-related ferroptosis alleviates renal cell injury in vitro and chronic kidney disease progression in vivo[J]. Antioxidants (Basel), 2023, 12(11): 1931. doi: 10.3390/antiox12111931 [12] Adam R J, Williams A C, Kriegel A J. Comparison of the surgical resection and infarct 5/6 nephrectomy rat models of chronic kidney disease[J]. Am J Physiol Renal Physiol, 2022, 322(6): F639-F654. doi: 10.1152/ajprenal.00398.2021 [13] Karbowska M, Kaminski T W, Znorko B, et al. Indoxyl sulfate promotes arterial thrombosis in rat model via increased levels of complex TF/VII, PAI-1, platelet activation as well as decreased contents of SIRT1 and SIRT3[J]. Front Physiol, 2018, 9: 1623. [14] Kuang H, Wang T, Liu L, et al. Treatment of early brain injury after subarachnoid hemorrhage in the rat model by inhibiting p53-induced ferroptosis[J]. Neurosci Lett, 2021, 762: 136134. doi: 10.1016/j.neulet.2021.136134 [15] Berg A H, Kumar S, Karumanchi S A. Indoxyl sulfate in uremia: An old idea with updated concepts[J]. J Clin Invest, 2022, 132(1): e155860. doi: 10.1172/JCI155860 [16] Cheng T H, Ma M C, Liao M T, et al. Indoxyl sulfate, a tubular toxin, contributes to the development of chronic kidney disease[J]. Toxins (Basel), 2020, 12(11): E684. [17] Takkavatakarn K, Phannajit J, Udomkarnjananun S, et al. Association between indoxyl sulfate and dialysis initiation and cardiac outcomes in chronic kidney disease patients[J]. Int J Nephrol Renovasc Dis, 2022, 15: 115-126. doi: 10.2147/IJNRD.S354658 [18] Persad K L, Lopaschuk G D. Energy metabolism on mitochondrial maturation and its effects on cardiomyocyte cell fate[J]. Front Cell Dev Biol, 2022, 10: 886393. doi: 10.3389/fcell.2022.886393 [19] Venditti P, Di Meo S. The role of reactive oxygen species in the life cycle of the mitochondrion[J]. Int J Mol Sci, 2020, 21(6): E2173. doi: 10.3390/ijms21062173 [20] Sies H. Findings in redox biology: From H2O2 to oxidative stress[J]. J Biol Chem, 2020, 295(39): 13458-13473. doi: 10.1074/jbc.X120.015651 [21] Di Meo S, Venditti P. Evolution of the knowledge of free radicals and other oxidants[J]. Oxid Med Cell Longev, 2020, 2020: 9829176. doi: 10.1155/2020/9829176 [22] Danieli M G, Antonelli E, Piga M A, et al. Oxidative stress, mitochondrial dysfunction, and respiratory chain enzyme defects in inflammatory myopathies[J]. Autoimmun Rev, 2023, 22(5): 103308. [23] Czabotar P E, Garcia-Saez A J. Mechanisms of BCL-2 family proteins in mitochondrial apoptosis[J]. Nat Rev Mol Cell Biol, 2023, 24(10): 732-748. doi: 10.1038/s41580-023-00629-4 [24] Luo X, O’Neill K L, Huang K. The third model of bax/bak activation: A bcl-2 family feud finally resolved?[J]. F1000Res, 2020, 9: F1000FacultyRev-F1000Faculty935. [25] Zhang L, Lu Z, Zhao X. Targeting bcl-2 for cancer therapy[J]. Biochim Biophys Acta Rev Cancer, 2021, 1876(1): 188569. doi: 10.1016/j.bbcan.2021.188569 [26] Wang J, Liu Y, Wang Y, et al. The cross-link between ferroptosis and kidney diseases[J]. Oxid Med Cell Longev, 2021, 2021: 6654887. [27] Zhang X, Li X. Abnormal iron and lipid metabolism mediated ferroptosis in kidney diseases and its therapeutic potential[J]. Metabolites, 2022, 12(1): 58. doi: 10.3390/metabo12010058 -
下载:
点击查看大图
图(6)
计量
- 文章访问数: 68
- HTML全文浏览量: 50
- PDF下载量: 13
- 被引次数: 0
