Effects of piRNA CFAPIR in doxorubicin-induced ferroptosis models of rat and human cardiomyocytes

doi:10.12307/2026.133

Abstract

Abstract: BACKGROUND: Ferroptosis plays a critical role in doxorubicin-induced cardiomyopathy; however, its specific regulatory mechanisms require further elucidation. Piwi-interacting RNA 413 (piRNA413) regulates ferroptosis in doxorubicin-induced cardiomyocytes, designated as cardiac ferroptosis-associated piRNA (CFAPIR). However, the specific regulatory mechanism needs to be further elucidated.
OBJECTIVE: To investigate the role and regulatory mechanism of piRNA CFAPIR in doxorubicin-induced cardiomyocyte ferroptosis and cardiomyopathy.
METHODS: (1) Intraperitoneal injection of doxorubicin was used to induce cardiomyopathy in mice. The myocardium was in situ injected with CFAPIR knocking down lentivirus. The body mass and survival rate of mice were monitored and recorded; cardiac function, heart volume and mass, inflammation, and fibrosis were evaluated. (2) Doxorubicin was used to induce ferroptosis in AC16 cardiomyocytes. CFAPIR inhibitor was transfected into cardiomyocytes. Cell injury and ferroptosis were assessed by measuring ferroptosis markers, intracellular iron level, malondialdehyde content, and reduced glutathione level, along with evaluating mitochondrial dysfunction. Additionally, the effect of CFAPIR on ABCB8 expression levels was analyzed.
RESULTS AND CONCLUSION: (1) The level of CFAPIR was significantly up-regulated in both doxorubicin-induced cardiomyopathy animal model (P < 0.000 1) and the cardiomyocyte ferroptosis model (P < 0.01). (2) At the animal level, CFAPIR knockdown markedly attenuated doxorubicin-induced cardiotoxicity, including the prevention of body mass loss (P < 0.05), improvement in survival rate, preservation of cardiac function (P < 0.01), reduction of cardiac atrophy (P < 0.05), inhibition of elevated lactate dehydrogenase activity (P < 0.05), and mitigation of cardiac fibrosis (P < 0.000 1). (3) At the cellular level, CFAPIR knockdown significantly improved doxorubicin-induced ferroptosis in cardiomyocytes, as evidenced by enhanced cell viability (P < 0.05), reduced lactate dehydrogenase activity (P < 0.01), upregulation of ferroptosis markers xCT (P < 0.01) and glutathione peroxidase 4 (P < 0.001), downregulation of prostaglandin-endoperoxide synthase 2 mRNA expression (P < 0.05), alleviation of iron overload (P < 0.05), decreased malondialdehyde levels (P < 0.05), restoration of reduced glutathione content (P < 0.01), reduced reactive oxygen species accumulation (P < 0.01), and improvement in mitochondrial membrane potential (P < 0.05). (4) CFAPIR knockdown significantly mitigated the doxorubicin-induced downregulation of the iron transport protein ABCB8 (P < 0.05). In conclusion, CFAPIR expression is markedly elevated in both the animal model of doxorubicin-induced cardiomyopathy and the cardiomyocyte ferroptosis model. CFAPIR knockdown significantly alleviates doxorubicin-induced cardiotoxicity and cardiomyocyte ferroptosis, potentially by targeting the mitochondrial iron transport protein ABCB8.

Key words: piRNA, CFAPIR, doxorubicin-induced cardiomyopathy, ferroptosis, mitochondrial dysfunction, iron transporter ABCB8

CLC Number:

Tao Xiangyu, Wang Shuang, Li Yuhan, Cao Jimin, Sun Teng. Effects of piRNA CFAPIR in doxorubicin-induced ferroptosis models of rat and human cardiomyocytes[J]. Chinese Journal of Tissue Engineering Research, 2026, 30(22): 5756-5769.

Figures/Tables 6

2.1 实验动物数量分析实验选用雄性C57BL/6J小鼠68只，全部进入结果。 2.2 在阿霉素诱导的小鼠心脏中CFAPIR水平显著升高阿霉素是常用的临床抗癌药物，在治疗过程中具有严重的心脏毒副作用，会造成心功能障碍和心肌损伤，从而诱发心肌病。为了探究piRNA CFAPIR是否参与阿霉素诱导心肌病的病理过程，构建了体内阿霉素诱导心肌病模型。 2.2.1 心肌病小鼠模型构建成功小鼠腹腔注射累积剂量为16 mg/mL的阿霉素后，通过检测小鼠体质量变化、生存率，以及小鼠心功能和心肌损伤指标评估造模效果。结果显示，与对照组相比，阿霉素处理组小鼠生存率降低；体质量显著减轻(P < 0.000 1)；心脏射血分数(P < 0.01)和短轴缩短率(P < 0.001)显著降低；心脏质量/胫骨长比值显著降低(P < 0.000 1)；心肌细胞横截面积显著减小(P < 0.000 1)；血清中乳酸脱氢酶活性显著增强(P < 0.001)；心脏纤维化程度显著加重(P < 0.001)，见图1-5。以上结果表明，阿霉素诱导心肌病小鼠模型构建成功。 2.2.2 阿霉素诱导心肌病模型中CFAPIR的水平分离小鼠心脏组织并提取其中总RNA，通过qRT-PCR实验检测CFAPIR的水平。结果显示，与生理盐水组相比，阿霉素组小鼠心脏组织中CFAPIR的水平显著升高14倍左右(P < 0.000 1)，见图6。该结果表明CFAPIR可能参与调控阿霉素诱导的心肌病。 2.3 敲低CFAPIR抑制阿霉素诱导的小鼠体质量减轻和生存率降低为了探究CFAPIR在阿霉素诱导心肌病中的作用，实验构建了CFAPIR敲低慢病毒，心脏原位注射CFAPIR敲低慢病毒或其阴性对照慢病毒后，每5 d腹腔注射生理盐水或阿霉素1次，持续2次，同时，每5 d监测并记录一次小鼠的体质量，以及每天观察小鼠的存活情况至实验结束，见图1A。结果显示，与生理盐水组相比，阿霉素组小鼠的体质量明显减轻(P < 0.000 1)，生存率显著降低。然而，与CFAPIR阴性对照+阿霉素组相比，CFAPIR敲低后显著增加了小鼠体质量(P < 0.05)和小鼠的生存率，见图1B，C。以上结果表明，敲低CFAPIR明显改善阿霉素诱导的小鼠体质量减轻和生存率降低。 2.4 敲低CFAPIR改善阿霉素诱导的心功能障碍为了探究CFAPIR在阿霉素诱导的小鼠心功能中的作用，通过超声心动图评价小鼠的心脏功能。结果显示，与生理盐水组相比，阿霉素组小鼠的射血分数(P < 0.01)和短轴缩短率(P < 0.001)显著降低。然而，与"

CFAPIR阴性对照+阿霉素组相比，CFAPIR敲低+阿霉素组小鼠的射血分数和短轴缩短率显著升高(P < 0.01)，见图2。以上结果表明，敲低CFAPIR显著改善了阿霉素诱导的心功能障碍。 2.5 敲低CFAPIR减弱阿霉素诱导的小鼠心脏萎缩为了探究CFAPIR在阿霉素诱导的小鼠心脏结构改变中的作用，测量记录小鼠心脏体积、质量和胫骨长度。结果显示，与生理盐水组相比，阿霉素组小鼠心脏体积明显减小，心脏质量/胫骨长比值显著降低(P < 0.000 1)，而这一现象被CFAPIR敲低显著减弱，见图3A，B。进一步通过心脏组织病理学染色进行验证，苏木精-伊红染色显示小鼠心脏横截面积显著减小，见图3C，苏木精-伊红染色(P < 0.000 1)和麦胚凝集素染色(P < 0.000 1)结果均显示心肌细胞横截面积显著减小，见图3D，E。然而，与CFAPIR阴性对照+阿霉素组相比，CFAPIR敲低后显著抑制了阿霉素诱导的心脏横截面积和心肌细胞横截面积(P < 0.000 1)的减小，见图3C-E。以上结果表明，敲低CFAPIR显著改善了阿霉素诱导的小鼠心脏萎缩和心肌细胞缩小。 2.6 敲低CFAPIR抑制阿霉素诱导的小鼠心脏毒性为了探究CFAPIR在阿霉素诱导心脏毒性中的作用，收集小鼠血清进行乳酸脱氢酶活性检测。结果显示，与生理盐水组相比，阿霉素组小鼠中乳酸脱氢酶活性显著增强(P < 0.000 1)，然而，与阴性对照+阿霉素组相比，CFAPIR敲低后显著抑制了阿霉素诱导的乳酸脱氢酶活性的增强(P < 0.05)，见图4。该结果表明，敲低CFAPIR显著减弱了阿霉素诱导的小鼠心脏毒性。 2.7 敲低CFAPIR减弱阿霉素诱导的小鼠心脏纤维化为了探究CFAPIR在阿霉素诱导小鼠心肌损伤中的作用，通过苏木精-伊红染色、Masson染色检测小鼠心脏组织的纤维化程度，通过天狼猩红染色检测小鼠心脏组织胶原蛋白含量。结果显示，与生理盐水组相比，阿霉素组小鼠的心脏纤维化面积显著增大(P < 0.000 1)，胶原蛋白含量显著增多(P < 0.000 1)。然而，与CFAPIR阴性对照+阿霉素组相比，CFAPIR敲低后显著抑制了阿霉素诱导的心脏纤维化程度加重，显著降低了心脏纤维化面积(P < 0.000 1)和胶原蛋白含量(P < 0.000 1)，见图5A-C。以上结果表明，敲低CFAPIR显著减弱了阿霉素诱导的心脏纤维化。 2.8 在阿霉素诱导的心肌细胞中CFAPIR水平显著升高铁死亡是阿霉素诱导心肌病的重要机制，为了探究CFAPIR调控阿霉素诱导心肌病是否靶向心肌细胞铁死亡，使用3 μmol/L阿霉素处理AC16人心肌细胞24 h，成功构建了阿霉素诱导心肌细胞铁死亡模型，见图7，8。随后检测CFAPIR的水平，结果显示，与对照组相比，阿霉素组细胞中CFAPIR水平显著上调平均50倍左右(P < 0.01)，见图9。以上结果提示CFAPIR可能参与了阿霉素诱导心肌细胞铁死亡的调控。 2.9 敲低CFAPIR减轻阿霉素诱导的心肌细胞损伤为了探究CFAPIR在阿霉素诱导心肌细胞损伤和心肌"

2.11 敲低CFAPIR减弱阿霉素诱导的心肌细胞活性氧积累由于氧化应激是铁死亡过程中发生脂质过氧化的重要因素，活性氧的大量生成促进铁死亡发生。因此，探究了CFAPIR在阿霉素诱导的心肌细胞活性氧积累中的作用。使用DCFH-DA荧光探针检测活性氧水平。结果显示，与对照组相比，阿霉素显著增加了心肌细胞中活性氧水平(P < 0.01)，而这一现象被CFAPIR的敲低显著减弱(P < 0.01)，见图11。 2.12 敲低CFAPIR抑制阿霉素诱导的线粒体功能障碍由于线粒体是铁代谢和活性氧生成的主要场所，二者都是参与铁死亡的关键因素，于是探究了CFAPIR在阿霉素诱导的心肌细胞线粒体功能障碍中的作用。通过JC-1荧光探针检测线粒体膜电位水平。正常细胞的线粒体膜电位较高，JC-1聚集在线粒体基质中，形成聚合物，可以产生红色荧光；当细胞受损线粒体膜电位较低时，JC-1则为单体，产生绿色荧光，因此，JC-1的红/绿荧光强度比值反映线粒体膜电位变化[44]。结果显示，与对照组相比，阿霉素显著减弱了JC-1的红/绿荧光强度比值(P < 0.05)，即线粒体膜电位减小。然而，与CFAPIR阴性对照+阿霉素组相比，CFAPIR敲低+阿霉素组中JC-1的红/绿荧光强度比值显著升高(P < 0.05)，即显著增大了阿霉素降低的线粒体膜电位，见图12。以上结果表明，敲低CFAPIR显著改善了阿霉素诱导的线粒体功能障碍。 2.13 CFAPIR调控铁转运蛋白ABCB8 为了进一步探索CFAPIR调控阿霉素诱导心肌细胞铁死亡和线粒体功能障碍的分子机制，鉴定了CFAPIR的下游靶标。ABCB8是一种线粒体铁转运蛋白，调控线粒体铁外流，有研究报道阿霉素诱导的心脏毒性和心脏铁超载均与ABCB8的耗竭密切相关。在阿霉素诱导心肌细胞铁死亡模型中，CFAPIR是否与ABCB8相互作用尚不清楚，故开展了相关研究。实验结果显示，与对照组相比，阿霉素处理的心肌细胞中ABCB8表达水平显著降低(P < 0.01)，而与CFAPIR阴性对照+阿霉素组相比，CFAPIR敲低显著上调了ABCB8的表达水平(P < 0.05)，见图13。以上结果表明，在阿霉素诱导心肌细胞铁死亡模型中，CFAPIR调控了ABCB8的表达，且可能通过靶向ABCB8发挥调控阿霉素诱导心肌细胞铁死亡和心肌病的作用。"

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