Dual-Response Functionalized Mitochondrial Fluorescent Probe for a Double Whammy Monitoring of Hypochlorite and Sulfur Dioxide in Heat Shock via Time Scales

【文献详情】 Hongshuai Cao, Feifei Yu, Kun Dou, Rui Wang, Yanlong Xing, Xianzhu Luo, and Fabiao Yu. Dual-Response Functionalized Mitochondrial Fluorescent Probe for a Double Whammy Monitoring of Hypochlorite and Sulfur Dioxide in Heat Shock via Time Scales. Anal. Chem. 2024, https://doi.org/10.1021/acs.analchem.4c05488

Abstract

Heat shock seriously affects the normal functioning of an organism and can lead to damage and even death in severe cases. To prevent or treat heat shock-related diseases, we require a better understanding of the mechanism of thermocytotoxicity. Here, we designed a functionalized dual-response fluorescent probe (HCy-SO2-HClO) that could individually or simultaneously detect hypochlorous acid (HClO) and sulfur dioxide (SO2) without interfering with each other and achieved the simultaneous tracing of both during the heat shock process for the first time. The introduction of the sulfonate group greatly increased the water solubility of the probe. Furthermore, the probe could effectively accumulate in the mitochondrial region. HCy-SO2-HClO could respond to HClO and SO2 within 10 s and 20 min, respectively, realizing a double whammy detection of both on the time scale. HCy-SO2-HClO exhibited high specificity and sensitivity for HClO and SO2. The highly biocompatible probe HCy-SO2-HClO successfully achieved the detection of endogenous and exogenous SO2 and HClO in living cells and in zebrafish. Moreover, the simultaneous detection of HClO and SO2 in heat shock cells and mouse intestines was realized for the first time. This probe has achieved the detection of dual markers, which enhanced the accuracy and precision of disease detection and could serve as an effective research tool to prevent heat stroke-related diseases.

Heat shock is an imbalance in thermoregulation caused by exposure to high temperatures, which in turn causes dysfunction of the central nervous and circulatory systems. Moreover, if heat shock is not treated in a timely manner, it may cause convulsions, kidney damage, or even death.  Although there are heat shock proteins in the body, individuals with low immunity or certain genetic diseases are prone to heat stroke, which can have a mortality rate of approximately 40%.  Therefore, understanding the pathogenesis of heat shock is critical. Currently, some reports demonstrate that mitochondrial dysfunction is induced when a person is exposed to high temperatures, leading to the disruption of intracellular redox homeostasis. Despite the severe impact of heat shock on health, research on its specific pathogenesis is relatively scarce, especially in terms of mitochondrial dysfunction and the disruption of intracellular redox homeostasis.

Hypochlorous acid (HClO), as a key intracellular active substance, participates in intracellular signal transduction and affects the growth, differentiation, and reproduction of cells. In addition, it is also an important component of the body’s immune system, providing an effective defense against bacteria and viruses.  However, an imbalance of intracellular HClO may trigger a series of diseases, such as inflammatory, neurological, cardiovascular, and cancer.Several recent reports exhibit that HClO is strongly associated with heat stroke (an inflammation-associated disease),  so investigating the important role that HClO plays in the balance of cellular redox homeostasis may help to understand heat shock diseases in greater depth. In contrast, sulfur dioxide (SO2), an indispensable intracellular antioxidant, assumes a crucial function in maintaining cellular homeostasis, preserving vasodilation, regulating cardiovascular disease, and combating blood pressure.  However, abnormal SO2 not only may cause respiratory illnesses, for instance, bronchitis and chronic obstructive pulmonary disease, but also may lead to organ damage. Therefore, the real-time measurement of SO2 in organisms is helpful in understanding its physiological role. Although some research has correlated SO2 or HClO with heat shock diseases,  simultaneous studies of both in heat shock diseases have not been reported. Therefore, we hope to further explore the link between intracellular redox homeostasis and heat shock through the fluctuation of intracellular HClO and SO2 levels in order to better prevent or treat heat shock diseases.

Currently, numerous methods for detecting HClO and SO2 have been developed, such as chromatography, colorimetry, and electrochemistry, among others.  Although these methods are able to accurately determine changes in HClO and SO2 levels, they are not well-suited for real-time detection in biological systems due to their lengthy preprocessing or analysis requirements and the potential for damaging biological tissues. Fluorescence imaging technology, with its high sensitivity, high resolution, and capability for noninvasive real-time detection of specific substances within biological systems, has gained widespread popularity.  Nowadays, fluorescence imaging technology is widely applied in biomedicine, disease diagnosis, surgical navigation, and environmental science.  Compared with single-response fluorescent probes, multiresponse fluorescent probes can obtain more effective information and avoid false-positive signals, thus improving the accuracy of detection and revealing the role of relevant substances in diseases, which is significant in promoting the use of fluorescent probes in biomedical fields.  Recently, several reviews have summarized the trends in dual-response probes.  Also, fluorescent probes for the dual response of HClO and SO2 have been developed.  However, most of the probes are not capable of detection both individually and simultaneously without interference.

Here, we proposed a mitochondria-targeted dual-responsive fluorescent probe (HCy-SO2-HClO) aimed at visualizing intracellular HClO and SO2. The probe was capable of efficiently and simultaneously monitoring HClO and SO2 through two different fluorescence channels across various time scales, effectively avoiding the problem of inaccurate detection of one substance due to the consumption of the probe by another substance. HCy-SO2-HClO exhibited high sensitivity and specificity for these two substances while also possessing excellent biocompatibility. Furthermore, we further delved into the interrelationship between intracellular redox homeostasis and the heat shock response via monitoring the dynamic changes of HClO and SO2. Notably, we successfully achieved the dual monitoring of HClO and SO2 for the first time in heat shock-treated cells and mouse models, suggesting that these two substances could serve as key biomarkers for heat shock. This finding not only provided important clues for insights into the biological mechanisms of heat shock but also offered a scientific basis and potential therapeutic strategies to prevent or treat heat shock-related diseases.

热休克是暴露在高温下引起的体温调节失衡，进而导致中枢神经和循环系统功能障碍。而且，如果热休克治疗不及时，可能会引起惊厥、肾脏损害，甚至死亡。虽然体内存在热休克蛋白，但免疫力低下或患有某些遗传病的个体易发生中暑，其死亡率可达40%左右。因此，了解热休克的发病机制至关重要。目前有研究表明高温暴露可诱发线粒体功能障碍，导致细胞内氧化还原稳态被破坏。尽管热休克对健康造成严重影响，但其具体发病机制的研究相对匮乏，尤其是在线粒体功能障碍和细胞内氧化还原稳态破坏方面。

次氯酸（HClO）作为一种关键的细胞内活性物质，参与细胞内信号转导，影响细胞的生长、分化和繁殖。此外，它也是人体免疫系统的重要组成部分，有效防御细菌和病毒。然而，细胞内HClO失衡可能引发一系列疾病，如炎症、神经、心血管和癌症等。近年来的研究表明，HClO与热射病（一种炎症相关性疾病）密切相关，因此研究HClO在细胞氧化还原稳态平衡中的重要作用有助于更深入地了解热射病。相比之下，细胞内不可或缺的抗氧化剂二氧化硫（SO₂）在维持细胞稳态、维持血管舒张、调节心血管疾病和抗血压方面发挥着至关重要的作用。然而，SO₂异常不仅可能引起支气管炎、慢性阻塞性肺疾病等呼吸系统疾病，还可能导致器官损伤。因此，对生物体中SO₂的实时测量有助于了解其生理作用。虽然有一些研究将SO₂或HClO与热射病联系起来，但在热射病中两者同时进行的研究尚未见报道。因此，海南医科大学于法标教授团队希望通过细胞内HClO和SO₂水平的波动，进一步探索细胞内氧化还原稳态与热休克之间的联系，以便更好地预防或治疗热休克疾病。目前已开发出多种检测HClO和SO₂的方法，如色谱法、比色法、电化学法等。虽然这些方法能够准确地确定HClO和SO₂水平的变化，但由于其漫长的预处理或分析要求以及对生物组织的潜在损害，它们不适合在生物系统中进行实时检测。荧光成像技术以其高灵敏度、高分辨率以及对生物系统内特定物质的无创实时检测能力而受到广泛欢迎。目前，荧光成像技术在生物医学、疾病诊断、手术导航和环境科学等领域得到了广泛应用。与单响应荧光探针相比，多响应荧光探针可以获得更有效的信息，避免假阳性信号，从而提高检测的准确性，揭示相关物质在疾病中的作用，对促进荧光探针在生物医学领域的应用具有重要意义。近年来，多项综述总结了双反应探针的发展趋势。此外，还开发了用于HClO和SO₂双重反应的荧光探针。然而，大多数探针都不能在不受干扰的情况下单独或同时检测。海南医科大学于法标教授团队提出了一种线粒体靶向的双响应荧光探针（HCy-SO₂-HClO），旨在可视化细胞内的HClO和SO₂。该探针能够在不同时间尺度上通过两个不同的荧光通道高效、同时监测HClO和SO₂，有效避免了由于探针被另一种物质消耗而导致一种物质检测不准确的问题。HCy-SO₂-HClO对这两种物质具有较高的敏感性和特异性，同时具有良好的生物相容性。此外，通过监测HClO和SO₂的动态变化，进一步探讨了细胞内氧化还原稳态与热休克反应之间的相互关系。值得注意的是，首次在热休克处理的细胞和小鼠模型中成功实现了HClO和SO₂的双重监测，表明这两种物质可以作为热休克的关键生物标志物。这一发现不仅为深入了解热休克的生物学机制提供了重要线索，也为预防或治疗热休克相关疾病提供了科学依据和潜在的治疗策略。

图1 探针的设计及作用机制

图2 光物理性质

图3 HeLa细胞中次氯酸和SO₂的成像

图4 探头的共定位实验

图5 热休克细胞中HClO和SO₂的荧光图像

图6 中暑小鼠小肠的荧光成像

总结：本研究构建了基于半花菁的线粒体靶向荧光探针HCy-SO₂-HClO，可实现HClO和SO₂在时间尺度上的一次性监测。该探针对HClO和SO₂均具有较高的特异性和敏感性。通过利用两者响应的时间差异实现对两者的有效检测，避免了HCy-SO₂-HClO的消耗而无法准确有效地检测其他物质。该探针不仅实现了活细胞内源性和外源性HClO和SO₂的检测，而且实现了热休克小鼠模型中HClO和SO₂的示踪，可作为预防或诊断热休克的有价值的标志物。通过检测HClO和SO₂，探究细胞氧化还原稳态与热休克的关系，为热休克相关疾病的早期预防或治疗提供有效策略。

Conclusions

We constructed mitochondria-targeted fluorescent probe HCy-SO2-HClO based on hemicarpocyanine, which could achieve one-shot monitoring of HClO and SO2 on a time scale. The probe exhibited a high specificity and sensitivity for both HClO and SO2. The effective detection of both was realized by leveraging the temporal differences in their responses, avoiding the depletion of HCy-SO2-HClO without the ability to accurately and effectively detect the other substance. The probe not only realized the detection of endogenous and exogenous HClO and SO2 in living cells but also achieved the tracking of HClO and SO2 in the mouse model of heat shock, which could be used as a valuable marker to prevent or diagnose heat shock. Through the detection of HClO and SO2, we explored the relationship between cellular redox homeostasis and heat shock, which provided an effective strategy for the early prevention or treatment of heat shock-related diseases.

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