Zheng Fan, Ma Yeshuo, Ding Jipeng, Huang Shuai, Zhang Shengwang, Huang Xueyan, Feng Bin, Zeng Hongliang, Chen Fei, Zeng Wenbin
Xiangya School of Pharmaceutical Sciences, Central South University, Changsha, 410013, People's Republic of China.
Hunan Key Laboratory of Diagnostic and Therapeutic Drug Research for Chronic Diseases, Central South University, Changsha, 410013, People's Republic of China.
Biomater Res. 2023 Jul 6;27(1):66. doi: 10.1186/s40824-023-00409-3.
Autophagy is a critical self-eating pathway involved in numerous physiological and pathological processes. Lysosomal degradation of dysfunctional organelles and invading microorganisms is central to the autophagy mechanism and essential for combating disease-related conditions. Therefore, monitoring fluctuations in the lysosomal microenvironment is vital for tracking the dynamic process of autophagy. Although much effort has been put into designing probes for measuring lysosomal viscosity or pH separately, there is a need to validate the concurrent imaging of the two elements to enhance the understanding of the dynamic progression of autophagy.
Probe HFI was synthesized in three steps and was developed to visualize changes in viscosity and pH within lysosomes for real-time autophagy tracking. Then, the spectrometric determination was carried out. Next, the probe was applied to image autophagy in cells under nutrient-deprivation or external stress. Additionally, the performance of HFI to monitor autophagy was employed to evaluate acetaminophen-induced liver injury.
We constructed a ratiometric dual-responsive probe, HFI, with a large Stokes shift over 200 nm, dual-wavelength emission, and small background interference. The ratiometric fluorescent signal (R = I /I ) of HFI had an excellent correlation with both viscosity and pH. More importantly, high viscosity and low pH had a synergistic promotion effect on the emission intensity of HFI, which enabled it to specially lit lysosomes without disturbing the inherent microenvironment. We then successfully used HFI to monitor intracellular autophagy induced by starvation or drugs in real-time. Interestingly, HFI also enabled us to visualize the occurrence of autophagy in the liver tissue of a DILI model, as well as the reversible effect of hepatoprotective drugs on this event.
In this study, we developed the first ratiometric dual-responsive fluorescent probe, HFI, for real-time revealing autophagic details. It could image lysosomes with minimal perturbation to their inherent pH, allowing us to track changes in lysosomal viscosity and pH in living cells. Ultimately, HFI has great potential to serve as a useful indicator for autophagic changes in viscosity and pH in complex biological samples and can also be used to assess drug safety.
自噬是一种关键的自我吞噬途径，参与众多生理和病理过程。功能失调的细胞器和入侵微生物的溶酶体降解是自噬机制的核心，也是对抗疾病相关状况所必需的。因此，监测溶酶体微环境的波动对于追踪自噬的动态过程至关重要。尽管在设计分别测量溶酶体粘度或pH值的探针方面已经付出了很多努力，但仍需要验证这两个要素的同时成像，以增强对自噬动态进展的理解。
探针HFI分三步合成，并被开发用于可视化溶酶体内粘度和pH值的变化，以实时追踪自噬。然后进行光谱测定。接下来，将该探针应用于在营养剥夺或外部应激条件下对细胞中的自噬进行成像。此外，利用HFI监测自噬的性能来评估对乙酰氨基酚诱导的肝损伤。
我们构建了一种比率型双响应探针HFI，其斯托克斯位移超过200nm，具有双波长发射且背景干扰小。HFI的比率荧光信号（R = I /I ）与粘度和pH值均具有良好的相关性。更重要的是，高粘度和低pH值对HFI的发射强度具有协同促进作用，使其能够特异性地照亮溶酶体而不干扰其固有微环境。然后，我们成功地利用HFI实时监测饥饿或药物诱导的细胞内自噬。有趣的是，HFI还使我们能够可视化药物性肝损伤（DILI）模型肝组织中自噬的发生，以及保肝药物对该事件的可逆作用。
在本研究中，我们开发了首个用于实时揭示自噬细节的比率型双响应荧光探针HFI。它能够以最小程度干扰溶酶体固有pH值的方式对溶酶体进行成像，使我们能够追踪活细胞中溶酶体粘度和pH值的变化。最终，HFI具有巨大潜力，可作为复杂生物样品中自噬相关粘度和pH值变化的有用指标，还可用于评估药物安全性。
