Near-Infrared in and out: Observation of Autophagy during Stroke via a Lysosome-Targeting Two-Photon Viscosity-Dependent Probe.

Fluorescence imaging using probes with two-photon excitation and near-infrared emission is currently the most popular in situ method for monitoring biological species or events, with a large imaging depth, low background fluorescence, low optical damage, and high spatial and temporal resolution. Nevertheless, current fluorescent dyes with near-infrared emission still have some disadvantages such as poor water solubility, low fluorescence quantum yield, and small two-photon absorption cross sections. These drawbacks are mainly caused by the structural characteristics of dyes with large conjugation surfaces but lacking strong and rigid structures. Herein, a lysosome-targeted and viscosity-sensitive probe (NCIC-VIS) is designed and synthesized. The protonation of morpholine not only helps anchor NCIC-VIS to the lysosome but also significantly enhances its water solubility. More importantly, its viscosity can increase the rigid structure of NCIC-VIS, which will improve the fluorescence quantum yield and the two-photon absorption cross section due to the imposed restrictions on molecular torsion. Based on the abovementioned characteristics, the real-time imaging of cellular autophagy (could increase the viscosity of lysosomes) was realized using NCIC-VIS. The results demonstrated that the level of autophagy was significantly enhanced in mice during stroke, while the inhibition of oxidative stress significantly reduced the degree of autophagy. The study corroborates that oxidative stress induced by stroke can lead to the development of autophagy.

An ultrasensitive polarity-specific two-photon probe for revealing autophagy in live cells during scrap leather-induced neuroinflammation process.

A polarity-sensitive fluorescence probe AMN was developed to demonstrate the role of autophagy inhibitory drugs in the process of leather residue-induced neuroinflammation, promoting the knowledge of the relationship between autophagy and neuroinflammation. AMN showed a turn-on fluorescent signal in the process of autophagy inhibition via two-photon confocal imaging, which is different from the current popular autophagy probes. Therefore, AMN can offer high-sensitive imaging analysis of the autophagy inhibition process to better understand the role of autophagy in the process of neuroinflammation. The model of scrap leather-induced neuroinflammation using PC12 cells demonstrated that neuroinflammation can induce autophagy by releasing reactive oxygen species (ROS), and autophagy can alleviate neuroinflammation significantly via ROS scavenging.

Observation of inflammation-induced mitophagy during stroke by a mitochondria-targeting two-photon ratiometric probe.

This study reports the development of a new, pH-sensitive, mitochondria-targeting two-photon ratiometric probe (Mito-BNO) for real-time tracking of mitophagy, a process that can be accelerated in brain tissue during stroke. Mito-BNO shows excellent capability for mitochondrial localisation (Pearson's correlation coefficient, r = 0.91), and can also effectively distinguish mitochondria from other subcellular organelles such as lysosomes and the endoplasmic reticulum (r = 0.40 and r = 0.33, respectively). Meanwhile, a rewarding pKa value (5.23 ± 0.03) and the pH reversibility suggest that Mito-BNO can track mitophagy in real time via confocal imaging. Most importantly, the relationship between mitophagy and neuroinflammation during stroke has been successfully demonstrated by evaluating the fluorescence of PC12 cells stained with Mito-BNO during an oxygen-glucose deprivation/reperfusion (OGD/R) process with and without anti-inflammatory treatment. The results indicate that the occurrence of mitophagy during stroke is caused by oxidative stress induced by neuroinflammation. This study will help further understanding stroke pathogenesis, can provide potential new targets for early diagnosis and treatment, and can also help to develop therapeutic drugs for stroke.

Near infrared in and out: Deep imaging for scrap leather induced autophagy in vivo by an ultrasensitive two-photon polarity probe.

As one of the important means for eukaryotic cells to maintain homeostasis, autophagy allows for transporting deformed biomacromolecules and damaged organelles to lysosome for digestion and degradation. The process of autophagy entails the merging of autophagosomes and lysosomes, culminating in the breakdown of biomacromolecules. This, in turn, leads to a change in lysosomal polarity. Therefore, fully understanding the changes of lysosomal polarity during autophagy is of significance to the study of membrane fluidity and enzymatic reaction. However, the shorter emission wavelength has greatly damaged the imaging depth, thus seriously limiting its biological application. Therefore, in this work, a near infrared in and out lysosome-targeted polarity-sensitive probe NCIC-Pola was developed. The fluorescence intensity of NCIC-Pola showed an approximate 1160-fold increase when the polarity decreased under two-photon excitation (TPE). In addition, the excellent fluorescence emission wavelength (692 nm) enabled the deep imaging analysis of scrap leather induced autophagy in vivo.

Imaging of hypochlorous acid in mitochondria using an asymmetric near-infrared fluorescent probe with large Stokes shift.

Small-molecule near-infrared (NIR) imaging facilitates deep tissue penetration, low autofluorescence, non-invasive visualization, and a relatively simple operation. As such it has emerged as a popular technique for tracking biological species and events. However, the small Stokes shift of most NIR dyes often results in a low signal-to-noise ratio and self-quenching due to crosstalk between the excitation and emission spectra. With this research, we developed a NIR-based fluorescent probe WD-HOCl for hypochlorous acid (HOCl) detection using the NIR dye TJ730 as the fluorophore, which exhibits a large Stokes shift of 156 nm, with no crosstalk between the excitation and emission spectra. It contains acyl hydrazide as the responsive group and a pyridinium cation as the mitochondria-targeting group. The fluorescence intensity of WD-HOCl was enhanced by 30.1-fold after reacting with HOCl. Imaging studies performed using BV-2 cells indicated that WD-HOCl could be used for endogenous HOCl detection and imaging in living cells exposed to glucose and oxygen deprivation/reperfusion. Finally, we demonstrated that inhibiting the expression of NOX2 reduced the HOCl levels and the severity of oxidative stress during stroke in a mouse model.

Near-infrared fluorescent probe for hydrogen sulfide: high-fidelity ferroptosis evaluation in vivo during stroke.

Ferroptosis is closely associated with cancer, neurodegenerative diseases and ischemia-reperfusion injury and the detection of its pathological process is very important for early disease diagnosis. Fluorescence based sensing technologies have become excellent tools due to the real-time detection of cellular physiological or pathological processes. However, to date the detection of ferroptosis using reducing substances as markers has not been achieved since the reducing substances are not only present at extremely low concentrations during ferroptosis but also play a key role in the further development of ferroptosis. Significantly, sensors for reducing substances usually consume reducing substances, instigating a redox imbalance, which further aggravates the progression of ferroptosis. In this work, a H2S triggered and H2S releasing near-infrared fluorescent probe (HL-H2S) was developed for the high-fidelity in situ imaging of ferroptosis. In the imaging process, HL-H2S consumes H2S and releases carbonyl sulfide, which is then catalyzed by carbonic anhydrase to produce H2S. Importantly, this strategy does not intensify ferroptosis since it avoids disruption of the redox homeostasis. Furthermore, using erastin as an inducer for ferroptosis, the observed trends for Fe2+, MDA, and GSH, indicate that the introduction of the HL-H2S probe does not exacerbate ferroptosis. In contrast, ferroptosis progression was significantly promoted when the release of H2S from HL-H2S was inhibited using AZ. These results indicate that the H2S triggered and H2S releasing fluorescent probe did not interfere with the progression of ferroptosis, thus enabling high-fidelity in situ imaging of ferroptosis.

Synergistic anticancer effect of exogenous wild-type p53 gene combined with 5-FU in human colon cancer resistant to 5-FU in vivo.

AIM: To investigate the anticancer effect of a recombinant adenovirus-mediated p53 (rAd-p53) combined with 5-fluorouracil (5-FU) in human colon cancer resistant to 5-FU in vivo and the mechanism of rAd-p53 in reversal of 5-FU resistance. METHODS: Nude mice bearing human colon cancer SW480/5-FU (5-FU resistant) were randomly assigned to four groups (n = 25 each): control group, 5-FU group, rAd-p53 group, and rAd-p53 + 5-FU group. At 24 h, 48 h, 72 h, 120 h and 168 h after treatment, 5 mice were randomly selected from each group and sacrificed using an overdose of anesthetics. The tumors were removed and the protein expressions of p53, protein kinase C (PKC), permeability-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1) (Western blot) and apoptosis (TUNEL) were determined. RESULTS: The area ratios of tumor cell apoptosis were larger in the rAd/p53 + 5-FU group than that in the control, 5-FU and rAd/p53 groups (P < 0.05), and were larger in the rAd/p53 group than that of the control group (P < 0.05) and the 5-FU group at more than 48 h (P < 0.05). The p53 expression was higher in the rAd/p53 and the rAd/p53 + 5-FU groups than that of the control and 5-FU groups (P < 0.05), and were higher in the rAd/p53 + 5-FU group than that of the rAd/p53 group (P < 0.05). Overexpression of PKC, P-gp and MRP1 was observed in the 5-FU and control groups. In the rAd/p53 + 5-FU group, the expression of P-gp and MRP1 was lower that of the control and 5-FU groups (P < 0.05), and the expression of PKC was lower than that of the control, 5-FU and rAd/p53 groups at more than 48 h (P < 0.05). In the rAd/p53 group, the expression of P-gp and MRP1 was lower that of the control and 5-FU groups at more than 48 h (P < 0.05), and the expression of PKC was lower than that of the control and 5-FU groups at more than 120 h (P < 0.05). CONCLUSION: 5-FU combined with rAd-p53 has a synergistic anticancer effect in SW480/5-FU (5-FU resistance), which contributes to reversal of 5-FU resistance.