A preliminary study was performed to investigate the staining characteristics of trabecula. endothelial cells with low density lipoprotein (LDL) and acetylated low density lipoprotein (Ac-LDL) labeled with a fluorescent probe, 1, 1`- dioctadecyl-3,3,3`, 3`- tetramethyl-indocarbocyanine perchlorate (Dil). Trabecular endothelial cells revealed a strong fluorescence with Dil-LDL, which was contradictory to the previous results obtained from other types of endothelial cells. These cells also showed a moderate fluorescence with Dil-Ac-LDL. Scleral fibroblasts and keratocytes showed a moderate to strong fluorescence with Dil-LDL and a weak fluorescence with Dil-Ac-LDL. Corneal endothelial cells revealed a very weak background fluorescence with Dil-LDL and a moderate fluorescence with Dil-Ar-LDL. Therefore, these four kinds of cells could not be definitely differentiated depending only on the staining characteristics with Dil-LDL and Dil-Ac-LDt.
Animals ; Cattle ; Endothelium/cytology ; Fluorescent Dyes/*diagnostic use ; Lipoproteins, LDL/*metabolism ; Trabecular Meshwork/*metabolism

As a classic fluorescent detect technique, fluorescence resonance energy transfer (FRET) has been widely used in biological researches. Researchers have developed a series of fluorescence detect probes which were based on FRET. Caspase family plays an important role in apoptosis pathway, especially Caspase-8 which located, at the initial of death receptor mediated apoptosis pathway, whose its activation can trigger subsequent precaspases' activation and lead to apoptosis. So it is of great significance to detect the activation of Caspase-8 in apoptosis assay. In this study, a fluorescent probe based on FRET has been designed which can detect the activity change of Caspase-8 in cells. To identify the effectiveness and specificity of the probe, we measure the Caspase-8 activity under the Caspase-8 specifically activated apoptosis inducer RGD-TRAIL with the flow cytometry FRET detection platform. The results show that the probe can respond to the activity change of Caspase-8 in apoptotic cells, and the change can be quantified rapidly by flow cytometry. The study provides a more efficient and convenient detection method of Caspase-8 activity in living cells.
Apoptosis ; Caspase 8 ; metabolism ; Flow Cytometry ; Fluorescence Resonance Energy Transfer ; Fluorescent Dyes ; Humans

OBJECTIVETo observe the influence of electroacupuncture (EA) on indocyanine green (ICG) metabolism in the liver and the action mechanism of acupuncture for hepatic regulation.

METHODSForty Kunming mice were randomly divided into an EA group and a control group, 20 cases in each one. Combined with classical hepatic metabolism test of ICG, after tail vein injection of ICG, twenty mice were treated with EA at "Zusanli" (ST 36) for 20 min. The ICG fluorescence intensity and distribution in the liver were observed with animal in vivo fluorescence imaging apparatus during 20 min of EA and 60 min of needle withdrawal. Twenty mice, taken as control group, were treated with ICG injection and no EA. The differences of fluorescence intensity and distribution in both groups were compared.

RESULTS(1) There was blocking and gathered fluorescent sign in both groups when ICG was injected immediately. With the extension of observation time, the fluorescence brightness and area were increased until 30 min of injection. This was in accordance with known metabolism activities of ICG in the liver. (2) At 10 min and 20 min of EA and after needle withdrawal, compared with the control group, the fluorescence intensity in the liver of the EA group was weaker and the distribution area was smaller. (3) According to quantitative statistics of fluorescence intensity, at each time point of treatment, the value in the control group was higher than that in the EA group, which appeared regularly all along. As for the total mean difference of ICG fluorescence intensity at all time points, there was statistical difference between the two groups (P < 0.05).

CONCLUSIONEA could reduce fluorescence intensity and lasting time of ICG, indicating that EA accelerates metabolic process of ICG in the liver. Also it is feasible to apply animal in vivo fluorescence imaging technology to the principle research of acupuncture effect.

Animals ; Electroacupuncture ; Female ; Fluorescent Dyes ; chemistry ; metabolism ; Indocyanine Green ; chemistry ; metabolism ; Liver ; chemistry ; metabolism ; Male ; Mice ; Whole Body Imaging

Redox signal transduction, especially the oxidative modification of proein thiols, correlates with many diseases and becomes an expanding research area. However, there was rare method for quick and specific detection of protein thiols and their oxidative modification in living cells. In this article, we review the current chemical strategies for the detection and quantification of protein thiols and related cysteine oxidation. We also look into the future of the development of fluorescent probes for protein thiols and their potential application in the research of reactive cysteine proteomes and early detection of redox-related diseases.
Animals ; Cysteine ; metabolism ; Fluorescent Dyes ; Humans ; Nitrosation ; Oxidation-Reduction ; Proteins ; chemistry ; metabolism ; Reactive Nitrogen Species ; metabolism ; Reactive Oxygen Species ; metabolism ; Sulfenic Acids ; analysis ; Sulfhydryl Compounds ; analysis ; chemistry ; metabolism

OBJECTIVE: To explore the mechanism by which berberine inhibits ferroptosis of mouse hippocampal neuronal cells (HT22). METHODS: Cultured HT22 cells were pretreated with 30 or 60 μmol/L berberine for 2 h before exposure to 0.5 μmol/L erastin for 8 h, and the cell proliferation, intracellular ferric iron level, changes in intracellular reactive oxygen species (ROS) and cell apoptosis were detected using CCK-8, Fe²⁺ fluorescent probe, fluorescent dye (DAPI) and fluorescent probe (H2DCFH-DA). RT-qPCR and Western blotting were used to detect the mRNA and protein expressions of Nrf2, HO-1 and GPX4 in the cells. We further tested the effects of treatments with 2 μmol/L ML385 (a Nrf2 inhibitor), 60 μmol/L berberine and erastin in the cells to explore the protective mechanism of berberine against erastin-induced ferroptosis in the neuronal cells. RESULTS: Treatment with 0.5 μmol/L erastin significantly lowered the viability of HT22 cells (P < 0.05) and increased the production of ROS, cell apoptosis rate and ferric iron level (P < 0.05). Pretreatment with 30 and 60 μmol/L berberine both significantly increased the vitality of erastin-exposed cells (P < 0.05) and lowered the levels of intracellular ROS and ferric iron content (P < 0.05). RT-qPCR and Western blotting showed that berberine obviously promoted the expressions of Nrf2, HO-1 and GPX4 in the cells (P < 0.05), and treatment with ML385 significantly inhibited the Nrf2-HO-1/GPX4 pathway, increased intracellular ROS and ferric iron contents and mitigated the protective effect of berberine against erastin-induced ferroptosis (P < 0.05). CONCLUSION Berberine can inhibit erastin-induced ferroptosis in HT22 cells possibly by activating the Nrf2-HO-1/ GPX4 pathway.
Animals ; Berberine/pharmacology* ; Ferroptosis ; Fluorescent Dyes ; Hippocampus/metabolism* ; Iron/metabolism* ; Mice ; NF-E2-Related Factor 2/metabolism* ; Piperazines ; Reactive Oxygen Species/metabolism*

Objective: To study the underlying mechanism of cadmium-induced apoptosis of mouse spermatocytes (GC-2 spd) . Methods: In March 2021, GC-2 spd cells were exposed to different concentrations of CdCl(2) for 24 hours, namely 5 μmol/L CdCl(2) (low-dose) group and 10 μmol/L CdCl(2) (high-dose) group, and unexposed GC-2 spd cells were used as control group. Mitochondrial morphology was observed in the cells stained with Mito-Track Red CMXRos fluorescent probes by confocal microscopy and the mitochrondrial membrane potential was measured by flow cytometry with JC-1 fluorescent probes. Mitochrondrial proteins, cytosolic proteins and total cellular proteins of GC-2 spd cells were extracted using cell mitochondria isolation kit and RIPA buffer, respectively. The expression of mitochondrial homeostasis regulatory proteins (FIS1 and OPA1), and apoptosis-related proteins (Cytochrome c and cleaved Caspase-3) were examined by Western blot. Results: Compared with the cells in the control group, the relative ratio of JC-1 red/green fluorescence signal in the cells of the low-dose and high-dose CdCl(2) groups decreased significantly (0.740±0.071, 0.570±0.028), with a statistically significant difference (P=0.017, 0.004) ; The morphology of mitochondria changed from long tube to point, and the proportion of cells containing point mitochondria increased significantly (45.1%±3.7% and 25.7%±4.9%), the difference was statistically significant (P=0.005, 0.001) ; The relative expression level of mitochondrial FIS1 in cells of low and high dose CdCl(2) groups was significantly higher (1.271±0.120, 1.693±0.155), the difference was statistically significant (P=0.046, 0.000) ; The relative expression level of OPA1 decreased significantly (0.838±0.050, 0.682±0.040), and the difference was statistically significant (P=0.049, 0.001). Compared with the control group, the relative expression level of cytochrome c protein in the cytoplasm of cells in the low dose group of CdCl(2) was not significantly increased (1.249±0.151), and the difference was not statistically significant (P=0.075). However, the relative expression level in the cytoplasm of cells in the high dose group of CdCl(2) was significantly increased (2.355±0.110), and the difference was statistically significant (P=0.000) ; The relative expression level of Cytochrome c in mitochondria of low and high dose CdCl(2) groups decreased significantly (0.681±0.043, 0.619±0.114), with a statistically significant difference (P=0.004, 0.001) ; Moreover, the level of cleaved Caspase-3 protein in cells gradually increased (5.486±0.544, 11.493±1.739), the difference was statistically significant (P=0.004, 0.000) . Conclusion: Cadmium induced cleaved Caspase-3 mediated apoptosis of GC-2 spd cells via promoting mitochrondrial fission and the release of Cytochrome c from the mitochrondria to the cytosol.
Male ; Mice ; Animals ; Mitochondrial Dynamics ; Caspase 3/metabolism* ; Cadmium/toxicity* ; Cytochromes c/metabolism* ; Fluorescent Dyes ; Apoptosis ; Apoptosis Regulatory Proteins/metabolism* ; Membrane Potential, Mitochondrial

OBJECTIVETo investigate uptake and accumulation of gentamicin by cells in the guinea pig inner ear after intratympanic injection using a fluorescent probe--gentamicin-Texas-red conjunction (GTTR).

METHODSAdult guinea pigs (n = 80) were administered a single dose of GTrR to the middle ear cavity through the intact membrane and survived for 12 h, 24 h, 48 h, 3 d, 4 d, 7 d, 14 d and 28 d. The distribution of GTTR in the cochlear and vestibular cells was observed after staining with phalloidin-alexa-488. Texas Red and DMSO were injected into the tympanum as control.

RESULTSDiffuse staining of gentamicin in the labyrinth was observed initially after local drug administration. At later time point the outer hair cells and sensory cells of vestibular organ were staining more densely than the support cells in the inner ear. The peak level of fluorescent density was reached 3 days after local injection. The GTTR was observed in the infracuticular zone.

CONCLUSIONSGTTR was a potential fluorescent probe to investigate the pharmacokinetics and mechanisms of gentamicin accumulation in local application.

Animals ; Anti-Bacterial Agents ; administration & dosage ; pharmacokinetics ; toxicity ; Ear, Inner ; metabolism ; Fluorescent Dyes ; Gentamicins ; administration & dosage ; pharmacokinetics ; toxicity ; Guinea Pigs ; Hair Cells, Auditory ; metabolism

OBJECTIVETo investigate the binding characteristics of endothelial cell (EC) with LPS free from the participation of serum factors.

METHODSLaser confocal microscope was employed in the observation of the binding of EC with FITC-LPS. The KD and the binding sites of each EC were calculated by radioligand binding assay of receptors (RBA) using [(3)H]-LPS.

RESULTSThe binding of EC with LPS was saturable, time and concentration dependent and it could be competed with overdosed LPS of the same type. The fluorescence mainly distributed in cytoplasm, especially near the nucleus, which could also be stained.

CONCLUSIONSThere might be some specific LPS binding sites existing on ECs and LPS could function intracellularily.

Cells, Cultured ; Dose-Response Relationship, Drug ; Endothelial Cells ; drug effects ; metabolism ; Fluorescein-5-isothiocyanate ; Fluorescent Dyes ; Humans ; Lipopolysaccharides ; metabolism ; Microscopy, Confocal ; Radioligand Assay ; Umbilical Veins ; cytology

The lipase labeled with the fluorescein isothiocyanat (FITC) was immobilized on the derivatives of the polyethylene glycol. The article discussed the effect of factors on the characters of lipase and analyzed the relationships among the activity of lipase, conformation, and fluorescence spectrum while the activity and the fluorescence spectrum of immobilized lipase were determined. The results demonstrated that polyethylene glycol 400-diacrylate could form appropriate network to improve the activity of enzyme. Adding ligand induced the lipase's catalytic conformation to increase the activity twice more than before. The active centre of lipase could be released by the extraction of ligand thus increasing the activity. After immobilization, the stability of labeled lipase improved greatly: immobilized lipases retained more than 70% and 60% of initial activity under conditions of 90 degrees C and strong acid or alkali, respectively. After immersing immobilized lipases into guanidine hydrochloride or urea for 15 days, the lipases retained upwards of 70% activity. The fluorescence spectrum could obviously reflect the changes of the activity and conformation of lipase. The fluorescence intensity was the minimum in the optimal pH and temperature. In the denaturing agent it declined as time passed. These results indicated that the unfolded processes of immobilized lipases are different under different conditions.
Dextrans ; chemistry ; Enzyme Stability ; Enzymes, Immobilized ; chemistry ; metabolism ; Fluorescein-5-isothiocyanate ; analogs & derivatives ; chemistry ; Fluorescent Dyes ; chemistry ; Lipase ; chemistry ; metabolism ; Polyethylene Glycols ; chemistry ; Protein Unfolding ; drug effects

Blood-brain barrier (BBB) is the major obstacle for drug delivery into the central nervous system (CNS). However, there is no ideal model animal for the study of BBB permeability till now. Currently zebrafish (Danio rerio) has emerged as a powerful model organism for the study of vertebrate biology. In this study, the feasibility of using zebrafish as model animal was investigated for BBB permeability by comparing the results of administration of BBB-penetrating peptide and protein to mouse and zebrafish. The results showed that the BBBs of mouse and zebrafish were similar in molecular permeability. Additionally, zebrafish has advantageous features as a model animal, such as small size, fertile and easy to breed. Therefore, it is suggested that zebrafish may be a favored model for the study of BBB permeability.
Animals ; Blood-Brain Barrier ; metabolism ; Brain ; metabolism ; Female ; Fluorescent Dyes ; pharmacokinetics ; Glycoproteins ; pharmacokinetics ; Green Fluorescent Proteins ; pharmacokinetics ; Male ; Mice ; Models, Animal ; Peptide Fragments ; pharmacokinetics ; Permeability ; Rhodamines ; pharmacokinetics ; Tissue Distribution ; Viral Proteins ; pharmacokinetics ; Zebrafish ; metabolism