Crosstalk among Oxidative Stress, Autophagy, and Apoptosis in the Protective Effects of Ginsenoside Rb1 on Brain Microvascular Endothelial Cells: A Mixed Computational and Experimental Study.

OBJECTIVE: Brain microvascular endothelial cells (BMECs) were found to shift from their usually inactive state to an active state in ischemic stroke (IS) and cause neuronal damage. Ginsenoside Rb1 (GRb1), a component derived from medicinal plants, is known for its pharmacological benefits in IS, but its protective effects on BMECs have yet to be explored. This study aimed to investigate the potential protective effects of GRb1 on BMECs. METHODS: An in vitro oxygen-glucose deprivation/reperfusion (OGD/R) model was established to mimic ischemia-reperfusion (I/R) injury. Bulk RNA-sequencing data were analyzed by using the Human Autophagy Database and various bioinformatic tools, including gene set enrichment analysis (GSEA), Gene Ontology (GO) classification and enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, protein-protein interaction network analysis, and molecular docking. Experimental validation was also performed to ensure the reliability of our findings. RESULTS: Rb1 had a protective effect on BMECs subjected to OGD/R injury. Specifically, GRb1 was found to modulate the interplay between oxidative stress, apoptosis, and autophagy in BMECs. Key targets such as sequestosome 1 (SQSTM1/p62), autophagy related 5 (ATG5), and hypoxia-inducible factor 1-alpha (HIF-1α) were identified, highlighting their potential roles in mediating the protective effects of GRb1 against IS-induced damage. CONCLUSION: GRbl protects BMECs against OGD/R injury by influencing oxidative stress, apoptosis, and autophagy. The identification of SQSTM1/p62, ATG5, and HIF-1α as promising targets further supports the potential of GRb1 as a therapeutic agent for IS, providing a foundation for future research into its mechanisms and applications in IS treatment.

Extraordinary acoustic transmission through annuluses in air and its applications in acoustic beam splitter and concentrator.

We report an extraordinary acoustic transmission through two layer annuluses made of metal cylinders in air both numerically and experimentally. The effect arises from the enhancement and reconstruction of the incident source induced by different Mie-resonance modes of the annuluses. The proposed system takes advantages of the consistency in the waveform between the input and output waves, the high amplitude amplification of output waves, and the easy adjustment of structure. More interestingly, we investigate the applications of the extraordinary acoustic transmission in the acoustic beam splitter and acoustic concentrator. Our finding should have an impact on ultrasonic applications.

Mitochondrial Q cycle-derived superoxide and chemiosmotic bioenergetics.

We examined the intrinsic relation between two interdependent and interacted processes, namely, chemiosmotic energy coupling partition and redox signaling involved in mitochondrial respiration. The following aspects of research were conducted and discussed: generation sites and release sidedness of superoxide from the Q cycle of complex III of the mitochondrial respiratory chain; the different physiological roles of PMF components, DeltaPsi and DeltapH (DeltapH(S)), of the Q cycle in mitochondrial superoxide generating and partitioning; and direct feedback effects of Q cycle-derived O(2)(*-) on PMF energy partition through its interaction with protons in DeltapH(S) to form HO(2)(*), leading to decreasing DeltapH(S) and ATP synthesis due to its increasing effects of basic proton leak of mitochondria. The present experimental data give new evidence for our hypothesis of reactive oxygen species cycle cooperation with Q cycle and H(+) cycle in respiratory chain in keeping PMF energy partition and its equilibrium with redox signaling regulation of mitochondrial respiration.

Evidence for DeltapH surface component (DeltapH(S)) of proton motive force in ATP synthesis of mitochondria.

BACKGROUND: One of the central debates in membrane bioenergetics is whether proton-dependent energy coupling mechanisms are mediated exclusively by protonic transmembrane electrochemical potentials, as delocalized pmf, DeltamicroH(+), or by more localized membrane surface proton pathways, as interfacial pmf, DeltamicroH(S). METHODS: We measure pH(S) in rat liver mitoplasts energized by respiration or ATP hydrolysis by inserting pH sensitive fluorescein-phosphatidyl-ethanolamine(F-PE) into mitoplast surface. RESULTS: In the presence of rotenone and Ap5A, succinate oxidation induces a bi-phasic interfacial protonation on the mitoplast membranes, a fast phase followed by a slow one, and an interfacial pH decrease of 0.5 to 0.9 pH units of mitoplast with no simultaneous pH changes in the bulk. Antimycin A, other inhibitors or uncouplers of mitochondrial respiration prevent the decrease of mitoplast pH(S), supporting that DeltamicroH(S) is dependent and controlled by energization of mitoplast membranes. A quantitative assay of ATP synthesis coupled with pH(S) of mitoplasts oxidizing succinate with malonate titration shows a parallel correlation between ATP synthesis, State 4 respiration and pH(S), but not with Psi(E). GENERAL SIGNIFICANCE: Our data substantiate pH(S) as the primary energy source of pmf for mitochondrial ATP synthesis. Evidence and discussion concerning the relative importance and interplay of pH(S) and Psi(E) in mitochondrial bioenergetics are also presented.

A study on permeability transition pore opening and cytochrome c release from mitochondria, induced by caspase-3 in vitro.

We recently described that there is a feedback amplification of cytochrome c release from mitochondria by caspases. Here we investigated how caspases impact on mitochondria to induce cytochrome c release and found that recombinant caspase-3 induced opening of permeability transition pore and reduction of membrane potential in vitro. These events were inhibited by Bcl-xL, cyclosporin A and z-VAD.fmk. Moreover, caspase-3 stimulated the rate of mitochondrial state 4 respiration, superoxide production and NAD(P)H oxidation in a Bcl-xL- and cyclosporin A-inhibitable manner. These results suggest that caspase-3 induces cytochrome c release by inducing permeability transition pore opening which is associated with changes in mitochondrial respiration and redox potential.

[Determination of reactive oxygen species in mitochondria by fluorometric probe].

AIM AND METHODS: Based on the reaction that 2',7'-dichlorodihydrofluorescein (DCFH) can be oxidized by reactive oxygen species (ROS) to yield the highly fluorescent 2',7'-dichlorofluorescin (DCF), ROS production in mitochondria can be observed dynamically as well as quantified directly by spectrofluorometer. RESULTS AND CONCLUSION: DCF fluorescence showed linear increase in State 4 mitochondria, which suggest ROS produced at constant rate. The slopes of the linear increase in fluorescence with time were computed performing a linear regression that took into account all relevant data points in selected time windows. The slopes were proportional to ROS production in mitochondria. Addition of sodium azide and malonic acid increased and decreased the rate of ROS production respectively during measurement. DCF fluorescence varied linearly with increasing concentration of mitochondria, which showed quantitative relations in definite range. Repeated measures showed low coefficients of variation. This method is reliable and efficient for determining ROS in mitochondria.

Electron Leak Causes Proton Leak in Skeletal Muscle Mitochondria in Exercise-induced Fatigue.

Using an animal model of exercise-induced fatigue by incremental exercise to exhaustion on a motor-driven treadmill, the changes of the mitochondrial proton leak and electron leak in mitochondria of skeletal muscle from rats immediately after exhaustive exercise were observed. The results showed that, in exercise-induced fatigue conditions, superoxide anions of rat skeletal muscle mitochondria increased State 4 respiration rate significantly accelerated and respiration control ratio clearly decreased. Results show that, in the condition of exercise-induced fatigue, the increased mitochondrial proton leak and electron leak may be important factors that cause the decrease in coupling of oxidative phosphorylation. Our results provide evidence supporting a hypothesis that electron leak causes proton leak in mitochondria.