OBJECTIVE: To explore the regulation of mitochondria on platelet apoptosis and activation, and the relationship between platelet apoptosis and activation. METHODS: Platelets were isolated from peripheral venous blood of healthy volunteers. Cyclosporin A (CsA), which has a protective effect on the function of platelet mitochondria, BAPTA, which can chelate calcium ions across membranes in platelets, and NAC, an antioxidant that reduces the level of intracellular reactive oxygen species, were selected for coincubation with washed platelets, respectively. By flow cytometry, platelet aggregator was used to detect the changes of platelet mitochondrial function and platelet activation indexes after different interventions. RESULTS: H89, staurosporine, and A23187 led to platelet mitochondrial abnormalities, while CsA could effectively reverse the decline of platelet mitochondrial membrane potential caused by them. Antioxidant NAC could reverse platelet mitochondrial damage correspondingly, and completely reverse platelet shrinkage and phosphatidylserine eversion induced by H89. BAPTA, prostaglandin E1, acetylsalicylic acid and other inhibitors could not reverse the decline of platelet mitochondrial membrane potential. CONCLUSION Mitochondrial function plays an important role in platelet apoptosis and activation. Abnormal mitochondrial function causes the imbalance of reduction/oxidation state in platelets, which leads to platelet apoptosis. Platelet apoptosis and activation are independent signal processes.
Humans ; Blood Platelets/metabolism* ; Antioxidants/pharmacology* ; Mitochondria/physiology* ; Platelet Activation ; Apoptosis ; Membrane Potential, Mitochondrial ; Reactive Oxygen Species/pharmacology*

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

OBJECTIVE: To explore the expression of microRNA-132 (miR-132) and its potential role in the development of atherosclerosis (AS). METHODS: Thirty AS samples and 30 samples of normal peripheral vessels were collected from atherosclerotic patients undergoing peripheral angiostomy in our hospital for detecting the expression level of miR-132 using RT-qPCR. The expression of miR-132 in human umbilical vein endothelial cells (HUVEC) was up-regulated by liposome transfection, and intracellular reactive oxygen species (ROS), localization relationship between ROS and mitochondria, functional changes of mitochondrial reactive oxygen superoxide species (mtROS), mitochondrial membrane potential (MMP) and opening of mitochondrial permeability transition pore (mPTP) were analyzed by flow cytometry and laser confocal microscopy. The activity of mitochondrial redox respiratory chain complex (type I, II, III, IV and V) in HUVECs was detected using ELISA, and the expression levels of key iron death proteins were detected with Western blotting. RESULTS: RT-qPCR results showed that miR-132 was significantly up-regulated in atherosclerotic plaques compared with normal vascular samples (P < 0.001). Compared with control HUVECs, HUVECs overexpressing miR-132 showed a significantly increased level of intracellular ROS (P < 0.001), and most of ROS was colocalized with mitochondria. HUVECs overexpressing miR-132 also showed significantly decreased MMP (P < 0.001) and obviously increased mtROS (P < 0.001) and opening of mPTP (P < 0.001), which led to mitochondrial REDOX respiratory chain stress disorder. The key iron death protein GPX4 was significantly down-regulated and the oxidized protein NOX4 was significantly increased in miR-132-overexpressing HUVECs (P < 0.001). CONCLUSION MiR-132 promotes atherosclerosis by inducing mitochondrial oxidative stress-mediated ferroptosis, which may serve as a promising therapeutic target for AS.
Apoptosis ; Atherosclerosis/genetics* ; Ferroptosis ; Human Umbilical Vein Endothelial Cells/metabolism* ; Humans ; Membrane Potential, Mitochondrial ; MicroRNAs/metabolism* ; Mitochondria/metabolism* ; Oxidation-Reduction ; Oxidative Stress ; Reactive Oxygen Species/metabolism*

The limited treatment options for advanced prostate cancer (PCa) lead to the urgent need to discover new anticancer drugs. Mannose, an isomer of glucose, has been reported to have an anticancer effect on various tumors. However, the anticancer effect of mannose in PCa remains unclear. In this study, we demonstrated that mannose inhibits the proliferation and promotes the apoptosis of PCa cells in vitro, and mannose was observed to have an anticancer effect in mice without harming their health. Accumulation of intracellular mannose simultaneously decreased the mitochondrial membrane potential, increased mitochondrial and cellular reactive oxygen species (ROS) levels, and reduced adenosine triphosphate (ATP) production in PCa cells. Mannose treatment of PCa cells induced changes in mitochondrial morphology, caused dysregulated expression of the fission protein, such as fission, mitochondrial 1 (FIS1), and enhanced the expression of proapoptotic factors, such as BCL2-associated X (Bax) and BCL2-antagonist/killer 1 (Bak). Furthermore, lower expression of mannose phosphate isomerase (MPI), the key enzyme in mannose metabolism, indicated poorer prognosis in PCa patients, and downregulation of MPI expression in PCa cells enhanced the anticancer effect of mannose. This study reveals the anticancer effect of mannose in PCa and its clinical significance in PCa patients.
Animals ; Apoptosis ; Cell Line, Tumor ; Humans ; Male ; Mannose ; Membrane Potential, Mitochondrial ; Mice ; Mitochondria ; Prostatic Neoplasms ; Reactive Oxygen Species

BACKGROUND AND OBJECTIVES: To reveal the detail mechanism of miR-484 on myocardial ischemia-reperfusion (MI/R) injury.METHODS: Rats model of MI/R injury was established based on control (Con; sham operate) group, ischemia-reperfusion (I/R) group, miR-484 treatment (miR) group, and I/R-negative control (IR-C) group, followed by pathological and interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IL-1β expression evaluation. Then the myocardial apoptosis, as well as the expression of miR-484, caspase-3, and caspase-9 in myocardium were examined. Finally, the regulatory relation between miR-484 and SMAD family member 7 (SMAD7) was predicated, followed by verification analysis.RESULTS: Compared with Con group, the expression of miR-484 in I/R and IR-C group was decreased. Compared with I/R and IR-C group, the expression of miR-484 was increased in miR group. Compared with Con group, the expression levels of IL-6, TNF-α, and IL-1β in cardiac myocytes of I/R group and IR-C group were increased. Compared with Con group, the apoptotic index, membrane potential of I/R, and the expression of caspase-3/9 were increased in IR-C group. Compared with the I/R and IR-C groups, the apoptotic index of myocardial cells in the ischemic region was decreased, the membrane potential was increased, and the expression of caspase-3/9 was decreased significantly in the miR group. SMAD7 was the target gene of miR-484.CONCLUSIONS: MiR-484 protected myocardial cells from I/R injury by suppressing caspase-3 and caspase-9 expression during cardiomyocyte apoptosis. MiR-484 reduced the expression of IL-6, TNF-α, and IL-1β in MI/R. MiR-484 might alleviate the decreasing of mitochondrial membrane potential in MI/R cells.
Animals ; Apoptosis ; Caspase 3 ; Caspase 9 ; Humans ; Interleukin-6 ; Interleukins ; Membrane Potential, Mitochondrial ; Membrane Potentials ; Myocardium ; Myocytes, Cardiac ; Rats ; Reperfusion Injury ; Tumor Necrosis Factor-alpha

Cell Cycle/drug effects* ; Cell Line, Tumor ; Chalcone/pharmacology* ; Humans ; Membrane Potential, Mitochondrial/drug effects* ; Neuronal Outgrowth/drug effects* ; Okadaic Acid/toxicity* ; Reactive Oxygen Species/metabolism* ; Tretinoin/pharmacology* ; tau Proteins/metabolism*

The organosulfur compound sulforaphane (SFN; C₆H₁₁NOS₂) is a potent cytoprotective agent promoting antioxidant, anti-inflammatory, antiglycative, and antimicrobial effects in in vitro and in vivo experimental models. Mitochondria are the major site of adenosine triphosphate (ATP) production due to the work of the oxidative phosphorylation (OXPHOS) system. They are also the main site of reactive oxygen species (ROS) production in nucleated human cells. Mitochondrial impairment is central in several human diseases, including neurodegeneration and metabolic disorders. In this paper, we describe and discuss the effects and mechanisms of action by which SFN modulates mitochondrial function and dynamics in mammalian cells. Mitochondria-related pro-apoptotic effects promoted by SFN in tumor cells are also discussed. SFN may be considered a cytoprotective agent, at least in part, because of the effects this organosulfur agent induces in mitochondria. Nonetheless, there are certain points that should be addressed in further experiments, indicated here as future directions, which may help researchers in this field of research.
Animals ; Antioxidants/pharmacology* ; Apoptosis/drug effects* ; Brain/ultrastructure* ; Carbon Monoxide Poisoning/metabolism* ; Cytoprotection ; Humans ; Isothiocyanates/pharmacology* ; Membrane Potential, Mitochondrial/drug effects* ; Mitochondria/metabolism* ; Sulfoxides

Enrofloxacin, a fluoroquinolone, is a broad-spectrum antibiotic widely used in veterinary medicine that inhibits the action of bacterial DNA gyrase, resulting in anti-bacterial effects. This study was performed to examine whether enrofloxacin has modulatory and anti-inflammatory activity on immune cells. A few studies have reported the anti-inflammatory effects of enrofloxacin. In this study, we used mouse spleen cells treated with lipopolysaccharide (LPS) and examined the effects of enrofloxacin. Several assays were performed in LPS-treated spleen cells after the enrofloxacin treatment. Enrofloxacin inhibited the metabolic activity and mitochondrial membrane potential of LPS-treated spleen cells significantly. On the other hand, enrofloxacin did not alter the proportion of the subsets in spleen cells, and did not induce cell death. The production of tumor necrosis factor-alpha in LPS-treated spleen cells was inhibited by enrofloxacin. Overall, enrofloxacin had modulatory activity in spleen cells treated with LPS. These data may broaden the use of enrofloxacin as an antibiotic with anti-inflammatory activity in veterinary clinics.
Animals ; Cell Death ; DNA, Bacterial ; Hand ; Hospitals, Animal ; Membrane Potential, Mitochondrial ; Mice ; Spleen ; Tumor Necrosis Factor-alpha ; Veterinary Medicine

Cyclosporin A (CsA) does not only exert a toxic effect on kidney parenchymal cells, but also protects them against necrotic cell death by inhibiting opening of mitochondrial permeability transition pore. However, whether CsA plays a role in hydrogen peroxide-induced kidney proximal tubular cell death is currently unclear. In the present study, treatment with CsA further increased apoptosis and necrosis in HK-2 human kidney proximal tubule epithelial cells during exposure to hydrogen peroxide. In addition, hydrogen peroxide-induced p53 activation and BH3 interacting-domain death agonist (BID) expression were higher in CsA-treated cells than those in non-treated cells, whereas hydrogen peroxide-induced activation of mitogen-activated protein kinases including p38, c-Jun N-terminal kinase, and extracellular signal-regulated kinase and activation of protein kinase B were not significantly altered by treatment with CsA. In oxidant-antioxidant system, reactive oxygen species (ROS) production induced by hydrogen peroxide was further enhanced by treatment with CsA. However, expression levels of antioxidant enzymes including manganese superoxide dismutase, copper/zinc superoxide dismutase, and catalase were not altered by treatment with hydrogen peroxide or CsA. Treatment with CsA further enhanced mitochondrial membrane potential induced by exposure to hydrogen peroxide, although it did not alter endoplasmic reticulum stress based on expression of glucose-regulated protein 78 and 94. Taken together, these data suggest that CsA can aggravate hydrogen peroxide-induced cell death through p53 activation, BID expression, and ROS production.
Apoptosis ; Catalase ; Cell Death ; Cyclosporine ; Endoplasmic Reticulum Stress ; Epithelial Cells ; Humans ; Hydrogen Peroxide ; Hydrogen ; JNK Mitogen-Activated Protein Kinases ; Kidney ; Membrane Potential, Mitochondrial ; Mitogen-Activated Protein Kinases ; Necrosis ; Permeability ; Phosphotransferases ; Proto-Oncogene Proteins c-akt ; Reactive Oxygen Species ; Superoxide Dismutase

This study sought to evaluate the effects of Asiatic acid in LPS-induced BV2 microglia cells and 1-methyl-4-phenyl-pyridine (MPP⁺)-induced SH-SY5Y cells, to investigate the potential anti-inflammatory mechanisms of Asiatic acid in Parkinson’s disease (PD). SH-SY5Y cells were induced using MPP⁺ to establish as an in vitro model of PD, so that the effects of Asiatic acid on dopaminergic neurons could be examined. The NLRP3 inflammasome was activated in BV2 microglia cells to explore potential mechanisms for the neuroprotective effects of Asiatic acid. We showed that Asiatic acid reduced intracellular production of mitochondrial reactive oxygen species and altered the mitochondrial membrane potential to regulate mitochondrial dysfunction, and suppressed the NLRP3 inflammasome in microglia cells. We additionally found that treatment with Asiatic acid directly improved SH-SY5Y cell viability and mitochondrial dysfunction induced by MPP⁺. These data demonstrate that Asiatic acid both inhibits the activation of the NLRP3 inflammasome by downregulating mitochondrial reactive oxygen species directly to protect dopaminergic neurons from, and improves mitochondrial dysfunction in SH-SY5Y cells, which were established as a model of Parkinson’s disease. Our finding reveals that Asiatic acid protects dopaminergic neurons from neuroinflammation by suppressing NLRP3 inflammasome activation in microglia cells as well as protecting dopaminergic neurons directly. This suggests a promising clinical use of Asiatic acid for PD therapy.
Cell Survival ; Dopaminergic Neurons ; In Vitro Techniques ; Inflammasomes ; Membrane Potential, Mitochondrial ; Microglia ; Mitochondria ; Neuroprotective Agents ; Reactive Oxygen Species