Objective: To establish a method for rapid determination of bongkrekic acid (BA) in plasma by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Methods: In November 2020, plasma samples were extracted by methanol and acetonitrile (1∶1) and purified directly. The samples were separated by C18 column. Gradient elution was carried out with 5 mmol/L ammonium acetate water acetonitrile solution as mobile phase. Under the optimized instrument conditions, the electrospray ionization multiple reaction monitoring (MRM) mode was used, and the external standard method was used for quantitative analysis. Results: The linear relationship of BA in plasma was good in the concentration range of 2-100 μg/L, the correlation coefficient was 0.9998, the average recovery was 83.7%-112.0%, the relative standard deviation within and between batches was less than 10%, the detection limit of the method was 0.7 μg/L and the lower limit of quantification was 2.0 μg/L. Conclusion: The method is simple, rapid, accurate and sensitive, and can meet the requirements for the determination of BA in blood samples of poisoning patients.
Bongkrekic Acid ; Chromatography, High Pressure Liquid ; Humans ; Solid Phase Extraction ; Tandem Mass Spectrometry

BACKGROUND: Numerous studies demonstrated that genistein induced the decrease of proliferation and apoptosis in a variety of cells. However, there is no report about the effect of genistein on proliferation and demise of mast cells. OBJECTIVE: This study was conducted to investigate genistein-induced aoptosis of mast cells as it pertains to both its basic drug mechanism and the potential therapeutics of the pathologic conditions accompanying mast cell proliferation. METHODS: p815 murine mastcytoma cell line was used to assess the effects of genistein treatment including viability and proliferation, morphlolgic study, DNA electrophoresis, the effect of caspase inhibitor, western blotting, and mitochondrial event. RESULTS: Genistein indeced many apoptotic manifestations as evidenced by changes in cell morphology, generation of DNA fragmentation, activation of caspase 3, and DNA hypoploidy. The reduction of mitochondrial membrae potential and the release of cytochrome c to cytosol were also demonstrated. However, reduction of mitochondrial membrane potential and cytochrome c release were not prevented by caspase inhibitors zVAD-fmk and BocD.fmk, or PTP(permeability transition pore) blockers such as bongkrekic acid and cyclosporin A. CONCLUSIONS: This in vitro study suggests that pathologic increases in mast cell number possibly be regulated in vivo by therapeutic strategy enhancing apoptosis by treatment of genistein.
Apoptosis* ; Blotting, Western ; Bongkrekic Acid ; Caspase 3 ; Caspase Inhibitors ; Cell Line ; Cyclosporine ; Cytochromes c ; Cytosol ; DNA ; DNA Fragmentation ; Electrophoresis ; Genistein ; Mast Cells ; Mastocytoma* ; Membrane Potential, Mitochondrial

3-Deazaadenosine (DZA), a cellular methylation blocker was reported to induce the caspase-3-like activities-dependent apoptosis in U-937 cells. In this study, we analyzed the activation pathway of the caspase cascade involved in the DZA-induced apoptosis using specific inhibitors of caspases. In the U-937 cells treated with DZA, cytochrome c release from mitochondria and subsequent activation of caspase-9, -8 and -3 were observed before the induction of apoptosis. zDEVD-Fmk, a specific inhibitor of caspase-3, and zLEHD-Fmk, a specific inhibitor of caspase-9, prevented the activation of caspase-8 but neither caspase-3 nor caspase-9, indicating that caspase-8 is downstream of both caspase-3 and caspase-9, which are activated by independent pathways. zVAD-Fmk, a universal inhibitor of caspases, kept the caspase-3 from being activated but not caspase-9. Moreover, ZB4, an antagonistic Fas-antibody, exerted no effect on the activation of caspase-8 and induction of apoptosis by DZA. In addition, zVAD-Fmk and mitochondrial permeability transition pore (MPTP) inhibitors such as cyclosporin A (CsA) and bongkrekic acid (BA) did not block the release of cytochrome c from mitochondria. Taken together, these results suggest that in the DZA-induced apoptosis, caspase-8 may serve as an executioner caspase and be activated downstream of both caspase-3 and caspase-9, independently of Fas receptor-ligand interaction. And caspase-3 seems to be activated by other caspses including IETDase-like enzyme and caspse-9 seems to be activated by cytochrome c released from mitochondria without the involvement of caspases and CsA- and BA- inhibitory MPTP.
Amino Acid Chloromethyl Ketones/pharmacology ; Apoptosis/*drug effects ; Bongkrekic Acid/pharmacology ; Caspases/*metabolism ; Cell Line ; Cyclosporine/pharmacology ; Cytochrome c/drug effects/metabolism ; Enzyme Activation ; Human ; Leukocytes, Mononuclear/cytology ; Ligands ; Membrane Glycoproteins/metabolism ; Tubercidin/*pharmacology ; U937 Cells

3-Deazaadenosine (DZA), a cellular methylation blocker was reported to induce the caspase-3-like activities-dependent apoptosis in U-937 cells. In this study, we analyzed the activation pathway of the caspase cascade involved in the DZA-induced apoptosis using specific inhibitors of caspases. In the U-937 cells treated with DZA, cytochrome c release from mitochondria and subsequent activation of caspase-9, -8 and -3 were observed before the induction of apoptosis. zDEVD-Fmk, a specific inhibitor of caspase-3, and zLEHD-Fmk, a specific inhibitor of caspase-9, prevented the activation of caspase-8 but neither caspase-3 nor caspase-9, indicating that caspase-8 is downstream of both caspase-3 and caspase-9, which are activated by independent pathways. zVAD-Fmk, a universal inhibitor of caspases, kept the caspase-3 from being activated but not caspase-9. Moreover, ZB4, an antagonistic Fas-antibody, exerted no effect on the activation of caspase-8 and induction of apoptosis by DZA. In addition, zVAD-Fmk and mitochondrial permeability transition pore (MPTP) inhibitors such as cyclosporin A (CsA) and bongkrekic acid (BA) did not block the release of cytochrome c from mitochondria. Taken together, these results suggest that in the DZA-induced apoptosis, caspase-8 may serve as an executioner caspase and be activated downstream of both caspase-3 and caspase-9, independently of Fas receptor-ligand interaction. And caspase-3 seems to be activated by other caspses including IETDase-like enzyme and caspse-9 seems to be activated by cytochrome c released from mitochondria without the involvement of caspases and CsA- and BA- inhibitory MPTP.
Amino Acid Chloromethyl Ketones/pharmacology ; Apoptosis/*drug effects ; Bongkrekic Acid/pharmacology ; Caspases/*metabolism ; Cell Line ; Cyclosporine/pharmacology ; Cytochrome c/drug effects/metabolism ; Enzyme Activation ; Human ; Leukocytes, Mononuclear/cytology ; Ligands ; Membrane Glycoproteins/metabolism ; Tubercidin/*pharmacology ; U937 Cells

Opening of mitochondrial permeability transition pore (mPTP) was found to have a critical role in cell death from ischemia/reperfusion (I/R) injury experimentally in the late 1980's. Thereafter, tremendous efforts have been made to define the molecular composition of mPTP and underlying mechanisms of its opening. mPTP opening, so far, has been demonstrated with the conformational changes of the mitochondrial protein components including cyclophilin-D, adenine nucleotide translocase, and voltage-dependent anion channel, which were induced by the modification of the levels of Ca2+, phosphate, mitochondrial membrane potential, intracellular pH and adenine nucleotide. At present, genetic modulation of the expression of protein components are being used in the investigation of its properties, presenting novel mechanisms of mPTP opening, including phosphate carrier. For therapeutic intervention, cyclosporin A and its analogues were first to be demonstrated to inhibit the opening of mPTP, affecting cyclophilin-D. There are numerous pharmacological substances that have direct or indirect effects on mPTP opening, including bongkrekic acid, reactive oxygen species scavengers, calcium channel blockers, and Na+/H+ exchanger-1 inhibitors, but only cyclosporin A was clinically tried to limit the myocardial infarction. Conditioning interventions, ischemic or anesthetic, have also been shown to be effective in limiting the detrimental effects of I/R injury. These interventions are commonly related to specific receptors on cell membrane and then signal transduction pathway consisting of many protein kinases, which eventually lead to mitochondria. And being presented are experimental evidences that inhibition of mPTP opening is a primary mechanism of these conditioning interventions. In conclusion, mPTP opening is now presented as primary mechanism and therapeutic target of I/R injury, but precise mechanism and standardized treatment method are needed to be clarified.
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine ; Adenine ; Bongkrekic Acid ; Calcium Channel Blockers ; Cell Death ; Cell Membrane ; Cyclosporine ; Hydrogen-Ion Concentration ; Membrane Potential, Mitochondrial ; Mitochondria ; Mitochondrial ADP, ATP Translocases ; Mitochondrial Membrane Transport Proteins ; Mitochondrial Proteins ; Myocardial Infarction ; Myocardium ; Permeability ; Protein Kinases ; Reactive Oxygen Species ; Reperfusion Injury ; Signal Transduction