scholarly journals Anti-Apoptotic Effect of Angelica Polysaccharide (APS) on Cryopreservation of Platelets

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3250-3250
Author(s):  
Mo Yang ◽  
Weiqing Su ◽  
Liuming Yang ◽  
Huimin Kong ◽  
Huiling Wei ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Caspase 3 ◽  
Conflicts Of Interest ◽  
Annexin V ◽  
Control Group ◽  
Apoptotic Effect ◽  
Vitro Effect

Abstract Background: Angelica Polysaccharide (APS) is from the root of Radix Angelicae Sinensis (Danggui). Danggui has been used for centuries to treat blood-deficiency related diseases. The hematopoietic effect of Danggui may be related to its constituent, polysaccharide. The effects of angelica polysaccharide on cryopreservation of platelets and megakaryocytes have not been well studied. This study focused on anti-apoptotic effect of APS and TPO on cryopreservation of platelets and megakaryocytes and provided new methods for prolonging the preservation time of platelets in vitro. Methods: The expression of platelet membrane glycoprotein CD41 and CD61, as well as the platelet apoptotic rate, Caspase 3 expression and mitochondrial membrane potential (MMP) were detected by flow cytometry; the anti-apoptotic mechanism of APS by PI3K /AKT signaling pathway was analyzed by Western blot assay. CFU assays were used to determine the effects of APS on megakaryocytic progenitor cells. Analyses of Annexin V, Caspase-3, and Mitochondrial Membrane Potential were conducted in megakaryocytic cell line M-07e. The effects of APS on cells treated with Ly294002, PI3K inhibitor and the effect of APS on the p-AKT were also studied. Results: The platelets were divided into 4 group: control group (4 ℃ stored platelets), APS group (APS-treated platelets stored at 4 ℃), LY294002 group (LY294002-treated platelets stored at 4 ℃) and LY294002+APS group (LY294002+APS treated platelets stored at 4 ℃). The apoptotic rate of platelets in LY294002 group was obviously increased. Compared with control group, the expression of CD41 and CD61 gradually decreased along with the enhancement of LY294002 concentrations (r=-0.953). The apoptotic rate of platelets in LY294002 group was enhanced significantly (P<0.05). While in LY294002+APS group, the apoptotic rate of platelets was significantly reduced (P<0.05) as compare with LY294002 group, which suggest that APS has an anti-apoptotic effect on the cryopreserved platelets. APS decreased the expression of Caspase-3 and inhibited the reduction of mitochondrial membrane potential induced by LY294002. Moreover, APS increased the activation of PI3K /AKT pathway in Platelets . We further analyzed the in vitro effect of APS on CFU-MK formation. APS (50 ug/ml) enhanced TPO (50 ng/ml) -induced CFU-MK formation (p=0.06, n=4). APS also significantly enhanced PDGF, bFGF and VEGF-induced CFU-MK formation (n=4). Moreover, the anti-apoptotic effect of APS in M-07e cells was also demonstrated by Annexin-V, Caspase-3, and JC-1 assays. Adding LY294002 alone increased the percentage of cells undergoing apoptosis. However, additional of APS to LY294002-treated cells reversed the percentage of cells undergoing apoptosis. Furthermore, addition of APS significantly increased the p-AKT. Conclusion: APS, like TPO, has an anti-apoptotic effect on the cryopreserved platelets and megakaryocytes through activating PI3K/AKT, decreasing the expression of Caspase-3 and inhibiting the reduction of MMP. Disclosures No relevant conflicts of interest to declare.

Supplementation of kaempferol to in vitro maturation medium regulates oxidative stress and enhances subsequent embryonic development in vitro

Zygote ◽  
2019 ◽  
Vol 28 (1) ◽  
pp. 59-64
Author(s):  
Yuhan Zhao ◽  
Yongnan Xu ◽  
Yinghua Li ◽  
Qingguo Jin ◽  
Jingyu Sun ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Reactive Oxygen Species ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Caspase 3 ◽  
Treated Group ◽  
Control Group ◽  
Oxygen Species

SummaryKaempferol (KAE) is one of the most common dietary flavonols possessing biological activities such as anticancer, anti-inflammatory and antioxidant effects. Although previous studies have reported the biological activity of KAE on a variety of cells, it is not clear whether KAE plays a similar role in oocyte and embryo in vitro culture systems. This study investigated the effect of KAE addition to in vitro maturation on the antioxidant capacity of embryos in porcine oocytes after parthenogenetic activation. The effects of kaempferol on oocyte quality in porcine oocytes were studied based on the expression of related genes, reactive oxygen species, glutathione and mitochondrial membrane potential as criteria. The rate of blastocyst formation was significantly higher in oocytes treated with 0.1 µm KAE than in control oocytes. The mRNA level of the apoptosis-related gene Caspase-3 was significantly lower in the blastocysts derived from KAE-treated oocytes than in the control group and the mRNA expression of the embryo development-related genes COX2 and SOX2 was significantly increased in the KAE-treated group compared with that in the control group. Furthermore, the level of intracellular reactive oxygen species was significantly decreased and that of glutathione was significantly increased after KAE treatment. Mitochondrial membrane potential (ΔΨm) was increased and the activity of Caspase-3 was significantly decreased in the KAE-treated group compared with that in the control group. Taken together, these results suggested that KAE is beneficial for the improvement of embryo development by inhibiting oxidative stress in porcine oocytes.

Volatile Anesthetics Induce Caspase-dependent, Mitochondria-mediated Apoptosis in Human T Lymphocytes In Vitro 

2005 ◽  
Vol 102 (6) ◽  
pp. 1147-1157 ◽  
Author(s):  
Torsten Loop ◽  
David Dovi-Akue ◽  
Michael Frick ◽  
Martin Roesslein ◽  
Lotti Egger ◽  
...  
Keyword(s):  
Membrane Potential ◽  
T Lymphocytes ◽  
Cytochrome C ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Caspase 3 ◽  
Volatile Anesthetics ◽  
Human T Lymphocytes ◽  
Induced Apoptosis

Background Volatile anesthetics modulate lymphocyte function during surgery, and this compromises postoperative immune competence. The current work was undertaken to examine whether volatile anesthetics induce apoptosis in human T lymphocytes and what apoptotic signaling pathway might be used. Methods Effects of sevoflurane, isoflurane, and desflurane were studied in primary human CD3 T lymphocytes and Jurkat T cells in vitro. Apoptosis and mitochondrial membrane potential were assessed using flow cytometry after green fluorescent protein-annexin V and DiOC6-fluorochrome staining. Activity and proteolytic processing of caspase 3 was measured by cleaving of the fluorogenic effector caspase substrate Ac-DEVD-AMC and by anti-caspase-3 Western blotting. Release of mitochondrial cytochrome c was studied after cell fractionation using anti-cytochrome c Western blotting and enzyme-linked immunosorbent assays. Results Sevoflurane and isoflurane induced apoptosis in human T lymphocytes in a dose-dependent manner. By contrast, desflurane did not exert any proapoptotic effects. The apoptotic signaling pathway used by sevoflurane involved disruption of the mitochondrial membrane potential and release of cytochrome c from mitochondria to the cytosol. In addition, the authors observed a proteolytic cleavage of the inactive p32 procaspase 3 to the active p17 fragment, increased caspase-3-like activity, and cleavage of the caspase-3 substrate poly-ADP-ribose-polymerase. Sevoflurane-induced apoptosis was blocked by the general caspase inhibitor Z-VAD.fmk. Death signaling was not mediated via the Fas/CD95 receptor pathway because neither anti-Fas/CD95 receptor antagonism nor FADD deficiency or caspase-8 deficiency were able to attenuate sevoflurane-mediated apoptosis. Conclusion Sevoflurane and isoflurane induce apoptosis in T lymphocytes via increased mitochondrial membrane permeability and caspase-3 activation, but independently of death receptor signaling.

Flavopiridol Decreases Mcl-1 and Initiates Early Mitochondrial Damage in Chronic Lymphocytic Leukemia (CLL) Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2098-2098
Author(s):  
David M. Lucas ◽  
Syed-Rehan A. Hussain ◽  
Amy J. Johnson ◽  
Lisa L. Smith ◽  
Amy J. Wagner ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mechanism Of Action ◽  
Mitochondrial Membrane ◽  
Tumor Lysis Syndrome ◽  
Annexin V ◽  
Caspase 8 ◽  
Adapter Protein ◽  
Tumor Lysis

Abstract We have noted impressive activity of the cyclin-dependent kinase inhibitor flavopiridol in advanced-stage CLL patients, including those with a deletion of chromosome 17p13. Using a novel, effective schedule of flavopiridol, substantial and sometimes dramatic evidence of tumor cell death is observed as early as 4–6 hours. This is accompanied by hyperkalemia, hyperphosphatemia, hypocalcemia, and dramatic elevation in LDH consistent with acute tumor lysis syndrome. Studies by several groups including our own have demonstrated that Mcl-1 protein and mRNA is down-regulated with flavopiridol. Mcl-1 is an important protein that contributes to mitochondrial membrane stability. We have therefore sought to determine the role of mitochondria disruption in the mechanism of action of flavopiridol. By flow cytometry using the voltage-sensitive dye JC-1, loss of mitochondrial membrane potential was detected in flavopiridol-treated whole blood as early as five hours, prior to the onset of annexin-V or propidium iodide staining. This is in contrast to in vitro studies using human serum, in which mitochondrial depolarization and annexin-V staining occurred simultaneously. In isolated CLL cells treated with flavopiridol in vitro, loss of mitochondrial membrane potential was not affected by inhibitors of caspases 8 or 9 or by the broad caspase inhibitor Z-VAD-fmk, although apoptosis was effectively blocked by Z-VAD-fmk and caspase-8 inhibitor, and to a lesser extent, caspase-9 inhibitor. Flavopiridol was also able to effectively induce apoptosis and mitochondrial membrane depolarization in Jurkat cell lines deficient in caspase-8 or its adapter protein FADD. Additionally, lymphoid cells overexpressing Bcl-2 are resistant to flavopiridol-mediated apoptosis relative to the vector control. This suggests there is not direct binding of flavopiridol or its metabolites to APAF-1 (cytosolic adapter protein) and apoptosome assembly, as this process is insensitive to Bcl-2 family proteins. Further mechanistic studies were undertaken using isolated liver mitochondria. While the electron transport system was not uncoupled in this system, potential mechanisms of mitochondrial injury in leukemic cells from CLL patients are currently under exploration. Taken together, these observations suggest that mitochondrial perturbation contributes significantly to the death process induced by flavopiridol. Further studies to identify the mechanism of mitochondrial perturbation will be essential to understanding flavopiridol’s mechanism of action and for predicting patients at risk for acute tumor lysis syndrome. (Support for this work was provided by the Samuel Waxman Foundation and the Leukemia & Lymphoma Society.)

scholarly journals Effects of Medical Diagnostic X-rays Delivered at 0.01 or 0.05 mGy on Human Blood Cells

2021 ◽  
Vol 20 (1) ◽  
pp. 136-144
Author(s):  
Benjamaporn Supawat ◽  
Jongchai Tinlapat ◽  
Rusleena Wongmahamad ◽  
Chuleekorn Silpmuang ◽  
Suchart Kothan ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Blood Cells ◽  
Mitochondrial Membrane ◽  
Low Dose ◽  
Medical Science ◽  
Control Group ◽  
X Rays ◽  
Medical Diagnostic

Background: Low-dose X-rays are commonly used in medical imaging to help in the diagnosis ofdiseases. However, the deleterious effects of exposure to medical diagnostic low-dose X-rays remaina highly debated topic. The objective was to study the effects of medical diagnostic X-rays on humanblood cells. Materials and Methods: We studied the effects of medical diagnostic low-dose X-rays (80kVp), i.e.,0.01 or 0.05 mGy, after the in vitro exposure of human red blood cells (RBCs) and peripheralblood mononucleated cells (PBMCs).Cells with no irradiation served as the control group. The biologicalendpoints that were used to determine the effects of medical diagnostic low-dose X-rays were hemolysisfor RBCs and mitochondrial membrane potential, lysosomes, and the cell cycle for PBMCs. Results: Ourresults showed no changes in the hemolysis of RBCs and mitochondrial membrane potential, lysosome, orcell cycle in cells exposed to these low doses of X-rays when compared to the corresponding nonirradiatedcells at all harvest timepoints. Conclusion: These results suggested that there were no deleterious effectsof diagnostic low-dose X-rays when human RBCs and PBMCs were exposed in vitro. Bangladesh Journal of Medical Science Vol.20(1) 2021 p.136-144

In vitro studies on the toxicity of isoniazid in different cell lines

2003 ◽  
Vol 22 (11) ◽  
pp. 607-615 ◽  
Author(s):  
Christina E Schwab ◽  
Helga Tuschl
Keyword(s):  
Cell Cycle ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mammalian Cells ◽  
Mitochondrial Membrane ◽  
Annexin V ◽  
Apoptotic Cells ◽  
Lymphoma Cells ◽  
Cytotoxicity Effects

The aim of the present study was to investigate in vitrothe mechanism of toxicity of isoniazid (-INH), the drug most widely used for treatment of tuberculosis. The human hepatoma line HepG2, the human lymphoblastoid line AHH-1 and the murine lymphoma cells YAC-1 were used as test systems. Active cell death (-apoptosis) and necrosis were detected by different flow cytometric methods: the binding of annexin V to the cell membrane and staining with propidium iodide (PI), the TUNEL assay for detection of DNA fragmentation and the occurrence of a sub G1 peak in cell cycle histograms. Mitochondrial membrane potential was analysed with the fluorescent probe JC-1. In addition to cytotoxicity, effects of INH on cell cycle were studied in HepG2 cells. The data of the present investigations indicate that INH induces cytotoxicity via apoptosis both in hepatoma and lymphoma cells. Twenty-four hours of application of INH in concentrations -26 mM led to a remarkable number of apoptotic cells positive for Annexin V. The induction of apoptosis was accompanied by a break down of the mitochondrial membrane potential and the occurrence of DNA strand breaks. At incubation times from 36 to 48 hours, a sub-G1 peak of late apoptotic cells was detected in cell cycle analysis. Furthermore, cell cycle studies showed a disruption of the cycle at low concentrations of INH which are only mildly cytotoxic. Thus the present study unequivocally demonstrated that INH induces cytotoxicity via apoptosis and can lead to a significant disturbance of the cell cycle in mammalian cells.

scholarly journals Anti-Cancer Effects of Clofazimine As a Single Agent and in Combination with Cisplatin in Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5897-5897 ◽  
Author(s):  
Ipek Durusu ◽  
Hazal Hepsen Husnugil ◽  
Heval Atas ◽  
Aysenur Biber ◽  
Selin Gerekci ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Membrane Potential ◽  
Fluorescence Microscopy ◽  
Cell Line ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Caspase 3 ◽  
Annexin V ◽  
Growth Inhibitory ◽  
U266 Cell

Abstract Multiple myeloma (MM) is a malignant neoplasm of bone marrow plasma B cells with high morbidity. Clofazimine (CLF) is an FDA-approved leprostatic, anti-tuberculosis, and anti-inflammatory drug that was previously shown to have growth suppression effects on various cancer types such as hepatocellular, lung, cervix, esophageal, colon, and breast cancers as well as melanoma, neuroblastoma, and leukemia cells. The objective of this study was to evaluate the anticancer effect of CLF on U266 resistant MM cell line. The relative cell viability of a panel of hematological cell lines (Jurkat, U266, Namalwa, K562, HL60) treated with 10 µM CLF after 24 h of treatment significantly reduced the viability in all cell lines, with percentages ranging between 28% (U266) and 38% (Jurkat) (p<0.001). IC50 value of CLF was found as 9.8 ± 0.7 µM on the U266 cell line. Previous studies showed that this level of CLF does not inhibit growth of healthy cells, which supports safety of CLF. CLF had both dose (2, 5, 10 µM) and time (12, 24, and 48 h) dependent growth inhibitory effect. Combination chemotherapy is an approach to increase the effectiveness of chemotherapeutics as well as overcome drug resistance and suppresses side effects of drugs. Therefore, we evaluate the combination effect of CLF in U266 cells and showed that combination with cisplatin led to a synergistic interaction between two compounds in all tested dose regimes, resulting in a 2.5-7.1 fold marked increase in cell death. Importantly this synergism was observed in U266 cells, which have mutant p53 at A161T showing resistance to cytotoxic agents such as platinum analogs (cisplatin etc.). <>Depolarization of the mitochondrial membrane is one of the first events in apoptosis. JC-1 is a lipophilic and cationic dye that reversibly changes color from green to red as the mitochondrial membrane potential increases (depolarization). JC-1 assay used in both flow cytometry analyses and fluorescence microscopy images have shown that relative to the control, CLF treatment results in the depolarization of mitochondrial membrane 15, 20.5, 14.3 fold respectively at 12, 24, and 48 h in U266 cell line (Figure 1). The caspase family of cysteine proteases plays an important role in apoptosis. Caspase-3 is a major protease activated during the early stages of programmed cell death. 10 µM CLF was applied for 12, 24, and 48 h and anti-active caspase-3 PE stained U266 cells were analyzed by flow cytometry. Caspase-3 activity is enhanced 5.6, 24.5 and 13.6-fold relative to untreated controls at 12h, 24h and 48 h respectively. Phosphatidylserine (PS) translocation to the outer leaflet of the cellular membrane is one of the key steps in early stages of apoptosis. To support our previous findings on apoptotic effect of CLF, we employed Annexin-V assay. CLF treatment caused a significant increase in the percentage of early and late apoptotic cells at 12 h (2.1 and 1.8 fold respectively), 24 h (4.1 and 12.3 fold) and 48 h (10.1 and 11.5 fold). Fluorescence microscopy images also supported flow cytometry data (Figure 2). Collectively, all three apoptosis assay results show that CLF significantly induces apoptosis in U266 cells. Our study is the first to show apoptotic and growth inhibitory effects of CLF on a p53-mutant resistant MM cell line U266. Our results also proved that combined therapy employing CLF together with chemotherapeutics seems to be a possible future therapeutic approach for MM. Further in vivo and clinical studies are warranted to evaluate its therapeutic potential for resistant MM treatment. Figure 1 Effect of 10 µM CLF on mitochondrial membrane potential. Flow cytometry fluorescence intensity A) Dot plots B) Bar plots of cells stained with JC-1 (n=3). C) Fluorescence microscopy image of JC-1-stained untreated cells indicating healthy mitochondria (red), D) In CLF-treated cells, green color shows diffusion of JC-1 from damaged mitochondria. Figure 1. Effect of 10 µM CLF on mitochondrial membrane potential. Flow cytometry fluorescence intensity A) Dot plots B) Bar plots of cells stained with JC-1 (n=3). C) Fluorescence microscopy image of JC-1-stained untreated cells indicating healthy mitochondria (red), D) In CLF-treated cells, green color shows diffusion of JC-1 from damaged mitochondria. Figure 2 Flow cytometry analysis of Annexin V-PE/7-AAD stained U266 cells treated with 10 µM CLF. A) Representative dot plots of Annexin V-PE vs 7-AAD signals gated as live, early apoptotic and late apoptotic quadrants B) Cell population bar graphs of corresponding dot plot quadrants (n=3). C) Early apoptotic U266 cell (right) stained with Annexin V-PE (green) and a late apoptotic U266 cell (left) stained with both Annexin V-PE (green) and nuclear dye PI (red) D) Close-up micrograph (160X) of a late apoptotic U266 cell. Figure 2. Flow cytometry analysis of Annexin V-PE/7-AAD stained U266 cells treated with 10 µM CLF. A) Representative dot plots of Annexin V-PE vs 7-AAD signals gated as live, early apoptotic and late apoptotic quadrants B) Cell population bar graphs of corresponding dot plot quadrants (n=3). C) Early apoptotic U266 cell (right) stained with Annexin V-PE (green) and a late apoptotic U266 cell (left) stained with both Annexin V-PE (green) and nuclear dye PI (red) D) Close-up micrograph (160X) of a late apoptotic U266 cell. Disclosures No relevant conflicts of interest to declare.

scholarly journals Staphylococcus-Induced Bacteriospermia In Vitro: Consequences on the Bovine Spermatozoa Quality, Extracellular Calcium and Magnesium Content

Animals ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 3309
Author(s):  
Michal Ďuračka ◽  
Kamila Husarčíková ◽  
Mikuláš Jančov ◽  
Lucia Galovičová ◽  
Miroslava Kačániová ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Dna Fragmentation ◽  
Mitochondrial Membrane Potential ◽  
Sperm Motility ◽  
Mitochondrial Membrane ◽  
Control Group ◽  
Sperm Dna Fragmentation ◽  
Bovine Semen ◽  
Sperm Dna

Bacterial contamination of bovine ejaculates intended for artificial insemination may be reflected in a significant economic loss due to unsuccessful fertilization as well as health issues of the recipients. The Staphylococcus genus represents a large part of bacteriocenosis of bovine ejaculates. Therefore, this study aims to get a closer look on the effects of Staphylococcus-induced bacteriospermia under in vitro conditions on bovine sperm quality. Prior to inducing bacteriospermia, spermatozoa were separated from each ejaculate using Percoll® Plus gradient medium in order to limit the effects only to the selected bacterial species. Seven Staphylococcus species previously isolated from bovine semen were used for our experiments at a turbidity of 0.5 McFarland (equivalent to 1.5 × 108 colony-forming units per mL). The contaminated semen samples were incubated at 37 °C and at times of 0, 2, and 4 h, motility, mitochondrial membrane potential, reactive oxygen species (ROS) generation, sperm DNA fragmentation, and magnesium (Mg) and calcium (Ca) extracellular concentration were analyzed and compared with the control group (uncontaminated). The results showed no significant changes at the initial measurement. However, significant adverse effects were observed after 2 h and 4 h of incubation. Most notably, the presence of S. aureus, S. warneri, S. kloosii, and S. cohnii caused a significantly increased ROS production, leading to sperm DNA fragmentation, changes in the mitochondrial membrane potential, and a decreased sperm motility. Furthermore, the presence of Staphylococcus species led to lower extracellular concentrations of Mg and Ca. In conclusion, the overgrowth of Staphylococcus bacteria in bovine semen may contribute to oxidative stress resulting in sperm DNA fragmentation, altered mitochondrial membrane potential, and diminished sperm motility.

scholarly journals In vitro effect of pulsed 900 MHz GSM radiation on mitochondrial membrane potential and motility of human spermatozoa

2008 ◽  
Vol 29 (4) ◽  
pp. 268-276 ◽  
Author(s):  
Nadia Falzone ◽  
Carin Huyser ◽  
Francois Fourie ◽  
Tim Toivo ◽  
Dariusz Leszczynski ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Human Spermatozoa ◽  
Vitro Effect
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