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Tranexamic acid (TXA) is widely used among young women because of its ability to whiten skin and treat menorrhagia. Nevertheless, its potential effects on oocyte maturation and quality have not yet been clearly clarified. Melatonin (MT) is an endogenous hormone released by the pineal gland and believed to protect cells from oxidative stress injury. In the present study, we used an in vitro maturation model to investigate the toxicity of TXA and the protective role of MT in mouse oocytes. Compared with the control group, the TXA-exposed group had significantly lower nuclear maturation (57.72% vs. 94.08%, P < 0.001) and early embryo cleavage rates (38.18% vs. 87.66%, P < 0.001). Further study showed that spindle organization (52.56% vs. 18.77%, P < 0.01) and chromosome alignment (33.23% vs. 16.66%, P < 0.01) were also disrupted after TXA treatment. Mechanistically, we have demonstrated that TXA induced early apoptosis of oocytes (P < 0.001) by raising the level of reactive oxygen species (P < 0.001), which was consistent with an increase in mitochondrial damage (P < 0.01). Fortunately, all these effects except the spindle defect were successfully rescued by an appropriate level of MT. Collectively, our findings indicate that MT could partially reverse TXA-induced oocyte quality deterioration in mice by effectively improving mitochondrial function and reducing oxidative stress-mediated apoptosis.NEW & NOTEWORTHY Tranexamic acid is increasingly used to whiten skin, reverse dermal damages, and treat heavy menstrual bleeding in young women. However, its potential toxicity in mammalian oocytes is still unclear. Our study revealed that tranexamic acid exposure impaired the mouse oocyte quality and subsequent embryo development. Meanwhile, melatonin has been found to exert beneficial effects in reducing tranexamic acid-induced mitochondrial dysfunction and oxidative stress.

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Despite efforts in osteosarcoma (OS) research, the role of inductive moderate hyperthermia (IMH) in delivering and enhancing the antitumor effect of liposomal doxorubicin formulations (LDOX) remains unresolved. This study investigated the effect of a combination treatment with LDOX and IMH on Saos-2 human OS cells. We compared cell viability using a trypan blue assay, apoptosis and reactive oxygen species (ROS) measured by flow cytometry and pro-apoptotic Bax protein expression examined by immunocytochemistry in response to IMH (42 MHz frequency, 15 W power for 30 min), LDOX (0.4 µg/mL), and LDOX plus IMH. The lower IC50 value of LDOX at 72 h indicated increased accumulation of the drug in the OS cells. LDOX plus IMH resulted in a 61% lower cell viability compared to no treatment. Moreover, IMH potentiated the LDOX action on the Saos-2 cells by promoting ROS production at temperatures of <42 °C. There was a 12% increase in cell populations undergoing early apoptosis with a less heterogeneous distribution of Bax after combination treatment compared to those treated with LDOX (p < 0.05). Therefore, we determined that IMH could enhance LDOX delivery and its antitumor effect via altered membrane permeabilization, ROS generation, and a lower level of visualized Bax heterogeneity in the Saos-2 cells, suggesting the potential translation of these findings into in vivo studies.

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Vincristine (VCR) is a microtubule-destabilizing chemotherapeutic agent commonly administered for the treatment of cancers in patients, which can induce severe side effects including neurotoxicity. In context of the effects on female fertility, ovarian toxicity has been found in patients and mice model after VCR exposure. However, the influence of VCR exposure on oocyte quality has not been elucidated. We established VCR exposure in vitro and in vivo model. The results indicated in vitro VCR exposure contributed to failure of oocyte maturation through inducing defects in spindle assembly, activation of SAC, oxidative stress, mitochondrial dysfunction, and early apoptosis, which were confirmed by using in vivo exposure model. Moreover, in vivo VCR exposure caused aneuploidy, reduced oocyte-sperm binding ability, and the number of cortical granules in mouse oocyte cortex. Taken together, this study demonstrated that VCR could cause meiotic arrest and poor quality of mouse oocyte.

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BACKGROUND: Breast cancer is a prevalent malignant tumor that affects women worldwide. The primary challenge in treating breast cancer is combating drug resistance, which contributes to relapse and metastasis. Jatrophone is a unique macrocyclic jatrophane diterpene found in various Jatropha and Euphorbia species. It possesses diverse biological and pharmacological activities, including anticancer activity. However, it is unclear whether jatrophone can overcome drug resistance in breast cancer. METHODS: This study includes the investigation of the cytotoxicity of jatrophone on doxorubicin-resistant breast cancer cells (MCF-7ADR) and the underlying molecular mechanisms. The effects of jatrophone on cell viability were determined using the sulforhodamine B (SRB) assay, while flow cytometry was used to evaluate cell cycle progression, apoptosis, and autophagy. A scratch assay was conducted to observe cell migration, and western blotting was used to measure downstream protein levels (PI3K, AKT, and NF-κB). Unpaired Student's t-tests were used for comparison between the two groups and the results were analyzed by one-way ANOVA with Tukey- Kremer post hoc test. RESULTS: It was shown that jatrophone exhibited potent cytotoxic activity on MCF-7ADR cells in a dose-dependent manner, with an IC50 value of 1.8 µM. It also significantly induced cell cycle S and G/M phase arrest. Interestingly, jatrophone induced both early and late apoptotic cell death, as well as autophagic cell death, with negligible necrosis. Furthermore, jatrophone treatment diminished the migration of MCF-7ADR cells. At the molecular level, jatrophone treatment significantly down-regulated the expression levels of PI3K, AKT, and NF-κB. ß. CONCLUSIONS: The results of the study suggest that jatrophone decreases the proliferation of MCF-7/ADR cells at a low micromolar concentration; induces cell cycle arrest; promotes apoptotic, and autophagic cell death; inhibits migration and EMT; and works on resistance by a mechanism involving the inhibition of the PI3K/Akt/ NF-κB pathway. These findings provide evidence of the potential of jatrophone to be a promising lead compound for targeting doxorubicin-resistant breast cancer cells and could be further investigated for its clinical application as a chemotherapy adjuvant.

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Tumor selectivity is yet a challenge in chemotherapy-based cancer treatment. A series of calixarenes derivatized at the lower rim with 3-phenyl-1H-pyrazole units with variable upper-rim substituent and conformations of macrocyclic core, alkyl chain length between heterocycle and core, as well as phenolic monomer (5-(4-tert-butylphenyloxy)methoxy-3-phenyl-1H-pyrazole) have been synthesized and characterized in a range of therapeutically relevant cellular models (M-HeLa, MCF7, A-549, PC3, Chang liver, and Wi38) from different target organs/systems. Specific cytotoxicity for M-HeLa cells has been observed in tert-butylcalix[4]arene pyrazoles in 1,3-alternate (compound 7b) and partial cone (compound 7c) conformations with low mutagenicity and haemotoxicity and in vivo toxicity in mice. Compounds 7b,c have induced mitochondrial pathway of apoptosis of M-HeLa cells through caspase-9 activation preceded by the cell cycle arrest at G0/G1 phase. A concomitant overexpression of DNA damage markers in pyrazole-treated M-HeLa cells suggests that calixarene pyrazoles target DNA, which was supported by the presence of interactions between calixarenes and ctDNA at the air-water interface.

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Repetitive low-level blast (rLLB) exposure is a potential risk factor for the health of soldiers or workers who are exposed to it as an occupational characteristic. Alveolar macrophages (AMs) are susceptible to external blast waves and produce pro-inflammatory or anti-inflammatory effects. However, the effect of rLLB exposure on AMs is still unclear. Here, we generated rLLB waves through a miniature manual Reddy-tube and explored their effects on MH-S cell morphology, phenotype transformation, oxidative stress status, and apoptosis by immunofluorescence, real-time quantitative PCR (qPCR), western blotting (WB) and flow cytometry. Ipatasertib (GDC-0068) or PDTC was used to verify the role of the Akt/NF-κB signaling pathway in these processes. Results showed that rLLB treatment could cause morphological irregularities and cytoskeletal disorders in MH-S cells and promote their polarization to the M1 phenotype by increasing iNOS, CD86 and IL-6 expression. The molecular mechanism is through the Akt/NF-κB signaling pathway. Moreover, we found reactive oxygen species (ROS) burst, Ca2+ accumulation, mitochondrial membrane potential reduction, and early apoptosis of MH-S cells. Taken together, our findings suggest rLLB exposure may cause M1 polarization and early apoptosis of AMs. Fortunately, it is blocked by specific inhibitors GDC-0068 or PDTC. This study provides a new treatment strategy for preventing and alleviating health damage in the occupational population caused by rLLB exposure.

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Bisphenol F (BPF), a substitute for bisphenol A (BPA), is progressively used to manufacture various consumer products. Despite the established reproductive toxicity of BPF, the underlying mechanisms remain to elucidate. This in-vitro study deep in sighted the BPF toxicity on mouse oocyte meiotic maturation and quality. After treating oocytes with BPF (300 µM), the oocyte meiotic progression was blocked, accentuated by a reduced rate in the first polar body extrusion (PBE). Next, we illustrated that BPF induced α-tubulin hyper-acetylation disrupted the spindle assembly and chromosome alignment. Concurrently, BPF resulted in severe oxidative stress and DNA damage, which triggered the early apoptosis in mouse oocytes. Further, altered epigenetic modifications following BPF exposure were proved by increased H3K27me3 levels. Concerning the toxic effects on spindle structure, oxidative stress, and DNA damage in mouse oocytes, BPF toxicity was less severe to oocyte maturation and spindle structure than BPA and induced low oxidative stress. However, compared with BPA, oocytes treated with BPF were more prone to DNA damage, indicating not less intense or even more severe toxic effects of BPF than BPA on some aspects of oocytes maturation. In brief, the present study established that like wise to BPA, BPF could inhibit meiotic maturation and reduce oocyte quality, suggesting it is not a safe substitute for BPA.

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Gossypol is a yellow natural polyphenolic compound extracted from the seeds, leaves, stems, and flower buds of the cotton plant. Several studies have shown that exposure to gossypol impacts reproductive health in both humans and animals. However, whether gossypol exposure would influence oocyte quality has not yet been determined. Here, we studied the effects of gossypol on the meiotic maturation of mouse oocytes in vitro. The results revealed that gossypol exposure did not affect germinal vesicle breakdown (GVBD) but significantly reduced polar body extrusion (PBE) rates. Moreover, we observed meiotic spindle organization and chromosome alignment were entirely disturbed after gossypol exposure. Further, gossypol exposure also caused mitochondrial dysfunction and abruptly decreased the levels of cellular ATP, and diminished the mitochondrial membrane potential (MMP). Accordingly, gossypol-induced oxidative stress was confirmed through an increased level of reactive oxygen species (ROS). Early apoptosis incidence also increased as identified by positive Annexin-V signaling. Collectively, the above findings provide evidence that gossypol exposure impaired oocyte meiotic maturation, disturbed spindle structure and chromosome dynamics, disrupted mitochondrial function, induced oxidative stress, and triggered early apoptosis. These findings emphasize gossypol's adverse effects on oocyte maturation and thus on female fertility.

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Epigenetic regulators of human spermatogonia stem cells (SSCs) remain largely unknown. We found that miRNA-122-5p was upregulated in human spermatogonia from obstructive azoospermia (OA) patients compared with non-obstructive azoospermia (NOA). MiRNA-122-5p stimulated the proliferation and DNA synthesis of human SSCs, whereas it inhibited the early apoptosis of human SSCs. CBL was predicted and identified as a direct target of miRNA-122-5p in human SSCs. CBL silencing led to an enhancement of cell proliferation and DNA synthesis and neutralized the effect of miRNA-122-5p inhibitor on the DNA synthesis of human SSCs. The decrease in the early apoptosis of human SSCs was observed after CBL knockdown. By comparing the profiles of lncRNAs between OA and NOA spermatogonia, CASC7 was significantly deficient in OA spermatogonia, and it had a direct association with miRNA-122-5p. LncRNA CASC7 competed with miRNA-122-5p, and it suppressed the inhibition of CBL. Collectively, these results implicate that miRNA-122-5p enhances the proliferation and DNA synthesis and inhibits the early apoptosis of human SSCs by targeting CBL and competing with lncRNA CASC7. Therefore, this study provides novel insights into epigenetic regulation of fate determinations of human SSCs, and it offers new targets for gene therapy of male infertility that is associated with aging.

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Photodynamic therapy (PDT) takes advantage of reactive oxygen species (ROS) to trigger the apoptosis for cancer therapy. Given that cell apoptosis is a form of programmed cell death involved with multiple suborganelles and cancer cells are more sensitive to ROS than normal cells, early confirmation of the apoptosis induced by ROS would effectively avoid overtreatment. Herein, we highlight an aggregation-induced emission (AIE)-based theranostic agent (TPA3) to in situ dynamically track mitophagy prior to late apoptosis. TPA3 showed high specificity to autophagy vacuoles (AVs), of which appearance is the signature event of mitophagy during early apoptosis and delivered photocytotoxicity to cancer cells and skin cancer tumors in nude mice under irradiation of white light. Furthermore, in situ monitoring of the dynamical mitophagy process involved with mitochondria, AVs, and lysosomes was performed for the first time under confocal microscopy, providing a real-time self-monitoring system for assessing the curative effect prior to late apoptosis. This fluorescence imaging guided PDT witness great advances for applying in the clinical application.

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Cord blood (CB) has been used as an alternative source for unrelated allogeneic hematopoietic stem cell transplantation. To determine which assay was useful for predicting the successful outcome of CB transplantation, CBs were grouped according to the temperature (4 °C, 24 °C, and 37 °C) and time (24, 48, and 72 h) after collection. The viability, early apoptosis, and colony forming units (CFUs) were ascertained for the total nucleated cells (TNCs) and CD34+ cells; in addition, the engraftment of infused CD34+ cells in NSG mice was determined. The viability of the TNCs and CD34+ cells and total CFUs were significantly decreased whereas the early apoptosis was significantly increased in the 72 h group at 37 °C compared to that of the 24 h group at 24 °C. The viability and early apoptosis of the TNCs correlated with those of CD34+ cells. In addition, the viability and early apoptosis correlated with the number of granulocyte/monocyte progenitor CFUs. In transplanted NSG mice, the frequency of human CD45+ cells decreased in the 72 h group at 24 °C compared to that of the 24 h group at 24 °C and was negatively correlated with early apoptosis of TNCs and CD34+ cells. This study demonstrated that the early apoptosis of TNCs and CD34+ cells constitutes a useful marker for predicting the engraftment of HSCs and may provide helpful data for standard assessment regarding CB quality by analyzing the correlation between in vitro and in vivo assays using NSG mice.

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INTRODUCTION: Scientific research is beginning to prove the connection between claims by African traditional medicine and the natural chemical specifics contained in medicinal plant Securidaca longipedunculata. Our previous studies showed that two natural saponin fractions (4A3 and 4A4) identified in the plant as triterpenoid glycosides are capable of activating apoptosis on cervical tumor cell lines. Considering this and some critical roles of human papillomavirus (HPV) E6 oncogene on cervical cells, by promoting carcinogenesis and cell survival, it became necessary to investigate the possible pathways for apoptosis transmission. METHODS: Tests conducted on relevant cervical tumor cell lines such as Caski and Bu25TK included the following: MTT assay; scratch assay (to determine cell migration/invasion); fluorescence microscopy with Annexin V-fluorescein isothiocyanate, muscle progenitor cell) and propidium iodide staining; and finally reverse transcriptase quantitative PCR (RT-qPCR) for gene analysis. RESULTS: Reduced cell proliferation was observed due to activities of 4A3 and 4A4 fractions, with half-maximal inhibitory concentration (IC50) of 7.03 and 16.39 µg/mL, respectively, on Caski cell line. A significant reduction in cell migration occurred within 48 and 72 hours, respectively, for Caski and Bu25TK cell lines. Late apoptosis was activated by 4A3, staining both Annexin V and PI, in contrast to 4A4's early apoptosis. RT-qPCR data revealed a fold change (FC) inhibition of antiapoptotic proteins such as MCL-1 and BCL2L1, with diminished level of AKT-3, VEGFA, MALAT1, etc. The expression of p53, proapoptotic BAD, and caspase-8 was nonsignificant. CONCLUSION: The low expression of AKT-3 and antiapoptotic proteins (MCL-1 and BCL2L1), as well as VEGFA, could simply be an indication for possible suppression of cell survival mechanisms via multiple channels. We therefore conclude that 4A3 and 4A4 fractions mediate activity via the inhibition of phosphatidylinositol-3-OH kinase (PI3K)-AKT/mTOR/NF-kB-dependent antiapoptotic stimuli. Further studies are ongoing to reveal the chemical structures and compositions of these two fractions.

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Inhibiting apoptotic cells helps ameliorate ischemic injury. Actually, only the apoptotic cells in early stage could be rescued. Molecular imaging of the early apoptosis would make sense in ischemic stroke; however, few of apoptosis molecular probes could specifically target early apoptosis. This study developed a small-molecule early apoptosis targeting probe CYS-F, which was synthesized by cystine with fluorescein isothiocyanate dyes. And the final molecular weight of CYS-F was only 1013 Da, which was much smaller than the traditional apoptosis marker annexin V. CYS-F showed excellent early apoptosis targeting ability both in vitro and in vivo. And CYS-F was cleared rapidly from the circulation with a blood half-life of 1.325 h. A favorable match was obtained between the images in fluorescence imaging and magnetic resonance imaging in stroke models. The target-to-background ratio of the lesions on 0 h was negative, which reflected the decreased blood flow. Multimodal molecular imaging showed the therapeutic effect of cystamine was dose dependence and CYS-F could also predict the outcome of ischemic stroke at an early stage. The versatility of CYS-F provides a comprehensive and convenient route for clinical decision-making in acute ischemic stroke.

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BACKGROUND: Common variable immunodeficiency (CVID) is a heterogeneous immune deficiency characterized by hypogammaglobulinemia. Since B cell maturation and differentiation is defective in this disorder, we evaluated apoptosis in B cells of patients with CVID compared with healthy donors (HD). METHODS: Determination of peripheral blood B-cell subsets in CVID and HDs, was performed using flow cytometry. We compared total apoptosis, early apoptosis and late apoptosis/necrosis in unstimulated and stimulated B-cells of patients with CVID and HDs. We also assessed the expression of the anti-apoptotic molecule BCL2 mRNA levels in B-cells by real-time PCR in CVID patients compared with HDs. RESULTS: Total B-cell apoptosis was increased in both unstimulated and stimulated B-cells from CVID patients compared with HDs (p=0.02 and p=0.004). Early apoptosis in stimulated B-cells (p=0.04) and late apoptosis/necrosis of B-cells in both unstimulated and stimulated B-cells (p=0.04 and p=0.03, respectively) were significantly higher in CVID patients compared with HDs. There was a significant inverse correlation between the percentages of post germinal center B-cells in the peripheral blood of CVID patients compared with percentage of apoptotic B-cells. However, anti-apoptotic BCL2 expression was not significantly reduced in B-cells from CVID patients compared with HDs (p=0.16). CONCLUSION: Increased apoptosis of B-cells may be a factor in abnormality of differentiated B-cell subsets and the impaired endogenous immunoglobulin production in CVID patients. Further studies of the expression of pro/anti-apoptotic mediators in B-cells of CVID patients may shed light on the mechanism behind this increased B-cell apoptosis, and present potential therapeutic interventions in the future.

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Pentachlorophenol (PCP) is a widespread, persistent environmental contaminant, and it is enzymatically activated to form a reactive metabolite, tetrachloro-1,4-benzoquinone (TCBQ). To our knowledge, there is no information about TCBQ toxicity on embryonic stem cells. Here, we demonstrated that TCBQ induced significantly apoptosis of mouse embryonic stem cells in a concentration-dependent manner. We also showed that TCBQ elevated genomic 5-hydroxymethylcytosine (5hmC) by affecting ten-eleven translocation (Tet) dioxygenases in mouse embryonic stem cells. We further investigated whether Tet dioxygenases were implicated in TCBQ-induced apoptosis. By depleting all three dioxygenases (Tet1-3), we found that Tet dioxygenases slightly inhibited both early and late apoptosis induced by TCBQ at a low concentration (30µmol/L). Meanwhile, treated by TCBQ at higher concentrations (40 and 50µmol/L), the total percentage of apoptotic cells was not affected by Tet dioxygenases. However, Tet dioxygenases tended to arrest mouse ES cells to be at early apoptotic stage and to reduce the cells to enter later apoptotic stage. These results indicate that Tet dioxygenases play a role in shaping TCBQ-induced apoptosis in mouse embryonic stem cells. Our study provides new insights into the toxicology of PCP and its reactive metabolite TCBQ.

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This study was aimed to observe the protective effects of components of water extract from Qi-Xue Bing-Zhi recipe (CWQB) on myocardial cells from hypoxia/reoxygenation (H/R) injury,by optimizing the metabolism of highenergy phosphates and then preventing cell damage and early apoptosis correlatively.Myocardial cells were separated and extracted from newborn SD rats.And then,H/R models were made by depriving oxygen for 3 hours and then regaining for 2 hours.Cardiomyocytes were divided into four groups,which were the control (normal oxygen) group,H/R (H/R model) group,TMZ (H/R model + 100 μmol/L Trimetazidine,TMZ) group,and CWQB (H/R model + 1 mmol/L CWQB) group.High-performance liquid chromatography (HPLC) was used in the content determination of adenosine monophosphate (AMP),adenosine diphosphate (ADP),adenosine triphosphate (ATP) in each group for the calculation of total adenylic acid (TAN) and energy charge (EC).Enzyme linked immunosorbent assay (ELISA) was used in the detection of myocardial damage markers,such as creatine kinase-MB (CK-MB) and cardiac troponin T (cTnT).The early apoptosis of myocardial cells were assessed by flow cytometry (FCM).The results showed that compared with H/R group,contents of AMP in the TMZ group and CWQB group were decreased,while contents of ADP,ATP,TAN and EC were all increased in both groups.The increasing degrees of ADP,ATP and TAN in TMZ group were higher than those of the CWQB group (P ＜ 0.05).Contents of CK-MB and cTnT were significantly decreased in the TMZ group and the CWQB group.Content of cTnT decreased more significantly in the CWQB group (P ＜ 0.05).In the TMZ group and the CWQB group,early apoptosis rate was decreased.The decreasing in the TH group was more significant (P ＜ 0.05).The correlation analysis showed that the level of CK-MB and concentration of ADP had significant negative correlation.The early apoptosis rate and AMP had significant positive correlation.The early apoptosis rate had negative correlation with concentrations of ADP and ATP (P ＜ 0.01).It was concluded that CWQB recipe can decrease the concentration of AMP in H/R cardiomyocytes,increase the concentrations of ADP,ATP,TAN and EC,and decrease myocardial damage makers such as CK-MB and cTnT,depress early apoptosis rate in H/R cardiomyocytes,in order to display its cardioprotective effects in H/R injury.

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Urokinase receptor (uPAR) is enhanced in many human cancer cells and is frequently an indicator of poor prognosis. Activation of [Formula: see text]1-integrin requires caveolin-1 and is regulated by uPAR. However, the underlying molecular mechanism responsible for the interaction between uPAR and [Formula: see text]1-integrin remains obscure. We found that modified regular Panax ginseng extract (MRGX) had a negative modulating effect on the uPAR/[Formula: see text]1-integrin interaction, disrupted the uPAR/integrin interaction by modulating caveoline-1, and caused early apoptosis in cancer cells. Additionally, we found that siRNA-mediated caveoline-1 downregulation inhibited uPAR-mediated [Formula: see text]1-integrin signaling, whereas caveoline-1 up-regulation stimulated the signaling, which suppressed p53 expression, thereby indicating negative crosstalk exists between the integrin [Formula: see text]1 and the p53 pathways. Thus, these findings identify a novel mechanism whereby the inhibition of [Formula: see text]1 integrin and the activation of p53 modulate the expression of the anti-apoptotic proteins that are crucially involved in inducing apoptosis in A549 lung cancer cells. Furthermore, MRGX causes changes in the expressions of members of the Bcl-2 family (Bax and Bcl-2) in a pro-apoptotic manner. In addition, MGRX-mediated inhibition of [Formula: see text]1 integrin attenuates ERK phosphorylation (p-ERK), which up-regulates caspase-8 and Bax. Therefore, ERK may affect mitochondria through a negative regulation of caspase-8 and Bax. Taken together, these findings reveal that MRGX is involved in uPAR-[Formula: see text]1-integrin signaling by modulating caveolin-1 signaling to induce early apoptosis in A549 lung-cancer cells and strongly indicate that MRGX might be useful as a herbal medicine and may lead to the development of new herbal medicine that would suppress the growth of lung-cancer cells.

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Magnetic Fe3O4 nanoparticles (MNPs) have shown promise as drug carriers for treating lung and liver tumors in vivo. However, little is known about the combined delivery of these MNPs with a second approach, extremely low frequency electro-magnetic field (ELFF) exposure, which has been shown to have value for in vitro treatment of tumor cells. Here, ELFF and MNPs were combined to treat healthy (HL-7702) and cancerous (Bel-7402, HepG2) hepatic cells lines to explore the potential therapeutic effects, bio-mechanisms, and potential toxicity of a combined drug-free treatment in vitro. Flow cytometry for anti-AFP (alpha fetal protein) antibody, which coated the MNPs, indicated that the combined treatment induced Bel-7402 and HepG2 hepatoma cells lines into early apoptosis, without significant effects on healthy hepatic cells. This effect appeared to be mediated through cellular membrane ion metabolism. The presence of AFP-loaded MNPs strengthened the effects of ELFF on tumor cells, inducing a higher frequency of early apoptosis, while having minimal toxic effects on healthy HL-7702 cells. Western blotting revealed that the apoptosis-triggering BCL proteins were up regulated in hepatoma cells compared to healthy cells. Flow cytometry and patch-clamp studies revealed that this resulted from a higher MNP uptake ratio and greater cellular membrane ion exchange current in tumor cells compared to HL-7702 cells. Further, patch-clamp results showed that combining MNPs with ELFF treatment induces cells into early apoptosis through an ion metabolism disturbance in cells, similar to ELFF treatment. In brief, the combination of ELFF and MNPs had beneficial effects on tumor cells without significant toxicity on healthy cells, and these effects were associated with cellular MNP uptake.

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Apoptosis is a programed cell death that is vital for tissue homeostasis. However, embryo apoptosis had been known to be related to embryo fragmentation which should be avoided in in vitro fertilization (IVF). The purpose of this study was to evaluate the relationship of embryo apoptosis with the grade of immature oocytes and cleavage stage of in vitro produced (IVP) cattle embryos. This study consisted of 345 oocytes collected through ovary slicing. Immature oocytes were graded as A, B and C. This grading was based on cumulus cell thickness and compactness. All oocytes then underwent an in vitro maturation (IVM) procedure. An IVF was done 24 h after IVM culture. Prior to staining, stage of cleaved embryos was determined and classified as either 2, 4, 8 or >8-cell embryo stage. Apoptosis status of cleaved IVP embryos was determined by using annexin V-FITC staining technique at 48 and 72 h post insemination (hpi). Apoptosis status for each embryo was classified as either early or late. The result showed that there was no significant difference (p > 0.05) of apoptosis status among grade A, B and C embryos. All grades of oocytes showed embryo apoptosis where 1.5% late apoptosis for grade A, 4.5% and 10.4% of early and late apoptosis for grade B and grade C. Early apoptosis was not seen in grade A embryo. We also noted no significant difference (p > 0.05) of apoptosis status between 2, 4, 8 and >8-cell embryo stage. Early apoptosis was also not seen in >8-cell stage. Even though there were no differences in apoptosis expression between the three classes, the cleavage rate of grade A oocytes was significantly higher (p < 0.01) than grade B and grade C. In conclusion, the apoptosis expression in the embryo can occur regardless of the oocyte quality and the cleavage stage of the embryo produced.

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BACKGROUND AND OBJECTIVE: Neutrophilic asthma is an important disease subgroup, including patients with severe phenotypes and erratic responses to standard treatments. Tamoxifen (TX), a selective estrogen receptor modulator (SERM) used as treatment of human breast cancer, has been shown to induce early apoptosis of equine blood and bronchoalveolar lavage fluid (BALF) neutrophils in vitro. Equine recurrent airway obstruction (RAO) is a naturally occurring neutrophilic condition, closely related with human asthma. Our purpose was to investigate the therapeutic potential of tamoxifen in horses with neutrophilic lung inflammation. METHODS: Twelve horses underwent acute lung inflammation through exposure to allergens known to cause RAO, after which they received treatment with either tamoxifen or dexamethasone. Outcome measures included evaluation of clinical signs, BALF cytology, and early apoptosis of blood and BALF neutrophils. RESULTS: Tamoxifen treatment decreased BALF neutrophil counts (65.3 ± 19.38% before treatment; 7.6 ± 4.5% 2 days post-treatment,; and 13.6 ± 9.3% 5 days post-treatment). A similar decrease was observed with dexamethasone treatment (48.6 ± 5.88% before treatment; 11.5 ± 8.1% 2 days post-treatment; 14.6 ± 10.3% 5 days post-treatment). Clinical and endoscopic scores improved in both treatment groups. Tamoxifen treatment significantly increased early apoptosis of peripheral blood neutrophils at 5 days post-treatment (27.04 ± 15.2%), and in BALF neutrophils at 2 and 5 days post-treatment (42.11 ± 11.67% and 48.98 ± 2.6%, respectively). CONCLUSION: Tamoxifen treatment in horses with induced acute pulmonary inflammation promoted early apoptosis of blood and BALF neutrophils, reduction in BALF neutrophils and improvement in the animals' clinical status.

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