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Ethnopharmacological Relevance: Shugan Xiaozhi (SGXZ) decoction is a traditional Chinese medicine used for treating nonalcoholic steatohepatitis (NASH). It has been used clinically for over 20 years and proved to be effective; however, the molecular mechanism underlying the effects of SGXZ decoction remains unclear., Aim of the Study: We analyzed the chemical components, core targets, and molecular mechanisms of SGXZ decoction to improve NASH through network pharmacology and in vivo experiments., Materials and Methods: The chemical components, core targets, and related signaling pathways of SGXZ decoction intervention in NASH were predicted using network pharmacology. Molecular docking was performed to verify chemical components and their core targets. The results were validated in the NASH model treated with SGXZ decoction. Mouse liver function was assessed by measuring ALT and AST levels. TC and TG levels were determined to evaluate lipid metabolism, and lipid deposition was assessed via oil red O staining. Mouse liver damage was determined via microscopy following hematoxylin and eosin staining. Liver fibrosis was assessed via Masson staining. Western blot (WB) and immunohistochemical (IHC) analyses were performed to detect inflammation and the expression of apoptosis-related proteins, including IL-1β, IL-6, IL-18, TNF-α, MCP1, p53, FAS, Caspase-8, Caspase-3, Caspase-9, Bax, Bid, Cytochrome c, Bcl-2, and Bcl-XL. In addition, WB and IHC were used to assess protein expression associated with the TLR4/MyD88/NF-κB pathway., Results: Quercetin, luteolin, kaempferol, naringenin, and nobiletin in SGXZ decoction were effective chemical components in improving NASH, and TNF-α, IL-6, and IL-1β were the major core targets. Molecular docking indicated that these chemical components and major core targets might interact. KEGG pathway analysis showed that the pathways affected by SGXZ decoction, primarily including apoptosis and TLR4/NF-κB signaling pathways, interfere with NASH. In vivo experiments indicated that SGXZ decoction considerably ameliorated liver damage, fibrosis, and lipid metabolism disorder in MCD-induced NASH mouse models. In addition, WB and IHC verified the underlying molecular mechanisms of SGXZ decoction as predicted via network pharmacology. SGXZ decoction inhibited the activation of apoptosis-related pathways in MCD-induced NASH mice. Moreover, SGXZ decoction suppressed the activation of TLR4/MyD88/NF-κB pathway in MCD-induced NASH mice., Conclusion: SGXZ decoction can treat NASH through multiple targets and pathways. These findings provide new insights into the effective treatment of NASH using SGXZ decoction., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Increase of the Ca 2+ -activated chloride channel TMEM16A is contribute to tumorigenesis. However, the expression level of TMEM16A and its underlying molecular mechanism for TMEM16Apromotingliver carcinogenesis is remains unknown., Methods: In the present study, the expression of TMEM16A in hepatocellular carcinoma (HCC) tissues were measured by quantitative reverse-transcription polymerase chain reaction (qRT-PCR), Western blot and immunohistochemical. Cell proliferation was detected using CCK-8, EdU staining and colony formation methods. Flow cytometry was carried out for detecting cell cycle distribution and apoptosis rate. Migration and invasion abilities were analyzed using transwell and wound healing assay. Western blot method was performed to analyze protein expression., Results: Here, we found TMEM16A was significantly increased in HCC tissues, and a higher TMEM16A expression levels were detected in larger tumor size, higher tumor grade, with distant metastasis and poor differentiation. Moreover, overexpression of TMEM16A promoted HCC growth, migration and invasion, and suppressed apoptosis in vitro and in vivo. Knockdown of TMEM16A inhibited HCC growth, migration and invasion, and induced apoptosis in vitro and in vivo. Furthermore, TMEM16A regulated PI3K/AKT-MAKP signaling pathway., Conclusion: Our data indicate that TMEM16A may represent a novel biomarker of HCC and may be a potential therapeutic target for diagnosis and therapy.

To observe changes in AMP-activated protein kinase (AMPK) activity and phosphorylation changes in AMPK signaling pathway in gastric smooth muscle cells of rats with diabetic gastroparesis (DGP), investigate the effect of AMPK on apoptosis and explore the underlying mechanism. After establishing rat model of DGP, rats were divided into normal control (NC) and DGP groups. The phosphorylation changes in AMPK pathway were detected by AMPK Signaling Phospho-Antibody Array, and the apoptosis-related proteins were determined. Rat gastric smooth muscle cells were cultured in vitro under different glucose conditions, and divided into normal and high glucose groups. The AMPK activity and intracellular Ca2+ changes in cells were observed. After AMPK silencing, cells were divided into high glucose-24h, high glucose-48h and high glucose-48h+siRNA groups. Changes in expression of apoptosis-related proteins were observed. AMPK activity and apoptosis rates were both increased in gastric smooth muscle tissues in DGP rats (P<0.05, P<0.001, respectively). A total of 14 apoptosis-related differentially phosphorylated proteins were identified. Under high-glucose condition, AMPK activity and intracellular Ca2+ concentrations in rat gastric smooth muscle cells were increased (both P<0.05). After AMPK silencing, p53 expression was decreased, Akt and p70 S6 ribosomal protein kinase (p70S6K) activities were were increased, Bcl-2 expression was increased, CaMKII activity was decreased in the high glucose-48h group. Under high-glucose condition, activated AMPK can directly or indirectly promote cells apoptosis by regulating the expression and activity of p53, Akt, p70S6K, Protein kinase A (PKA), Phospholipidol C (PLC)-β3, CaMKII, CaMKIV and eukaryotic translation initiation factor 4E binding protein1 (4E-BP1) in rat gastric smooth muscle cells., (© 2019 The Author(s).)

Through the roles of vitamin B1 and B12 in neuroprotection and in improving cerebral palsy symptoms have been previously noticed, the action mechanism is still unclear. This study aims to investigate the protective effect of vitamin B1 and B12 on neuron injury in cerebral palsy and to clarify the mechanism of vitamin B1 and B12 inhibiting neurons apoptosis, and to focus on the role of lncRNA MALAT1 in this process. In order to investigate the effect of vitamin B1 and B12 on neurons injury in vivo and on neuron apoptosis in vitro , we, respectively, introduced vitamin B1 and B12 into cerebral palsy rat and in apoptosis-induced N2A neurons by Oxygen Glucose Deprivation/reoxygenation (OGD/R). Our results demonstrated that vitamin B1 and B12 treatment improved the motor and memory functions and ameliorated the neurons injury in cerebral palsy rats. OGD/R treatment repressed the expression of MALAT1 and BDNF and the phosphorylation of PI3K and Akt, and enhanced the miR-1 expression, which were all reversed by vitamin B1 and B12 treatment in N2A neurons. Vitamin B1 and B12 inhibited miR-1 expression through MALAT1, promoted BDNF expression and activated PI3K/Akt signaling through the MALAT1/miR-1 axis. Vitamin B1 and B12 suppressed neuron apoptosis by up-regulating BDNF via MALAT1/miR-1 pathway. MALAT1 interference abolished the neuroprotective effect of vitamin B1 and B12 in cerebral palsy rats. Collectively, vitamin B1 and B12 up-regulates BDNF and its downstream PI3K/Akt signaling through MALAT1/miR-1 axis, thus suppressing neuron apoptosis and mitigating nerve injury in cerebral palsy rats.

Fipronil (FPN) is a widely used phenylpyrazole pesticide that can kill pests by blocking γ-aminobutyric acid (GABA)-gated chloride channels. In addition, there are lack of studies on the effects of FPN on the female mammalian gametes. In this study, porcine oocytes were used to investigate the effects of FPN on the oocyte maturation process. The results showed that the first polar body extrusion rate significantly decreased (100 μM FPN vs. control, 18.64 ± 2.95% vs. 74.90 ± 1.50%, respectively), and oocytes were arrested at the germinal vesicle stage in 100 μM FPN group. Meanwhile, the FPN caused a significant increase in reactive oxygen species (ROS) levels and severe DNA damage inside the oocytes. Furthermore, apoptosis was enhanced along with decreases in mitochondrial membrane potential, BCL-xL, and the release of cytochrome C in FPN-treated group. Additionally, low CDK1 activity and delayed cyclin B1 degradation during germinal vesicle breakdown were found in the FPN-treated group, which resulted from the activation of ATM-P53-P21 pathway. In conclusion, FPN induces apoptosis and cell cycle arrest in porcine oocyte maturation because of increased ROS levels and DNA damage. This suggests that the FPN in the environment may have potential detrimental effects on the female mammalian reproductive system.

Objective: To investigate the underlying mechanism of lncRNA myocardial infarction-associated transcript (MIAT) in hypoxic-ischemic (HI)-induced neonatal cerebral palsy., Materials and Methods: Neonatal rat model of HI injury was established to detect the motor function. LncRNA MIAT, miR-211, glial cell line-derived neurotrophic factor (GDNF) and caspase-3 expressions were measured by qRT-PCR or western blot. The apoptosis of Neuro2A cells was detected by flow cytometry. RNA immunoprecipitation (RIP) and RNA pull-down assays were performed to confirm the interaction between MIAT and miR-211., Results: Compared with control group, lncRNA MIAT and GDNF were downregulated in striatal tissues of neonatal rats in HI group and oxygen glucose deprivation (OGD)-induced ischemic injury of Neuro2A cells, whereas miR-211 was up-regulated in striatal tissues of HI group and OGD-induced ischemic injury of Neuro2A cells. LncRNA MIAT interacted with miR-211, and lncRNA MIAT overexpression reduced neuron apoptosis through miR-211. Besides, GDNF expression was positively regulated by lncRNA MIAT and negatively regulated by miR-211 in Neuro2A cells. In vivo experiment proved MIAT promoted motor function and relieved HI injury., Conclusion: MIAT overexpression reduced apoptosis of Neuro2A cells through miR-211/GDNF, which relieved HI injury of neonatal rats.

Objective: To investigate the protective effects and the possible mechanisms of simvastatin on myocardial injury induced by diabetes., Methods: Twenty-four SD rats (180~220)g were randomly divided into control group (control, n =8) and modeled groups( n =16), the modeled groups were injected with streptozotocin intraperitoneally to induce diabetes. Then the modeled rats were randomly divided into diabetes mellitus group (DM group, n =8) and diabetes mellitus + simvastatin group (DM+S group, n =8). Rats in DM+S group were treated with simvastatin at the dose of 40 mg/(kg·d)by gavage for 4 weeks, and the other two groups were treated with the same amount of saline. At the end of experiments, the heart tissues were collected for further observation. The content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) in heart tissues were measured by spectrophotometry; HE staining of rat heart slides was used to observe the pathological changes; TUNEL assay was used to determine the apoptosis index of myocardial cells in each groups; The distribution of p53 in the heart tissues was evaluated by immunohistochemistry; Western blot was used to detect the expressions of p53, p53-phospho-serine 15, Bax and Bcl-2 in the heart tissues., Results: ①Compared with control group, the content of malondialdehyde (MDA) was increased while the activity of superoxide dismutase (SOD) was decreased significantly in DM group ( P <0.01). After simvastatin administration, the activity of SOD was increased and the content of MDA was decreased significantly ( P <0.01). ② HE staining results showed that the myocardial cells in the DM group were disorganized, with unclear morphological structure and a large number of inflammatory cells infiltration. Compared with DM group, the myocardial morphology in DM+S group was improved significantly. ③TUNEL staining results showed that the apoptosis index of myocardial cells in DM group was increased significantly compared with that of control group, and the apoptosis index was decreased significantly after the treatment of simvastatin ( P <0.01).④ Immunohistochemistry showed that compared with control group,the expression of p53 in DM group was increased significantly, and was expressed in both cytoplasm and nucleus, while the expression of p53 in DM+S group was decreased and the expression of p53 in nucleus was decreased significantly ( P <0.01). ⑤ The results of Western blot showed that the expression levels of p53, p53-phospho-serine15 and Bax were higher than those in control group, and the expression of Bcl-2 was lower than that in control group ( P <0.01). After simvastatin administration, the expression levels of p53,p53-phospho-serine 15 ( P <0.01) and Bax were decreased significantly ( P <0.05) and the expression of Bcl-2 was increased ( P <0.05)., Conclusions: Simvastatin exerted protective effects on myocardial injury caused by diabetes through improving the abnormal morphological changes of diabetic myocardium, alleviating oxidative stress and inhibiting apoptosis of myocardial cells. The mechanism is related to the regulation of apoptosis pathway mediated by p53.

Fatty acid synthase (FAS) is an important enzyme involved in the de novo synthesis of long-chain fatty acids. During development, the function of FAS in growth is greater than that in energy storage pathways; therefore, we hypothesized that knockout of FAS would affect early embryonic development owing to the induction of endoplasmic reticulum (ER) stress. In the present study, the function of FAS was studied using the CRISPR (clustered regularly interspaced short palindromic repeats)/ CRISPR-associated protein 9 (Cas9) system. Cas9 and single-guide RNA (sgRNA) were injected into parthenotes to decrease the number of FAS-positive embryos. The efficiency of knockout was assayed by DNA sequencing. We found that FAS knockout caused excessive production of reactive oxygen species (ROS). Excess ROS induced ER stress, resulting in activation of the adaptive unfolded protein response (UPR). FAS knockout caused splicing of the X-box binding protein 1 gene (XBP1) and expression of spliced XBP1 mRNA. In addition, FAS knockout caused phosphorylation of PKR-like ER kinase (PERK), and an increase in the mRNA expression of the ER stress-regulated genes, activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP). Finally, Ca 2+ was released from the ER and taken up by the mitochondria. As the ER stress became intolerable, apoptosis was initiated. These results demonstrate that FAS knockout induced ROS generation, which mediated the activation of UPR via the ER stress, ultimately leading to apoptosis induction., (© 2017 Wiley Periodicals, Inc.)

Objective: To explore whether targeting phosphoglycerate kinase 1 (PGK1) can enhance the sensitivity of BRAF V600E mutation melanoma cells to vemurafenib., Methods: The methods of cell biology, molecular biology and pharmacology(MTT assay, Western blot, FCM, Colongenic assay) were used in this study., Results: ① Silencing of PGK1 expression increased the efficacy of vemurafenib in melanoma cells, as evidenced by greater killing in the tumor cells subjected to combined treatment of vemurafenib with siPGK1; ②The mechanism of enhanced sensitivity of melanoma cells to vemurafenib was associated with activation of apoptotic signaling pathway., Conclusions: Targeting of PGK1 may represent a novel strategy of sensitizing melanoma cells to vemurafinib.

Deoxynivalenol (DON) is a widespread trichothecene mycotoxin which contaminates agricultural staples and elicits a complex spectrum of toxic effects on humans and animals. It has been shown that DON impairs oocyte maturation, reproductive function and causes abnormal fetal development in mammals; however, the mechanisms remain unclear. In the present study, we investigate the possible reasons of the toxic effects of DON on porcine oocytes. Our results showed that DON significantly inhibited porcine oocyte maturation and disrupted meiotic spindle by reducing p-MAPK protein level, which caused retardation of cell cycle progression. In addition, up-regulated LC3 protein expression and aberrant Lamp2, LC3 and mTOR mRNA levels were observed with DON exposure, together with Annexin V-FITC staining assay analysis, these results indicated that DON treatment induced autophagy/apoptosis in porcine oocytes. We also showed that DON exposure increased DNA methylation level in porcine oocytes through altering DNMT3A mRNA levels. Histone methylation levels were also changed showing with increased H3K27me3 and H3K4me2 protein levels, and mRNA levels of their relative methyltransferase genes, indicating that epigenetic modifications were affected. Taken together, our results suggested that DON exposure reduced porcine oocytes maturation capability through affecting cytoskeletal dynamics, cell cycle, autophagy/apoptosis and epigenetic modifications., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Cathepsin B, a lysosomal cysteine protease of the papain family, has recently been implicated in the quality and developmental competence of bovine preimplantation embryos. In this study, to determine whether inhibition of cathepsin B activity can improve porcine oocyte maturation and early embryo developmental competence, we supplemented in vitro maturation or embryo culture media with E-64, a cathepsin B inhibitor. Cathepsin B activity was high in poor quality germinal vesicle stage oocytes, but no differences in mRNA expression or protein localization were observed between good and poor quality oocytes, which were categorized based on morphology. Following treatment with 1 μM E-64, cathepsin B activity sharply decreased in 4-cell and blastocyst stage embryos. E-64 had no effect on cell number but significantly (P < 0.05) increased blastocyst formation and decreased the number of apoptotic cells in blastocysts. It also significantly (P < 0.05) enhanced mitochondrial membrane potential in blastocysts, reducing the release of cytochrome c and resulting in decreased expression of Caspase-3 and Caspase-9. In conclusion, inhibition of cathepsin B activity in porcine parthenotes using 1 μM E-64 resulted in attenuation of apoptosis via a reduction in the release of cytochrome c from mitochondria.

This study examined the role of CRH-induced ovarian cell apoptosis in the restraint stress (RS)-induced impairment of oocyte competence. Oocyte percentages of apoptotic cumulus cells (CCs) did not differ between stressed and control mice before in vitro maturation (IVM) but became significantly higher in stressed mice after IVM without serum, growth factor, and hormone. The level of Bcl2 mRNA decreased significantly in mural granulosa cells (MGCs) and ovarian homogenates after RS. Whereas ovarian estradiol, testosterone, and IGF1 decreased, cortisol and progesterone increased significantly following RS. RS increased the level of CRH in serum, ovary, and oocyte while enhancing the expression of CRHR1 in CCs, MGCs, and thecal cells. RS down-regulated ovarian expression of glucocorticoid receptor and brain-derived neurotrophic factor. Furthermore, CRH supplementation to IVM medium impaired oocyte developmental potential while increasing apoptotic CCs, an effect that was completely overcome by addition of the CRHR1 antagonist antalarmin. Results suggest that RS impaired oocyte competence by increasing CRH but not glucocorticoids. Increased CRH initiated a latent apoptotic program in CCs and oocytes during their intraovarian development, which was executed later during IVM to impair oocyte competence. Thus, elevated CRH interacted with increased CRHR1 on thecal cells and MGCs, reducing the production of testosterone, estrogen, and IGF1 while increasing the level of progesterone. The imbalance between estrogen and progesterone and the decreased availability of growth factors triggered apoptosis of MGCs and facilitated CC expression of CRHR1, which interacted with the oocyte-derived CRH later during IVM to induce CC apoptosis and reduce oocyte competence.

Background: The effect of Doxorubicin, a widely used chemotherapeutic agent, on early mouse embryonic development has not been previously characterized., Materials and Methods: Expression of apoptosis-related genes and poly(ADP-ribose) polymerase (PARP) family genes were assessed by real-time reverse transcription polymerase chain reaction (RT-PCR). Apoptosis in mouse blastocysts was tested using the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Cleaved (c)-PARP was analyzed by western blot., Results: A 20 h exposure to doxorubicin caused rapid cytoplasmic fragmentation, DNA condensation and disruption of the cytoskeleton in mouse embryos. Doxorubicin altered the expression of genes involved in DNA repair and apoptosis and blocked early embryonic development, suggesting that doxorubicin affects DNA synthesis and repair. Furthermore, the effect of doxorubicin on early embryo development was determined by assessing the rates of development to different stages and an apoptosis index. Both assays confirmed that doxorubicin altered embryonic development. In conclusion, doxorubicin blocked pre-implantation development in early mouse embryos by altering apoptosis-related gene expression and inactivating DNA repair by PARP.

Autophagy, an essential process for cellular maintenance, cell viability, and development, is the bulk degradation of proteins and organelles. This study investigated the expression levels of autophagy-related genes and the effect of 3-methyladenine (3-MA, an autophagy inhibitor) or rapamycin (an autophagy inducer) on maternal gene degradation and apoptosis in porcine parthenotes developing in vitro. LC3, which is essential for the formation of autophagosomes, was widely expressed in porcine parthenotes. High levels of autophagy-related genes, Atg5, Beclin1 and Lc3 transcripts were expressed in the 1-cell (1C) stage and gradually decreased through the 2-cell (2C) to blastocyst stages. The mRNA expression of Gdf9, c-mos and cyclin B maintained high levels in 2C and 4-cell (4C) embryos treated with 3-MA compared with the control. The Bmp15 and cyclin B mRNA levels were significantly reduced in embryos treated with rapamycin compared with the control. These results suggest that autophagy influences the degradation of these maternal genes. Furthermore, 3-MA-treated embryos exhibited significantly reduced developmental rates, decreased total cell numbers and increased rates of apoptosis. Expression of Atg5, Beclin1 and Lc3 and synthesis of LC3 protein were significantly reduced at the blastocyst stage. Although rapamycin treatment did not affect the developmental rate, it decreased the cell number and increased the rate of apoptosis, and the expression of Atg5, Beclin1 and Lc3 and LC3 protein synthesis were increased. Finally, blastocysts derived following treatment with 3-MA or rapamycin exhibited significantly decreased expression of selected transcription factors, including Pou5f1, Sox2 and Nanog. In conclusion, our results demonstrate that autophagy influences maternal mRNA degradation and apoptosis at the blastocyst stage and suggest that autophagy plays an important role in early embryo development in the pig.

Mitochondria are important regulators of both apoptosis and autophagy. One of the triggers for mitochondrial-mediated apoptosis is the production of reactive oxygen species (ROS), which include hydrogen peroxide, superoxide, hydroxyl radical, nitric oxide and peroxynitrite. Recently, several studies have indicated that ROS may also be involved in the induction of autophagy. In the present study, we used H(2)O(2) to induce mitochondrial stress, examined apoptotic- and autophagic-related gene expression and observed LC3 protein (autophagosome presence marker) expression in porcine parthenotes developing in vitro. In porcine four-cell parthenotes cultured for 5 days in NCSU37 medium containing 0.4% BSA, the developmental rate and mitochondrial distribution did not differ from that of the group supplemented with 100 µM H(2)O(2) but was significantly decreased in the group supplemented with 500 µM H(2)O(2) (P<0.05). Transmission electron microscopy (TEM) indicated that whereas normal shaped mitochondria were observed in blastocysts from the control group, abnormal mitochondria (mitophagy) and autophagic vacuoles were observed in blastocysts from the group that received 500 µM H(2)O(2). Furthermore, addition of H(2)O(2) (100 µM and 500 µM) decreased cell numbers (P<0.05) and increased both apoptosis (P<0.05) and LC3 protein expression in the blastocysts. Real-time RT-PCR showed that H(2)O(2) significantly decreased mRNA expression of anti-apoptotic gene Bcl-xL but increased pro-apoptotic genes, Caspase 3 (Casp3) and Bak, and autophagy-related genes, microtubule-associated protein 1 light chain 3 (Map1lc3b) and lysosomal-associated membrane protein 2 (Lamp2). However, the addition of H(2)O(2) had no effect on mRNA expression levels in nuclear DNA-encoded mitochondrial-related genes, cytochrome oxidase (Cox) 5a, Cox5b and Cox6b1, in blastocysts. These results suggest that H(2)O(2) leads to mitochondrial dysfunction that results in apoptosis and autophagy, which is possibly related to porcine early embryo development.

Objective: Investigate whether preconditioning with diazoxide (DZ), a mitochondrial ATP-sensitive potassium channel opener, could enhance Akt protein to up-regulate Bcl-2/Bax protein ratio against apoptosis., Methods: Cultured for 9 - 10 d in vitro, the hippocampal neurons of Sprague-Dawley rats were assigned to the following 5 groups randomly: Control (Group A), Anoxia (Group B), Anoxia + DZ100 micromol/L (Group C), Anoxia + DZ100 micromol/L + 5-hydroxydecanoate (Group D), Anoxia + DZ100 micromol/L + LY294002 50 micromol/L (Group E). Prior to oxygen deprivation, the hippocampal neurons were treated with DZ for 1 h per day and this treatment was persisted for 3 d. Each experiment was repeated for three times and each group contained 16 wells or 2 dishes of neurons for each time. Thereafter, neurons were derived of oxygen for 4 h. At 48 h of reoxygenation the neuronal optical density (A) were measured by MTT method, while the apoptotic rates were assayed by annexin V-FITC staining. The expressions of Akt, Bcl-2 and Bax proteins were detected and evaluated by Western blot., Results: Compared with other pretreatment, DZ 100 micromol/L led to the elevation of A, whereas promoted the decrease of apoptotic rate (P < 0.05). Compared with those in other anoxic groups, the expressions of Akt protein and Bcl-2 protein in Group C were increased significantly (P < 0.05), whereas the Bax density were reduced significantly (P < 0.05). Preceding administration of 100 micromol/L DZ took protective effects on the neurons induced by anoxia-reoxygenation., Conclusions: DZ 100 micromol/L increased Akt protein to up-regulate Bcl-2/Bax protein ratio against apoptosis induced by anoxia-reoxygenation.

Objective: To screen for novel gene(s) associated with tumor metastasis, and to investigate the effect of overexpression of phosphoprotein associated with glycosphingolipid microdomains 1 (PAG1) on the biological behaviors of human prostatic cancer cell line PC-3M-1E8 in vitro., Methods: Four cDNA microarrays were constructed using cDNA library of prostatic cancer cells PC-3M-1E8 (high metastatic potential), PC-3M-2B4 (low metastatic potential), lung cancer cells PG-BE1 (high metastatic potential)and PG-LH7 (low metastatic potential)to screen genes which were differentially expressed according to their different metastatic properties. From a battery of differentially expressed genes, PAG1, which was markedly downregulated in both high metastatic sublines of PC-3M and PG was chosen for further investigation. Real-time PCR and Western blot were used to confirm the gene expression of PAG1 at mRNA and protein levels. Full-length coding sequence of human PAG1 was subcloned into plasmid pcDNA3.0 and the recombinant plasmids were stably transfected into PC-3M-1E8. The cell proliferation ability, anchorage-independent growth, cell cycle distribution, apoptosis rates and invasive ability were detected by MTT, and in addition, soft agar colony formation, flow cytometry analysis and matrigel invasion assay using Boyden chamber were also carried out respectively. All experiments contained pcDNA3.0-PAG1-transfected clones, vector transfected clones and non-transfected parental cells., Results: A total of 327 differentially expressed genes were obtained between the high and low metastatic sublines of PC-3M cells, including 123 upregulated and 204 downregulated genes in PC-3M-1E8. A total of 281 genes, including 167 upregulated and 114 downregulated genes were obtained in PG-BE1 cells. Nine genes were simultaneously downregulated and 8 genes were upregulated in both high metastatic cell lines of PC-3M and PG. The expression of PAG1 at mRNA and protein level were decreased in the high metastatic subline PC-3M-1E8. Western blot revealed that PAG1 protein was downregulated in PC-3M-1E8 cell line which was in agreement with the gene expression at mRNA level. The proliferation ability and clonogenicity of PAG1 overexpression cells by stable transfection were markedly decreased in comparing with that of the control cells (P < 0.05). Colonies formed in stably PAG1-transfected cells, the vector-transfected clones and parental cells were 26.7 ± 5.2, 47.2 ± 3.2 and 52.3 ± 3.4 respectively (P < 0.05). Flow cytometry analysis showed that the stable PAG1-transfected cells at G₀-G₁ phase were significantly more than that of the control cells (P < 0.05). However, no difference of the apoptosis rate was found between PAG1-transfected cells and control cells (P > 0.05). The number of cells passing through the matrigel and multipore membrane was also decreased in the stable PAG1-transfected cells (35.1 ± 4.9) compared with those of the vector-transfected clones (127.6 ± 6.6) and parental cells (135.0 ± 5.0, P < 0.05)., Conclusions: Using cDNA microarray technique and differential gene expression analysis of sublines of the parental cancer cell lines enable of revealing the metastasis-related genes, among which PAG1 represents one of those under-expressed genes in the high metastatic subline PC-3M-1E8. Transfection expression of PAG1 suppresses cell proliferation, anchorage-independent growth and invasive ability of PC-3M-1E8 cells in vitro. Conclusively, PAG1 may play an important role in inhibiting the proliferation, invasion and metastasis of the cancer cells.

Objective: To investigate the effects of tumor metastasis suppressor gene 1 (TMSG-1) overexpression on the proliferation, invasion and apoptosis of breast cancer cells and to determine possible correlations of TMSG-1 and metastasis of breast cancer., Methods: Full-length human TMSG-1 coding sequences were cloned into plasmid pcDNA3.0-FLAG. The recombinant plasmids constructs were transfeced into MDA-MB-231, a highly malignant breast cancer cell line. Parental, vector-only stable transfectant and TMSG-1 stable transfectant clones were tested by MTT, soft agar colony formation and Boyden chamber assays. At twenty-four hours and forty-eight hours post transient transfection, double staining with Annexin-V-FITC and PI were employed to distinguish apoptotic cells from living cells by flow cytometry analysis., Results: Three TMSG-1 overexpression clones were selected. Compared with the control cells, TMSG-1 overexpression MDA-MB-231 cells showed strong inhibition of proliferation and decreased clonogenicity in soft agar (P<0.05). Transfection of TMSG-1 into MDA-MB-231 cells significantly suppressed the cell invasion ability in vitro (decreased numbers of cells trespassing the matrigel in three experiments: 72.3+/-8.1, 85.0+/-4.2, and 73.5+/-7.8) in comparison with nave cells without transfection (187.5+/-2.1) and cells transfected with the control vector (162.3+/-6.8) (P<0.01). Transient transfection of TMSG-1 into MDA-MB-231 cells could promote cell apoptosis at 24 and 48 hours after transfection (P<0.05)., Conclusions: TMSG-1 protein may have multiple functions in the regulation of proliferation, invasion and apoptosis of metastatic breast cancer cells, likely as a metastasis suppressor gene.

Objective: To observe the pathological changes and investigate the correlation of hepatocyte apoptosis with cytochrome P450 2E1(CYP2E1) expression and oxygen free radical in alcoholic liver disease (ALD) in rat., Methods: 40% ethanol in the dose of 8 g/kg body weight was given to rats by gavage twice daily for 8 weeks in model group (n=37), and rats in control group (n=33) received same volume of saline by gavage. At the end of the 8th week, the contents of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined. The pathological changes in the liver was observed under light microscope with hematoxylin and eosin (HE) staining, and hepatocyte apoptosis was detected by the terminal deoxynucleotidyl transferase mediated dUTP biotin nick and labeling (TUNEL) method. Expression of serum CYP2E1 was determined by polymerase chain reaction (PCR), the contents of malondialdehyde (MDA) and activities of superoxide dismutase (SOD) in serum were detected by thibabituric acid (TBA) quantifying method., Results: The TUNEL positive cells were located around the central vein, and spotty and focal necrosis was found in the ALD group. The apoptotic index (AI) in the ALD group was significantly higher than in the control group (P<0.05). To express in CYP2E1, the allelic frequency of c1 and c2 were 91.65%, and 8.35% respectively, in control group, while the allelic frequency of c1 and c2 were 53.35% and 46.65%, respectively, in ALD group, and there were significantly differences between two groups (all P<0.05). The content of MDA in serum had positive correlation with hepatocyte apoptosis index (MDA vs. AI, r( MDA ) =0.644), and the activity of SOD in serum had negative correlation with AI (SOD vs. AI, r( SOD ) =-0.511, all P<0.05) in the ALD group, and there was negative correlation between MDA and SOD (r=-0.582, P<0.05)., Conclusion: Chronic alcohol administration induced alcoholic liver disease and liver dysfunction, and hepatocyte apoptosis is enhanced. Rsa I and Pst I RFLPs are related with ALD in model rats, and c2 gene might be related with the development of ALD. The content of MDA and activity of SOD play an important role in the process of hepatocyte apoptosis and lipid peroxidation process.

Successful in vitro development of embryos is dependent upon maintenance of cellular function in the embryonic microenvironment. However, the molecular aspects involved in the thermoprotection of embryos, against heat and cold stress it is not clear. The aim of this study was to determine the effects of heat and cold shock on the viability and development of porcine diploid parthenotes developing in vitro. Exposure of two-cell stage embryos to 41 degrees C did not affect further cleavage. However, prolonged heat shock, greater than 12h, reduced the percentage of blastocysts that developed from two-cell stage parthenotes, as well as the total number of nuclei in the blastocysts that formed. Furthermore, the degree of apoptosis was increased (P<0.05) in these blastocyst stage parthenotes. In contrast, exposure of two-cell parthenotes to cold (30 degrees C) for 24h did not affect the cleavage rates, development to blastocyst, nor the total cell numbers per blastocyst. Real time PCR revealed that quantitative expression of the Bcl-xL gene was not different, but amounts of HSP 70.2, Bak, and Caspase 3mRNA were significantly increased in the heat shocked embryos, as compared with untreated controls. These results suggest that porcine embryos are more tolerant to cold shock than to heat shock. Heat stress seems to induce apoptosis related gene expression in porcine parthenotes developing in vitro, which results in diminished parthenote viability.

Survivin, an inhibitor of apoptotic protein containing a single baculoviral inhibit apoptotic protein repeat domain, is a bifunctional protein that suppresses apoptosis and regulates cell division. Thus, we used double stranded RNA (dsRNA) interference to manipulate survivin expression in bovine embryos and analyze its role in blocking apoptosis and facilitating development of pre-implantation embryos. In vitro fertilized embryos (1-cell) were injected with survivin dsRNA, and expression of survivin mRNA was evaluated by real-time quantitative RT-PCR. To analyze survivin protein expression, we performed immunocytochemistry using a rabbit anti-bovine suvivin antibody. Expression levels of survivin mRNA and protein were decreased in the dsRNA group compared to the sham group. Rates of in vitro blastocyst development were lower in the survivin dsRNA-injected group than in the sham-injected group. Also, the total cell number seen in blastocysts was decreased in the dsRNA group. TUNEL assays of DNA fragmentation indicated an increased apoptotic index in the dsRNA group compared to the sham group. These results indicate that survivin is important for optimal development of bovine blastocysts and confirm that survivin expression suppresses apoptosis of pre-implantation embryos., (Copyright (c) 2006 Wiley-Liss, Inc.)

High mobility group box 1 (HMGB1) is considered a component of chromatin and membranes with a role in a variety of biologically important processes. The aim of this study was to determine the effects of HMGB1 on the viability and development of porcine diploid parthenotes cultured in vitro. In vitro derived 4-cell parthenotes were cultured to blastocysts, with or without recombinant HMGB1, in the presence or absence of BSA. The addition of 1, 10, 100 or 1000ng/mL HMGB1 into NCSU 23 medium containing 0.4% BSA did not enhance the development of 4-cell parthenotes to the blastocyst stage and did not change the total number of nuclei in the blastocysts. However, addition of 10 or 100ng/mL HMGB1 into NCSU 23 medium in the absence of BSA increased (P<0.05) both the development rate of parthenotes to the blastocyst stage and total cell numbers. When cultured in NCSU23 medium supplemented with 10 or 100ng/mL HMGB1 and without BSA, apoptosis in parthenotes at the blastocyst stage was decreased (P<0.05). Based on real time RT-PCR, the addition of HMGB1 to the culture medium in the absence of BSA decreased mRNA expression of pro-apoptotic genes Bak (P<0.005) or Caspase3 (Casp3, P<0.01), but not Bcl-xL (Bcl2l2). In conclusion, we inferred that recombinant HMGB1 in the culture medium in the absence of BSA prevented apoptosis of porcine parthenotes and enhanced porcine embryo viability.

Evaluation of apoptosis and expression level of apoptosis-related genes is useful for examining the variation in embryo quality according to environmental change. The objective of this study was to investigate DNA fragmentation and apoptosis-related gene expression patterns in frozen-thawed bovine blastocysts. In vitro produced day 7 blastocysts were frozen by two different vitrification methods (conventional 0.25 ml straw or MVC straw). After thawing, DNA fragmentation of surviving embryos was examined by TUNEL assay, and the expression patterns of their apoptotic genes (survivin, Fas, Hsp 70 and caspase-3) were evaluated using real-time quantitative reverse transcriptase polymerase chain reaction. In vitro survival rates of frozen-thawed embryos were higher following the MVC vitrification method (88.2% re-expanded at 24 h, 77.1% hatching at 48 h) than the conventional (C) vitrification method (77.0% re-expanded at 24 h, 66.7% hatching at 48 h). However, both vitrified methods resulted in a significantly higher apoptotic index (C vitrification method 11.9%, MVC vitrification method 11.0%) than in non-frozen embryos (3.0%). Expression levels of survivin, Fas, caspase-3, and Hsp 70 were also increased in the frozen-thawed embryos compared with non-frozen embryos. These results indicate that the cryopreservation procedure might cause damage that results in an increase in DNA fragmentation and apoptosis-related gene transcription, reducing developmental capacity of frozen-thawed embryos.

To investigate the role of nuclear encoded genes in mitochondrial function during oocyte maturation and early embryogenesis we examined the expression pattern and function of the cytochrome oxidase (Cox) subunits, Cox5a, 5b, and 6b1 during oocyte maturation and early embryo development. Transcription of Cox5a, 5b, or 6b1 was observed in oocytes and during early development; their expression levels were abundant in mature oocytes (MII) and zygotes (1C), and lowest at the 2-cell stage (2C), gradually increasing from 4-cell to blastocyst stage. Immunocytochemical studies revealed that COX5A, 5B, or 6B1 proteins were expressed in all blastomeres of the blastocyst. Silencing of mRNA expression by RNA interference (siRNA) did not inhibit oocyte maturation or developmental events up to the morula and blastocyst stages, but disrupted mitochondrial distribution. Significantly higher apoptosis and lower cell numbers were observed in siRNA-treated blastocysts. Real time RT-PCR revealed that silencing of Cox5a, 5b, or 6b1 did not alter mRNA levels of Bcl-xL (Bcl2l1), but increased transcription levels of proapoptotic genes, Bax and caspase 3 (Casp3). Furthermore, mRNA and protein levels of E-cadherin (CDH1) were decreased in siRNA microinjected blastocysts. These results suggest that gene expression of the Cox subunits, Cox5a, 5b, and 6b1 is not required for embryo developmental events up to the blastocyst stage. The loss of these genes leads to mitochondrial dysfunction that results in apoptosis of the blastocyst stage embryos.

Polyamines, namely putrescine, spermidine, and spermine, are biogenic low-molecular-weight aliphatic amines which play essential roles in cell growth and proliferation. The aim of this study was to determine the effects of polyamines on the viability and development of porcine diploid parthenotes developing in vitro. The addition of 0.1 or 1.0 microM of putrescine, spermidine, or spermine, individually, to the culture medium did not enhance the development of 2-cell parthenotes to the blastocyst stage and did not change the total number of nuclei in the blastocysts. However, combined addition of these three compounds increased developmental rate to blastocyst and total cell numbers. Apoptosis in blastocyst stage parthenotes was decreased in the presence of exogenous polyamines. Real time PCR revealed that addition of polyamines to the culture media decreased the ratio of mRNA expression of Bak/Bcl-xL, Fas/Bcl-xL, and caspase 3, and enhanced mRNA expression of ornithine decarboxylase (ODC) and spermidine synthase, enzymes of polyamine biosynthesis. In the presence of L-alpha-difluoromethyl ornithine (an inhibitor of ODC) or cyclohexylamine (an inhibitor of spermidine synthase) development of porcine parthenotes decreased, apoptosis increased, and mRNA expression of the ratio of Bak/Bcl-xL and Fas/Bcl-xL, and caspase 3 increased. These results suggest that exogenous polyamines in the culture medium prevent apoptosis of porcine parthenotes and results in the net enhancement of porcine embryo viability., (2005 Wiley-Liss, Inc.)

The aim of this study was to determine the effects of IGF-I on the viability and development of porcine presumptive diploid parthenotes developing in vitro. In vitro derived 2-cell parthenotes were cultured to blastocysts with or without exogenous IGF-I in the presence or absence of BSA. The addition of 10 or 100 ng/ml IGF-I into NCSU 23 medium did not enhance the development of 2-cell parthenotes to the blastocyst stage. However, IGF-I increased cell numbers (P < 0.001) in the presence of BSA. Similarly, IGF-I alone did not affect apoptosis in the blastocysts but when combined with BSA, it reduced apoptosis (P < 0.05). Real time reverse-transcription polymerase chain reaction revealed that IGF-I significantly decreased the mRNA expression (pro-apoptotic gene Fas or Bak and anti-apoptotic gene Bcl-xL) ratios of both Fas/Bcl-xL (P < 0.01) and Bak/Bcl-xL (P < 0.01) in the presence of 0.4% BSA. These data suggest that IGF-I synergies with BSA to act as a mitogen and apoptotic survival factor in porcine preimplantation parthenotes.

The objective of this study was to determine developmental pattern, total cell number, apoptosis and apoptosis-related gene expression in haploid and diploid embryos following parthenogenetic activation. In vitro-matured porcine oocytes were activated by electrical pulses and cultured in the absence or presence of cytochalasin B for 3 h. Zygotes with two polar bodies (haploid) and one polar body (diploid) were carefully selected and were further cultured in NCSU 23 medium containing 0.4% bovine serum albumin (BSA) for 7 days. The percentage of development to blastocyst stage was higher (p < 0.01) in the diploid than in the haploid parthenotes. In haploid blastocysts, average total cell number was significantly reduced (p < 0.05) and apoptosis was increased at day 7. The relative abundance of Bcl-xL and Bak mRNA in the diploid blastocysts was similar to that of in vivo-fertilized embryos. However, Bcl-xL was significantly decreased, and Bak mRNA was significantly increased (p < 0.05) in haploid parthenotes compared with the diploid parthenotes. These results suggest that the haploid state affects apoptosis-related gene expression which results in increased apoptosis and decreased developmental competence of haploid parthenotes.

This study was conducted to determine the effects of polyvinyl alcohol (PVA), fetal bovine serum (FBS) and bovine serum albumin (BSA) on blastocoel formation, total cell number, apoptosis and Bcl-xL and Bak gene expression in porcine presumptive diploid parthenotes developing in vitro. The addition of 0.4% BSA to the culture medium enhanced the development of 2-cell or late 4-cell stage parthenotes to the blastocyst stage (P < 0.01) while FBS decreased the incidence of blastocoel formation. FBS also reduced the frequency of blastocysts developed from both 2-cell (P < 0.001) and late 4-cell (P < 0.05) embryos and increased the percentage of blastocysts undergoing apoptosis (P < 0.001). The relative abundance of Bcl-xL mRNA in presumptive diploid parthenotes in the control, PVA- and BSA-supplemented medium was similar to that of in vivo-derived embryos, but was significantly higher than in parthenotes cultured with FBS supplement (P < 0.05). Bak mRNA significantly increased at the blastocyst stage in FBS-supplemented cells (P < 0.01). These results suggest that apoptosis-related gene expression is significantly affected by FBS, and that this may result in alteration of apoptosis and embryo viability of porcine embryos developing in vitro.

The aim of this study was to determine the interactive effects of bovine serum albumin (BSA) and epidermal growth factor (EGF) on the viability and development of porcine presumptive diploid parthenotes developing in vitro. The addition of 0.1 and 0.4% BSA to the culture medium enhanced the development of 4-cell parthenotes to the blastocyst stage but EGF had no effect. However, while BSA also increased cell numbers, it did so only when EGF was also present. Either agent on its own had no effect. Similarly, apoptosis in the blastocysts was not influenced by either agent on its own but was reduced when both BSA and EGF were present. Furthermore, semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that EGF enhanced the mRNA expression of Bcl-xL in the presence of 0.4% BSA but BSA and EGF alone had no effect. EGF and/or BSA did not influence Bak gene expression in the blastocyst stage parthenotes. These results suggest that EGF and BSA synergistically enhance Bcl-xL gene expression, which may result in a net increase in cell number in porcine presumptive diploid parthenotes developing in vitro., (Copyright 2003 Wiley-Liss, Inc.)

Glucose-regulated proteins 75(grp75) is a member of hsp70 family. The expression of grp75 is upregulated during glucose starvation (such as ischemia). To evaluate grp75 function, CHL cells were cultured with glucose-free media for 20 h (A) and glucose-free media for 12 h + glucose-containing media for 8 h (ischemia reperfusion) (B). A constructed rat grp75 cDNA expression vector (pcDNA/grp75) was transfected into CHL cells and a cell strain that stably overexpressed grp75 was obtained. The transfected cells and untransfected cells(control group) were cultured with A or B. By MTT, LDH leakage measurement and flow cytometry analysis, growth rate of untransfected cells in B is significantly lower than that in glucose-containing media for 20 h (C) (p < 0.05) and A (p < 0.05). Growth rate of transfected cells is apparently higher than that of control group in B (p < 0.01). LDH liberation percentage of untransfected cells in B is obviously higher than that in C(p < 0.01) and it is not different from A(p > 0.05). LDH liberation percentage of transfected cells is apparently lower than that of control group in B(p < 0.01). Apoptosis of transfected cells is obviously lower by flow cytometry analysis. These results provide evidence for the cytoprotective function of grp75 during glucose starving and ischemia reperfusion.

Heat stress (HS) is a long-standing hurdle that animals face in the living environment. Alpha-lipoic acid (ALA) is a strong antioxidant synthesized by plants and animals. The present study evaluated the mechanism of ALA action in HS-induced early porcine parthenotes development. Parthenogenetically activated porcine oocytes were divided into three groups: control, high temperature (HT) (42 °C for 10 h), and HT + ALA (with 10 µM ALA). The results show that HT treatment significantly reduced the blastocyst formation rate compared to the control. The addition of ALA partially restored the development and improved the quality of blastocysts. Moreover, supplementation with ALA not only induced lower levels of reactive oxygen species and higher glutathione levels but also markedly reduced the expression of glucose regulatory protein 78. The protein levels of heat shock factor 1 and heat shock protein 40 were higher in the HT + ALA group, which suggests activation of the heat shock response. The addition of ALA reduced the expression of caspase 3 and increased the expression of B-cell lymphoma-extra-large protein. Collectively, this study revealed that ALA supplementation ameliorated HS-induced apoptosis by suppressing oxidative and endoplasmic reticulum stresses via activating the heat shock response, which improved the quality of HS-exposed porcine parthenotes. [ABSTRACT FROM AUTHOR]

Rotenone is a commonly used insecticidal chemical in agriculture and it is an inhibitor of mitochondrial complex Ⅰ. Previous studies have found that rotenone induces the production of reactive oxygen species (ROS) by inhibiting electron transport in the mitochondria of somatic and germ cells. However, there is little precise information on the effects of rotenone exposure in porcine oocytes during in vitro maturation, and the mechanisms underlying these effects have not been determined. The Cumulus-oocyte complexes were supplemented with different concentrations of rotenone to elucidate the effects of rotenone exposure on the meiotic maturation of porcine oocytes during in vitro maturation for about 48 hours. First, we found that the maturation rate and expansion of cumulus cells were significantly reduced in the 3 and 5 μM rotenone-treated groups. Subsequently, the concentration of rotenone was determined to be 3 μM. Also, immunofluorescence, western blotting, and image quantification analyses were performed to test the rotenone exposure on the meiotic maturation, total and mitochondrial ROS, mitochondrial function and biogenesis, mitophagy and apoptosis in porcine oocytes. Further experiments showed that rotenone treatment induced mitochondrial dysfunction and failure of mitochondrial biogenesis by repressing the level of SIRT1 during in vitro maturation of porcine oocytes. In addition, rotenone treatment reduced the ratio of active mitochondria to total mitochondria, increased ROS production, and decreased ATP production. The levels of LC3 and active-caspase 3 were significantly increased by rotenone treatment, indicating that mitochondrial dysfunction induced by rotenone increased mitophagy but eventually led to apoptosis. Collectively, these results suggest that rotenone interferes with porcine oocyte maturation by inhibiting mitochondrial function. [ABSTRACT FROM AUTHOR]

Fatty acid synthase (FASN) is an enzyme responsible for the de novo synthesis of long-chain fatty acids. During oncogenesis, FASN plays a role in growth and survival rather than acting within the energy storage pathways. Here, the function of FASN during early embryonic development was studied using its specific inhibitor, C75. We found that the presence of the inhibitor reduced blastocyst hatching. FASN inhibition decreased Cpt1 expression, leading to a reduction in mitochondria numbers and ATP content. This inhibition of FASN resulted in the down-regulation of the AKT pathway, thereby triggering apoptosis through the activation of the p53 pathway. Activation of the apoptotic pathway also leads to increased accumulation of reactive oxygen species and autophagy. In addition, the FASN inhibitor impaired cell proliferation, a parameter of blastocyst quality for outgrowth. The level of OCT4, an important factor in embryonic development, decreased after treatment with the FASN inhibitor. These results show that FASN exerts an effect on early embryonic development by regulating both fatty acid oxidation and the AKT pathway in pigs. [ABSTRACT FROM AUTHOR]

Melamine is a nitrogen heterocyclic triazine compound which is widely used as an industrial chemical. Although melamine is not considered to be acutely toxic with a high LD50 in animals, food contaminated with melamine expose risks to the human health. Melamine has been reported to be responsible for the renal impairment in mammals, its toxicity on the reproductive system, however, has not been adequately assessed. In the present study, we examined the effect of melamine on the follicle development and ovary formation. The data showed that melamine increased reactive oxygen species (ROS) levels, and induced granulosa cell apoptosis as well as follicle atresia. To further analyze the mechanism by which melamine induces oxidative stress, the expression and activities of two key antioxidant enzymes superoxide dismutase (SOD) and glutathi-one peroxidase (GPX) were analyzed, and the concentration of malondialdehyde (MDA) were compared between control and melamine-treated ovaries. The result revealed that melamine changed the expression and activities of SOD and GPX in the melamine-treated mice. Therefore, we demonstrate that melamine causes damage to the ovaries via oxidative stress pathway. [ABSTRACT FROM AUTHOR]

ABSTRACT Zearalenone (ZEN) is a metabolite of Fusarium and is a common contaminant of grains and foodstuffs. ZEN acts as a xenoestrogen and is considered to be cytotoxic, tissue toxic, and genotoxic, which causes abortions and stillbirths in humans and animals. Since estrogens affect oocyte maturation during meiosis, in this study we investigated the effects of ZEN on mouse oocyte meiotic maturation and granulosa cell proliferation. Our results showed that ZEN-treated oocyte maturation rates were decreased, which might be due to the disrupted cytoskeletons: (1) ZEN treatment resulted in significantly more oocytes with abnormal spindle morphologies; (2) actin filament expression and distribution were also disrupted after ZEN treatment, which was confirmed by the aberrant distribution of actin regulatory proteins. In addition, cortical granule-free domains (CGFDs) were disrupted after ZEN treatment, which indicated that ZEN may affect mouse oocyte fertilization capability. ZEN reduced mouse granulosa cell proliferation in a dose-dependent manner as determined by MTT assay and TUNEL apoptosis analysis, which may be another cause for the decreased oocyte maturation. Thus, our results demonstrated that exposure to zearalenone affected oocyte meiotic maturation and granulosa cell proliferation in mouse. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 1226-1233, 2015. [ABSTRACT FROM AUTHOR]

Iron is an important trace element involved in several biological processes. The role of iron in porcine early embryonic development remains unknown. In the present study, we depleted iron (III, Fe3+) with deferoxamine (DFM), a specific Fe3+ chelator, in cultured porcine parthenotes and monitored embryonic development, apoptosis, mitochondrial membrane potential, and ATP production. Results showed biphasic function of Fe3+ in porcine embryo development. 0.5 μM DFM obviously increased blastocyst formation (57.49 ± 2.18% vs. control, 43.99 ± 1.72%, P < 0.05) via reduced (P < 0.05) production of reactive oxygen species (ROS), further increased mitochondrial membrane potential and ATP production in blastocysts (P < 0.05). 0.5 μM DFM decreased mRNA expression of Caspase 3 (Casp3) and increased Bcl-xL. However, results showed a significant reduction in blastocyst formation in the presence of 5.0 μM DFM compared with the control group (DFM, 21.62 ± 3.92% vs. control, 43.99 ± 1.73%, P < 0.05). Fe3+ depletion reduced the total (DFM, 21.10 ± 8.78 vs. control, 44.09 ± 13.65, P < 0.05) and increased apoptotic cell number (DFM, 11.10 ± 5.24 vs. control, 2.64 ± 1.43, P < 0.05) in the blastocyst. An obvious reduction in mitochondrial membrane potential and ATP level after 5.0 μM DFM treatment was observed. Co-localization between mitochondria and cytochrome c was reduced after high concentration of DFM treatment. In conclusion, Fe3+ is essential for porcine embryonic development via mitochondrial function maintenance, but redundant Fe3+ impairs the function of mitochondria. [ABSTRACT FROM AUTHOR]

The feasibility of using the modified-cut standard straw (M-CSS) method for the vitrification of immature mouse oocytes has been tested. The effects of different vitrification methods on oocyte survival, cytoskeletal organization, the distribution of cortical granules (CGs), and apoptosis have also been compared. Immature mouse oocytes were vitrified-thawed using electron microscope grid or M-CSS method, and cultured to meiosis II (MII) stage. Oocyte development, cytoskeletal organization, CG distribution, and the expression of apoptosis-related genes were evaluated. Rates of recovery (91.7 vs. 74.9%) and survival (89.0 vs. 62.6%) were significantly higher in M-CSS group than in EM grid group. The number of oocytes with normal chromosome alignment at the spindle and spindle morphology were similar in both groups. However, the actin cap was significantly degraded in EM grid groups (52.6 vs. 35.1%, respectively). Abnormal release of CGs also frequently occurred in EM grid groups (42.6 vs. 32.7%, respectively). Pro-apoptosis-related gene expression levels of Bax, caspase 3 were expressed lower than control in MII stage oocytes derived from M-CSS group; anti apoptosis-related genes, survivin and heat shock factor-1 (Hsf-1) were slightly increased. However, all genes expression was significantly increased in MII stage oocytes derived from EM grid groups. Vitrification reduces the survival rate of immature mouse oocytes, alters cytoskeletal organization and CG distribution, and promotes apoptosis. However, these effects are less pronounced in vitrified oocytes generated by M-CSS than in those generated by EM grid method. Therefore, the novel M-CSS is a feasible approach for the cryopreservation of immature mouse oocytes. [ABSTRACT FROM AUTHOR]

In this study, we investigated the effects of various durations of dibutyryl cyclic AMP (dbcAMP) treatment on the in vitro maturation (IVM) and subsequent development of parthenogenetically activated embryos. Immature porcine oocytes were cultured with or without 1 mM dbcAMP during the first 20, 28, or 36 h of culture, and then incubated for an additional 24 h without dbcAMP. The expression ofWee1B,Myt, andCdc25Band the level of maturation promoting factor (MPF) in metaphase II oocytes were analyzed by real-time PCR (qRT-PCR) and enzyme linked immunosorbent assay (ELISA), respectively. The distribution of actin microfilaments in oocytes was also assessed. Subsequently, apoptotic cells in blastocysts from each group were visualized by transferase-mediated dUTP nick-end labeling staining. Results showed that oocytes extruded the first polar body between 12 and 18 h after being released from dbcAMP. MPF activity in oocytes at 28 + 24 h and 36 + 24 h after dbcAMP treatment was higher than that in the control group. Significantly more blastocysts were present among embryos in 28 + 24 h (54.28% vs. 39.11%,P< 0.05) and 36 + 24 h (47.24% vs. 32.94%,P< 0.05) groups than among embryos cultured in the absence of dbcAMP. However, the number of total and apoptotic cells was not significantly different between groups. The distribution of actin microfilaments was abnormal in oocytes cultured for 60 h without dbcAMP. In addition, the expression ofWee1B,Myt, andCdc25Bwas higher in the control group at 44 h than in the dbcAMP group, but there were no differences in expression at the other time points. In conclusion, dbcAMP treatment delays oocyte maturation and maintains oocyte quality. [ABSTRACT FROM AUTHOR]

• Astragaloside IV contributed to the enhancement of miR-150−5p level in HCC cells. • AS-IV modulated miR-150−5p or β-catenin levels to facilitate HCC cells apoptosis in HCC. • MiR-150−5p directly targeted and negatively regulated β-catenin in HCC. • MiR-150−5p regulated cell apoptosis and tumor growth through β-catenin in HCC. Hepatocellular carcinoma (HCC), a common malignant tumor, has been regarded as a leading cause of cancer-related deaths globally. Astragaloside IV (AS-IV) was reported to participate in the regulation of multiple tumors. However, the role of AS-IV in HCC was still unclear in HCC. Bioinformatics analysis and function or mechanism experiments including RT-qPCR, MTT assay, flow cytometry, Western blot, luciferase reporter assay and xenografts assays were applied to investigate the function of AS-IV, miR-150−5p and CTNNB1. We discovered that AS-IV treatment was supposed to significantly increase miR-150−5p level. In addition, AS-IV accelerated cell apoptosis by inducing miR-150−5p in vitro and in vivo. Furthermore, AS-IV increased cell apoptosis rate through reducing β-catenin level in vitro and in vivo. In detail, AS-IV triggered a decline of Bax and a rise of Bcl-2 in HCC cells and xenograft tissues. In mechanism, we validated the combination between miR-150−5p and CTNNB1. Moreover, miR-150−5p could negatively regulate CTNNB1 level by binding to its3'UTR. Finally, rescue assay demonstrated that CTNNB1 overexpression partially rescued the inhibitive effect on tumor growth and promotive influence on cell apoptosis caused by miR-150−5p amplification. The up-regulation of miR-150−5p induced by AS-IV suppressed the progression of HCC by repressing β-catenin, providing a new molecular target for the utilization of AS-IV In the treatment of HCC. [ABSTRACT FROM AUTHOR]

Ethnopharmacological relevance Oryeongsan (ORS, Wulingsan) has been reported to possess renal protective effects from renal diseases such as diabetes-induced renal damage, and nephrocalcinosis. Aim of the study This study was conducted to evaluate the quantitative analysis and the inhibitory effect of ORS on hypertonic stress-induced water channel and apoptosis in murine inner medullary collecting duct cell line (mIMCD-3). Materials and methods Chromatographic and NMR spectroscopic analysis were performed and water balance regulation was determined by Western blot, RT-PCR, and immunofluorescnece. Results Seven active principles (5-hydroxymethylfurfural, alismoxide, methyl − trans-cinnamate, adenine, guanosine, adenosine, and ferulic acid) in ORS were isolated and the structures were identified mainly by NMR spectroscopic analysis. In addition, contents of these metabolites in ORS were evaluated by HPLC analysis. Pretreatment with ORS significantly attenuated the hypertonic stress (175 mM NaCl)-induced increase in protein levels of AQP2 and apical membrane insertion. ORS also attenuated osmolyte sodium- myo -inositol transporter (SMIT) expression and tonicity-responsive enhancer binding protein (TonEBP) mRNA under hypertonic stress. Those actions of ORS presented the similar effect of PKA inhibitor which AQP2 expression throughout the inhibition of vasopressin-mediated cAMP/PKA signal pathway. On the other hand, pretreatment with ORS attenuated hypertonic stress-induced cell death. Hypertonic stress-induced Bax or caspase-3 expression was decreased by ORS, resulting in anti-apoptotic effect. Conclusions The present data suggest that the beneficial effect of ORS in water balance and apoptosis against hypertonic stress of renal collecting ducts. [ABSTRACT FROM AUTHOR]

In vitro porcine embryo production systems have been established and well characterized. However, the efficiency of embryo development during IVC is still very low. In the present study, we have investigated the development of parthenogenetic porcine embryos in the well-known PZM-5 medium for porcine embryos, which was modified by replacing glutamine with the GlutaMAX supplement. We revealed that blastocyst apoptosis was significantly lower in the presence of GlutaMAX, which reduced the release of mitochondrial cytochrome c. Furthermore, the expression of apoptosis genes was significantly lower during GlutaMAX treatment (P < 0.05). The modified medium was also examined for the eventual loss of its efficacy in the presence of GlutaMAX. Three, 6, and 12 months after medium preparation, blastocyst formation in the GlutaMAX-supplemented medium was significantly higher than the number of blastocysts in the medium containing glutamine. After a long period of storage, ammonia concentration was significantly increased in the glutamine medium, whereas it was not statistically different in the GlutaMAX medium. Elevated ammonia concentrations reduced the mitochondrial membrane potential and ATP content of blastocysts in the glutamine medium. These results demonstrate that GlutaMAX can reduce blastocyst apoptosis via inhibition of the cytochrome c pathway and significantly extend the shelf life of the culture medium to at least 1 year. [ABSTRACT FROM AUTHOR]

Nobiletin is a natural flavonoid found in citrus fruits with beneficial effects, including anti-inflammatory, anti-cancer and anti-oxidation effects. The aim of this study was to investigate whether nobiletin improves mitochondrial function in porcine oocytes and examine the underlying mechanism. Oocytes enclosed by cumulus cells were cultured in TCM-199 for 44 h with 0.1% dimethyl sulfoxide (control), or supplemented with 5, 10, 25, and 50 μM of nobiletin (Nob5, Nob10, Nob25, and Nob50, respectively). Oocyte maturation rate was significantly enhanced in Nob10 (70.26 ± 0.45%) compared to the other groups (control: 60.12 ± 0.47%; Nob5: 59.44 ± 1.63%; Nob25: 63.15 ± 1.38%; Nob50: 46.57 ± 1.19%). The addition of nobiletin reduced the levels of reactive oxygen species and increased glutathione levels. Moreover, Nob10 promoted mitochondrial biogenesis by upregulating the protein levels of sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor-gamma coactivator 1 α (PGC-1α). This resulted in an increase in the number of active mitochondria, mitochondrial DNA copy number, mitochondrial membrane potential, and ATP production, thereby enhancing mitochondrial function. The protein level of p53 decreased, followed by the phosphorylation of B-cell lymphoma 2, suggesting a reduction in mitochondria-mediated apoptosis in the Nob10 group. Additionally, the release of cytochrome c from the mitochondria was significantly diminished along with a decrease in the protein expression of caspase 3. Thus, nobiletin has a great potential to promote the in vitro maturation of porcine oocytes by suppressing oxidative stress and promoting mitochondrial function through the upregulation of the SIRT1/PGC-1 α signaling pathway. • Nobiletin enhanced meiotic maturation in porcine oocytes. • Nobiletin reduced oxidative stress during oocyte maturation. • Nobiletin promoted mitochondrial function by regulating SIRT1/PGC-1α signaling pathway in porcine oocytes. • Nobiletin attenuated apoptosis by inhibiting p53/Bcl-2 signaling pathway in porcine oocytes. [ABSTRACT FROM AUTHOR]

Abstract Thiamethoxam (TMX) is a neonicotinoid insecticide. It has specific high toxicity to insects. Residues of TMX have been detected in various crops. Early embryo quality is vital for fertility. Excessive production of reactive oxygen species (ROS) can override embryonic antioxidant defenses, producing oxidative stress that triggers apoptosis, necrosis, and/or permanent DNA damage responses in the early embryo. Comparative studies have indicated that TMX hepatotoxicity is significant in mammals in acute tests, but little is known about accumulated chronic toxicity in early embryonic development. Porcine embryos were obtained here by the parthenogenetic activation of meiosis II oocytes and cultured in the PZM-5 medium with or without TMX. These embryos were evaluated by various methods. The expansion and hatching of blastocysts treated with TMX decreased by 21.73% and 16.71%, respectively, as compared with controls. In an analysis of 5-bromo-2-deoxyuridine (BrdU) incorporation, the rate of cell proliferation was 44.33% lower as compared with expanded blastocysts of the control group. ROS and γH2AX levels were higher in the TMX group than in the control group. Real-time reverse-transcription polymerase chain reaction showed that Sod1 expression increased and the expression of Mnsod, Gpx1, Igta5, and Cox2 decreased. A CDK1 kinase assay revealed that maturation-promoting factor (MPF) activity diminished by 31.41% in expanding blastocysts. In conclusion, these results suggest that TMX inhibits blastocyst expansion and hatching by ROS-induced DNA damage checkpoint activation, which inhibits the activation of MPF and cell cycle progression in porcine blastocysts. Highlights • TMX results in oxidation-reduction reaction out of balance and increased ROS which induces subsequent DNA damage. • The DNA damage accumulates greatly because of rapid cell proliferation of blastocysts at score 3. • The ROS-dependent DNA damage response arrests cell cycle of blastocysts by ATM-CHK1-CDC25C and ATM-p53-p21 pathways. [ABSTRACT FROM AUTHOR]

Aflatoxin B1 (AFB1) is a type of mycotoxin produced by the fungi Aspergillus flavus and Aspergillus parasiticus . AFB1 is considered as the most toxic mycotoxin owing to its toxic effect on health. In the present study, the toxic effect of AFB1 on early porcine embryonic development and its possible mechanism were investigated. Blastocyst formation was impaired with treatment of 1 nM AFB1 compared with control, 0.01, 0.1 group (40.13 ± 2.10%, 28.21 ± 1.62%, 32.34 ± 2.07% vs 19.01 ± 1.06%). Further study showed that the presence of AFB1 induced the generation of reactive oxygen species (ROS). And excessive ROS caused DNA damage which confirmed by the comet assay. Additionally, AFB1 also disrupted the DNA damage repair through the regulation of 53BP1. The AFB1 treatment significantly increased the γH2A foci and decreased the 53BP1 foci. TUNEL assay confirmed the generation of apoptosis, further resulting in the occurrence of autophagy. Moreover, AFB1 significantly increased the expression of pro-apoptosis genes Bax and Casp3 and reduced the expression of anti-apoptotic genes Bcl2 and Bcl-xl . In addition, the AFB1 also significantly increased the expression of autophagy related genes Lc3 and Beclin1. The presence of AFB1 significantly impaired the cell proliferation, a parameter of blastocyst quality for outgrowth. These results showed that the presence of AFB1 impaired porcine early embryonic development through oxidative stress, as well as DNA damage and repair, apoptosis, autophagy. [ABSTRACT FROM AUTHOR]