Heat stress impairs oocyte maturation through ceramide-mediated apoptosis in pigs
Heat stress (HS) is an emerging issue that greatly impairs the reproductive performance of animals and humans. In particular, disruption of oocyte maturation due to HS is considered a major cause of impaired reproductive performance. HS is known to induce ceramide generation, which causes reactive oxygen species (ROS) production and mitochondrial dysfunction, thereby inducing apoptosis. Therefore, we investigated whether inhibition of ceramide generation ameliorates HS-induced apoptosis in porcine cumulus-oocyte complexes (COCs) using specific inhibitors of the de novo (fumonisin B1, FB1) and hydrolytic pathways (desipramine, Des) of ceramide formation. We investigated the effects of FB1 and Des supplementation under HS conditions (41.5 °C for 44 h) on in vitro maturation (IVM) of porcine COCs. After IVM, HS significantly reduced proportion of COCs exhibiting fully expanded cumulus cells and the rate of metaphase II in oocytes. After parthenogenetic activation (PA), HS significantly reduced the rates of cleavage and blastocyst formation with a lower total cell number and a higher percentage of apoptosis in blastocysts.
However, FB1 or Des supplementation under HS avoided detrimental effects of HS on expansion of cumulus cells, nuclear maturation of oocytes, and embryonic development after PA including the rates of cleavage and blastocyst formation, total cell number, and the percentage of apoptosis in blastocysts. Furthermore, FB1 or Des addition under HS, compared with HS alone, significantly decreased ceramide generation, ROS production, cytochrome C expression, and apoptosis and increased mitochondrial membrane potential in COCs, reaching levels comparable with those of the control. Taken together, our results indicate that HS impaired oocyte maturation through ceramide-mediated apoptosis.
Finally, the interaction of SCGN with ATF4 was computationally predicted and then validated by a co-immunoprecipitation assay. We found that ox-LDL treatment increased the levels of ER stress markers, such as phosphorylated protein kinase-like endoplasmic reticulum kinase, phosphorylated eukaryotic initiation factor 2 alpha, activating transcription factor 4 (ATF4), and transcription factor CCAAT-enhancer-binding protein homologous protein, and promoted MIN6 cell apoptosis; in addition, the expression of SCGN was downregulated. siRNA-mediated SCGN knockdown exacerbated β-cell ER stress by increasing ATF4 expression.
Tetrahedral Framework Nucleic Acid Inhibits Chondrocyte Apoptosis and Oxidative Stress through Activation of Autophagy
Osteoarthritis (OA) is a degenerative articular cartilage pathogenic process that is accompanied by excessive chondrocyte apoptosis. The occurrence of chondrocyte death and OA is related to decreased autophagy. Tetrahedral framework nucleic acid (TFNA), a potent bioactive DNA nanomaterial, exerts antiapoptotic and antioxidative effects in various diseases, resulting in autophagy promotion and inhibition of the Wnt/β-catenin-signaling pathway. Here, we aimed to elucidate the therapeutic effects of TFNA on OA and its potential molecular mechanism of action. TFNA was synthesized and characterized by established methods. An interleukin (IL)-1β stimulated OA cell model was established and treated with TFNA. Cellular uptake of TFNA and intracellular reactive oxygen species levels were examined via immunofluorescence and flow cytometry.
Apoptotic cell death was documented by the Cell Counting Kit-8 (CCK8) assay and flow cytometry. Transmission electron microscopy was applied to view the autophagosomes. The expression of BCL2, BAX, caspase-3, Nrf2, HO-1, LC3-II, Beclin1, Atg7, β-catenin, Lef-1, and CyclinD1 was detected by immunofluorescence and western blotting. TFNA was successfully synthesized and effectively entered chondrocytes in the absence or presence of IL-1β without the help of transfection agents. TFNA treatment in IL-1β-induced chondrocytes reduced apoptosis by activating the BCL2/BAX/caspase-3 pathway, inhibited oxidative stress by regulating the Nrf2/HO-1-signaling pathway, and enhanced autophagy through upregulated LC3-II, Beclin1, and Atg7. Moreover, TFNA showed chondroprotective effects by regulating the Wnt/β-catenin-signaling pathway. Overall, TFNA may have utility as a therapeutic nanomedicine for OA.
Heat stress impairs oocyte maturation through ceramide-mediated apoptosis in pigs
Raddeanin A suppresses lung cancer cell proliferation via induction of apoptosis and increased production of ROS
At present, in vitro cell experiments have confirmed that RaddeaninA can effectively inhibit the proliferation of some tumor cells, but the effect of RaddeaninA on lung cancer cells has not been observed. Therefore, this study explored its effect on lung cancer cells and its mechanism of action. Human lung cancer cell lines were treated with serum-free medium and varied concentrations of Raddeanin A. Cell proliferation and apoptosis were determined using MTT, and flow cytometric assays, respectively. The intracellular level of ROS was determined using DCFH-DA assay. Protein and mRNA expressions of bax, bcl-2 and cyt c were measured using Western blotting and qRT-PCR. RaddeaninA treatment can promote PC-9 cell apoptosis in a time and dose-dependent manner (p<0.05).
Treatment of PC-9 cells with Raddeanin significantly and dose-dependently increased the activities of caspase-9 and caspase-3 (p<0.05), and led to significant and dose-dependent increases in ROS levels. Treatment of PC-9 cells with Raddeanin A led to significant and dose-dependent decreases in mitochondrial membrane potential. It significantly and dose-dependently upregulated bax mRNA and protein expressions, but down-regulated bcl-2 mRNA and protein expressions significantly and dose-dependently (p<0.05). On the other hand, Raddeanin significantly and dose-dependently down-regulated cytoplasmic bax protein expression, while upregulating cyt c expression (p<0.05).
Lyophilized powder containing 10 mM Sodium Phosphate, 0.15 M Sodium Chloride, 1% (w/v) BSA and 0.1% (v/v) Kathon CG. Protease/IgG free, pH 7.2. Rehydrate with 0.55 ml of deionized water. Prepare fresh working dilution daily.
Description: Goat anti Human kappa chain secondary antibody (HRP)
Lyophilized powder containing 10 mM Sodium Phosphate, 0.15 M Sodium Chloride, 1% (w/v) BSA and 0.1% (v/v) Kathon CG. Protease/IgG free, pH 7.2. Rehydrate with 0.55 ml of deionized water. Prepare fresh working dilution daily.
Description: Goat anti Human kappa chain secondary antibody (HRP)
Lyophilized from 10 mM Sodium Phosphate, 0.15 M Sodium Chloride, 1% (w/v) BSA and 0.05% (w/v) Sodium Azide. Protease/IgG free, pH 7.2. Dilute in dH2O to a concentration of 1 mg/ml
Description: Goat anti Human kappa chain secondary antibody (rhodamine)
Goat Anti-Human kappa ( chain specific) was previously known under catalogue number 25-787-278169 was previously known under catalogue number 25-787-278169
Goat Anti-Human kappa ( chain specific) was previously known under catalogue number 25-787-278166 was previously known under catalogue number 25-787-278166
Goat Anti-Human kappa ( chain specific) was previously known under catalogue number 25-787-278167 was previously known under catalogue number 25-787-278167
Goat Anti-Human kappa ( chain specific) was previously known under catalogue number 25-787-278162 was previously known under catalogue number 25-787-278162
Similarly, bax protein expression was significantly and dose-dependently upregulated in mitochondria, but the corresponding cyt c expression was significantly and dose-dependently down-regulated (p<0.05). Raddeanin A is a potential and effective lung cancer chemotherapy drug, which can induce lung cancer cell apoptosis and inhibit proliferation.
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