Anti-Human CASP3 Polyclonal Antibody

Objective: This study aimed to investigate the effects of cigarette smoke (extract) on autophagy and apoptosis in oral mucosa epithelial cells. Methods: The effects of cigarette smoke extract (CSE) on autophagy and apoptosis in oral epithelial cells were studied in vivo and in vitro. Leuk-1 cells were administered cigarette smoke extract or chloroquine (CQ) and rapamycin (RAPA) at different concentrations. Immunoblotting, immunofluorescence, Western blotting and flow cytometry were used to detect autophagy-related protein and apoptosis levels, screen the optimal concentration and stimulation time, and verify the effect of CSE stimulation on autophagy and apoptosis in leuk-1 cells. Meanwhile, autophagy expression in epithelial cells from the local oral tissues of mice who had smoked for 5 months was detected. Results: Under CS stimulation, LC3-II and Beclin-1, the key proteins of leuk-1 autophagy, were upregulated in a concentration- and time-dependent manner. In addition, CS significantly upregulated the expression of Cleaved caspase-3 (C-casp3), a protein involved in apoptosis. However, under stimulation with CQ, autophagy in leuk-1 cells was inhibited and the level of C-casp3 and the apoptosis rate were increased. The autophagy activator RAPA significantly reduced the level of C-casp3 and apoptosis rate in leuk-1 cells. Conclusion: The results of this study indicate that CS can simultaneously activate autophagy and apoptosis in mouse and human oral epithelial cells, that autophagy inhibition can aggravate the CSE-triggered apoptosis of oral epithelial cells, and that autophagy induction can inhibit the CSE-triggered apoptosis of oral epithelial cells. Autophagy is suggested to play a protective role in the CSE-induced apoptosis of oral epithelial cells. Further studies are needed to explore the concrete mechanisms underlying the regulatory effects of CS-induced apoptosis and to gain in-depth insight into the complex interactions between apoptosis and autophagy.

Genistein is an isoflavone that has estrogen (E-2)-like activity and is beneficial for follicular development, but little is known regarding its function in oxidative stress (OS)-mediated granulosa cell (GC) injury. Here, we found that after exposure to H2O2, Genistein weakened the elevated levels of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA), which were regarded as the biomarkers for OS, and rescued glutathione (GSH) content and GSH/GSSG ratio accompanying with a simultaneous increase in cyclic adenosine monophosphate (cAMP) level, whereas addition of protein kinase A (PKA) inhibitor H89 impeded the effects of Genistein on the levels of ROS and MDA. Further analysis evidenced that Genistein enhanced the activities of antioxidant enzymes superoxide dismutase (SOD), GSH-peroxidase (GSH-Px), and catalase (CAT) in H2O2-treated GCs, but this enhancement was attenuated by H89. Under OS, Genistein improved cell viability and lessened the apoptotic rate of GCs along with a reduction in the activity of Casp3 and levels of Bax and Bad messenger RNA (mRNA), while H89 reversed the above effects. Moreover, Genistein treatment caused an obvious elevation in mitochondrial membrane potential (MMP) followed by a decline in the levels of intracellular mitochondrial superoxide, but H89 inhibited the regulation of Genistein on MMP and mitochondrial superoxide. Supplementation of Genistein promoted the secretion of E-2 and increased the expression of Star and Cyp19a1 mRNA, whereas suppressed the level of progesterone (P-4) accompanied with a decline in the level of Hsd3b1 mRNA expression. H89 blocked the regulation of Genistein on the secretion of E-2 and P-4, and alleviated the ascending of Star and Cyp19a1 elicited by Genistein. Collectively, Genistein protects GCs from OS via cAMP-PKA signaling.