Z-VAD-FMK

Recent reports considered that it was the disturbance of calcium homeostasis and the accumulation of misfolded proteins in the endoplasmic reticulum (ER) that activated hepatic stellate cells (HSCs) apoptosis and promoted fibrosis resolution. However, the signal-transducing events that are activated by ER stress after HSCs activation were incompletely understood. In this study, we induced ER stress with thapsigargin (TG), and determined the activation of calpain and the cleavage of caspase by analyzing the protein levels and the correspondingly increased intracellular calcium levels and the induction of the proapoptotic transcription factor CHOP. Moreover, the phosphorylation of JNK and p38 MAPK were followed by the activation of the executioner caspases, caspase-3. As expected, preventing an increase in intracellular calcium levels using intracellular calcium chelators, EGTA, and BAPTA/AM, could substantially inhibit the phosphorylation of JNK and p38 MAPK, abolish the activation of calpains, namely caspase-12, caspase-9, and caspase-3, and provide significant protection for TG-treated activated HSCs. Interestingly, pretreatment with p38 MAPK inhibitor SB202190, JNK inhibitor SP600125, the pan-caspase inhibitor z-VAD-FMK, or calpain inhibitors calpeptin, significantly reduced the cell apoptosis and the cleavage of caspase-12 and caspase-3.

Rhus verniciflua Stokes has been widely used as a traditional medicinal plant with a variety of pharmacological activities. We investigated the mechanisms involved in mediating the effects of Rhus verniciflua Strokes (R. verniciflua) extract in human chronic myelogenous leukemia K562 cells, including caspase-dependent apoptotic pathways related to cell-cycle arrest, as well as the inhibition of nuclear factor NF-κB activation and upregulation of the mitogen-activated protein kinase (MAPK) signaling pathway. R. verniciflua extract suppressed the abnormal cellular proliferation of K562 cells in a dose- and time‑dependent manner and increased the quantitative proportions of cells involved in the early and late process of apoptosis. Furthermore, R. verniciflua extract significantly mediated the mRNA levels of pro-apoptotic and anti-apoptotic regulators, such as Bcl-2, Bax, Mcl-1 and survivin in apoptotic cells. Particularly, the treatment of K562 cells with R. verniciflua extract augmented the caspase‑3 activity and increased the expression of caspase‑3 protein, while co-treatment with R. verniciflua extract and the permeant pan‑caspase inhibitor Z-VAD-FMK and caspase‑3 inhibitor Z-DEVD-FMK inversely enhanced the proliferation of K562 cells.

Reactive oxygen species (ROS) are fundamental for intracellular signalling. In spermatozoa, they are involved both to apoptosis and to capacitation, and changes in ROS levels can alter the balance between these two processes. Oestrous sheep serum (OSS) is considered an efficient agent for in vitro capacitation of ram spermatozoa. We have explored the effects of OSS on ram sperm physiology, especially on ROS production, during in vitro capacitation. Semen samples from 15 rams were cryopreserved. After thawing, samples were submitted to four treatments: control (CTL), 10% OSS supplementation for in vitro sperm capacitation, caspase inhibitor (INH, Z-VAD-FMK 100 μM) and OSS (10%) plus caspase inhibitor (I + E). Sperm samples were incubated for 30 min at 38.5°C and 5% CO2 and evaluated motility and kinetic parameters by computer-assisted semen analysis (CASA) and viability (propidium iodide), apoptotic-like membrane changes (YO-PRO-1), acrosomal status (PNA-FITC), intracellular calcium (FLUO-3), membrane fluidity (M540) and ROS production (CM-H2 DCFDA) by flow cytometry. OSS induced changes in kinetic parameters compatible with capacitation, with a decrease in the percentage of progressive motility and linearity, and an increase in the amplitude of the lateral displacement of the sperm head (p < .