cytochrome-c-t and Cicatrix

We previously demonstrated that FK506, a generally applied immunosuppressant in organ transplantation, could promote peripheral nerve regeneration through reducing scar formation. However, little is known about how FK506 reduces scar formation. Herein we investigated the influence of FK506 on fibroblast proliferation and its correlation with scar formation after sciatic nerve injury in rats, and further explored the effect of FK506 on fibroblast proliferation and apoptosis in vitro. Masson staining and immunohistochemistry revealed that scar area and fibroblast number in the nerve anastomosis of sciatic nerve-injured rats were significantly reduced after FK506 administration. The scar area had a significant positive correlation with the fibroblast number, as detected by linear correlation analysis. CCK-8 assay and flow cytometry indicated that FK506 also inhibited proliferation and induced apoptosis of fibroblasts in vitro. It was primarily phosphorylation of JNK and ERK that were activated during the apoptosis of fibroblast. Pretreatment of cells with JNK inhibitor, SP600125, or ERK inhibitor, PD98059, could inhibit FK506-induced fibroblast apoptosis, respectively. Moreover, simultaneous application of both inhibitors had additive roles in cell protection from apoptosis. These results suggest that FK506-induced fibroblast apoptosis contributes to the suppression of fibroblast proliferation and then results in the reduction of scar formation in sciatic nerve-injured rat, and that JNK and ERK are involved in FK506-induced fibroblast apoptosis.

Topics: Anastomosis, Surgical; Animals; Anthracenes; Apoptosis; Caspase 3; Cell Proliferation; Cells, Cultured; Cicatrix; Cytochromes c; Cytoprotection; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Flavonoids; Immunosuppressive Agents; JNK Mitogen-Activated Protein Kinases; Male; Phosphorylation; Rats; Rats, Sprague-Dawley; Sciatic Nerve; Tacrolimus

Hydroxycamptothecin (HCPT) has been proven to induce apoptosis in fibroblasts. In this study, we investigated whether the PRKR-like ER kinase (PERK) pathway is implicated in apoptotic signaling of human Tenon's capsule fibroblasts (HTCFs) by HCPT.. Normal and PERK-knockdown HTCFs were used in this study. Apoptosis was determined by the cell viability assay, Annexin V/propidium iodide (PI) dual-staining, cell cycle analysis in HTCFs treated with HCPT in various doses and for various durations. Endoplasmic reticulum (ER) stress markers and sensor proteins were detected by Western blot analysis. Mitochondrial dysfunction was measured by detecting the mitochondrial membrane potential (ΔΨm) and measuring the expression of cytochrome c (cyt c).. HCPT induced apoptosis in the HTCFs, which was characterized as decreased cell viability and sub-S fraction of the cell cycle and increased apoptosis rate by Annexin V/PI dual-staining. The activity levels of caspase-3 and caspase-9 were significantly increased and were accompanied by cytosolic release of cyt c and decreased ΔΨm in response to HCPT. Treatment with HCPT increased the expression of glucose-regulated protein 78 (GRP78), phospho-PERK, activating transcription factor 6 (ATF6), phosphoinositol-requiring kinase 1 (IRE1), C/EBP homologous protein (CHOP), Bax, and phospho-c-Jun N-terminal kinase (JNK) and decreased the expression of Bcl-2. Knockdown of PERK attenuates HCPT-induced apoptosis in HTCFs, dependent upon both ER stress and the mitochondrial apoptotic pathway.. This study suggests that the ER stress response and mitochondrial dysfunction are involved in apoptosis induced by HCPT in HTCFs, which might be mediated by PERK; thus, this study offers new insight into preventing postoperative scarring via treatment with HCPT.

Topics: Apoptosis; Biomarkers; Blotting, Western; Camptothecin; Caspases; Cell Cycle; Cell Survival; Cells, Cultured; Cicatrix; Cytochromes c; eIF-2 Kinase; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Fibroblasts; Humans; Membrane Potential, Mitochondrial; Mitochondria; Signal Transduction; Stress, Physiological; Tenon Capsule