Son E, et al. Korean J Physiol Pharmacol. 2020 Mar;24(2):173-183. https://doi.org/10.4196/kjpp.2020.24.2.173

Heart ischemia/reperfusion (I/R) injury is related to iron content. However, the occurrence and mechanism of changes in labile iron pool (LIP) during I/R is debatable. Moreover, the identity of the iron form dominant in LIP during I/R is unclear. Herein, we measured changes of LIP during simulated ischemia (SI) and reperfusion (SR), in which ischemia was simulated in vitro with lactic acidosis and hypoxia. Total LIP did not change in lactic acidosis, whereas LIP, especially Fe³⁺, increased in hypoxia. Under SI, accompanied by hypoxia with acidosis, both Fe²⁺ and Fe³⁺ were significantly increased. Increased total LIP was maintained at 1 h post-SR. However, the Fe²⁺ and Fe³⁺ portion was changed. The increased Fe²⁺ was decreased, and conversely the Fe³⁺ was increased. BODIPY oxidized signal increased and through the time-course these changes correlated with blebbing of cell membrane and SR-induced LDH release. These data suggested lipid peroxidation occurred via Fenton’s reaction. The experiments using bafilomycin A1 and zinc protoporphyrin suggested no role of ferritinophagy or heme oxidation in the increase of LIP during SI. The extracellular source, transferrin assessed using serum transferrin bound iron (TBI) saturation showed that the depletion of TBI reduced SR-induced cell damages and additive saturation of TBI accelerated SR-induced lipid peroxidation. Furthermore, Apo-Tf dramatically blocked the increase of LIP and SR-induced damages. In conclusion, Tf-mediated iron induces the increase of LIP during SI, and it causes Fenton reaction-mediated lipid peroxidation during the early phase of SR.

The lack of effective anti-fibrotic agents remains a significant unmet need in the treatment of intestinal fibrosis, a condition largely driven by transforming growth factor-β1 (TGF-β1)-mediated myofibroblast activation. Phosphodiesterase 4 (PDE4) inhibitors, known to increase intracellular cAMP levels, exhibit anti-inflammatory and potential anti-fibrotic properties, though their efficacy in intestinal fibrosis is poorly defined. In this study, we investigated the anti-fibrotic effects and underlying mechanisms of PDE4 inhibitors (rolipram, roflumilast, piclamilast, and mesopram) in human intestinal myofibroblasts (HIMFs) stimulated with TGF-β1. All PDE4 inhibitors significantly reduced TGF-β1-induced mRNA expression of collagen1A1 (COL1A1) and fibronectin (FN1), with mesopram additionally reducing α-smooth muscle actin (ACTA2) mRNA expression. At the protein level, however, only roflumilast significantly decreased procollagen1A1 (Procol1A1) and FN expression. Mechanistically, roflumilast, rolipram, and mesopram significantly reduced serum response factor (SRF) mRNA expression, whereas piclamilast and mesopram significantly inhibited myocardin-related transcription factor A (MRTFA) mRNA expression. Rolipram and roflumilast exhibited a decreasing trend in MRTFA mRNA expression without statistical significance. Notably, roflumilast demonstrated superior inhibitory effects, significantly decreasing both MRTF-A and SRF protein expression and effectively reducing nuclear localization of MRTF-A. Additionally, rolipram and roflumilast significantly attenuated TGF-β1-induced phosphorylation of Smad2. These findings indicate that PDE4 inhibitors, particularly roflumilast, effectively suppress TGF-β1-induced fibrogenic activation in HIMFs by modulating MRTF/SRF and Smad-dependent pathways, highlighting the value of roflumilast for further preclinical investigation as a potential therapeutic strategy for intestinal fibrosis.