Induction of ferroptosis in human keratinocyte HaCaT cells by squalene hydroperoxide: Possible prevention of skin ferroptosis by botanical extracts.

Ever since the proposal of ferroptosis, it has been studied as a nonapoptotic cell death caused by iron ion-dependent phospholipid (PL) peroxidation. We previously showed that treatment of human hepatoma cell line HepG2 with prepared PL hydroperoxide (PLOOH) resulted in ferroptosis. However, in human sebum, the major hydroperoxide is not PLOOH but squalene hydroperoxide (SQOOH), and to our knowledge, it is not established yet whether SQOOH induces ferroptosis in the skin. In this study, we synthesized SQOOH and treated human keratinocyte HaCaT cells with SQOOH. The results showed that SQOOH induces ferroptosis in HaCaT cells in the same way that PLOOH causes ferroptosis in HepG2 cells. Some natural antioxidants (botanical extracts) could inhibit the ferroptosis in both the cell types. Consequently, future research focus would revolve around the involvement of SQOOH-induced ferroptosis in skin pathologies as well as the prevention and treatment of skin diseases through inhibition of ferroptosis by botanical extracts.

Protective effect of ebselen on bleomycin-induced lung fibrosis: analysis of the molecular mechanism of lung fibrosis mediated by oxidized diacylglycerol.

The molecular mechanisms underlying the development of pulmonary fibrosis remain unknown, and effective treatments have not yet been developed. It has been shown that oxidative stress is involved in lung fibrosis. Oxidized diacylglycerol (DAG) produced by oxidative stress is thought to play an important role in lung fibrosis. This study assessed the effect of oxidized DAG in an animal model of pulmonary fibrosis induced by aspiration of bleomycin (BLM) into the lungs. The inhibitory effect of ebselen on pulmonary fibrosis was also investigated. In lung fibrotic tissue induced by BLM, an increase in lipid peroxides and collagen accumulation was observed. Moreover, the levels of oxidized DAG, which has strong protein kinase C (PKC) activation activity, were significantly increased over time following the administration of BLM. Western blotting showed that phosphorylation of PKCα and δ isoforms was increased by BLM. Oral administration of ebselen significantly suppressed the increase in oxidized DAG induced by BLM and improved lung fibrosis. PKCα and δ phosphorylation were also significantly inhibited. The mRNA expression of α-smooth muscle actin and collagen I (marker molecules for fibrosis), as well as the production of transforming growth factor-ß and tumor necrosis factor-α(a potentially important factor in the fibrotic process), were increased by BLM and significantly decreased by ebselen. The administration of BLM may induce lipid peroxidation in lung tissue, while the oxidized DAG produced by BLM may induce overactivation of PKCα and δ, resulting in the induction of lung fibrosis.

Protective Effect of Ebselen on Ischemia-reperfusion Injury in Epigastric Skin Flaps in Rats.

The purpose of this study was to determine the role of oxidized diacylglycerol (DAG) and the molecular mechanism underlying ischemia-reperfusion (I/R) injury in rat skin flaps. The protective effect of ebselen on the viability of rat skin flaps with I/R injury was investigated. Flaps were designed and raised in the left inguinal region. Then, a microvascular clamp was applied to the vascular pedicle and reperfused after 6 hr. After 7 days of I/R (I/R group), the skin flap survival area ratio was significantly reduced compared to the normal skin. The administration of ebselen significantly improved the ratio compared to the I/R group. The flap survival area ratio of the I/R + ebselen group was significantly improved compared to the I/R + vehicle group. In the I/R + ebselen group, the oxidized DAG content and intensity of phosphorylated PKCα and PKCδ were significantly lower compared to the I/R + vehicle group. Furthermore, the inflammatory response was suppressed in the I/R + ebselen group compared to the I/R + vehicle group. These results indicate that ebselen is useful as a preventive and therapeutic agent for skin flap necrosis caused by I/R, because of reduction and elimination of oxidized DAG.

Cellular Uptake of Pyrroloquinoline Quinone in Its Intact and Derivatized Forms from the Cell Culture Medium of 3T3-L1.

Following a growing interest in the physiological effects of pyrroloquinoline quinone (PQQ), more cell culture experiments have begun to elucidate its mechanism of action. However, to our knowledge, no reports have used instrumental analysis, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), to study cellular uptake of PQQ. In addition, despite the propensity of PQQ to react with amino acids and other compounds, only a handful of cell culture experiments have been conducted on PQQ derivatives. In the present study, we prepared PQQ derivatives by reacting PQQ with various amino acids and used them as reference standards for optimizing the LC-MS/MS analysis conditions to detect PQQ and its derivatives. Using this method, we evaluated the uptake of PQQ into mouse 3T3-L1 cells and found that most PQQ added to the medium was taken up by the cells in its unchanged form, while some PQQ reacted with amino acids in the medium and was taken up by the cells as PQQ derivatives. These results suggest that PQQ derivatives may contribute to the physiological effects of PQQ. To further elucidate the function of PQQ, it is necessary for future studies to clarify the activity of PQQ derivatives and to evaluate the types of PQQ present in food, animal, and cell samples in more detail.

Structural Analysis and Anti-Inflammatory Effect of a Digalactosyldiacylglycerol-Monoestolide, a Characteristic Glycolipid in Oats.

Digalactosyldiacylglycerol- (DGDG-) monoestolide is a characteristic glycolipid in oats. DGDG-monoestolides possess a unique structure whereby a fatty acid of DGDG is replaced by a fatty acid ester of hydroxy fatty acid (FAHFA). While the physiological effects of DGDG and FAHFA have been reported previously, the effects of DGDG-monoestolides are unknown. Hence, we isolated a major DGDG-monoestolide molecular species from oats, analyzed its structure, and evaluated its anti-inflammatory effect. Based on GC-MS, MS/MS, and NMR analyses, the isolated compound was identified as a DGDG-monoestolide that contains the linoleic acid ester of 15-hydroxy linoleic acid (LAHLA) and linoleic acid (i.e., DGDG-LAHLA). The isolated DGDG-LAHLA was evaluated for its anti-inflammatory effect on LPS-stimulated RAW264 cells. The production of nitric oxide and cytokines (IL-6, TNF-α, and IL-10) were significantly decreased by DGDG-LAHLA, suggesting the anti-inflammatory effect of DGDG-LAHLA for the first time. In addition, our data showed a pronounced uptake of DGDG-LAHLA by cells. Some compounds corresponding to the predicted DGDG-LAHLA metabolites were also detected, suggesting that both intact DGDG-LAHLA and its metabolites may contribute to the above anti-inflammatory activities. These results are expected to expand the availability of oats as a functional food.

Possible Glutathione Peroxidase 4-Independent Reduction of Phosphatidylcholine Hydroperoxide: Its Relevance to Ferroptosis.

Ferroptosis is mainly caused by iron-mediated peroxidation of phospholipids and has recently attracted attention due to its involvement in various diseases. At the center of it is supposedly the inability of glutathione peroxidase 4 (GPX4) to reduce excess peroxidized phospholipids (e.g., phosphatidylcholine hydroperoxide (PCOOH)) that trigger ferroptosis. However, the involvement of enzymes other than GPX4 in ferroptosis is scarcely known. To elucidate this matter, we evaluated the uptake of PCOOH in a GPX4 knockout (KO) human hepatoma cell line HepG2 generated using CRISPR-Cas9. After confirming that GPX4 expression in the KO cells was below the detection limit, we cultured both wild-type (WT) and GPX4 KO HepG2 cells in a medium containing 50 µM PCOOH for 1-8 hours. By analyzing the level of PCOOH and its reduction product (phosphatidylcholine hydroxide, PCOH) in cells using liquid chromatography-tandem mass spectrometry, we detected the cellular uptake of PCOOH. On top of this, we detected a large amount of PCOH not only in WT HepG2 but also in GPX4 KO HepG2, thus indicating the notable involvement of enzymes other than GPX4 (e.g., other GPX family, glutathione S-transferase, thioredoxin, or peroxiredoxin) in reducing PCOOH. Further corroboration of these findings hopefully leads to the development of novel methods to prevent ferroptosis-related diseases by targeting enzymes other than GPX4.

Determination of pyrroloquinoline quinone by enzymatic and LC-MS/MS methods to clarify its levels in foods.

Pyrroloquinoline quinone (PQQ) is believed to be a new B vitamin-like compound, and PQQ supplementation has received attention as a possible treatment for diseases including dementia and diabetes. However, the distribution of PQQ in foods is unclear, due to the difficulty in analyzing the compound. Therefore, in this study, enzymatic and LC-MS/MS methods were optimized to enable an accurate analysis of PQQ in foods. The optimized methods were applied to the screening of foods, in which PQQ contents were identified in ng/g or ng/mL levels. Furthermore, we newly found that some foods related to acetic acid bacteria contain PQQ at 1.94~5.59 ng/mL higher than beer, which is known to contain relatively high amounts of PQQ. These results suggest that the optimized methods are effective for the screening of foods containing PQQ. Such foods with high PQQ content may be valuable as functional foods effective towards the treatment of certain diseases.