Indigo Leaves-Induced Pulmonary Arterial Remodeling without Right Ventricular Hypertrophy in Rats.

Indigo naturalis (IN), derived from the leaves of the indigo plant, is a traditional Chinese medicine that has historically been used for its anti-inflammatory properties in the treatment of various diseases, including ulcerative colitis (UC). However, long-term use of IN in UC patients is incontrovertibly associated with the onset of pulmonary arterial hypertension (PAH). To investigate the mechanisms by which IN induces PAH, we focused on the raw material of IN, indigo leaves (IL). Only the condition of long-term chronic (6 months) and high-dose (containing 5% IL in the control diet) administration of IL induced medial thickening in the pulmonary arteries without right ventricular hypertrophy in our rat model. IL administration for a month did not induce pulmonary arterial remodeling but increased endothelin-1 (ET-1) expression levels within endothelial cell (EC) layers in the lungs. Gene Expression Omnibus analysis showed that ET-1 is a key regulator of PAH and that the IL component indican and its metabolite IS induced ET-1 mRNA expression via reactive oxygen species-dependent mechanism. We identified the roles of indican and IS in ET-1 expression in ECs, which were linked to pulmonary arterial remodeling in an animal model.

Changes in intestinal microbiota and biochemical parameters in patients with inflammatory bowel disease and irritable bowel syndrome induced by the prolonged addition of soluble fibers to usual drug therapy.

OBJECTIVES: Partially hydrolyzed guar gum (PHGG) is a soluble dietary fiber;in addition to improving bowel movements, it maintains intestinal health by producing short-chain fatty acids. However, majority of clinical studies on PHGG have been concluded within a month and excluded usual drug therapy. Hence, this study aimed to determine the effects of long-term consumption of PHGG, in combination with drug therapy, on gut bacteria ratios, laboratory values for inflammatory response, and fecal characteristics. METHODS AND RESULTS: The study was performed in patients with irritable bowel syndrome (IBS), Crohn's disease (CD), and ulcerative colitis (UC), by the administration of PHGG for six months while they continued their usual treatment. PHGG treatment caused significant changes in patients with IBS, including an increase in the abundance of short-chain fatty acid-producing bacteria, a significant decrease in Bacteroides abundance, and normalization of the Bristol scale of stool. In patients with UC, non-significant normalization of soft stools and decrease in fecal calprotectin were observed. Adverse events were not observed in any of the groups. CONCLUSION: Thus, it would be beneficial to include PHGG in the usual drug therapies of patients with IBS. J. Med. Invest. 71 : 121-128, February, 2024.

Dauferulins A-L, daucane-type sesquiterpenes from the roots of Ferula communis: Their structures and biological activities.

Phytochemical study on the roots of a medicinal plant Ferula communis L. (Apiaceae) resulted in the isolation of 20 sesquiterpenes including 12 previously undescribed compounds, dauferulins A-L (1-12). The detailed spectroscopic analysis revealed 1-12 to be daucane-type sesquiterpenes with a p-methoxybenzoyloxy group at C-6. The absolute configurations of 1-12 were deduced by analysis of the ECD spectra. Dauferulins A-L (1-12), known sesquiterpenes (13-20), and analogues (14a-14l) derived from 6-O-p-methoxybenzoyl-10α-angeloyloxy-jeaschkeanadiol (14) were evaluated for their effects on AMPK phosphorylation in human hepatoma HepG2 cells as well as inhibitory activities against erastin-induced ferroptosis on human hepatoma Hep3B cells and IL-1ß production from LPS-treated murine microglial cells.

CA9 and PRELID2; hypoxia-responsive potential therapeutic targets for pancreatic ductal adenocarcinoma as per bioinformatics analyses.

A strong hypoxic environment has been observed in pancreatic ductal adenocarcinoma (PDAC) cells, which contributes to drug resistance, tumor progression, and metastasis. Therefore, we performed bioinformatics analyses to investigate potential targets for the treatment of PDAC. To identify potential genes as effective PDAC treatment targets, we selected all genes whose expression level was related to worse overall survival (OS) in The Cancer Genome Atlas (TCGA) database and selected only the genes that matched with the genes upregulated due to hypoxia in pancreatic cancer cells in the dataset obtained from the Gene Expression Omnibus (GEO) database. Although the extracted 107 hypoxia-responsive genes included the genes that were slightly enriched in angiogenic factors, TCGA data analysis revealed that the expression level of endothelial cell (EC) markers did not affect OS. Finally, we selected CA9 and PRELID2 as potential targets for PDAC treatment and elucidated that a CA9 inhibitor, U-104, suppressed pancreatic cancer cell growth more effectively than 5-fluorouracil (5-FU) and PRELID2 siRNA treatment suppressed the cell growth stronger than CA9 siRNA treatment. Thus, we elucidated that specific inhibition of PRELID2 as well as CA9, extracted via exhaustive bioinformatic analyses of clinical datasets, could be a more effective strategy for PDAC treatment.

Role of ferroptosis in cisplatin-induced acute nephrotoxicity in mice.

BACKGROUND: Cisplatin is widely used as an antitumor drug for the treatment of solid tumors. However, its use has been limited owing to nephrotoxicity, a major side effect. The mechanism of cisplatin-induced nephrotoxicity (CIN) has long been investigated in order to develop preventive/therapeutic drugs. Ferroptosis is a newly identified form of non-apoptotic regulated cell death induced by iron-mediated lipid peroxidation and is involved in the pathophysiology of various diseases. In this study, we examined the role of ferroptosis in CIN. METHODS: We evaluated the role of ferroptosis in CIN by in vivo experiments in a mouse model. RESULTS: Cisplatin increased the protein expressions of transferrin receptor-1 and ferritin, and iron content in the kidney of mice. In addition, treatment with cisplatin augmented renal ferrous iron and hydroxyl radical levels with co-localization. Mice administered cisplatin demonstrated kidney injury, with renal dysfunction and increased inflammatory cytokine expression; these changes were ameliorated by Ferrostatin-1 (Fer-1), an inhibitor of ferroptosis. The expression of the ferroptosis markers, COX2 and 4-hydroxynonenal (4-HNE), increased with cisplatin administration, and decreased with the administration of Fer-1. By contrast, cisplatin-induced apoptosis and necroptosis were inhibited by treatment with Fer-1. Moreover, deferoxamine, an iron chelator, also inhibited CIN, with a decrease in the expression of COX-2 and 4-HNE. CONCLUSION: Ferroptosis is involved in the pathogenesis of CIN and might be used as a new preventive target for CIN.

Diphenhydramine may be a preventive medicine against cisplatin-induced kidney toxicity.

Cisplatin is widely used as an anti-tumor drug for the treatment of solid tumors. Unfortunately, it causes kidney toxicity as a critical side effect, limiting its use, given that no preventive drug against cisplatin-induced kidney toxicity is currently available. Here, based on a repositioning analysis of the Food and Drug Administration Adverse Events Reporting System, we found that a previously developed drug, diphenhydramine, may provide a novel treatment for cisplatin-induced kidney toxicity. To confirm this, the actual efficacy of diphenhydramine was evaluated in in vitro and in vivo experiments. Diphenhydramine inhibited cisplatin-induced cell death in kidney proximal tubular cells. Mice administered cisplatin developed kidney injury with significant dysfunction (mean plasma creatinine: 0.43 vs 0.15 mg/dl) and showed augmented oxidative stress, increased apoptosis, elevated inflammatory cytokines, and MAPKs activation. However, most of these symptoms were suppressed by treatment with diphenhydramine. Furthermore, the concentration of cisplatin in the kidney was significantly attenuated in diphenhydramine-treated mice (mean platinum content: 70.0 vs 53.4 µg/g dry kidney weight). Importantly, diphenhydramine did not influence or interfere with the anti-tumor effect of cisplatin in any of the in vitro or in vivo experiments. In a selected cohort of 98 1:1 matched patients from a retrospective database of 1467 patients showed that patients with malignant cancer who had used diphenhydramine before cisplatin treatment exhibited significantly less acute kidney injury compared to ones who did not (6.1 % vs 22.4 %, respectively). Thus, diphenhydramine demonstrated efficacy as a novel preventive medicine against cisplatin-induced kidney toxicity.

[Conjugation to Branched Glycerol Oligomers, a Novel Strategy for Extremely Hydrophobic Agents].

Ascertaining the absorption, distribution, metabolism, and excretion (ADME) profile of drugs is one of the most crucial factors in the process of drug discovery. Since it is important to combine water solubility and cell permeability within the compound to achieve the desired ADME properties, an appropriate balance between lipophilicity and hydrophilicity is required. It is often necessary to facilitate hydrophilicity of very hydrophobic candidates, because quite lipophobic molecules are rarely hit as positive in molecular-targeted or cell-based screenings. For that purpose, it has been popular to conjugate hydrophobic molecules with polyethylene glycol (PEG). However, PEG is a polymer, and PEG-conjugated molecules are not uniform. Besides, the dosage should be much increased compared with the original molecule due to the increase in molecular weight. Therefore we have been developing alternative ways to endow hydrophobic compounds with extra hydrophilicity by conjugating with symmetrically branched glycerol oligomers. This technology is versatile and easily applicable to various hydrophobic compounds. Water-solubility of fenofibrate, one of the most hydrophobic medicines in clinical use, was facilitated by a factor of more than 2000, and its lipid-lowering effect in vivo improved more than ten-fold, by simply conjugating with branched glycerol trimer, for instance. Here we will briefly introduce the basic concepts and our successful experiences of applying branched glycerol oligomers including antitumor agents in terms of water-solubility, pharmacological effects, and pharmacokinetics, and merits and current issues will be discussed in this review.

[Metabolic beneficial effects of Citrus Sudachi].

Sudachi (Citrus Sudachi) is a sour fruit and is a popular seasoning for Japanese dishes, Washoku. It can grow only in Tokushima and the surrounding areas for some reason, and therefore, Sudachi is a specialty of Tokushima prefecture in Japan. We usually use only its juice, and the size of the fruit is so small that a huge amount of pomace has been an industrial problem of the region. Therefore, we undertook exploratory studies of the Sudachi peel in terms of health promotion with making a kind of collaborative consortium for the study of acid citruses. Our recent activities on Sudachi research including the consortium were shared and discussed in the symposium, and the metabolic effects of Sudachi peel will be briefly introduced in the current manuscript.

Deletion of H-ferritin in macrophages alleviates obesity and diabetes induced by high-fat diet in mice.

AIMS/HYPOTHESIS: Iron accumulation affects obesity and diabetes, both of which are ameliorated by iron reduction. Ferritin, an iron-storage protein, plays a crucial role in iron metabolism. H-ferritin exerts its cytoprotective action by reducing toxicity via its ferroxidase activity. We investigated the role of macrophage H-ferritin in obesity and diabetes. METHODS: Conditional macrophage-specific H-ferritin (Fth, also known as Fth1) knockout (LysM-Cre Fth KO) mice were used and divided into four groups: wild-type (WT) and LysM-Cre Fth KO mice with normal diet (ND), and WT and LysM-Cre Fth KO mice with high-fat diet (HFD). These mice were analysed for characteristics of obesity and diabetes, tissue iron content, inflammation, oxidative stress, insulin sensitivity and metabolic measurements. RAW264.7 macrophage cells were used for in vitro experiments. RESULTS: Iron concentration reduced, and mRNA expression of ferroportin increased, in macrophages from LysM-Cre Fth KO mice. HFD-induced obesity was lower in LysM-Cre Fth KO mice than in WT mice at 12 weeks (body weight: KO 34.6 ± 5.6 g vs WT 40.1 ± 5.2 g). mRNA expression of inflammatory cytokines and infiltrated macrophages and oxidative stress increased in the adipose tissue of HFD-fed WT mice, but was not elevated in HFD-fed LysM-Cre Fth KO mice. However, WT mice fed an HFD had elevated iron concentration in adipose tissue and spleen, which was not observed in LysM-Cre Fth KO mice fed an HFD (adipose tissue [µmol Fe/g protein]: KO 1496 ± 479 vs WT 2316 ± 866; spleen [µmol Fe/g protein]: KO 218 ± 54 vs WT 334 ± 83). Moreover, HFD administration impaired both glucose tolerance and insulin sensitivity in WT mice, which was ameliorated in LysM-Cre Fth KO mice. In addition, energy expenditure, mRNA expression of thermogenic genes, and body temperature were higher in KO mice with HFD than WT mice with HFD. In vitro experiments showed that iron content was reduced, and lipopolysaccharide-induced Tnf-α (also known as Tnf) mRNA upregulation was inhibited in a macrophage cell line transfected with Fth siRNA. CONCLUSIONS/INTERPRETATION: Deletion of macrophage H-ferritin suppresses the inflammatory response by reducing intracellular iron levels, resulting in the prevention of HFD-induced obesity and diabetes. The findings from this study highlight macrophage iron levels as a potential therapeutic target for obesity and diabetes.

Novel Hydrophilic Camptothecin Derivatives Conjugated to Branched Glycerol Trimer Suppress Tumor Growth without Causing Diarrhea in Murine Xenograft Models of Human Lung Cancer.

Camptothecin possesses broad antitumor spectra on various cancers. In spite of its marked tumor-suppressing potency, camptothecin is too hydrophobic to be solved in water and therefore not currently in clinical use. CPT-11 (irinotecan) is one of the hydrophilic analogues of camptothecin and widely prescribed. However, its water solubility is still low and furthermore evokes severe diarrhea. Therefore, we designed and synthesized novel highly hydrophilic camptothecin derivatives by conjugating SN38 with branched glycerol trimer (SN38-BGL), which we have been developing as a unique strategy to endow hydrophobic molecule with much hydrophilicity, to maximize the benefit of CPT-11 and minimize the adverse effects. The SN38-BGLs exhibited equivalent or slightly stronger tumor-suppressing effects in murine xenograft human lung cancer models compared to CPT-11. However, neither early- nor late-onset diarrhea was observed when SN38-BGL was administered. Heights of villi in jejunum and ileum were bigger than those from CPT-11-treated mice, indicating that SN38-BGL is less harmful than CPT-11. Ex vivo digestion by liver microsome did not yield SN38 but a couple of other molecules against our expectations, which suggests the involvement of other active metabolites than SN38 and may explain the differences. Hence, SN38-BGLs can be a novel hydrophilic camptothecin derivative without causing severe diarrhea.

Proton pump inhibitors block iron absorption through direct regulation of hepcidin via the aryl hydrocarbon receptor-mediated pathway.

Proton pump inhibitors (PPIs) have been used worldwide to treat gastrointestinal disorders. A recent study showed that long-term use of PPIs caused iron deficiency; however, it is unclear whether PPIs affect iron metabolism directly. We investigated the effect of PPIs on the peptide hepcidin, an important iron regulatory hormone. First, we used the FDA Adverse Event Reporting System database and analyzed the influence of PPIs. We found that PPIs, as well as H2 blockers, increased the odds ratio of iron-deficient anemia. Next, HepG2 cells were used to examine the action of PPIs and H2 blockers on hepcidin. PPIs augmented hepcidin expression, while H2 blockers did not. In fact, the PPI omeprazole increased hepcidin secretion, and omeprazole-induced hepcidin upregulation was inhibited by gene silencing or the pharmacological inhibition of the aryl hydrocarbon receptor. In mouse experiments, omeprazole also increased hepatic hepcidin mRNA expression and blood hepcidin levels. In mice treated with omeprazole, protein levels of duodenal and splenic ferroportin decreased. Taken together, PPIs directly affect iron metabolism by suppressing iron absorption through the inhibition of duodenal ferroportin via hepcidin upregulation. These findings provide a new insight into the molecular mechanism of PPI-induced iron deficiency.

Iron accumulation causes impaired myogenesis correlated with MAPK signaling pathway inhibition by oxidative stress.

Skeletal muscle atrophy is caused by disruption in the homeostatic balance of muscle degeneration and regeneration under various pathophysiological conditions. We have previously reported that iron accumulation induces skeletal muscle atrophy via a ubiquitin ligase-dependent pathway. However, the potential effect of iron accumulation on muscle regeneration remains unclear. To examine the effect of iron accumulation on myogenesis, we used a mouse model with cardiotoxin (CTX)-induced muscle regeneration in vivo and C2C12 mouse myoblast cells in vitro. In mice with iron overload, the skeletal muscles exhibited increased oxidative stress and decreased expression of satellite cell markers. Following CTX-induced muscle injury, these mice also displayed delayed muscle regeneration with a decrease in the size of regenerating myofibers, reduced expression of myoblast differentiation markers, and decreased phosphorylation of MAPK signaling pathways. In vitro, iron overload also suppressed the differentiation of C2C12 myoblast cells but the suppression could be reversed by superoxide scavenging using tempol. Excess iron inhibits myogenesis via oxidative stress, leading to an imbalance in skeletal muscle homeostasis.-Ikeda, Y., Satoh, A., Horinouchi, Y., Hamano, H., Watanabe, H., Imao, M., Imanishi, M., Zamami, Y., Takechi, K., Izawa-Ishizawa, Y., Miyamoto, L., Hirayama, T., Nagasawa, H., Ishizawa, K., Aihara, K.-I., Tsuchiya, K., Tamaki, T. Iron accumulation causes impaired myogenesis correlated with MAPK signaling pathway inhibition by oxidative stress.

Methanol extraction fraction from Citrus Sudachi peel exerts lipid reducing effects in cultured cells.

Ectopic fat accumulation is associated with insulin resistance and type 2 diabetes mellitus. Citrus sudachi is an evergreen tree that is found mainly in Tokushima Prefecture in Japan. Previously, it was demonstrated that Citrus sudachi could inhibit the rising trend of blood glucose and fatty acid in human subjects. In the current study, we illustrated the function of methanol extracts from sudachi peel and investigated the mechanism of this effect. We got the five kinds of methanol extracts by using diaion HP-20, and those were named by hydrophobicity from M-F1 to M-F5. Among the 5 kinds of sudachi methanol extracts, only M-F4 significantly decreased the intracellular triglyceride of C2C12 cells. It augmented the AMPK activity and increased the transcription of PPARα and its downstream targets CPT-1b and UCP2. In conclusion, M-F4 improved the lipid metabolism possibly through AMPK, PPARα and their downstream targets like CPT-1b and UCP2. Furthermore, this extract may be useful for preventing obesity and diabetes related diseases. J. Med. Invest. 65:225-230, August, 2018.

[AMPK as a Metabolic Intersection between Diet and Physical Exercise].

　Physical exercise is well known to be beneficial to our health. Therapeutic exercise is widely applicable to metabolic disorders, including obesity and diabetes. In addition, recent studies have suggested its potential benefit in the treatment of more various diseases such as mental disorders and cancer. 5'AMP-activated protein kinase (AMPK), which is an intracellular central metabolic sensor as well as a regulator, has been demonstrated to play significant roles in the contracting skeletal muscles, suggesting that AMPK should be one of the key molecules mediating metabolic effects during physical exercise. Therefore, AMPK is a desirable therapeutic target for drug discovery. In the symposium S41 held in the 137th Annual Meeting of the Pharmaceutical Society of Japan, our data on the molecular mechanisms of isoform-specific postprandial suppression of AMPK activity were shared, and we discussed potential roles of AMPK as an intersection where metabolic signals by physical exercise and feeding status crosstalk. Here, I would like to introduce basic knowledge related to AMPK and recent findings regarding how AMPK activity is regulated in response to physiological and pharmacological stimulation.

[Sodium-Glucose Transporters as a Therapeutic Target for Diabetes from the Viewpoint of Drug Discovery and Pharmacotherapy].

　Sodium-glucose transporter (SGLT)-2 inhibitors, which are currently in clinical use in most of the world, are unique as their hypoglycemic effects are completely independent of insulin action. Potential benefits and indications for the treatment of other diseases like circulatory and renal disorders are attracting attention. SGLT2 inhibitors not only reduce blood glucose levels but also alter the whole-body energy balance to lower body weight, which should result in the amelioration of multiple metabolic disorders like metabolic syndrome. In the symposium, we briefly introduced the physiological as well as biological functions of SGLTs and discussed strategies for drug design by looking back at the history of drug discovery for SGLT2 inhibitors. We also shared our recent data on their combined usage with other hypoglycemic agents and effects on glucagon secretion, which are current clinical topics relevant to SGLT2 inhibitors. Among those topics, strategies for drug discovery of SGLT2 inhibitors are discussed in this review.

Renoprotective effects of a factor Xa inhibitor: fusion of basic research and a database analysis.

Renal tubulointerstitial injury, an inflammation-associated condition, is a major cause of chronic kidney disease (CKD). Levels of activated factor X (FXa), a blood coagulation factor, are increased in various inflammatory diseases. Therefore, we investigated the protective effects of an FXa inhibitor against renal tubulointerstitial injury using unilateral ureteral obstruction (UUO) mice (a renal tubulointerstitial fibrosis model) and the Food and Drug Administration Adverse Events Reporting System (FAERS) database. The renal expression levels of FX and the FXa receptors protease-activated receptor (PAR)-1 and PAR-2 were significantly higher in UUO mice than in sham-operated mice. UUO-induced tubulointerstitial fibrosis and extracellular matrix expression were suppressed in UUO mice treated with the FXa inhibitor edoxaban. Additionally, edoxaban attenuated UUO-induced macrophage infiltration and inflammatory molecule upregulation. In an analysis of the FAERS database, there were significantly fewer reports of tubulointerstitial nephritis for patients treated with FXa inhibitors than for patients not treated with inhibitors. These results suggest that FXa inhibitors exert protective effects against CKD by inhibiting tubulointerstitial fibrosis.

Mechanisms of the pH- and Oxygen-Dependent Oxidation Activities of Artesunate.

Artemisinin was discovered in 1971 as a constituent of the wormwood genus plant (Artemisia annua). This plant has been used as an herbal medicine to treat malaria since ancient times. The compound artemisinin has a sesquiterpene lactone bearing a peroxide group that offers its biological activity. In addition to anti-malarial activity, artemisinin derivatives have been reported to exert antitumor activity in cancer cells, and have attracted attention as potential anti-cancer drugs. Mechanisms that might explain the antitumor activities of artemisinin derivatives reportedly induction of apoptosis, angiogenesis inhibitory effects, inhibition of hypoxia-inducible factor-1α (HIF-1α) activation, and direct DNA injury. Reactive oxygen species (ROS) generation is involved in many cases. However, little is known about the mechanism of ROS formation from artemisinin derivatives and what types of ROS are produced. Therefore, we investigated the iron-induced ROS formation mechanism by using artesunate, a water-soluble artemisinin derivative, which is thought to be the underlying mechanism involved in artesunate-mediated cell death. The ROS generated by the coexistence of iron(II), artesunate, and molecular oxygen was a hydroxyl radical or hydroxyl radical-like ROS. Artesunate can reduce iron(III) to iron(II), which enables generation of ROS irrespective of the iron valence. We found that reduction from iron(III) to iron(II) was activated in the acidic rather than the neutral region and was proportional to the hydrogen ion concentration.