Recent pharmacological studies have been developed based on finding new disease-related genes, accompanied by the production of gene-manipulated disease model animals and high-affinity ligands for the target proteins. However, the emergence of this gene-based strategy in drug development has led to the rapid depletion of drug target molecules. To overcome this, we have attempted to utilize clinical big data to explore a novel and unexpected hypothesis of drug–drug interaction that would lead to drug repositioning. Here, we introduce our data-driven approach in which adverse event self-reports are statistically analyzed and compared in order to find and validate new drug targets. The hypotheses provided by such a data-driven approach will likely impact the style of future drug development and pharmaceutical study.

Selenoprotein P (SeP) is one of the 25 human selenocysteine (Sec)-containing proteins, and is generally thought to function as a plasma carrier of the trace element selenium in the body. Recent studies, however, indicate unsuspected pivotal roles of SeP in human diseases, particularly in type 2 diabetes mellitus (T2DM) and pulmonary arterial hypertension (PAH). In this review, we will summarize the characteristics of SeP and recent advances in the field, especially focusing on the emerging roles of SeP in pathophysiological conditions. We will also discuss potential medical/pharmaceutical applications targeting SeP.

Recent progress in the fields of tissue engineering, micro-electro mechanical systems, and materials science have greatly improved cell culture systems, which were traditionally performed in a static two-dimensional manner. This progress has led to a number of new cell culture concepts represented by organ-on-a-chip, three dimensional (3D)-tissues, and microphysiological systems, among others. In this review, these culture models are categorized as reconstituted human organ models, which recapitulate human organ-like structure, function, and responses with physiological relevance. In addition, we also describe the expectations of reconstituted organ models from the viewpoint of a pharmaceutical company based on recent concerns expressed in drug discovery and development. These models can be used to assess the pharmacokinetics, safety and efficacy of new molecular entities (NMEs) prior to clinical trials. They can also be used to conduct mechanistic investigations of events that arise due to administration of NMEs in humans. In addition, monitoring biomarkers of organ function in these models will aid in the translation of their changes in humans. As the majority of reconstituted human organ models show improved functional characteristics and long-term maintenance in culture, they are valuable for modeling human events. An example is described using the three-dimensional bioprinted human liver tissue model in this article. Implementation of reconstituted human organ models in drug discovery and development can be accelerated by encouraging collaboration between developers and users. Such efforts will provide significant benefits for delivering new and improved medicines to patients.

Transporter gene knockout models are a practical and widely used tool for pharmacokinetic studies in drug discovery. P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) are major efflux transporters that control absorption and bioavailability, and are important when determining oral drug disposition. To the best of our knowledge, beyond the rule of five (bRo5) molecules launched on the market to date tend to be substrates for efflux transporters. The purpose of this study is to evaluate in vivo the impact of efflux transporters on the oral absorption process and systemic clearance using rats which lack P-gp and/or Bcrp expression. We administered five bRo5 substrates (asunaprevir, cyclosporine, danoprevir, ledipasvir, and simeprevir) intravenously or orally to wild-type and Mdr1a, Bcrp, and Mdr1a/Bcrp knockout rats, calculated the clearance, oral bioavailability, and absorption rate profile of each substrate, and compared the results. Systemic clearance of the substrates in knockout rats changed within approximately ±40% compared to wild-types, suggesting the efflux transporters do not have a significant influence on clearance in rats. On the other hand, the oral absorption of substrates in the knockout rats, especially those lacking Mdr1a, increased greatly—between 2- and 5-fold more than in wild-types. This suggests that rat efflux transporters, especially P-gp, greatly reduce the oral exposure of these substrates. Moreover, results on the absorption rate–time profile suggest that efflux transporters are constantly active during the absorption period in rats. Transporter knockout rats are a useful in vivo tool for estimating the transporter-mediated disposition of bRo5 molecules in drug discovery.

The aim of this study was to prepare binary supercooled liquid (SCL) by intermolecular interaction and apply this formulation to transdermal drug delivery. Ketoprofen (KET) and ethenzamide (ETH) were selected as binary SCL component. Thermal analysis of physical mixtures of KET and ETH showed decreases in melting points and glass transition below room temperature, thereby indicating formation of KET–ETH SCL. Intermolecular interactions between KET and ETH in the SCL were evaluated from Fourier transform (FT)-IR spectra. KET–ETH SCL maintained SCL state at 25°C with silica gel over 31 d and at 40°C/89% relative humidity (RH) over 7 d. KET SCL and KET–ETH SCL showed similar permeability of KET for hairless mice skin, which was two-fold higher than that of KET aqueous suspension. Our findings suggest that the SCL state could enhance the skin permeation of drugs and the binary SCL formed by intermolecular interaction could also improve the stability of the SCL. The binary SCL system could become a new drug form for transdermal drug delivery.

Immune checkpoint inhibitors (ICIs) exert beneficial effects in non-small cell lung cancer (NSCLC) patients. However, ICIs are only advantageous for a limited population of NSCLC patients. Therefore to enhance their effects, combination therapies with ICIs have been developed. To identify preferable chemotherapy to combine with ICIs against lung cancer, we examined immunological effects of docetaxel compared with epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI). We found no difference in peripheral lymphocyte counts and ratio of their subpopulations in lung cancer patients before and after both treatments. On the other hand, plasma levels of high-mobility group box 1 (HMGB1), a damage-associated molecular pattern (DAMP) protein, showed significant increase after docetaxel treatment. Furthermore, we investigated effects of HMGB1 on tumor-infiltrating immune cells obtained from surgically resected tumor tissue from NSCLC patients. When the tumor infiltrating cells were stimulated with HMGB1, CD11c⁺ cells showed increased expression of activation markers. These findings imply that docetaxel could be involved in anti-tumor immunity via HMGB1. Therefore docetaxel might be a candidate for combination treatment with ICIs.

The aim of this study was to investigate the effects of egg yolk powder enriched with astaxanthin (ASX-E) on blood pressure in spontaneously hypertensive rats (SHR) and to verify the benefits of ASX-E as a functional food. To investigate the antihypertensive effect, SHR were fed with an ASX-E mixed diet before hypertension development. Blood pressures were determined periodically during the study by the tail-cuff method. At the end of the study, animals were euthanized, and their thoracic aortas were collected to determine vascular conductance. The thoracic aorta tension was measured with a force displacement transducer. Concentration-dependent response relationships were determined by cumulative addition of 10⁻⁹–10⁻⁴ M Carbamoylcholine (Cch). Blood pressures of the SHR in the ASX-E mixed diet group were ASX-dose-dependently lower than that of those in the control group. In SHR fed with an ASX-E mixed diet, Cch induced vasorelaxation in the thoracic aorta with endothelium lining but not without endothelium. However, the antihypertensive effect of ASX-E was not observed on blood pressures in SHR that were fed with ASX-E only after the development of hypertension. Results suggest that ASX-E protects endothelial function and thereby prevents the development of hypertension. Hence, the results of our research indicate that daily consumption of ASX-E has a potential benefit on human health.

Forest bathing is suggested to have beneficial effects on various aspects of human health. Terpenes, isoprene based-phytochemicals emitted from trees, are largely responsible for these beneficial effects of forest bathing. Although the therapeutic effects of terpenes on various diseases have been revealed, their effects on neuronal health have not yet been studied in detail. Here, we screened 16 terpenes that are the main components of Korean forests using Drosophila Alzheimer’s disease (AD) models to identify which terpenes have neuroprotective effects. Six out of the 16 terpenes, ρ-cymene, limonene (+), limonene (−), linalool, α-pinene (+), and β-pinene (−), partially suppressed the beta amyloid 42 (Aβ42)-induced rough eye phenotype when fed to Aβ42-expressing flies. Among them, limonene (+) restored the decreased survival of flies expressing Aβ42 in neurons during development. Limonene (+) treatment did not affect Aβ42 accumulation and aggregation, but did cause to decrease cell death, reactive oxygen species levels, extracellular signal-regulated kinase phosphorylation, and inflammation in the brains or the eye imaginal discs of Aβ42-expressing flies. This neuroprotective effect of limonene (+) was not associated with autophagic activity. Our results suggest that limonene (+) has a neuroprotective function against the neurotoxicity of Aβ42 and, thus, is a possible therapeutic reagent for AD.

Conjugation with polyethylene glycol (PEG) is performed to increase serum half-life of the Fab for clinical applications. However, current designs for recombinant Fab only allow PEGylation at the interchain SS bond (disulfide bond) at the C-terminal end of the heavy chain and light chain of the Fab, which the decrease of thermostability occurred by partial reduction of the interchain SS bond. An adalimumab Fab mutant with a novel interchain SS bond (CH₁ : C177–CL : C160) and one cysteine at the C-terminal end (mutSS Fab_SH) was designed to maintain Fab thermostability and for site-specific PEGylation. MutSS Fab_SH was expressed in Pichia pastoris and purified mutSS Fab_SH was conjugated with 20-kDa PEG targeted at the free cysteine. Based on enzyme-linked immunosorbent assay (ELISA), PEGylation did not affect the binding capacity of the mutSS Fab_SH. To confirm the influence of PEGylation on the pharmacokinetic behavior of the Fab, PEGylated mutSS Fab_SH was administered to rats via tail vein injection. Analysis of the mean serum concentration of the PEGylated mutSS Fab_SH versus time through ELISA indicated an increase in half-life compared to that of non-PEGylated wild-type Fab. Consequently, we have successfully demonstrated that a Fab mutant with a novel interchain SS bond and one free cysteine at the C-terminal end can be PEGylated without changes in functionality. This design can potentially be used as a platform for modification of other recombinant Fabs.

Ferroptosis is characterized by the accumulation of iron-derived reactive oxygen species (ROS). Ferroptosis causes neuronal death in multiple neurological disorders. Dexmedetomidine (Dex), an extensively used anesthetic, has neuroprotective effects against ROS, but its effect on iron metabolism remains unknown. In this study, SK-N-SH cells were treated with Dex for 24 h before treatment with 100 µM tert-butyl hydroperoxide (t-BHP; an ROS inducer) for 1 h. Afterward, intracellular ROS and labile ferrous iron [Fe(II)] levels were assessed. Dex hindered the increase in cellular ROS and labile Fe(II) levels caused by t-BHP, although Dex alone had no effect on labile Fe(II) level. t-BHP increased the expression of iron importers, transferrin receptor-1 and divalent metal transporter-1, and iron regulatory protein 1 and 2. These effects were abrogated by Dex treatment and SP-1 knockdown. t-BHP increased the phosphorylation of c-Jun N-terminal kinase (JNK) and signal transducer and activator of transcription 4 (STAT4), the primary up-stream activators of SP-1, but Dex decreased this. This study, for the first time, revealed that the antioxidative effect of Dex is partly associated to the inhibition of intracellular iron accumulation induced by t-BHP. Dex regulates iron metabolism by regulating iron importers and exporters through JNK/Sp1 and Stat4/Sp1 signaling. It is worth investigating whether Dex can protect neurons from ferroptosis.

Salvia przewalskii Maxim is a traditional Chinese herbal medicine and is known to have antibacterial, antiviral, anti-oxidant, anti-thrombotic and anti-depressant properties. However, the major active components of S. przewalskii and its anti-hypoxic effects are still unclear. This study probed the major active component and anti-hypoxic activity of S. przewalskii. The major active components of S. przewalskii were detected by HPLC. The anti-hypoxic effects of S. przewalskii were detected in mice and a rat model of hypoxic preconditioning. The results showed that there are eight active components, including sodium danshensu, rosmarinic acid, lithospermic acid, salvianolic acid B, dihydrotanshinone I, cryptotanshinone, tanshinone I and tanshinone IIA, and each component showed a certain anti-hypoxic effect. Moreover, S. przewalskii enhanced anti-hypoxia in mice, which was manifested as prolonged survival time in acute hypoxic preconditioning and the amelioration of acute hypoxia-induced changes in the activity of superoxide dismutase (SOD) and lactate dehydrogenase (LDH). In addition, S. przewalskii also repaired tissue damage in chronic hypoxia by downregulating hypoxia inducible factor-1α (HIF-1α), proliferating cell nuclear antigen (PCNA), Bcl-2, CDK4, CyclinD1 and P27Kip1 and inhibiting pro-inflammatory cytokines and the RhoA–Rho-associated protein kinase (ROCK) signalling pathway. Our findings provide new insight into the anti-hypoxic effect of S. przewalskii as a promising agent for high-altitude pulmonary hypertension treatment.

There is significant cultivation of persimmon (Diospyros kaki) in East Asia, a plant whose fruit has abundant nutrients, including vitamins, polyphenols, and dietary fiber. Persimmon dietary supplements can benefit health by amelioration of diabetes, cardiovascular disease, and obesity. There are also persimmon-based beverages produced via fermentation, such as wines and vinegars, and increasing consumption of these products in East Asia. Although there is great interest in functional foods, the health effects of fermented persimmon extract (FPE) are completely unknown. We examined the effects of FPE on the metabolic parameters of mice fed a high-fat diet (HFD). Our results indicated that FPE supplementation led to an approx. 15% reduction of body weight, reduced abdominal and liver fat, and reduced serum levels of triglycerides, total cholesterol, and glucose. FPE also blocked the differentiation of murine 3T3-L1 pre-adipocyte cells into mature adipocytes. We suggest that gallic acid is a major bioactive component of FPE, and that AMP-activated protein kinase mediates the beneficial effects of FPE and gallic acid.

Resveratrol (Res) is a natural active antioxidant that is effective in relieving inflammatory bowel disease (IBD). However, the specific mechanism for its function is unknown. In our study, dextran sodium sulfate (DSS)-induced mouse IBD disease model was constructed. All mice were randomly divided into three groups. The treatment effects of resveratrol on IBD were evaluated by observing the body weight, fecal traits, colon/spleen gross appearance, tissue hematoxylin–eosin (H&E)/immunohistochemistry (IHC) and inflammatory factors. The expression of small ubiquitin-like modifier protein 1 (SUMO1) and its Wnt/β-catenin pathway-related genes was analyzed by IHC, Western blot, Real-time PCR (RT-PCR) and Immunofluorescence. The outcome indicated that resveratrol treatment significantly relieved the symptoms of IBD. The expression level of anti-inflammatory cytokines was increased while that of pro-inflammatory cytokines was decreased in both colon and spleen tissues of resveratrol-treated mice. SUMO1 expression and Wnt/β-catenin pathway were suppressed in colon and spleen tissues of IBD mice treated with resveratrol. In addition, we provided evidence that resveratrol inhibited SUMO1 and β-catenin expression and their nuclear localization in human colonic epithelial cell line (FHC). Moreover, we found that SUMO1 and β-catenin had higher expression levels in colorectal cancer patients than in health and colitis patients. In conclusions, resveratrol alleviates DSS-induced IBD by modulating SUMO1 through Wnt/β-catenin pathway.

Edoxaban is used to prevent and treat stroke or systemic embolism such as venous thromboembolism. Although bleeding is the most common complication of anticoagulants, only a few studies have addressed the safety of direct oral anticoagulants in East Asian patients. In this study, we investigated the risk factors for bleeding in Japanese patients receiving edoxaban. A retrospective review of the records of 198 patients who received 30 mg/d edoxaban in our hospital between April 2015 and March 2017 was performed. Subsequently, these patients were followed up to 1 year. Seven (3.5%) and 22 (11.1%) patients developed major bleeding and clinically relevant bleeding, respectively. In the univariate Cox regression analyses, low baseline hemoglobin levels (p = 0.002) and low baseline creatinine clearance (p = 0.020) were significantly associated with major bleeding. Multivariate Cox regression analysis revealed that a low baseline hemoglobin level was a significant risk factor for major bleeding and clinically relevant bleeding [hazard ratio 1.67 per 1 g/dL decrease (95% confidence interval 1.14–2.56, p = 0.008) and hazard ratio 1.31 per 1 g/dL decrease (95% confidence interval 1.06–1.62, p = 0.013), respectively]. Baseline hemoglobin level in quartiles also showed a quartile-dependent decrease in major bleeding and clinically relevant bleeding event. These results suggest that low baseline hemoglobin level is a significant risk factor for both major bleeding and clinically relevant bleeding in Japanese patients receiving edoxaban. Thus, these patients should be carefully monitored.

Choline as a quaternary amine nutrient is metabolized to trimethylamine by gut microbiota and subsequently oxidized to circulating trimethylamine-N-oxide (TMAO), a gut-derived metabolite associated with liver toxicity and cardiovascular disease. The study was to probe the possible vasoprotective and hepatoprotective effects of total saponins of Gynostemma pentaphyllum (TSGP) in 3% high-choline water-feeding mice. The purified TSGP was obtained with content of 83.0% saponins, and its antioxidant activities were evaluated in vitro. Furthermore, the mice fed with high choline for 8 weeks significantly expressed vascular endothelial dysfunction and liver oxidative stress (p < 0.01 vs. Normal). Administration of TSGP at 400 and 800 mg/kg·body weight (b.w.) significantly lowered the serum total cholesterol (TC), triglyceride (TG), low density lipoprotein-cholesterol (LDL-C), endothelin-1 (ET-1) and thromboxane A₂ (TXA₂) levels, as well as hepatic malondialdehyde (MDA) formation, but effectively elevated the serum nitric oxide (NO), endothelial nitric oxide synthase (eNOS) and prostaglandin I₂ (PGI₂) levels, as well as alanine aminotransferase (ALT), aspartate aminotransferase (AST), T-superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities in high choline-fed mice. Hematoxylin–eosin (H&E) and oil red O staining also suggested that TSGP could exert the significant protection against endothelial dysfunction and liver injury in high choline-treated mice. These findings suggest that TSGP is of the saponins-enriched extract, and is a good candidate of dietary supplement and therapeutic application in vascular and hepatic oxidative injury.

Creatine transporter (CRT) deficiency (CRT-D) results in a significant reduction of brain creatine levels, which causes various neurological symptoms in early childhood, and diagnosis of the severity of CRT-D based on the residual CRT transport activity in liquid biopsy samples would be beneficial for early intervention. The apparent reduction in creatine transport activity in CRT-D is thought to be due to reduced intrinsic CRT-mediated creatine transport per CRT protein and/or reduced absolute CRT protein expression on the plasma membranes. The purpose of this study was thus to determine the normal level of intrinsic CRT-mediated creatine transport activity based on absolute CRT protein quantification using rat CRT-overexpressing HEK293 cells (CRT/HEK293 cells), and to clarify creatine transport in erythrocyte- and leukocyte-enriched fractions isolated from the circulating blood of rats. The intrinsic creatine transport rate was calculated to be 0.237 µL/(min·fmol CRT) based on the initial uptake rate and the absolute CRT protein level in CRT/HEK293 cells. Taking into account Avogadro’s constant, the creatine transport activity per CRT protein is estimated to be 1190 creatine/(min·CRT molecule) in the presence of [¹⁴C]creatine at an extracellular concentration of 5 µM. Isolated leukocyte-enriched fraction exhibited mRNA expression of CRT and partially Na⁺-dependent [¹⁴C]creatine transport, whereas erythrocytes showed neither. These characteristics suggest that the leukocytes contain the CRT-mediated creatine uptake system, and are available for evaluation of residual CRT transport activity in CRT-D patients.

Ferroptosis is a form of necrosis caused by iron-induced accumulation of lipid hydroperoxide, involving several molecular events, and has been implicated in Parkinson’s disease. Gastrodin is a component of Gastrodia elata Blume with strong antioxidant activity. We examined whether gastrodin can prevent H₂O₂-induced cytotoxicity in rat glioma cell line C6. For this purpose, C6 cells were pretreated with gastrodin (1, 5, 25 µM) and then exposed to 100 µM H₂O₂. Results showed that pretreatment of C6 cells with gastrodin decreased H₂O₂-induced lactate dehydrogenase (LDH) release and cell death. Moreover, gastrodin decreased intracellular malondialdehyde (MDA) level, whereas increased glutathione peroxidase (GPX) activity and glutathione (GSH) level after H₂O₂ treatment. In addition, treatment of deferoxamine (DFO), ferrostatin-1, and liproxstatin-1 abolished ferroptosis induced by H₂O₂ or erastin pretreatment. Treatment with gastrodin attenuated H₂O₂-induced ferroptosis and decreased lipid reactive oxygen species (ROS) (C11-BODIPY) production in C6 cells. Moreover, gastrodin increased the protein expression of nuclear factor erythroid 2-related factor 2 (Nrf2), GPX4, ferroportin-1 (FPN1), and heme oxygenase-1 (HO-1) in C6 cells treated with H₂O₂. RSL3, a GPX4 inhibitor, inhibited GPX4 protein level in cells co-treated with gastrodin and 100 µM H₂O₂. These findings indicate that gastrodin can inhibit H₂O₂-induced ferroptosis through its antioxidative effect in C6 cells.

The intravenous injection of bendamustine often induces venous irritation, which reduces patients’ QOL. We previously reported that the dilution of the final volume of bendamustine from 250 to 500 mL significantly decreased the incidence of venous irritation. However, the influence of this change on the therapeutic efficacy of bendamustine remains unclear. Therefore, the aim of this study was to evaluate the efficacy and safety profiles of bendamustine at different dilutions of the final volume, comparing with the correspondences of previous studies. Thirty-four patients, who received a total of 161 courses of bendamustine and rituximab chemotherapy, were included in this study. The overall response rate of this regimen was 94.1% in this study, which was comparable to that reported in the BRB study (94.2%, a phase II study of bendamustine plus rituximab therapy in Japanese patients). Additionally, the median progression-free survival was not inferior to that reported in the BRB study. Bendamustine-induced venous irritation was observed in 17.6% of the patients during the first treatment cycle administered at a final volume of 500 mL, and was found to be lower than that observed in the control, where bendamustine was administered at a final volume of 250 mL (85.7%). These results suggest that diluting bendamustine to 500 mL, but not to 250 mL, reduces the incidence of venous irritation without a negative impact on its therapeutic efficacy; thus, this simple strategy may be beneficial to ensure efficacy and safety in patients receiving regimens including bendamustine.

The β-adrenoceptor (β-AR)-mediated pharmacological effects of catecholamine (CA) metabolites are not well known. We examined the effects of seven CA metabolites on smooth muscle relaxation in mouse and guinea pig (GP) tracheas and rat thoracic aorta. Among them, metadrenaline (MA) significantly relaxed GP trachea (β₂-AR dominant), even in the presence of clorgiline, a monoamine oxidase-A inhibitor. In mouse trachea (β₁-AR dominant), normetadrenaline (NMA) and MA (10⁻⁴ M each) apparently did not affect isoprenaline (ISO)-induced relaxation, but significantly inhibited it in the presence of clorgiline. ISO-induced relaxation was also unaffected by 3,4-dihydroxyphenylglycol (DHPG) (10⁻⁴ M), but significant suppression was observed with the addition of 3,5-dinitrocatechol, a catechol-O-methyltransferase inhibitor. In GP trachea, NMA, MA, 3,4-dihydroxymandelic acid (DOMA), and DHPG (10⁻⁴ M each) significantly augmented ISO-induced relaxation. However, in the presence of clorgiline plus 3,5-dinitrocatechol, both NMA and MA (10⁻⁴ M) significantly suppressed ISO-induced relaxation. DHPG (10⁻⁴ M) also significantly suppressed ISO-induced relaxation in the presence of 3,5-dinitrocatechol. In rat thoracic aorta, DHPG (10⁻⁴ M) significantly suppressed relaxation induced by CGP-12177 A (a β₃-AR partial agonist) in the presence of 3,5-dinitrocatechol plus propranolol. Our findings indicate that 1) MA may possess β₂-AR agonistic action; 2) NMA and MA augment β₂-AR-mediated tracheal relaxation in the absence of CA metabolic inhibitors, though themselves possessing β₁-, β₂-AR antagonistic action (β₂ > β₁); 3) DHPG exhibits β₁-, β₂-, β₃-AR antagonistic action, and this is particularly marked for β₃-AR. Our observations may help explain some of the pathologies associated with pheochromocytoma, which is characterized by increased CA metabolite levels.

Obesity elevates the risk of cardiovascular disease and has been strongly associated with increases in the incidence of many metabolic diseases. Therefore, prevention of obesity leads to the prevention of metabolic diseases. In light of this, substances that exert anti-obesity effects are crucial for the prevention of obesity. Indirubin, a 3,2′ bisindole isomer of indigo, is the active component of the traditional Chinese medicine used for the treatment of chronic myelocytic leukemia. In particular, indirubin-3′-oxime (1) was shown to inhibit the differentiation of adipocytes. In this study, we investigated the inhibitory effects of nine indirubin-3′-oxime derivatives against lipid accumulation during differentiation in 3T3-L1 cells. Among the compounds tested, 5-methoxyindirubin-3′-oxime (2) and 6-bromoindirubin-3′-oxime (7) at 5 µM exhibited significantly stronger inhibitory activity than indirubin-3′-oxime (1). Furthermore, 5-methoxyindirubin-3′-oxime (2) and 6-bromoindirubin-3′-oxime (7) markedly suppressed the expression of CCAAT/enhancer-binding protein α, peroxisome proliferator activator γ2, and adipocyte protein 2, both of which are key adipogenic regulators at the intermediate stage of adipocyte differentiation. Our results demonstrate that 5-methoxyindirubin-3′-oxime (2) and 6-bromoindirubin-3′-oxime (7) significantly down-regulated lipid accumulation during differentiation of 3T3-L1 cells, suggesting their potential as novel therapeutic drugs against the development of obesity.

Acute pancreatitis (AP) is one kind of acute surgical abdominal disease in the world. It causes intestinal damage with subsequent bacterial migration, endotoxemia and secondary pancreatic infections. In this investigation, we determined that edaravone (EDA) reduces pancreatic and intestinal injury after AP in mice. This was demonstrated by a reduction in histological score, apoptosis, interleukin (IL)-6, IL-1β and tumor necrosis factor (TNF)-α, along with obstructing activation of Toll-like receptor 4 (TLR4) and nuclear factor-κB (NFκB). Our study results suggested that EDA exerts its protective effects against pancreatic and intestinal injury after AP via regulation of the TLR4/NFκB pathway. Our findings provide the basis for EDA to treat AP-induced pancreatic and intestinal injury, even might develop as a potential therapy for other inflammatory diseases.

It is therapeutically important to elucidate the factors involved in the radiation resistance of tumors. We previously showed that ATP is released from mouse melanoma B16 cells in response to γ-irradiation, but the role of adenosine, a metabolite of ATP, is still unclear. Here, we show that the adenosine A2B receptor is involved in DNA damage repair and radioresistance in mouse melanoma B16 cells. The DNA damage response after γ-irradiation was attenuated by pretreatment with A2B receptor antagonists, such as PSB603, while it was enhanced by pretreatment with A2B receptor agonists, such as BAY60-6583. γ-Irradiation decreased the cell survival rate, and pretreatment with PSB603 further reduced the survival rate. On the other hand, pretreatment with BAY60-6583 increased the cell survival rate after irradiation. The DNA damage response and the cell survival rate after γ-irradiation were both decreased in A2B-knockdown cells. In vivo experiments in mice confirmed that tumor growth was suppressed and delayed in the irradiated group pretreated with PSB603, compared with the irradiation-alone group. Our results indicate that adenosine A2B receptor contributes to radioresistance, and could be a new target for the development of agents to increase the efficacy of radiotherapy.

Peroxisome proliferator-activated receptor γ (PPARγ), the molecular target for antidiabetic thiazolidinediones (TZDs), is a master regulator of preadipocyte differentiation and lipid metabolism. The adverse side effects of TZDs, arising from their potent agonistic activity, can be minimized by PPARγ partial agonists or PPARγ non-agonists without loss of insulin sensitization. In this study, we reported that WSF-7, a synthetic chemical derived from natural monoterpene α-pinene, is a partial PPARγ agonist. We found that WSF-7 binds directly to PPARγ. Activation of PPARγ by WSF-7 promotes adipogenesis, adiponectin oligomerization and insulin-induced glucose uptake. WSF-7 also inhibits obesity-mediated PPARγ phosphorylation at serine (Ser)273 and improves insulin sensitivity of 3T3-L1 adipocytes. Our study suggested that WSF-7 activates PPARγ transcription by a mechanism different from that of rosiglitazone or luteolin. Therefore, WSF-7 might be a potential therapeutic drug to treat type 2 diabetes.

Renal interstitial fibrosis (RIF) is a common pathological characteristic associated with end-stage renal disease. However, treatment strategies for RIF are still very limited. In this study, we reported that kaempferol, a classic flavonoid, exhibited strong and widely inhibitory effect on the expression of fibrosis related genes in transforming growth factor beta 1 (TGF-β1) treated NRK-52E cells. Further studies revealed that kaempferol inhibited TGF-β1 induced epithelial–mesenchymal transition (EMT) process of NRK-52E cells and improved renal function deterioration and RIF in unilateral ureteral obstruction (UUO) rats. After exploring the underlying mechanisms, we found that kaempferol was able to activate the BMP-7-Smad1/5 pathway, rather than the TGF-β1-Smad2/3 pathway. To further validate these results, DMH1 and BMP-7 knockdown were utilized at the cellular level and the results showed that both methods were able to antagonize the effects of kaempferol on the EMT process of NRK-52E cells induced by TGF-β1. In UUO rats, inhibition of BMP-7 signaling by DMH1 also reversed the effects of kaempferol on renal function decline and RIF. Taken together, our findings demonstrated that kaempferol could be a good candidate for renal fibrosis treatment.

CD81 is a highly conserved four-transmembrane protein in mammals and widely expressed on many tissues. It belongs to the tetraspanin family and forms complexes with various cell surface membrane proteins. It also functions in cell migration and B-cell activation, which is induced by CD81 complexing with CD19, CD21 and the B-cell receptor. Thus, CD81 is thought to play a key role in regulating cell function and fate. However, little is known about the degradation mechanism of CD81. Here we found that CD81 on the plasma membrane is degraded by the lysosome pathway via endocytosis. The expression levels of CD81 in HEK293T cells treated with a proteasome inhibitor (lactacystin) and lysosome inhibitors (chloroquine and bafilomycin A1) were analyzed by flow cytometry. The expression of CD81 on the cell surface was increased by the lysosome inhibitors, but not lactacystin. A pulldown assay revealed that CD81 was conjugated with a K63- and K29-linked poly-ubiquitin chain before its degradation, and the poly-ubiquitination site was Lys8 at the N-terminal intracellular domain of CD81. Furthermore, mutant CD81, in which Lys8 was substituted with alanine (Ala), extended the CD81 half-life compared with wildtype. CD81 was mainly localized on the plasma membrane in normal cells, but also co-localized with lysosomal LAMP1 and early endosomal EEA1 in chloroquine-treated cells. Furthermore, a clathrin-mediated endocytosis inhibitor, chlorpromazine, stabilized CD81 expression on the cell surface. Hence, we demonstrated that CD81 is internalized by clathrin-mediated endocytosis and subsequently degraded via a lysosome pathway requiring the K63- and K29-linked poly-ubiquitination of CD81.

Women with estrogen deficiency are at the risk of suffering from neurological symptoms such as memory impairment. In the present study, we investigated the effect of garlic, Allium sativum L. (Asparagales: Amaryllidaceae), treated with subcritical water on memory impairment in ovariectomized (OVX) rats. OVX rats were administered garlic powder for 84 d. Hippocampus-dependent spatial memory was assessed using the Morris water maze test. Escape latency of the OVX rats increased compared with that of sham-operated rats. The prolonged escape latency of the OVX rats decreased to the level of that of sham-operated rats upon the administration of garlic powder (0.5% in feed). The weights of the body, uterus, and brain were not affected by the garlic powder administration. These results suggest that garlic powder treated with subcritical water mitigates memory impairment in OVX rats.

Equol, an intestinal metabolite of daidzein, inhibited more potently mushroom tyrosinase in vitro than other inhibitors, genistein and kojic acid. We investigated the mechanism underlying tyrosinase inhibition by equol. Treating racemic equol with tyrosinase produced 3ʹ-hydroxyequol. Because the optical activity of the product showed <25% enantiomeric excess, the reaction was not highly stereospecific. Using enzyme-linked immunosorbent assays with an anti-equol monoclonal antibody, we observed that equol bound to pre-coated tyrosinase in a dose-dependent manner. Our results suggested the formation of a stable equol–tyrosinase complex.

The mechanism underlying the improvement in hepatic function by liver hydrolysate (LH) after ethanol-induced hepatic injury is unclear. Therefore, we investigated the effects of LH administration on chronic ethanol-induced hepatic injury in normal rats and the mechanism underlying the improvement of its symptoms by LH. LH attenuated liver damage and reduced oxidative stress after chronic ethanol-induced hepatic injury in normal rats. LH treatment reduced hepatic injury biomarkers of plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT). LH treatment also decreased levels of 8-hydroxy-deoxyguanosine (8-OHdG) as oxidative stress marker. LH may prove beneficial to prevent the liver damage of chronic ethanol, at least in part, by alleviating oxidative stress.

Diabetic kidney disease (DKD) is the leading cause of chronic kidney disease. Current therapies for DKD are insufficient. Therefore, there is an urgent need for identifying new therapies. An increasing number of micro RNAs (miRNAs) and long noncoding RNAs (lncRNAs) have been demonstrated to modulate the progression of diabetic kidney disease. Nevertheless, until now, there have been few reports evaluating the relevance of circular RNAs (circRNAs) in DKD. circRNAs have been reported to regulate the occurrence and development of multiple diseases. In this study, we intended to explore the circRNA expression profiles and determine the role of circRNA in DKD. We identified a series of dysregulated circRNAs in glucose-stressed HK-2 cells using circRNA microarray analysis. Among the candidate circRNAs, we found that circACTR2 was upregulated and may be involved in inflammation and pyroptosis. Knockdown of circACTR2 significantly decreased pyroptosis, interleukin (IL)-1β release and collagen IV and fibronectin production, indicating the effective regulation by circACTR2 of cell death and inflammation. Overall, our study identified a new circRNA, circACTR2, that regulates high glucose-induced pyroptosis, inflammation and fibrosis in proximal tubular cells. The present study preliminarily explores the role of circRNAs in pyroptosis of tubular cells, and provides novel insight into the pathogenesis of DKD and new therapeutic strategies.

Bile acid sequestrants are used as medicinal drugs to treat dyslipidemia and type 2 diabetes. We found that cholestyramine, a bile acid sequestrant, increases cecal short-chain fatty acid (SCFA) production and intestinal immunoglobulin A (IgA) in C57BL/6J mice. In a 12-week high-fat diet study, feeding cholestyramine (2% (w/w)) significantly promoted C2–C4 SCFAs in the cecum by approximately 1.6-fold and fecal IgA by 1.8-fold. In an 8-week normal-fat diet study, feeding cholestyramine (1 and 2%) increased the cecal propionic acid content by approx. 2.0-fold. Fecal IgA was also significantly increased at 4 weeks (1%: 1.7-fold; 2%: 2.1-fold) and 8 weeks (1%: 1.8-fold; 2%: 2.0-fold) in the normal-fat diet study. These results indicate that bile acid sequestrants may exert their physiological functions, such as intestinal IgA production, through SCFA-dependent signaling pathways.

Although substantial evidence suggests that an increase in the level of trimethylamine-N-oxide (TMAO) is associated with the risk of cardiovascular diseases, including atherosclerosis, chronic kidney diseases, and hypertension, the direct effect of TMAO on vascular endothelial function remains unclear. Therefore, we investigated the acute effects of TMAO on endothelium-dependent relaxation induced by acetylcholine (ACh) in the superior mesenteric arteries and femoral arteries of rat. In endothelium-intact preparations, it was observed that TMAO (300 µmol/L for 60 min) did not affect ACh-induced relaxation in either of the two arteries. In endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation under nitric oxide synthase (NOS) and cyclooxygenase (COX) inhibitions by N^ω-nitro-L-arginine (L-NNA) and indomethacin, respectively, TMAO specifically impairs the relaxation in femoral arteries but not in the superior mesenteric arteries. Under the inhibitory actions of NOS and as well as blockade of intermediate-conductance calcium-activated potassium channel (IK_Ca) (by TRAM-34) and small-conductance calcium-activated potassium channel (SK_Ca) (by apamin), which are putative sources of EDHF, ACh-induced relaxation was low, and there were no differences between the control and TMAO-treated groups with respect to both arteries. In femoral arteries, TMAO slightly reduces ACh-induced relaxation in the presence of indomethacin (preserved NO and EDHF signals) but does not affect ACh-induced NO-mediated relaxation under the combined presence of indomethacin, TRAM-34, and apamin. These results suggest that acute treatment with TMAO specifically impairs EDHF-mediated relaxation in the femoral arteries but not in the superior mesenteric arteries. These novel observations show that TMAO is a causative factor in the development of peripheral arterial disease.