Our study explored the therapeutic effect and the mechanism of quercetin against hypoxia/reoxygenation (H/R)-induced injury in human coronary artery endothelial cells (CAECs). Quercetin was selected as a potential component for the BuShenKangShuaiPian formula (BSKSP) treatment via the Network pharmacology analysis. Cell viability and reactive oxygen species (ROS) production were measured by CCK8 assay and immunofluorescence, respectively. The expression of Bax, Bcl-2, Cle-caspase-3, cytochrome c (Cyt-C), NF-E2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) protein was quantified by western blotting. The superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA) activity, mtDNA copy number, and ATP production were measured via corresponding kits. Quercetin was selected from the BSKSP for its high degree value (Degree value: 22). Besides, quercetin protected CAECs against H/R-induced cytotoxicity and apoptosis. The H/R-induced increased ROS level, ATP production, Cyt-C release, and decreased mtDNA copy number were removed by the quercetin. Moreover, quercetin upregulated the Nrf2/ HO-1 axis, SOD, and CAT activity, and downregulated MDA levels in H/R treated CAECs, while knockdown Nrf2 reversed the protection of quercetin against H/R-induced oxidative stress, mitochondrial damage, and apoptosis. Quercetin protects CAECs against H/R-induced mitochondrial apoptosis via the Nrf2/HO-1 axis, which innovatively suggests the therapeutic potential of quercetin for coronary heart disease (CHD) treatment.

Seizure-like burst activities are induced by blockade of GABAA and/or glycine receptors in various spinal ventral roots of brainstem-spinal cord preparation from neonatal rodents. We found that this is not applicable to the phrenic nerve and that a new inhibitory descending pathway may suppress seizure-like activity in the phrenic nerve. Experiments were performed in brainstem-spinal cord preparation from newborn rats (age: 0-1 day). Left phrenic nerve and right C4 activities were recorded simultaneously. When GABAA and glycine receptors were blocked by 10 μM bicuculline and 10 μM strychnine (Bic+Str), seizure-like burst activities appeared in the fourth cervical ventral root (C4) but not the phrenic nerve. After making a transverse section at C1, the inspiratory burst activity disappeared from both C4 and the phrenic nerve, whereas seizure-like activity appeared in both nerves. We hypothesized that inhibitory descending pathways other than those via GABAA and/or glycine receptors (from the medulla to the spinal cord) work to avoid disturbance of regular respiratory-related diaphragm contraction by seizure-like activity. We found that cannabinoid receptor antagonist, AM251 was effective for the induction of seizure-like activity by Bic+Str in the phrenic nerve in brainstem-spinal cord preparation. Cannabinoid receptors may be involved in this descending inhibitory system.

Dipeptidyl peptidase 4 (DPP4), a serine protease expressed on luminal and apical cell membrane, is identical to the lymphocyte cell surface protein CD26. DPP4 rapidly deactivates hormones and cytokines by cleaving their NH2-terminal dipeptides. Its functions are based on membrane digestion and/or binding of bioactive peptides, signal molecules, and extracellular matrix components. The soluble form is also present in body fluids such as serum, urine, semen, and synovial fluid. The extremely broad distribution of CD26/DPP4 indicates its divergent roles depending on cell type and activated conditions. The cellular localization was earlier examined by enzyme histochemistry and subsequently by immunohistochemistry. Although immunohistochemical analyses are higher in specificity and easier to use at electron microscopic levels than enzyme histochemistry, the immunoreaction is considerably affected by the animal species, types of tissue sections, and specificity of antibodies. Understanding of the functional significance and advancement of its clinical use (diagnosis and treatment of diseases) require precise information on the cellular distribution including subcellular localization and pathological changes. This short review summarizes in particular immunohistochemical findings on CD26/DPP4.

Osteocytes, osteoblasts (bone-forming cells), and osteoclasts (bone-resorbing cells) are the primary types of cells that regulate bone metabolism in mammals. Sclerostin produced in bone cells activates osteoclasts, inhibiting bone formation; excess production of sclerostin, therefore, leads to the loss of bone mass. Fish scales have been reported to have morphological and functional similarities to mammalian bones, making them a useful experimental system for analyzing vertebrate bone metabolism in vitro. However, whether fish scales contain cells producing sclerostin and/or osteocytes has not been determined. The current study demonstrated, for the first time, that sclerostin-containing cells exist in goldfish scales. Analysis of the distribution and shape of sclerostin-expressing cells provided evidence that osteoblasts produce sclerostin in goldfish scales. Furthermore, our results found that osteocyte-like cells exist in goldfish scales, which also produce sclerostin. Finally, we demonstrated that microgravity in outer space increased the level of sclerostin in the scales of goldfish, a finding suggesting that the induction of sclerostin is the mechanism underlying the activation of osteoclasts under microgravity.

Osteoporosis disturbs the balance of bone metabolism, and excessive bone resorption causes a decrease in bone density, thus increasing the risk of fracture. (-)-Epigallocatechin-3-gallate (EGCG) is the most abundant catechin contained in green tea. EGCG has a variety of pharmacological activities. Recently, it was reported that EGCG inhibits osteoclast differentiation, but the details of the mechanism underlying the EGCG-mediated suppression of osteoclastogenesis are unknown. In this study, we investigated the effects of EGCG on several signaling pathways in osteoclastogenesis. EGCG suppressed the expression of the nuclear factor of activated T cells cytoplasmic-1 (NFATc1), the master regulator of osteoclastogenesis. EGCG decreased the expression of cathepsin K, c-Src, and ATP6V0d2 and suppressed bone resorption. We also found that EGCG upregulated heme oxygenase-1 (HO-1) and suppressed the extracellular release of high-mobility group box 1 (HMGB1). In addition, EGCG decreased the expression of the receptor for advanced glycation end products (RAGE), which is the receptor of HMGB1, in osteoclastogenesis. In summary, our study showed that EGCG could inhibit osteoclast differentiation through the downregulation of NFATc1 and the suppression of the HO-1-HMGB1-RAGE pathway. EGCG might have the potential to be a lead compound that suppresses bone resorption in the treatment of osteoporosis.

As chondrocytes fail to retain their chondrogenic potential in two-dimensional monolayer cultures, several three-dimensional culture systems have been employed for investigating the physiology and pathophysiology in articular cartilage tissues. In this study, we introduced a polyethylene glycol-coated microfabricated chip that enables spheroid formation from ATDC5 cell line, commonly used as a model for in vitro chondrocyte research. ATDC5 cells cultured in our devices aggregated immediately and generated a single spheroid per well within 24 h. Most cells in spheroids cultured in differentiation medium were viable and the circular shape and smooth surface of the spheroid were maintained up to 14 d in culture. We also detected potent hypoxia conditions, a key factor in chondrogenesis, in whole lesions of ATDC5 spheroids. Expression of chondrogenesis-related genes and type X collagen protein was significantly increased in ATDC5 spheroids grown in differentiation medium, compared with monolayer-cultured ATDC5 cells. We also demonstrated that the differentiation medium-induced Akt protein phosphorylation was upregulated in ATDC5 cells cultured in our spheroid device, suggesting that enhancement of chondrogenic potential in ATDC5 spheroids results from PI3/Akt signaling activation. These results indicated that our spheroid culture system could constitute a high-throughput strategy approach towards elucidating the molecular mechanisms that regulate chondrogenesis.

Sodium trisulfide (Na2S3) releases hydrogen polysulfide (H2Sn) and is useful for the investigation of the effects of H2Sn on the cell functions. In the present study, we first examined the effects of Na2S3 on the gene expression of IEC-6 cells, a rat intestinal epithelial cell line. Microarray analysis and reverse transcription-polymerase chain reaction analysis revealed that Na2S3 increased the gene expression of early growth response 1 (EGR1) and Kruppel-like transcription factor 4 (KLF4). It was interesting that U0126, an inhibitor of the activation of extracellular signal-regulated kinase 1 (ERK1), ERK2, and ERK5, inhibited the Na2S3-induced gene expression of EGR1 and KLF4. Na2S3 activated ERK1 and ERK2 (ERK1/2) within 15 min. In addition to ERK1/2, Na2S3 activated ERK5. We noticed that the electrophoretic mobility of ERK5 was decreased after Na2S3 treatment. Phos-tag analysis and in vitro dephosphorylation of the cell extracts indicated that the gel-shift of ERK5 was due to its phosphorylation. The gel-shift of ERK5 was inhibited completely by both U0126 and ERK5-IN-1, a specific inhibitor of ERK5. From these results, we concluded that the gel-shift of ERK5 was induced through autophosphorylation by activated ERK5 after Na2S3 treatment. The present study suggested that H2Sn affected various functions of intestinal epithelial cells through the activation of the ERK1/2 and ERK5 pathways.

The intestinal ecosystem is involved in the pathogenesis of mood disorders such as depression. Intestinal microbes can affect the central nervous system through the gut-brain axis, which raises the possibility of using probiotics for preventing depression. In this study, we examined the effect of heat-inactivated Lactobacillus gasseri CP2305 (CP2305) in a subchronic and mild social defeat stress (sCSDS) mouse model. sCSDS suppressed food intake. However, dietary CP2305 intake rescued it, suggesting that CP2305 improved the decreased appetite in sCSDS mice. sCSDS did not alter the gene expression of brain-derived neurotrophic factor, nerve growth factor, and neurotrophin-3 in the hippocampus. However, dietary CP2305 provided following sCSDS increased the gene expression of these neurotrophins in the hippocampus. These findings suggest that CP2305 supplementation would aid in preventing psychosocial stress-induced disorders.

For a long time, many types of vaccines have been useful for the prophylaxis of many infectious diseases. Thus far, many adjuvants that enhance the effects of vaccines have been explored. However, very few adjuvants are being used for humans worldwide. In this study, we investigated the adjuvant activity of various substances, and found citrulline to have high potential as an adjuvant. Citrulline is a type of amino acid present in the body of many organisms. A number of biological activities of citrulline have been reported; however, no adjuvant activity has been reported thus far. Aluminum salts, which are commonly used as adjuvants are not water soluble; therefore, some difficulties are encountered while using them as vaccine adjuvants. Citrulline is easy to use because of its water solubility. In this study, we showed for the first time the adjuvant activity of citrulline by using viral antigens and amyloid β peptide. Water-soluble citrulline, which is present in our body, is a potential adjuvant candidate.

The tumor suppressor gene TP53 (gene) codes for a transcription factor which transactivates its target genes responsible for cell cycle arrest, DNA repair, apoptosis, and senescence. TP53 is well known to be the most frequent target of genetic mutations in nearly half of human cancers including oral squamous cell carcinoma (OSCC). Many p53 mutants including R248Q and R248W not only lose its tumor-suppressor activities, but also interfere with the functions of wild-type p53; this is so-called dominant-negative (DN) mutation. The DN p53 mutation is a predictor of poor outcome in patients with various cancers, and also a risk factor for metastatic recurrence in patients with OSCC. Recently it has been reported that DN p53 mutants acquire new oncogenic activities, which is named gain-of-function (GOF). This study aimed at determining whether R248Q and R248W were involved in OSCC cells' acquiring aggressive phenotypes, using SAS, HSC4 and Ca9-22 cell lines. First, two mutants p53, R248Q and R248W, were respectively transfected into SAS cells harboring recessive-type p53 (E336X). As a result, SAS cells expressing R248Q showed highly spreading, motile and invasive activities compared to parent or mock-transfected cells whereas those expressing R248W did not increase those activities. Secondly, in HSC4 cells harboring R248Q and Ca9-22 cells harboring R248W, expressions of the mutants p53 were inhibited by the transfection with siRNAs targeting p53. The inhibition of the mutants p53 decreased spreading, motile and invasive activities of HSC4 cells whereas it did not affect those activities of Ca9-22 cells. These findings suggest that R248Q p53 mutation, but not R248W p53 mutation, induces more motile and invasive potentials in human OSCC cells.