Seung Yun Han

All-Trans-Retinoic Acid Rescues Neurons After Global Ischemia by Attenuating Neuroinflammatory Reactions

Retinoic acid (RA) plays an important role in the developing mammalian nervous system. Based on this concept, some studies have demonstrated the beneficial effects of RA administration on neurogenesis in neuropathological diseases. Some investigations have revealed the anti-inflammatory effects of RA treatment in multiple systems, in addition to its role in neurogenesis. To date, however, the neuroprotective efficacy of RA after cerebral ischemia, especially in the context of its anti-inflammatory effects, has been poorly demonstrated. Additionally, to the best of our knowledge, experiments of the therapeutic efficacy of RA treatment in a transient global ischemic model in the Mongolian gerbil have been lacking worldwide. Here, we studied the neuroprotective effects and neurobehavioral outcomes of intraperitoneally administered all-trans-RA (ATRA; a synthetic form of RA) on brains with transient global ischemia that was induced with the bilateral common carotid artery occlusion and reperfusion (BCCAO/R) model in the gerbil. In order to identify whether these neuroprotective mechanisms were due to the anti-inflammatory effects of ATRA, in vivo hippocampal expression of proinflammatory cytokines including tissue necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) after ATRA injection and in vitro levels of release of nitric oxide, TNF-α and IL-6 from lipopolysaccharide (LPS)-stimulated BV2 microglial cells after ATRA treatment were evaluated. The results showed that ATRA can protect pyramidal neurons in the hippocampal CA1 region against BCCAO-induced neuronal apoptosis and significantly reduce the extent of astrocytosis and microglial activation. In addition, the ischemia-induced neurobehavioral changes were normalized by ATRA injection. Consistent with these phenotypic data, we observed the diminishing effects of ATRA treatment on the production of proinflammatory mediators (e.g., TNF-α and IL-6) in hippocampal homogenates and LPS-stimulated BV2 cells, and these effects were dose-dependent. These results suggest a beneficial role of ATRA in the attenuation of global cerebral ischemia due to its anti-inflammatory properties, resulting in, at least partly, the inhibition of microglial secretion of variable proinflammatory cytokines.

Autophagy enhancement contributes to the synergistic effect of vitamin D in temozolomide‑based glioblastoma chemotherapy

Temozolomide (TMZ), an alkylating agent, is recommended as the initial treatment for high-grade glioblastoma. TMZ is widely used, but its short half-life and the frequency of tumor resistance limit its therapeutic efficacy. In the present study, the anticancer effect of vitamin D (VD) combined with TMZ upon glioblastoma was determined, and the underlying mechanism of this effect was identified. Through cell viability, clonogenic and wound healing assays, the current study demonstrated that treatment of a C6 glioblastoma cell line with TMZ and VD resulted in significantly increased in vitro antitumor effects compared with either VD or TMZ alone. Autophagy, hypothesized to be the dominant mechanism underlying TMZ-based tumor cell death, was maximally activated in TMZ and VD co-treated C6 cells. This was demonstrated by ultrastructural observations of autophagosomes, increased size and number of microtubule-associated protein 1 light chain 3 (LC3) puncta and increased conversion of LC3-I to LC3-II. However, the extent of apoptosis was not significantly different between cells treated with TMZ and VD and those treated with TMZ alone. Addition of the autophagy inhibitor 3-methyladenine markedly inhibited the anticancer effect of TMZ and VD treatment, indicating that the chemosensitizing effect of VD in TMZ-based glioblastoma therapy is generated through enhancement of cytotoxic autophagy. TMZ and VD co-treatment also significantly inhibited tumor progression and prolonged survival duration in rat glioblastoma orthotopic xenograft models when compared with TMZ treatment alone. These in vivo results are concordant with the aforementioned in vitro results, together revealing that the combined use of TMZ and VD exerts synergistic antitumor effects on rat models of glioblastoma and may represent an effective therapeutic strategy.

Omega-3 Polyunsaturated Fatty Acids May Attenuate Streptozotocin-Induced Pancreatic β-Cell Death via Autophagy Activation in Fat1 Transgenic Mice

Background Inflammatory factors and β-cell dysfunction due to high-fat diets aggravate chronic diseases and their complications. However, omega-3 dietary fats have anti-inflammatory effects, and the involvement of autophagy in the etiology of diabetes has been reported. Therefore, we examined the protective effects of autophagy on diabetes using fat-1 transgenic mice with omega-3 self-synthesis capability. Methods Streptozotocin (STZ) administration induced β-cell dysfunction in mice; blood glucose levels and water consumption were subsequently measured. Using hematoxylin and eosin (H&E) and Massons trichrome staining, we quantitatively assessed STZ-induced changes in the number, mass, and fibrosis of pancreatic islets in fat-1 and control mice. We identified the microtubule-associated protein 1A/1B light chain 3-immunoreactive puncta in β-cells and quantified p62 levels in the pancreas of fat-1 and control mice. Results STZ-induced diabetic phenotypes, including hyperglycemia and polydipsia, were attenuated in fat-1 mice. Histological determination using H&E and Massons trichrome staining revealed the protective effects of the fat-1 expression on cell death and the scarring of pancreatic islets after STZ injection. In the β-cells of control mice, autophagy was abruptly activated after STZ treatment. Basal autophagy levels were elevated in fat-1 mice β-cells, and this persisted after STZ treatment. Together with autophagosome detection, these results revealed that n-3 polyunsaturated fatty acid (PUFA) enrichment might partly prevent the STZ-related pancreatic islet damage by upregulating the basal activity of autophagy and improving autophagic flux disturbance. Conclusion Fat-1 transgenic mice with a n-3 PUFA self-synthesis capability exert protective effects against STZ-induced β-cell death by activating autophagy in β-cells.

Protective efficacy of an Ecklonia cava extract used to treat transient focal ischemia of the rat brain

Phlorotannins (marine algal polyphenols) have been reported to exhibit beneficial biological activities, serving as both antioxidants and anti-inflammatory agents. Among marine algae, Ecklonia cava, a member of the Laminariaceae, is a very popular food regarded as healthy in Korea and Japan. Recently, benefits afforded by phlorotannins in the treatment of various clinical conditions have been reported, but any therapeutic effects of such materials in the treatment of neurodegenerative diseases such as stroke remain unclear. Also, the mechanisms of action of the algal components remain poorly understood. In the present in vivo study, administration of Ecklonia cava polyphenols (ECP) at 10 mg/kg and 50 mg/kg intraperitoneally (i.p.) significantly decreased infarct size and the extent of brain edema in the rat after induction of transient focal ischemia via middle cerebral artery occlusion (MCAO). Further, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay revealed dose-dependent blockage of neuronal apoptosis upon intravenous ECP treatment. Neurobehavioral tests performed over the 6 days after MCAO revealed a reduction in neurological motor performance in control animals, but administration of ECP (50 mg/kg i.p.) prevented this decline. In vitro, a significant neuroprotective effect of ECP was evident when cell viability was assayed after induction of H2O2-mediated oxidative stress, upon retinoic acid treatment, in the differentiated neuroblastoma cell line SH-SY5Y. Interestingly, ECP blocked the rise in cytosolic calcium, in a dose-dependent manner, in differentiated SH-SY5Y cells exposed to H2O2. Together, the results suggest that ECP exerts neuroprotective effects in the focally ischemic brain by reducing Ca2+-mediated neurotoxicity.

Pes anserinus and anserine bursa: anatomical study

This study investigated the boundary of anserine bursa with the recommended injection site and shape on the insertion area of pes anserinus (PA), with the aim of improving clinical practice. Eighty six legs from 45 Korean cadavers were investigated. The mixed gelatin solution was injected to identify the shape of anserine bursa, and then the insertion site of the PA tendons was exposed completely and carefully dissected to identify the shape of the PA. The sartorius was inserted into the superficial layer and gracilis, and the semitendinosus was inserted into the deep layer on the medial surface of the tibia. The number of the semitendinosus tendons at the insertion site varied: 1 in 66% of specimens, 2 in 31%, and 3 in 3%. The gracilis and semitendinosus tendons were connected to the deep fascia of leg. Overall, the shape of the anserine bursa was irregularly circular. Most of the anserine bursa specimens reached the proximal line of the tibia, and some of the specimens reached above the proximal line of the tibia. In the medial view of the tibia, the anserine bursa was located posteriorly and superiorly from the tibias midline, and it followed the lines of the sartorius muscle. The injection site for anserine bursa should be carried out at 20° from the vertical line medially and inferiorly, 15 or 20 mm deeply, and at the point of about 20 mm medial and 12 mm superior from inferomedial point of tibial tuberosity.

Blood-brain barrier-permeable fluorone-labeled dieckols acting as neuronal ER stress signaling inhibitors

We studied the blood-brain barrier (BBB) permeability and intracellular localization of a fluorescein isothiocyanate (FITC)-labeled dieckol (1) and a rhodamine B-labeled dieckol (7), for exploring the possible therapeutic application of fluorone-labeled dieckols in neurodegenerative diseases. Both compounds (1 &7) were synthesized through a click reaction and were found to be localized in the endoplasmic reticulum (ER) of the two types of brain cell lines (SH-SY5Y and BV-2 cells) tested; they also reduced ER stress in the SH-SY5Y human neuroblastoma cells. In addition, 1 and 7 were shown to pass the BBB in rats upon intravenous administration. Altogether, our study demonstrates, for the first time, that targeted ER-stress reduction in brain cells can be achieved by introducing fluorone-dieckol conjugates into systemic circulation. Therefore, 1 and 7 provide a novel and promising ER-targeting therapeutic strategy for neurodegenerative diseases.

Impaired autophagy promotes bile acid-induced hepatic injury and accumulation of ubiquitinated proteins

Chronic exposure to hydrophobic bile acids such as chenodeoxycholic acid (CDCA) and cholic acid (CA) in the liver during cholestasis causes hepatotoxicity and inflammatory response. However, the detailed mechanisms regarding the role of autophagy in cholestatic hepatotoxicity remain largely unknown. Here we determined autophagic clearance in livers of bile duct-ligated mice, in which bile acids accumulate, and in human hepatoma HepG2 cells treated with CDCA and CA. The accumulation of bile acids caused defective autophagic clearance, shown by the accumulation of insoluble p62 and ubiquitinated proteins and cell death accompanied by caspase-3 processing. Hepatocytes exposed to bile acids also showed the accumulation of autophagosomes via suppressed autophagy flux. Treatment of CDCA markedly suppressed Beclin-1 expression, which exhibits a higher cytotoxicity than CA. Moreover, pharmacological or genetic inhibition of autophagy enhanced bile acid-induced cell death. Finally, inxa0vivo, bile duct ligation led to aberrant accumulation of p62 and ubiquitinated proteins in the liver. Our data demonstrate that inhibited autophagy is an essential component of liver injury during cholestasis.

Rhus verniciflua Stokes attenuates cholestatic liver cirrhosis-induced interstitial fibrosis via Smad3 down-regulation and Smad7 up-regulation

Cholestatic liver cirrhosis (CLC) eventually proceeds to end-stage liver failure by mediating overwhelming deposition of collagen, which is produced by activated interstitial myofibroblasts. Although the beneficial effects of Rhus verniciflua Stokes (RVS) on various diseases are well-known, its therapeutic effect and possible underlying mechanism on interstitial fibrosis associated with CLC are not elucidated. This study was designed to assess the protective effects of RVS and its possible underlying mechanisms in rat models of CLC established by bile duct ligation (BDL). We demonstrated that BDL markedly elevated the serological parameters such as aspartate aminotransferase, alanine transaminase, total bilirubin, and direct bilirubin, all of which were significantly attenuated by the daily uptake of RVS (2 mg/kg/day) for 28 days (14 days before and after operation) via intragastric route. We observed that BDL drastically induced the deterioration of liver histoarchitecture and excessive deposition of extracellular matrix (ECM), both of which were significantly attenuated by RVS. In addition, we revealed that RVS inhibited BDL-induced proliferation and activation of interstitial myofibroblasts, a highly suggestive cell type for ECM production, as shown by immunohistochemical and semi-quantitative detection of α-smooth muscle actin and vimentin. Finally, we demonstrated that the anti-fibrotic effect of RVS was associated with the inactivation of Smad3, the key downstream target of a major fibrogenic cytokine, i.e., transforming growth factor β (TGF-β). Simultaneously, we also found that RVS reciprocally increased the expression of Smad7, a negative regulatory protein of the TGF-β/Smad3 pathway. Taken together, these results suggested that RVS has a therapeutic effect on CLC, and these effects are, at least partly, due to the inhibition of liver fibrosis by the downregulation of Smad3 and upregulation of Smad7.

Extract of Rhus verniciflua stokes protects against renal ischemia-reperfusion injury by enhancing Nrf2-mediated induction of antioxidant enzymes

Ischemia-reperfusion injury (IRI) may cause acute kidney disease (AKD) by mediating the oxidative stress-induced apoptosis of parenchymal cells. The extract of Rhus verniciflua Stokes (RVS) is used as a traditional herbal medicine as it exhibits anti-oxidant, anti-apoptotic and anti-inflammatory properties. Therefore, the current study investigated the therapeutic effect and the underlying mechanism of RVS on IRI-induced AKD in vivo and in vitro. The current study assessed the effects of RVS on a mouse model of renal IRI and in hypoxic human renal tubular epithelial HK-2 cells. The results demonstrated that the IRI-induced elevation of blood urea nitrogen, serum creatinine and lactate dehydrogenase was significantly attenuated by the intraoral administration of RVS (20 mg/kg/day) for 14 days prior to surgery. It was demonstrated that IRI surgery induced histological damage and cellular apoptosis in renal parenchyma, which were attenuated by pretreatment with RVS. Furthermore, in HK-2 cells incubated with 300 µM CoCl2 to induce chemical hypoxia, it was demonstrated that RVS treatment significantly inhibited cell death and the production of reactive oxygen species (ROS). Furthermore, RVS treatment upregulated the levels of endogenous antioxidant enzymes, including heme oxygenase-1 and catalase, as well as their upstream regulator nuclear factor erythroid 2-related factor 2, in HK-2 cells. Taken together, these results suggested that the intraoral administration of RVS induces a therapeutic effect on IRI-induced AKD. These effects are at least partly due to the attenuation of ROS production via upregulation of the antioxidant defense system in renal tubular cells.

Protective effect of Rhus verniciflua Stokes extract in an experimental model of post-menopausal osteoporosis

Post-menopausal osteoporosis (PMO) is a major global human health concern. Owing to the need for therapeutic drugs without side effects, natural extracts containing various polyphenolic compounds that may exert estrogenic effects have been studied in depth. Rhus verniciflua Stokes (RVS), which has been used as a traditional herbal medicine for centuries in Korea, was recently revealed to exert estrogenic effects attributable to its bioactive ingredients sulfuretin and butein, which have strong estrogen receptor–binding affinities. In this study, the protective potential of RVS in PMO was evaluated by using an experimental animal model of PMO, which was established by ovariectomy (OVX) of female Sprague Dawley rats. The oral administration of RVS at 20 mg/kg or 100 mg/kg for 8 weeks markedly protected against OVX-induced atrophy of the uterine tube and reversed the elevation in the ratio of serum receptor activator of nuclear factor-κB ligand to osteoprotegerin, which is a marker of disease severity. In addition, RVS inhibited OVX-induced tibia bone loss, activated osteogenic activity, and suppressed osteoclastic activity in the tibial epiphyseal plate, a region of bone remodeling. Collectively, these factors indicated that the oral intake of RVS might be beneficial for the prevention of PMO.