Sheng-I Lue

Multi-laminated metal hydroxide nanocontainers for oral-specific delivery for bioavailability improvement and treatment of inflammatory paw edema in mice

Multiple layers of pH-sensitive enteric copolymers were coated over layered double hydroxide (LDH) nanoparticles for controllable drug release and improved solubility of hydrophobic drugs. The nano-sized LDH carriers significantly improved the accessibility of sulfasalazine molecules that have positively charged frameworks. In addition, the successful encapsulation of negatively charged enteric copolymers was achieved via electrostatic attractions. The multi-layered enteric polymer coating could potentially protect nanoparticle dissolution at gastric pH and accelerate the dissolution velocity, which would improve the drug bioavailability in the colon. Next, biological studies of this formulation indicated a highly protective effect from the scavenging of superoxide free radicals and diethyl maleate (DEM) induced lipid peroxidation, which are major cell signalling pathways for inflammation. The histological view of the liver and kidney sections revealed that the nanoformulation is safe and highly biocompatible. The animal studies conducted via paw inflammation induced by complete Freunds adjuvant (CFA) revealed that enteric-coated LDH-sulfasalazine nanoparticles provided a sustained release that maintained the sulfasalazine concentrations in a therapeutic window. Therefore, this nanoformulation exhibited preferential efficacy in reducing the CFA-induced inflammation especially at day 4.

CGS 26303 upregulates mRNA expression of heme oxygenase-1 in brain tissue of rats subjected to experimental subarachnoid hemorrhage.

Previous studies indicate that intravenous infusion of CGS 26303, an endothelin-converting enzyme inhibitor, prevents and reverses cerebral vasospasm after experimental subarachnoid hemorrhage. Attenuation of the vasospastic response could result from enhanced production of nitric oxide via activation of endothelial nitric oxide synthase, neuronal nitric oxide synthase, or inducible nitric oxide synthase in brain tissue. Carbon monoxide has the same attenuation effect and is synthesized by inducible hemeoxygenase- 1 or constitutive heme-oxygenase-2. In this study, we investigated the effect of endothelin-converting enzyme inhibitor on mRNA expression of endothelial nitric oxide synthase, neuronal nitric oxide synthase, inducible nitric oxide synthase, hemeoxygenase- 1 and heme-oxygenase-2 in brain tissue of rats subjected to subarachnoid hemorrhage using semi-quantitative reverse transcription-polymerase chain reaction. The results showed that gene expression of inducible nitric oxide synthase or HSP70 was not detected in all groups of rats (n = 5/group). Expression of endothelial nitric oxide synthase, neuronal nitric oxide synthase or heme-oxygenase-2 mRNA in brain tissue in the groups of subarachnoid hemorrhage or subarachnoid hemorrhage treated with endothelin-converting enzyme inhibitor appeared to be the same as compared with control rats. The subarachnoid hemorrhage rats treated with endothelin-converting enzyme inhibitor showed a significant increase in the levels of heme-oxygenase-1 mRNA expression as compared with both subarachnoid hemorrhage and control rats. These data suggest that the reduction of cerebral vasospasm by CGS 26303 in rats subjected to experimental subarachnoid hemorrhage may result from both over-expression of heme-oxygenase-1 in brain tissue and suppression of endothelin biosynthesis in basilar arteries.

The Hormonal Regulation of Color Changes in the Sexually Dichromatic Frog Buergeria robusta

During the breeding season, dynamic changes in body coloration are regularly observed in the male brown tree frog Buergeria robusta. This study investigated the hypothesis that this sexual dichromatism in male B. robusta is mediated through hormonal regulation. Frogs were exogenously injected with testosterone (T) or estradiol (E2). This manipulation revealed that the body coloration (hue, brightness, and saturation) of the male frog increased significantly (i.e., the brilliant yellow color developed) in response to T but not in response to E2. Concurrently, the levels of expression of brain-derived neurotrophic factor (BDNF) and pituitary adenylate cyclase-activating polypeptide (PACAP) in the pituitary gland were reduced in frogs whose coloration was pale brown on a yellow background. In particular, the weakest expressions of BDNF, PACAP, and PACAP type II receptors (VPAC-1R) were found in male frogs with a brilliant yellow body color during the breeding season regardless of background color. These changes may decrease α-melanocyte-stimulating hormone production associated with the PACAP receptors (VPAC-1R), resulting in the aggregation of black pigment in melanophores and the production of a brilliant yellow body color. The effects of hormones on skin coloration were further examined in isolated skin in vitro. The results of this investigation showed that the dispersion of xanthophores was stimulated by T or prolactin (PRL) and that the melanophores were aggregated by melatonin (MEL) but not by E2. Furthermore, yellow pigments in the xanthophores were significantly dispersed following the PRL+T treatment. In the T+MEL, PRL+MEL, and T+PRL+MEL treatments, xanthophores were dispersed, and melanophores were aggregated and subsequently moved to the low spongiosum layer of the dorsal skin, causing the increase in yellow coloration. These results reveal that multiple hormones play major roles in the regulation of the brilliant yellow coloration of male B. robusta by high plasma T during the breeding season.

Early inactivation of PKCε associates with late mitochondrial translocation of Bad and apoptosis in ventricle of septic rat

BACKGROUND Sepsis is usually accompanied by cardiomyocyte apoptosis and myocardial depression. Protein kinase C (PKC) has been reported to be important in regulating cardiac function and apoptosis; however, which PKC isoform is involved in sepsis-induced myocardial apoptosis remains unknown. MATERIALS AND METHODS A rat model of sepsis by cecal ligation and puncture was used. Early and late sepsis refers to those rats sacrificed at 9 and 18 h after cecal ligation and puncture, respectively. Ventricular septum (Sep), left ventricle (LV), and right ventricle were fractionated into membrane, mitochondrial, and cytosolic fractions, individually. The protein levels of PKC isoforms (-α, -β, -δ, -ε, -ζ, -ι, -λ, and -μ) and mitochondrial translocation of Bad were quantified by Western blot analysis. Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP in situ nick-end labeling. The morphology of mitochondria was examined by electron microscopy. RESULTS The membrane/cytosol ratio of PKCε was predominantly higher in the Sep, LV, and right ventricle under physiological conditions. At early sepsis, the membrane/cytosol ratio of PKCε was significantly decreased in Sep and LV. At late sepsis, cardiomyocyte apoptosis associated with severe mitochondrial swelling and crista derangement were observed in Sep and LV at late sepsis. Additionally, mitochondria/cytosol ratio of Bad was significantly increased in Sep and LV. CONCLUSIONS The early inactivation of PKCε in the ventricle may affect the mitochondrial translocation of Bad and subsequent mitochondrial disruption and apoptosis at late sepsis. This finding opens up the prospect for a potential therapeutic strategy targeting PKCε activation to prevent myocardial depression in septic patients.

Role of Exogenous Hsp72 on Liver Dysfunction during Sepsis

This study examined the role of exogenous heat shock protein 72 (Hsp72) in reversing sepsis-induced liver dysfunction. Sepsis was induced by cecal ligation and puncture. Liver function was determined on the basis of the enzymatic activities of serum glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT). Apoptosis was determined using terminal deoxynucleotidyl transferase dUTP nick end labeling staining. B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), cleaved caspase-3 and caspase-9, and cleaved poly (ADP-ribose) polymerase (PARP) protein expressions were analyzed using Western blotting. Results showed GOT and GPT levels increased during sepsis, and levels were restored following the administration of human recombinant Hsp72 (rhHsp72). Increased liver tissue apoptosis was observed during sepsis, and normal apoptosis resumed on rhHsp72 administration. The Bcl-2/Bax ratio, cleaved caspase-3, caspase-9, and PARP protein expressions in the liver tissues were upregulated during sepsis and normalized after rhHsp72 treatment. We conclude that, during sepsis, exogenous Hsp72 restored liver dysfunction by inhibiting apoptosis via the mitochondria-initiated caspase pathway.

Release of endogenous heat shock protein 72 on the survival of sepsis in rats.

BACKGROUND This study was undertaken to clarify the role of extracellular heat shock protein 72 on the survival of sepsis and to determine possible factor(s) that may be responsible for it. MATERIALS AND METHODS Sepsis was induced by cecal ligation and puncture. Changes in serum levels of heat shock protein (Hsp72) and cytokines were determined during sepsis, and the results were correlated with the survival. Effects of heat pretreatment on Hsp72 expression in septic rat leukocytes and those of septic rat serum, lipopolysaccharide (LPS), and certain cytokines on the release of Hsp72 in macrophage NR8383 cells were determined. RESULTS Circulating Hsp72 levels were increased during the progress of sepsis (0, 5.5, 6.5, 10, and 6.5 ng/mL at 0, 3, 6, 9, and 18 h after cecal ligation and puncture, respectively) and the increases were correlated positively with survival rates. LPS triggered the release of Hsp72 in heat pretreated animals. Heat pretreatment increased Hsp72 expression in nonsepsis (+535%, P < 0.01) and sepsis (+116%, P<0.01%) rat leukocytes. Incubation of sepsis rat serum with NR8383 cells increased levels of extracellular heat shock protein 72 in cultured medium. Cytokine profiling revealed that among the 19 cytokines screened, four of them were increased as follows: cytokine-induced neutrophil chemoattractant 3 (+211.3%, P < 0.05), interleukin 10 (+147%, P < 0.05), MCP-1 (+49.6%, P < 0.05), and tumor necrosis factor alpha (+51.8%, P < 0.05). MCP-1 and LPS were capable of releasing Hsp72 from NR8383 cells. CONCLUSIONS These results demonstrate that the increases in the levels of circulating Hsp72 had a beneficial effect in improving animal survival during the progress of sepsis. The increases in circulating Hsp72 may be mediated via MCP-1 and/or LPS.

The Immediate Mitochondrial Stress Response in Coping with Systemic Exposure of Silver Nanoparticles in Rat Liver

Silver nanoparticles (Ag-nps) induce hepatotoxicities via oxidative stress. The oxidative stress-induced mitochondrial damages are known to be regulated by mitochondrial unfolded protein response (mtUPR) and autophagy/mitophagy systems. The present study was undertaken to investigate role of mtUPR and autophagy/mitophagy on the Ag-nps induced mitochondrial dysfunction. Experiments consisted of two groups of Sprague Dawley rats: control and Ag-nps treated. The Ag-nps treated group received an intraperitoneally injection of 1.5 ml deionized water containing 500 mg/ kg of Ag-nps. Control group received equal volume of deionized water. All animals were sacrificed at different time points (6, 12, 18 and 24 hr post treatment), followed by removal of livers for determination of ATP content and protein expression. The results show that multi-ubiquitinated proteins (Ub-proteins) in liver mitochondria were increased at 12 hr and the increases were sustained up to 24 hr following Ag-nps treatment. Expressions of mtUPR-associated proteins including transcriptional factors (C/EBP homologous protein (CHOP) and CAAT/enhancer-binding protein-β (C/EBPβ)), molecular chaperones (heat shock protein (Hsp) 70 and Hsp60) and protease (caseinolytic Clp protease (ClpP)) remained unchanged during the entire experimental period, except that Hsp10 was upregulated. Expression of LC3-II (autophagy marker) and BNIP3 (mitophagy marker) were upregulated at 6 hr and the upregulation remained throughout the experimental period. ATP content was reduced at 6 hr after Ag-nps exposure and the reductions were sustained during the entire experimental period. These results demonstrate that activation of autophagy/mitophagy markers co-existed with upregulation of Ub-proteins and reduction of ATP content without changing mtUPR in rat liver following Ag-nps administration. These findings indicate that protective effects of autophagy/mitophagy markers were overwhelmed by detrimental actions of Ub-proteins on the control of mitochondrial function, and the counter-balance of the two systems eventually resulting in impaired mitochondrial function, i.e., reduction of ATP content.

Pyrogallol abates VSMC migration via modulation of Caveolin-1, matrix metalloproteinase and intima hyperplasia in carotid ligation mouse

Migration of vascular smooth muscle cells (VSMCs) contributes to intimal hyperplasia and other vascular diseases. Caveolin-1 (Cav-1) has been recognized as a proliferative inhibitor of VSMCs and is likely to be an important regulator of VSMC migration. The underlying mechanism of pyrogallol on the VSMC migration is not fully understood. This study attempted to dissect the role of Cav-1 and matrix metalloproteinase (MMP) in VSMC migration and to investigate the effect of pyrogallol on VSMC mobility during carotid artery ligation mice. The mRNA expression of MMP-3 and MMP-13 was down-regulated in cultured VSMC prepared from Cav-1-deficient (Cav-1 KO) mice whereas MMP-14 expression was up-regulated. Pyrogallol effectively inhibited the migration of Cav-1 KO VSMC by promoting the expression of tissue inhibitors of metalloproteinase (TIMP)-2. Pyrogallol also inhibited the migration of Cav-1 wild type (WT) VSMC, however, by increasing TIMP-1 expression and repressing MMP-2 activity. In a parallel in vivo study, intra-peritoneal (ip) of pyrogallol to carotid artery ligated mice significantly suppressed intima formation in mice carotid artery. Furthermore, the proMMP-9 activity in pyrogallol-treated mice serum significantly increased from Day 0 to Day 2 and decreased from Day 2 to Day 7 in a time-dependent manner. In addition, WT mice treated with pyrogallol had significantly reduced neointima formation, whereas no differences were observed in Cav-1 knock out (KO) mice. These results suggest that pyrogallol not only inhibited VSMC migration but also effectively diminishes the severity of neointima hyperplasia, implying that pyrogallol possesses potential anti-atherogenic effects for the treatment of vascular diseases.

Attenuation of intercellular adhesion molecule-1 and cerebral vasospasm in rabbits subjected to experimental subarachnoid haemorrhage by CGS 26303

Endothelin converting enzyme (ECE) inhibitor CGS26303 has been shown to be effective on SAH-induced cerebral vasospasm. However, there is no data related to CGS 26303 having a blocking effect of adhesion molecules. Therefore, the aim of this study was to investigate the effect of CGS 26303 on SAH-induced vasospasm and the plasma levels of ICAM, VCAM, and E-selectin. Male rabbits (n=36) were allocated into four groups (9 in each group): 1) control group (group C), no SAH; 2) group of SAH (group Sa), SAH only; 3) group of SAH plus vehicle (group Sv) and; 4) group of SAH plus CGS 26303 (group Sc). Administration of CGS 26303 30 mg/kg i.v. was initiated 1 h after SAH, and subsequently effected at 12, 24, and 36 h post SAH. All animals were sacrificed 48 h post SAH, and plasma levels of intercellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), and E-selectin levels were examined. ICAM-1 level in group 4 (SAH with CGS 26303 treatment) was significantly lower than those in groups 2 (SAH only) and 3 (SAH plus vehicle) (p<0.001). ICAM-1 level in the SAH with CGS 26303 treatment group (group 4) did not differ significantly from that of the control group. However, VCAM-1 levels showed no significant difference between the groups and CGS26303 administration did not decrease the E-selectin level following SAH. We concluded that the anti-spastic effect of CGS 26303 may partially be mediated by reversing increased ICAM-1 levels after SAH.

The effect of KMUVS-1 on experimental subarachnoid haemorrhage-induced cerebrovasospasm

Cerebral vasospasm is the leading cause of mortality and morbidity in patients suffering aneurysmal subarachnoid haemorrhage (SAH). In this study, we plan to investigate the effect of a Chinese medicinal formula, KMUVS-1, on vasospasm. Experimental SAH was induced in New Zealand white rabbits by injecting autogenous blood into cisterna magna. Animals were divided into the following groups: control (no SAH), SAH only, SAH plus low-dose (1 mg/kg), medium-dose (500 mg/kg), and high-dose (1000 mg/kg) oral KMUVS-1. Oral KMUVS-1 was given to animals 30min, 12, 24, and 36 h after induction of SAH. Animals were sacrificed 48 h after SAH. Basilar arteries were removed for analysis. Compared to the healthy controls, the average cross-sectional areas of the lumen were reduced by 46% in SAH group. The magnitude of cerebral vasospasm was significantly and dose-dependently attenuated in animals treated with KMUVS-1. The average cross-sectional areas were reduced by 31%, 19%, and 12% in animals receiving low, medium, and high-dose KMUVS-1, respectively, compared to those of healthy controls. KMUVS-1 effectively attenuates cerebral vasospasm in this pilot study. Because this formula contains several Chinese herbal drugs which all have individually been shown to have vasodilatation effects, we will further examine which component is most effective for alleviation of vasospasm.