Ching-Wen Liu

Preparation and characterization of gelatin-based mucoadhesive nanocomposites as intravesical gene delivery scaffolds

This study aimed to develop optimal gelatin-based mucoadhesive nanocomposites as scaffolds for intravesical gene delivery to the urothelium. Hydrogels were prepared by chemically crosslinking gelatin A or B with glutaraldehyde. Physicochemical and delivery properties including hydration ratio, viscosity, size, yield, thermosensitivity, and enzymatic degradation were studied, and scanning electron microscopy (SEM) was carried out. The optimal hydrogels (H), composed of 15% gelatin A175, displayed an 81.5% yield rate, 87.1% hydration ratio, 42.9 Pa·s viscosity, and 125.8 nm particle size. The crosslinking density of the hydrogels was determined by performing pronase degradation and ninhydrin assays. In vitro lentivirus (LV) release studies involving p24 capsid protein analysis in 293T cells revealed that hydrogels containing lentivirus (H-LV) had a higher cumulative release than that observed for LV alone (3.7-, 2.3-, and 2.3-fold at days 1, 3, and 5, resp.). Lentivirus from lentivector constructed green fluorescent protein (GFP) was then entrapped in hydrogels (H-LV-GFP). H-LV-GFP showed enhanced gene delivery in AY-27 cells in vitro and to rat urothelium by intravesical instillation in vivo. Cystometrogram showed mucoadhesive H-LV reduced peak micturition and threshold pressure and increased bladder compliance. In this study, we successfully developed first optimal gelatin-based mucoadhesive nanocomposites as intravesical gene delivery scaffolds.

Mechanism of bombesin-induced tonic contraction of the porcine lower esophageal sphincter.

Gastroesophageal reflux disease (GERD) is a disorder that is related to an incompetent lower esophageal sphincter (LES). Previous studies showed that bombesin could increase LES pressure in humans and opossums. The aim of the present study was to characterize the effects of bombesin on porcine LES contraction. We used the selective agonists, neuromedin B (NMB), gastrin-releasing peptide (GRP), and [D-Tyr6,Apa-4Cl11,Phe13,Nle14]bombesin-(6-14) (DTACPN-BN), as well as receptor antagonists of bombesin receptor subtype 2 (BB2), and 3 (BB3) for ex vivo contraction studies. Atropine, nifedipine, tetrodotoxin, and ω-conotoxin GVIA were used to explore the agonist-induced LES contraction mechanism. Reverse transcription polymerase chain reaction and immunohistochemistry were applied to detect bombesin receptor expression. Our results indicate that GRP and DTACPN-BN, but not NMB, induced tonic contractions of the porcine LES in a dose-dependent manner, and the contractions were inhibited with selective BB2 and BB3 antagonists. The GRP-induced contraction is mainly caused by L-type Ca2+ channel-mediated Ca2+ influx. However, DTACPN-BN-induced contractions are associated with neuronal conduction. RT-PCR and immunohistochemistry revealed that BB2 and BB3 were expressed in the porcine LES. Bombesin-induced tonic contraction of the LES is mediated through BB2 and BB3. Bombesin, BB2, and BB3 agonists might have the potential to treat GERD.

Salvia miltiorrhiza causes tonic contraction in rat ileum through Ca2+-calmodulin pathway

ETHNOPHARMACOLOGICAL RELEVANCE Danshen, root of Salvia miltiorrhiza (SM), has been traditionally used in Chinese medicine for the treatment of heart, abdomen, gurgling in the intestines, and relieving fullness. However, the effects of SM on intestine have rarely been done to date. AIM OF THE STUDY To investigate the contraction effect of SM on isolated rat ileum and its mechanisms involved. MATERIALS AND METHODS The isometric contractions of ileum segments were investigated in organ baths for spontaneous activity and response to ethanolic extracts of SM. To determine the contraction mechanism caused by SM extracts, atropine (a muscarinic receptor antagonist), tetrodotoxin (TTX, a sodium channel blocker), nifedipine (a calcium channel blocker), Ca(2+) free Krebs solution with EGTA, or trifluoperazine (TFP, a calmodulin blocker) was administered and its response to cumulative dosages of SM extracts were examined. The effect of SM extracts on Ca(2+) signaling in the intestinal epithelial cell-6 (IEC-6) was examined using fura-2 as a Ca(2+) indicator. RESULTS SM extracts caused dose-dependent tonic contraction on rat ileum in ex vivo organ bath studies. The contraction induced by SM extracts was not inhibited by atropine, TTX, nifedipine, or in Ca(2+) free solution. However, the ileal contractions induced by SM extracts were significantly inhibited by TFP in a dose-dependent manner. In IEC-6 cells, the SM extracts induced extracellular Ca(2+) entry and massive intracellular Ca(2+) release in Ca(2+)-contained medium, and induced intracellular Ca(2+) release in Ca(2+)-free medium. CONCLUSION These results demonstrate that SM extracts cause ileal contraction through the Ca(2+)-calmodulin pathway.

Salvia miltiorrhiza Induces Tonic Contraction of the Lower Esophageal Sphincter in Rats via Activation of Extracellular Ca2+ Influx

Up to 40% of patients with gastroesophageal reflux disease (GERD) suffer from proton pump inhibitor refractory GERD but clinically the medications to strengthen the lower esophageal sphincter (LES) to avoid irritating reflux are few in number. This study aimed to examine whether Salvia miltiorrhiza (SM) extracts induce tonic contraction of rat LES ex vivo and elucidate the underlying mechanisms. To investigate the mechanism underlying the SM extract-induced contractile effects, rats were pretreated with atropine (a muscarinic receptor antagonist), tetrodotoxin (a sodium channel blocker), nifedipine (a calcium channel blocker), and Ca2+-free Krebs-Henseleit solution with ethylene glycol tetraacetic acid (EGTA), followed by administration of cumulative dosages of SM extracts. SM extracts induced dose-related tonic contraction of the LES, which was unaffected by tetrodotoxin, atropine, or nifedipine. However, the SM extract-induced LES contraction was significantly inhibited by Ca2+-free Krebs-Henseleit solution with EGTA. Next, SM extracts significantly induce extracellular Ca2+ entry into primary LES cells in addition to intracellular Ca2+ release and in a dose-response manner. Confocal fluorescence microscopy showed that the SM extracts consistently induced significant extracellular Ca2+influx into primary LES cells in a time-dependent manner. In conclusion, SM extracts could induce tonic contraction of LES mainly through the extracellular Ca2+ influx pathway.

Negative correlation between serum uric acid and kidney URAT1 mRNA expression caused by resveratrol in rats

SCOPE This study established a hyperuricemic rat model to elucidate the effect of resveratrol on the transport of UA in the kidney. METHODS AND RESULTS Hyperuricemia was induced in rats through daily oral gavage of a potassium oxonate and UA mixture over 3 weeks. Our results revealed that resveratrol significantly reduced the serum UA levels but not creatinine, c-creative protein, alanine aminotransferase, or aspartate aminotransferase levels in these rats. Furthermore, renal URAT1 and OAT1 mRNA expression were significantly higher in the rats treated with allopurinol than in those with no treatment. Therefore, allopurinol not only inhibited UA production but also mediated renal URAT1 and OAT1 expression. The correlation analysis revealed that UA levels correlated negatively with renal IL-6 mRNA expression in rats treated with allopurinol. Moreover, URAT1 showed strong immunoreactivity in the distal convoluted tubule of rats treated with allopurinol or resveratrol and in hyperuricemic treated with allopurinol. Finally, in the rats treated with resveratrol, UA levels correlated negatively with renal URAT1 mRNA expression; thus, resveratrol reduced URAT1 mRNA expression under high UA levels, thereby reducing UA reabsorption in renal cells. CONCLUSION Resveratrol contributes to URAT1 expression, which is potentially useful in therapeutic strategies aimed at treating hyperuricemia.

Mechanism of resveratrol-induced relaxation in the human gallbladder

BackgroundResveratrol is a polyphenolic compound extracted from plants and is also a constituent of red wine. Resveratrol produces relaxation of vascular smooth muscle and may prevent cardiovascular diseases. Although resveratrol has been reported to cause relaxation of the guinea pig gallbladder, limited data are available about the effect of resveratrol on the gallbladder smooth muscle in humans. The purpose of this study was to investigate the relaxation effects of resveratrol in human gallbladder muscle strips.MethodsWe studied the relaxant effects of resveratrol in human gallbladder. In addition, we also investigated mechanism of resveratrol-induced relaxation in human gallbladder by tetraethylammonium (a non-selective potassium channels blocker), iberiotoxin (an inhibitor of large conductance calcium-activated potassium channel), glibenclamide (an ATP-sensitive potassium channel blocker), charybdotoxin (an inhibitor of large conductance calcium-activated potassium channels and slowly inactivating voltage-gated potassium channels), apamine (a selective inhibitor of the small conductance calcium-activated potassium channel), KT 5720 (a cAMP-dependent protein kinase A inhibitor), KT 5823 (a cGMP-dependent protein kinase G inhibitor), NG-Nitro-L-arginine (a competitive inhibitor of nitric oxide synthase), tetrodotoxin (a selective neuronal Na+ channel blocker), and ω-conotoxin GVIA (a selective neuronal Ca2+ channel blocker).ResultsThe present study showed that resveratrol has relaxant effects in human gallbladder muscle strips. In addition, we found that resveratrol-induced relaxation in human gallbladder is associated with nitric oxide, ATP-sensitive potassium channel, and large conductance calcium-activated potassium channel pathways.ConclusionsThis study provides the first evidence concerning the relaxant effects of resveratrol in human gallbladder muscle strips. Furthermore, these results demonstrate that resveratrol is a potential new drug or health supplement in the treatment of biliary colic.

APOBEC3G exerts tumor suppressive effects in human hepatocellular carcinoma.

In this study, we collected 44 hepatitis B virus surface antigen positivity HBsAg (+) tumor and nontumor hepatocellular tissues from hepatocellular carcinoma (HCC) patients during hepatectomy, and quantified the APOBEC3G (A3G) mRNA by using a real-time PCR. Our results showed higher expression of A3G mRNA in the nontumor tissues than in the tumor tissues of the HBsAg (+) HCC patients. To further investigate this phenomenon, we constructed a pLV-A3G vector and transfected it into the human HCC cell line, Hep 3B. The results of an immunofluorescence analysis showed the overexpression of A3G in the cytoplasm. We then evaluated A3G cytotoxicity by using a cell viability assay (MTS assay), the results of which showed that Hep 3B cell viability was 88 and 58% after the transfection of pLV and pLV-A3G, respectively, indicating the growth inhibitory effects of A3G on Hep 3B cells. To further evaluate the tumor suppressive effects of A3G, we used a plastic pipette tip to scratch Hep 3B cells grown on a culture dish (to 70–80% confluence) after transfection with pLV-A3G. Our data indicated a ratio of wound closure of 100% in the control cells and in the pLV-expressing cells, compared with 43% in the pLV-A3G-overexpressing cells, 72 h after the wound scratch, as observed using phase-contrast microscopy. These results indicated that A3G inhibits wound healing in Hep 3B cells. Overall, our results suggest that A3G inhibits the growth of human hepatoma cells.

Mechanism of resveratrol-induced relaxation of the guinea pig fundus

BACKGROUND Resveratrol is a polyphenolic compound that can be isolated from plants and also is a constituent of red wine. Resveratrol induces relaxation of vascular smooth muscle and may prevent cardiovascular diseases. PURPOSE Impaired gastric accommodation plays an important role in functional dyspepsia and fundic relaxation and is a therapeutic target of functional dyspepsia. Although drugs for fundic relaxation have been developed, these types of drugs are still rare. The purpose of this study was to investigate the relaxant effects of resveratrol in the guinea pig fundus. STUDY DESIGN We studied the relaxant effects of resveratrol in the guinea pig fundus. In addition, we investigated the mechanism of resveratrol-induced relaxation on the guinea pig fundus by using tetraethylammonium (a non-selective potassium channel blocker), apamine (a selective inhibitor of the small conductance calcium-activated potassium channel), iberiotoxin (an inhibitor of large conductance calcium-activated potassium channels), glibenclamide (an ATP-sensitive potassium channel blocker), KT 5720 (a cAMP-dependent protein kinase A inhibitor), KT 5823 (a cGMP-dependent protein kinase G inhibitor), NG-nitro-L-arginine (a competitive inhibitor of nitric oxide synthase), tetrodotoxin (a selective neuronal Na+ channel blocker), ω-conotoxin GVIA (a selective neuronal Ca2+ channel blocker) and G-15 (a G-protein coupled estrogen receptor antagonist). RESULTS The results of this study showed that resveratrol has potent and dose-dependent relaxant effects on the guinea pig fundic muscle. In addition, the results showed that resveratrol-induced relaxation of the guinea pig fundus occurs through nitric oxide and ATP-sensitive potassium channels. CONCLUSION This study provides the first evidence concerning the relaxant effects of resveratrol in the guinea pig fundic muscle strips. Furthermore, resveratrol may be a potential drug to relieve gastrointestinal dyspepsia.

A Hydrogel-Based Epirubicin Delivery System for Intravesical Chemotherapy

This study aimed to examine the efficacy of epirubicin-loaded gelatin hydrogel (EPI-H) in the treatment of superficial urothelium carcinoma. Hydrogel was prepared by Schiff base-crosslinking of gelatin with glutaraldehyde. EPI-H exhibited high entrapment efficiency (59.87% ± 0.51%). EPI-H also increased epirubicin accumulation in AY-27 cells when compared with the effect of aqueous solutions of epirubicin (EPI-AQ); respective epirubicin-positive cell counts were 69.0% ± 7.6% and 38.3% ± 5.8%. EPI-H also exhibited greater cytotoxicity against AY-27 cells than that of EPI-AQ; IC50 values were 13.1 ± 1.1 and 7.5 ± 0.3 μg/mL, respectively. Cystometrograms showed that EPI-H reduced peak micturition, threshold pressures, and micturition duration, and that it increased bladder compliance more so than EPI-AQ. EPI-H enhanced epirubicin penetration into basal cells of urothelium in vivo, whereas EPI-AQ did so only to the umbrella cells. EPI-H inhibited tumor growth upon intravesical instillation to tumor-bearing bladder of F344 rats, inducing higher levels of caspase-3 expression than that observed with EPI-AQ treatment; the number of caspase-3 positive cells in treated urothelium carcinoma was 13.9% ± 4.0% (EPI-AQ) and 34.1% ± 1.0%, (EPI-H). EPI-H has value as an improved means to administer epirubicin in intravesical instillation treatments for bladder cancer.