Li-Ching Chang

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.

Estradiol mediates relaxation of porcine lower esophageal sphincter

Graphical abstract Figure. No caption available. HighlightsEstradiol causes relaxation in the porcine lower esophageal sphincter (LES).The mechanism was related to the potassium channel.G protein‐coupled estrogen receptor (GPER) was detected in the porcine LES.GPER mediates estradiol‐induced relaxation in the porcine LES. ABSTRACT Most pregnant women have symptoms of gastroesophageal reflux disease (GERD) during pregnancy. Postmenopausal hormone replacement therapy is associated with GERD. The effects of estradiol on lower esophageal sphincter (LES) motility and GERD are not clearly known. The purpose of this study is to investigate the effects of estradiol on the motility of the porcine LES. Relaxations of clasp and sling strips of porcine LES caused by estradiol were measured using isometric transducers. We investigated the mechanism of estradiol‐induced relaxation of the porcine LES using tetraethylammonium, apamine, iberiotoxin, glibenclamide, KT5720, KT5823, NG‐nitro‐l‐arginine, tetrodotoxin, and ω‐conotoxin GVIA. Reverse transcription polymerase chain reaction (PCR) analysis and immunohistochemistry (IHC) were performed to determine the existence of the G protein‐coupled estrogen receptor (GPER) in the porcine LES. In endothelin‐1‐precontracted porcine LES strips, estradiol caused marked relaxations in a concentration‐dependent manner. The mechanism of estradiol‐induced relaxation on the porcine LES was associated with the potassium channel. Reverse transcription PCR analysis and IHC revealed that GPER was expressed in the sling and clasp fibers of the porcine LES. This finding suggests that GPER mediates the relaxation of the porcine LES. Estradiol may play a role in LES motility.

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.