| Summary: | 博士 === 高雄醫學大學 === 藥學系博士班 === 104 === Proton pump inhibitors (PPIs) are widely prescribed for the management of gastroesophageal reflux disease (GERD), and up to 40% of patients with GERD have symptoms refractory to PPIs. Few medications are available to strengthen the lower esophageal sphincter (LES) to avoid irritant reflux. The purpose of this study is to explore medications to strengthen the LES and elucidate the underlying mechanisms.
The chapter one of this study was aimed at examining the effects of Salvia miltiorrhiza (SM) extract inducing tonic contraction of rat LES, and elucidating the underlying mechanisms. To investigate the mechanism underlying SM extract-induced contractile effects, rat LES was pretreated with atropine (a muscarinic receptor antagonist), tetrodotoxin (a neuronal sodium channel blocker), nifedipine (a calcium channel blocker), and Ca2+-free Krebs-Henseleit solution, followed by administration of SM extract and collection of contractile range. In addition, primary LES smooth muscle cells were isolated. Spectrofluorophotometery, confocal microscopy, and CellR Xcellence System were used to determine the mechanism underlying changes in Ca2+ signaling in the cells treated with SM extract.
SM extract induced tonic contraction of the LES in a dose-dependent manner, which was unaffected by tetrodotoxin, atropine, or nifedipine. However, the extract-induced LES contraction was significantly inhibited by Ca2+-free Krebs-Henseleit solution, which showed extracellular Ca2+ influx was related to SM extract induced-contraction of the LES. The fluorescent calcium indicator Fura-2 showed that the extract significantly induced extracellular Ca2+ influx into primary LES cells in a dose-response manner. Further, confocal microscopy showed increased extracellular Ca2+ fluorescence intensity of primary LES cells, which was induced by the extract. The CellR System showed that Ca2+ influx was related to store-operated calcium channels. Thus, SM extract could induce tonic contraction of the LES mainly through store-operated calcium channel-mediated Ca2+ influx pathway.
The chapter two of this study was aimed at investigating the effect of bombesin on porcine LES and its underlying mechanism. Selective agonists neuromedin B (NMB), gastrin-releasing peptide (GRP), and [D-Tyr6,Apa-4Cl11,Phe13,Nle14]bombesin-(6-14) (DTACPN-BN), and antagonists of bombesin receptor subtypes 2 (BB2) and 3 (BB3) were used to study the mechanism of bombesin-induced contraction of LES ex vivo. Atropine, nifedipine, tetrodotoxin, and ω-conotoxin GVIA (a neuronal calcium channel blocker) were used to investigate the mechanisms of agonist-induced LES contraction. Reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry staining were used to detect the expression of bombesin receptors in porcine LES.
The results showed that GRP and DTACPN-BN, but not NMB, caused tonic contractions of porcine LES in a dose-dependent manner, and these contractions were suppressed separately by selective BB2 and BB3 antagonists. GRP-induced contraction was mainly associated with L-type calcium channel-mediated calcium influx. However, DTACPN-BN induced-contraction was related to neuronal conduction. RT-PCR and immunohistochemistry staining showed BB2 and BB3 expression in porcine LES. Therefore, bombesin-induced tonic contraction of the LES is mediated by BB2 and BB3.
Our findings suggest that SM extract, bombesin, and BB2 and BB3 agonists have the potential to be developed as new drugs for GERD.
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