Effects of Tegoprazan, a Novel Potassium-Competitive Acid Blocker, on Rat Models of Gastric Acid-Related Disease.

Tegoprazan, a novel potassium-competitive acid blocker (P-CAB), is a next-generation therapeutics developed for the treatment of acid-related gastrointestinal diseases such as gastroesophageal reflux disease (GERD) and peptic ulcers. In the present study, the in vitro and in vivo pharmacological properties of tegoprazan were compared with those of esomeprazole, a representative proton pump inhibitor. In vitro enzyme assays were performed using ion-leaky vesicles containing gastric H+/K+-ATPases isolated from pigs. The in vivo efficacies of tegoprazan were evaluated in rat models of GERD and peptic ulcer. Tegoprazan inhibited the activity of porcine H+/K+-ATPase with an IC50 value of 0.53 µM in a reversible manner, whereas esomeprazole showed weak and irreversible inhibition with an IC50 value of 42.52 µM. In a GERD model, tegoprazan showed dose-dependent efficacy in inhibiting esophageal injury and gastric acid secretion with an ED50 of 2.0 mg/kg, which was 15-fold more potent than that of esomeprazole. In peptic ulcer models, tegoprazan exhibited superior antiulcer activity compared with esomeprazole. The ED50 of tegoprazan in the naproxen-, ethanol-, and water-immersion restraint stress-induced peptic ulcer models were 0.1, 1.4, and 0.1 mg/kg, respectively. In the acetic acid-induced peptic ulcer model, the curative ratio of tegoprazan at 10 mg/kg was higher than that of esomeprazole at 30 mg/kg (44.2% vs. 32.7%, respectively), after 5 days of repeated oral administration. Thus, tegoprazan is a novel P-CAB that shows potent and reversible inhibition of gastric H+/K+-ATPase and may provide stronger efficacy compared with previous proton pump inhibitors.

New phytopharmaceutical agent CJ-20001 modulates stress-induced inflammatory infiltration into gastric mucosa.

BACKGROUND/AIMS: CJ-20001 is a phytopharmaceutical agent and currently being investigated in a Phase II trial for the treatment of acute and chronic gastritis patients in Korea. In this study we addressed the protective effects of CJ-20001 against water immersion restraint stress (WIRS)-induced gastric injury in rats and studied the underlying mechanisms. METHODOLOGY: To evaluate the protective effect of CJ-20001 on stress-induced gastric lesions, rats were exposed to water immersion restraint stress. Inflammatory infiltration into gastric mucosa was examined by immunohistochemistry and in vitro invasion assay. Expression of proinflammatory cytokines was detected with reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Pretreatment with CJ-20001 dose-dependently attenuated the WIRS-induced gastric lesions as demonstrated by gross pathology and histology. WIRS increased infiltration of mast cells and macrophages into the gastric mucosa and submucosal layer, whereas the inflammatory infiltration was markedly inhibited by CJ-20001 administration. An in vitro cell invasion assay showed that treatment with CJ-20001 decreased the migration of macrophages. CJ-20001 suppressed the expression of proinflammatory cytokines, IL-18, IP-10 and GRO/KC, in lipopolysaccharides (LPS)-treated macrophages. CONCLUSIONS: These data suggest that novel phytopharmaceutical agent CJ-20001 has the potent anti-inflammatory properties through inhibition of inflammatory infiltration in psycho-physiological stress-induced gastric injury.

Galpha13 regulates methacholine-induced contraction of bronchial smooth muscle via phosphorylation of MLC20.

Reversible airway constriction is induced by an increase in airway smooth muscle contractility in response to methacholine likely as a bronchospastic stimulus. Despite the finding of Galpha12 and Galpha13 up-regulation in airway hyperresponsive animals, their functional role of contraction in airway smooth muscle has not been directly explored. This study investigated the differential regulatory role of Galpha12/Galpha13 in methacholine-induced contraction of trachea and bronchus in Galpha12 or Galpha13 gene knockout mice after ovalbumin sensitization and challenges. Organ bath assays and videomicroscopy revealed that Galpha13 deficiency delayed methacholine-induced contractile response of bronchiolar smooth muscle, but not that of tracheal smooth muscle. In primary bronchial smooth muscle cells, knockdown of Galpha13 blocked methacholine-induced phosphorylation of 20 kDa regulatory light chain of myosin II (MLC20), a prerequisite step for the contractile initiation of actin and myosin. Galpha13-dependent MLC20 phosphorylation was confirmed in murine embryonic fibroblasts. After ovalbumin sensitization and challenges, wild type mice exhibited methacholine-induced bronchial contraction of lung tissue. Heterozygous absence of the Galpha13 gene abrogated methacholine-induced contractions, whereas homozygous absence of the Galpha12 gene failed to do so. Our findings indicate that Galpha13, but not Galpha12, specifically regulates cholinergic bronchial contraction in airway responsiveness via controlling phosphorylation of MLC20 by methacholine.

Regulation of thymus-dependent and thymus-independent production of immunoglobulin G subclasses by Galpha12 and Galpha13.

BACKGROUND: A previous study from this laboratory showed that Galpha12 members participate in the production of inflammatory cytokines. In spite of the identification of B cell homeostasis responses regulated by Galpha13, the functional roles of Galpha12 members in the production of immunoglobulin (Ig) isotypes remained unknown. This study investigated whether Galpha12 members are involved in the Ig isotype antibody production with the purpose of establishing their functions in thymus-dependent and thymus-independent humoral responses. RESULTS: Mice lacking Galpha12 and/or Galpha13 showed an impaired antigen-specific antibody production promoted by challenge(s) of ovalbumin or trinitrophenyl-lipopolysaccharide (TNP-LPS), used for thymus-dependent and thymus-independent stimuli, respectively. Homozygous knockout (KO) of Galpha12 or double heterozygous KO of Galpha12/Galpha13 significantly reduced the antigen-specific total IgG level after multiple ovalbumin immunizations with decreases in the production of IgG1, IgG2a and IgG2b subclasses, as compared to wild type control. In contrast, IgM production was not decreased. Moreover, mice deficient in Galpha12 or partially deficient in Galpha13 or Galpha12/Galpha13 showed significantly low production of IgG2b in response to TNP-LPS. In TNP-LPS-injected mice, IgG1 and IgG2a productions were unaffected by the G protein KOs. CONCLUSION: Our results demonstrate that both Galpha12 and Galpha13 are essentially involved in thymus-dependent and independent production of IgG subclasses, implying that the G-proteins contribute to the process of antigen-specific IgG antibody production.

Liquiritigenin, an aglycone of liquiritin in Glycyrrhizae radix, prevents acute liver injuries in rats induced by acetaminophen with or without buthionine sulfoximine.

Glycyrrhizae radix has been used as one of the oldest and most frequently employed botanicals in both western and oriental countries. Previously, we showed that liquiritigenin (LQ), an aglycone of liquiritin in G. radix, exerts cytoprotective effects against heavy metal-induced toxicity in vitro. This study investigated in vivo protective effects of LQ against acute liver injuries induced by acetaminophen (APAP) or APAP plus buthionine sulfoximine (BSO). Liver injuries were assessed by blood biochemistry and histopathology in rats administered with LQ purified from the acid hydrolyates of liquiritin singly (p.o. or i.v., 2-4 days) or in combination with dimethyl-4,4'-dimethoxy-5,6,5',6'-dimethylenedioxybiphenyl-2,2'-dicarboxylate (DDB), a synthetic derivative of Schisandrin C in Fructus shizandrae, and exposed to APAP or APAP + BSO. LQ treatments (oral) effectively decreased liver injuries induced by a single dose of APAP, as evidenced by decreases in hepatic necrosis and inflammation as well as plasma alanine aminotransferase and lactate dehydrogenase activities. LQ, when intravenously applied, enhanced hepatoprotective effect with a greater potency. APAP + BSO led to severe liver injuries, resulting in lethality. LQ pretreatments significantly reduced the potentiated liver necrosis, decreasing mortality. In spite of the improvement in blood biochemistry, DDB failed to protect the liver from injuries induced by APAP or APAP + BSO. Combined treatments of rats with LQ and DDB showed some additive protective effect. The present study demonstrates that LQ efficaciously protects the liver from acute injuries induced by APAP or from APAP-induced severe injuries during GSH deficiency, indicating that LQ is one of the principal cytoprotective components comprised in G. radix.

Seungma-galgeun-tang attenuates proinflammatory activities through the inhibition of NF-kappaB signal pathway in the BV-2 microglial cells.

Seungma-galgeun-tang (SGT) has been used for treatment of chronic diseases in the folk medicine recipe. Since nitric oxide (NO) is one of the major inflammatory parameters, we first studied the effects of SGT on NO production in lipopolysaccharide (LPS)-stimulated BV-2 microglia. SGT inhibited the secretion of NO in BV-2 microglia, without affecting cell viability. The protein level of inducible nitric oxide synthase (iNOS) was decreased by SGT and SGT also inhibited production of PGE(2) and expression of Cox-2. Proinflammatory cytokines, such as TNF-alpha, IL-1beta and IL-12, were inhibited by SGT in a dose-dependent manner and SGT blocked the activation of NF-kappaB, which was considered to be a potential transcription factor for the expression of iNOS, COX-2 and proinflammatory cytokines. SGT also blocked the degradation of IkappaB and activation (decrease of cytosolic p65) of NF-kappaB, p65. These results suggest that SGT could exert its anti-inflammatory actions by suppressing the synthesis of NO through inhibition of NF-B activity.

Comparison of RAPD, AFLP, and EF-1α Sequences for the Phylogenetic Analysis of Fusarium oxysporum and Its formae speciales in Korea.

Although Fursarium oxysporum causes diseases in economically important plant hosts, identification of F. oxysporum formae speciales has been difficult due to confusing phenotypic classification systems. To resolve these complexity, we evaluated genetic relationship of nine formae speciales of F. oxysporum with random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), and translation elongation factor-1 alpha (EF-1α) gene. In addition, the correlation between mycotoxin content of fusaric acid and isolates based on molecular marker data was evaluated using the modified Mantel's test. According to these result, these fusaric acid-producing strains could not identify clearly, and independent of geographic locations and host specificities. However, in the identification of F. oxysporum formae speciales, especially, AFLP analysis showed a higher discriminatory power than that of a the RAPD and EF-1α analyses, all three techniques were able to detect genetic variability among F. oxysporum formae speciales in this study.

Repression by oxidative stress of iNOS and cytokine gene induction in macrophages results from AP-1 and NF-kappaB inhibition mediated by B cell translocation gene-1 activation.

Previously, we reported that oxidative stress caused by sulfur amino acid deficiency (SD) induces B cell translocation gene-1 (Btg-1), which belongs to the Apro family, in hepatocytes. In view of the impairment of immune function by protein restriction that causes SD, this study investigated whether SD or other oxidative stress inhibits iNOS and cytokine expression and induces Btg-1 in macrophages and explored the causal relationship of Btg-1 induction and repression of the genes. When macrophages were incubated in sulfur amino acid-deprived medium, lipopolysaccharide induction of iNOS, TNFalpha, IL-1beta, and IL-6 was significantly decreased compared to control. Because AP-1 and NF-kappaB are the common transcription factors that regulate the genes encoding iNOS and cytokines, we examined AP-1 and NF-kappaB DNA binding activities and transactivation of the iNOS gene containing the DNA binding elements. Induction of the reporter gene pGL-miNOS-1588 comprising the -1.6 kb iNOS promoter in lipopolysaccharide-activated macrophages was inhibited 30-70% by SD or treatment with pro-oxidants, including tert-butylhydroxyquinone, buthionine sulfoximine, and 3-morpholinosydnonimine. Oxidative stress increased Btg-1 mRNA. SD-induced oxidative stress activated Btg-1 in macrophages, as evidenced by nuclear translocation of endogenous or green fluorescent protein-tagged Btg-1, which localized in the cytoplasm in the resting state. Expression of Btg-1 inhibited lipopolysaccharide-inducible AP-1 and NF-kappaB activities, repressing transactivation of the target gene pGL-miNOS-1588. These results provide evidence that oxidative stress induced by SD or pro-oxidants inhibits the expression of iNOS and cytokines in macrophages with Btg-1 activation and that the gene repression by oxidative stress may result from Btg-1-mediated inhibition of AP-1 and NF-kappaB activities.