Volume 119, Issue 6, Pages 1610-1622 (December 2000) Neutrophil chemoattractant 2β regulates expression of the Reg gene in injured gastric mucosa in rats  Hideaki Kazumori, *, Shunji Ishihara, *, Eiichi Hoshino, ‡, Kousaku Kawashima, *, Nobuyuki Moriyama, *, Hiroshi Suetsugu, *, Hiroshi Sato, *, Kyoichi Adachi, *, Ryo Fukuda, *, Makoto Watanabe, *, Shin Takasawa, §, Hiroshi Okamoto, §, Hirokazu Fukui, ∥, Tsutomu Chiba, ∥, Yoshikazu Kinoshita, *  Gastroenterology  Volume 119, Issue 6, Pages 1610-1622 (December 2000) DOI: 10.1053/gast.2000.20262 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Effects of AG-041R or ranitidine on Reg mRNA expression in WRS-induced gastric lesions. Total RNA (20 μg) was extracted from rat glandular stomachs at the indicated times after induction of ulcers and underwent Northern blot analysis with an Reg cDNA probe. The data represent 4 separate experiments. Relative signal intensities of the Reg mRNA level to the β-actin mRNA level were quantified with an image analyzer. Results are expressed as mean ± SEM of 4 rats. *P < 0.05, **P < 0.01, vs. control. aP < 0.05, bP < 0.01, vs. vehicle treated at the indicated times. Gastroenterology 2000 119, 1610-1622DOI: (10.1053/gast.2000.20262) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Time course changes of serum gastrin concentration after WRS. Results are expressed as mean ± SEM of 4 rats. *P < 0.05 vs. control. Gastroenterology 2000 119, 1610-1622DOI: (10.1053/gast.2000.20262) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Dose-dependent effects of intraduodenal administration of AG-041R and ranitidine on unstimulated gastric acid secretion. Results are expressed as mean ± SEM of 7 rats. *P < 0.05, **P < 0.01, vs. control. □, Control; ●, AG-041R; ■, ranitidine. Gastroenterology 2000 119, 1610-1622DOI: (10.1053/gast.2000.20262) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Effects of AG-041R and ranitidine on (A) gastric acid secretion and (B) ulcer indices and serum gastrin concentrations after WRS. Results are expressed as mean ± SEM of (A) 5 rats and (B) 4 rats. *P < 0.05 vs. control. □, Control; ●, AG-041R; ■, ranitidine. Gastroenterology 2000 119, 1610-1622DOI: (10.1053/gast.2000.20262) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Effects of AG-041R and ranitidine on (A) ulcer indices, serum gastrin concentrations, and (B) Reg gene expression in stomachs with indomethacin-induced gastric lesions. (A) Results are expressed as mean ± SEM of 4 rats. *P < 0.05 vs. control. □, Control; ●,AG-041R; ■, ranitidine. (B) The blots represent 4 separate experiments. Gastroenterology 2000 119, 1610-1622DOI: (10.1053/gast.2000.20262) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 Time course changes of Reg mRNA expression in rat stomachs with acetic acid–induced gastric ulcers. Total RNA (20 μg) was extracted from glandular stomachs at the indicated times after the induction of ulcers and underwent Northern blot analysis with an Reg cDNA probe. The blots represent 4 separate experiments. Relative signal intensities of Reg mRNA level to β-actin mRNA level were quantified with an image analyzer. Results are expressed as mean ± SEM of 4 rats. *P < 0.05, **P < 0.01; vs. control. Gastroenterology 2000 119, 1610-1622DOI: (10.1053/gast.2000.20262) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 Effects of AG-041R and ranitidine on Reg mRNA expression in rat stomachs with acetic acid–induced gastric lesions. Total RNA (20 μg) was extracted from the glandular stomachs on the fourth day after the induction of ulcers and underwent Northern blot analysis. The blots represent 4 separate experiments. Gastroenterology 2000 119, 1610-1622DOI: (10.1053/gast.2000.20262) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 8 Time course changes of expression of CINC-2β, IL-1β, TNF-α, and Reg mRNA in WRS-induced gastric lesions. Total RNA (20 μg) was extracted at the indicated times after WRS and underwent Northern blot analysis with Reg, CINC-2β, IL-1β, or TNF-α cDNA probes. (A) The data represent 4 separate experiments. (B) Relative signal intensities of Reg, CINC-2β, IL-1β, and TNF-α mRNA levels to β-actin mRNA levels were quantified with an image analyzer. Results are expressed as mean ± SEM of 4 rats. *P < 0.05, **P < 0.01; vs. control. Gastroenterology 2000 119, 1610-1622DOI: (10.1053/gast.2000.20262) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 9 (A) Time course changes of Reg protein production after the induction of WRS-induced gastric lesions. Protein (50 μg) was extracted from rat glandular stomachs at the indicated times after the induction of ulcers and underwent Western blot analysis. The data represent 4 separate experiments. Relative signal densities of Reg protein production were quantified by laser densitometry. Results are expressed as mean ± SEM of 4 rats. *P < 0.01 vs. control. (B) Time course changes of CINC-2β protein production after the induction of WRS-induced gastric lesions. Results are expressed as mean ± SEM of 4 rats. *P < 0.05 vs. control. Gastroenterology 2000 119, 1610-1622DOI: (10.1053/gast.2000.20262) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 10 Effects of CINC-2β on Reg gene expression in isolated cultured ECL cells. After incubation with various concentrations of CINC-2β for 12 hours, total RNA (10 μg) was extracted and underwent Northern blot analysis with a Reg cDNA probe. The data represent 4 separate experiments. Relative signal intensities of Reg mRNA level to that of β-actin were quantified with an image analyzer. Results are expressed as mean ± SEM of 4 samples. *P < 0.05, **P < 0.01; vs. control. Gastroenterology 2000 119, 1610-1622DOI: (10.1053/gast.2000.20262) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 11 Effect of various proinflammatory cytokines on Reg gene expression in cultured isolated ECL cells. After incubation with 10−9 mol/L of various proinflammatory cytokines for 12 hours, total RNA (10 μg) was extracted and underwent Northern blot analysis with a Reg cDNA probe. The data represent 4 separate experiments. Relative signal intensities of Reg mRNA level to β-actin mRNA level were quantified with an image analyzer. Results are expressed as mean ± SEM of 4 samples. *P < 0.05 vs. control. Gastroenterology 2000 119, 1610-1622DOI: (10.1053/gast.2000.20262) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 12 Effect of various growth factors on Reg gene expression in cultured isolated ECL cells. After incubation with 10−9 mol/L of various growth factors for 12 hours, total RNA (10 μg) was extracted and underwent Northern blot analysis with a Reg cDNA probe. The data represent 4 separate experiments. Relative signal intensities of Reg mRNA level to β-actin mRNA level were quantified with an image analyzer. Results are expressed as mean ± SEM of 4 samples. Gastroenterology 2000 119, 1610-1622DOI: (10.1053/gast.2000.20262) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 13 Effect of CINC-2β on Reg protein production in cultured isolated ECL cells. After incubation with various concentrations of CINC-2β for 12 hours, protein (30 μg) was extracted and underwent Western blot analysis. The data represent 4 separate experiments. Relative signal densities of Reg protein production were quantified by laser densitometry. Results are expressed as mean ± SEM of 4 samples. *P < 0.05, **P < 0.01; vs. control. Gastroenterology 2000 119, 1610-1622DOI: (10.1053/gast.2000.20262) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 14 Effects of anti-CINC-2β antibody on Reg gene expression in stomachs with WRS-induced gastric lesions. Total RNA (20 μg) was extracted and underwent Northern blot analysis with a Reg cDNA probe. The data represent 4 separate experiments. Relative signal intensities of Reg mRNA level to β-actin mRNA level were quantified with an image analyzer. Results are expressed as mean ± SEM of 4 rats. *P < 0.01 vs. rats treated with IgG without WRS, **P < 0.05 vs. rats treated with IgG with WRS. Gastroenterology 2000 119, 1610-1622DOI: (10.1053/gast.2000.20262) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 15 RT-PCR analysis of the expression of CXCR-2. Lane 1, lung; lane 2, total glandular stomach; lane 3, isolated ECL cells, obtained by separate experiments. NC, identical volumes of the PCR reaction mixtures without the addition of cDNA, which served as a negative control, produced no DNA bands. Gastroenterology 2000 119, 1610-1622DOI: (10.1053/gast.2000.20262) Copyright © 2000 American Gastroenterological Association Terms and Conditions