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Impact of Helicobacter pylori (HP)-infection on heat shock protein response in gastric mucosa
April 1998
significantly larger (by about 30%) than that in animals inoculated with type II Hp strain. This marked prolongation of ulcer healing in Hp-inoculated mice was associated with increased apoptosis rate reaching in group A: 2.7 - 0.24, B: 2.5 -+ 0.2 and C: 1.6 -+ 0.18, while PCNA(%) was significantly reduced in group A: 34.80%+ 1.95% and B: group as compared to group C: 39.3 -+ 1.75). In type I unlike in type 1I Hp inoculated mice, there was a rise in expression of both apoptosis related proteins bax and bcl-2 (bax/bcl-2 ratio > 1). Conclusion: Infection with cagA+ Hp strains delays ulcer healing via decrease in cell proliferation at the ulcer margin and increased cell loss due to apoptosis and increased expression of apoptosis related proteins. • G0765 GASTRIC MUCOSA REPEATEDLY EXPOSED TO HELICOBACTER PYLORI (ItP) LIPOPOLYSACCHARIDE (LPS) SHOWS INCREASED TOLERANCE TO INJURY VIA INOS, COX-2 AND HSP 70 MECHANISMS. PC. Konturek*, T. Brzozowski #, SJ Konturek#, A. Taut*, P. Pierzchalski #, E.G. Hahn*.Dept. of Med. I, Univ. Erlangen, Germany*; Inst. Physiol. Univ. Sch. Med., Krakow, Poland #. Background: Hp-LPS is known to impair intestinal integrity but its influence on gastric mucosa has been little studied.Bacterial LPS stimulate the local production of nitric oxide (NO) and prostaglandins (PG), at least partly, through the induction of inducible form of cyclooxygenase (COX-2) and nitric oxide synthase (iNOS). Heat shock proteins (HSP) play a pivotal role in protecting mucosal cells from different types of injury including the Hp infection. Aim: This study was undertaken: 1) to determine the effects of repeatedly given Hp-LPS on the gastric damage induced by 100% ethanol or water immersion and restraint stress (WRS); 2) to assess the expression of iNOS, COX-2 and HSP 70 after Hp-LPS treatment and 3) to examine the role of gastric mucosal blood flow (GBF) in the LPS-induced gastroprotection. Methods: Rats received either lmg/kg of Hp-LPS (i.p.) or vehicle (saline) once daily for 5 days. After 5 days of repeated Hp-LPS administration, rats were challenged with strong irritants such as 100% ethanol or with WRS for 3,5h. The mRNA and protein expression of iNOS, COX-2 and HSP-70 in gastric mucosa were assessed by reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blot, respectively. Results: After 5 days of treatment with Hp-LPS no macroscopic lesions in the gastric mucosa were visible, but the subsequent challenge with 100% ethanol or WRS resulted in significant reduction in lesion area as compared with respective values obtained in vehicle-treated rats. This reduction in gastric lesions after Hp-LPS treatment was associated by a significant enhancement in GBF accompanied by a significant increase in protein and mRNA expression for iNOS, COX-2 and HSP 70. Conclusion: 1) Repeated administration of Hp-LPS enhances gastric mucosal defense against noxious factors (100% ethanol, stress) 2) this enhanced mucosal defense may be mediated, at least in part, by increased expression of iNOS, COX-2 and HSP 70.
• G0766 IMPACT OF HELICOBACTER PYLORI (HP)-INFECTION ON HEAT SHOCK PROTEIN RESPONSE IN GASTRIC MUCOSA. PCh Konturek*, H. Fischer #, EG Hahn*, W. Domschke #, J. Neumann##, W. Schmitz##, JW Konturke #. *Dept. Med. I, Univ. Erlangen-Nuremberg and #Dept. Med. B and ##Inst. of Pharmacol. Univ.Muenster, Germany. Background: Heat shock proteins (Hsp) play an important role in protecting mucosal cells from different types of injury. Although Hp is considered as a major cause of gastritis, there is little information about the heat shock protein response to Hp infection. The aim of the present study was to determine the mRNA and protein expression for Hsp 70, one of the member of Hsp-family, in Hp-positive patients before and after eradication of Hp as well as in mice infected with cagA and vacA positive Hp-strains. Material and Methods: In 15 patients with Hp-infection and chronic type B gastritis and in 8 normal controls with normal mucosa (Hp-), the expression of mRNA and protein for Hsp 70 was analyzed before and 4 weeks after eradication therapy (omeprazole 20 mg bid + clarithromycin 500 mg bid + metronidazole 500 mg bid for 7 days) using reverse transcriptase polymerase chain reaction (RT-PCR) and Western-blot, respectively. The activity and severity of gastritis in Hp-infected patients were assessed according to Sydney classification. Additionally, the mRNA expression of Hsp 70 was determined by RT-PCR in the gastric mucosa of mice inoculated three times (2x109 CFU) with Hp expressing cagA and vacA cytotoxins. The animals were sacrificed 14 days after first inoculation with Hp. The total RNA was isolated from gastric mucosa and then reverse transcribed to eDNA using reversetranscription kit (Stratagene). RT-generated eDNA was amplified by PCR using specific primers for Hsp70 and GAPDH as internal standard. PCR products were separated on 1,5% agarnse gel and their intensity was determined using video image analysis (Kodak Science, US). The Hsp70 signal was normalized to the GAPDH signal from the same RNA and expressed as Hsp70/GAPDH ratio. Results: The eradication of Hp resulted in a significant improvement of activity and severity of gastritis as well as in a significant increase in mRNA and protein expression (8.7 -+ 1.2x10'* phosphor imager units before eradication vs. 12 -+ 1.5x104 phosphor imager units after eradication) for Hsp70. The gastric infection of mice with Hp assessed by histology and the culture of gastric homogenates resulted in a significant decrease in expression of mRNA for Hsp 70 as compared to vehicle (saline) treated controls (HSP70/GAPDH ratio: 0.97 + 0.05 in infected mice vs. 1.62 -+ 0.07 in the control group). Conclusion: 1) Hp infection is associated
Esophageal, Gastric, and Duodenal Disorders A187
with a reduced protein expression and synthesis of Hsp 70 in gastric mucosal cells that in humans was partly reversed by Hp eradication. 2) The alteration in Hsp 70 expression in gastric mucosa infected by Hp may contribute to decreased cell resistance to the injury caused by Hp. G0767 PANCREATIC SECRETORY TRYPSIN INHIBITOR (PSTI) IN HEALING OF STRESS-INDUCED GASTRIC LESIONS IN RATS. PC. Konturek*, T. Brzozowski #, SJ. Konturek #, A. Taut*, G. Elia÷, S.Yagi**, E.G. Hahn*. Dept. Med. I, Univ. Erlangen, Germany*; Inst. Physiol. Univ. Sch. Med. Krakow, Poland#; ICRF, London+; Shionogi &Co, Japan**.
Background: PSTI is a serine protease inhibitor that prevents excessive digestion of the gastrointestinal mucus, but its role in the mechanism of mucosal defense has been little studied. The aim of this study was to determine the effect of exogenous PSTI on gastric H÷ secretion and healing of gastric lesions induced by stress and to examine the immunohistochemical and gene expression of PSTI in gastric mucosa during healing of these lesions. Material and Methods: Stress ulcerations were induced by exposure of rats to a 3.5 h of water immersion and restraint stress (WRS) with or without pretreatment with vehicle or PSTI (0.1 mg/kg s.c.). Rats were then sacrificed immediately (0 h) and at 6h or 12h after the termination of stress. The gastric blood flow (GBF) was measured by H2-gas clearance method at each time period and the gastric mucosal samples were taken for PSTI immunohistochemistry and expression of mRNA for PSTI using reverse transcriptase polymerase chain reaction (RT-PCR) and Southern hybrydization. Results: Stress produced numerous gastric lesions and decreased GBF by about 30% as compared to that in vehicle-treated mucosa in non-stressed rats. PSTI given s.c. in graded doses (0.01-1 mg/kg) inhibited dose dependently gastric H+ and pepsin outputs in conscious rats with gastric fistula and accelerated significantly healing of WRS-induced gastric lesions. The healing effect of PSTI (0.1 mg/kg s.c.) recorded at 6h and 12h after WRS was accompanied by a significant rise in the GBF. The expression of PSTI mRNA in the intact mucosa was weak, but following exposure to stress it increased significantly to reach maximum at 6 h after the termination of WRS. Conclusions: 1) exogenous PSTI accelerates healing of stress-induced gastric lesions, at least in part, due to its antisecretory activity and enhancement of gastric microcirculation. 2) mucosal healing results in the induction of gene expression for PSTI that might play an important role in the mechanism of mucosal recovery from lesions induced by stress. Supported in part by grant from IZKF-Erlangen G0768 HELICOBACTER PYLORI (HP) INFECTION DELAYS HEALING OF ISCHEMIA-REPERFUSION (I/R)-INDUCED GASTRIC ULCERATIONS: A NEW MODEL FOR STUDYING PATHOGENESIS OF HPINFECTION IN THE RAT STOMACH. S.J. Konturek, T. Brzozowski, S. Kwieciefi, R. Pajdo, Z. ~liwowski, P.C. Konturek, E.G. Hahn. Dept. Physiol. Univ. Med. Sch. Cracow, Poland, Dept. Med. I. Univ. Erlangen-Nuremberg, Erlangen, Germany. Background: Hp infection is considered as a major risk factor in of gastric ulcerations but studies on Hp pathogenecity are limited due to lack of adequate animal model. In this study we developed the new model with gastric Hp infection in rat gastric mucosa with acute gastric erosions induced by I/R that progressed to chronic ulcers. Methods: I/R lesions were produced in rats by clamping the celiac artery for 0.5 h followed by 1 h of reperfusion and gastric inoculation with type I Hp expressing cagA cytotoxin (cultured from flesh clinical isolates) (2x109 CFU, i.g.) was applied immediately after 1 h of reperfusion (R). In separate group of rats, equipped with gastric fistula the effect of Hp without of with I/R on gastric secretion was determined. Rats were sacrificed at 0, 3, 12 h and then after 1, 3, 7, 9, 12 and 14 days after I/R without or with Hp inoculation. Gastric lesion area was determined planimetrically and gastric blood flow (GBF) was measured by H2-gas clearance technique. The blood was withdrawn for measurement of plasma IL-113 by ELISA and gastrin by RIA and gastric biopsy were taken for histology, rapid urease test and Hp-culture. Results: Gastric I alone by celiac artery clamping resulted in almost complete suppression of GBF and gastric H+ secretion but no gastric lesions were observed. When I was followed by 1 h of R the area of erosions induced by I/R averaged 34 -+ 6 mm2 and this was further significantly increased at 3 h to reach maximum at 12 h after I/R. The erosions progressed to ulcers after 3-7 days but then after 7 days the area of these ulcers declined progressively. Viable Hp were found to gradually increase from 1 to 14 days and as confirmed by histology, Hp staining, CLOtest and culture. Hp inoculation significantly delayed healing of I/R ulcers at each time interval tested and this effect was accompanied by decreased GBF and increased plasma IL-113 and gastrin levels. Gastric H+ that was completely inhibited up to 12 h after I/R but returned to control value after 2 days upon I/R, this effect being significantly decreased in Hp-infected rats and accompanied by higher increment in plasma gastrin and IL-l~3. Conclusions: 1) Gastric infection with Hp expressing cagA cytotoxin delays healing of I/R gastric injury due to impairment of gastric microcirculation and excessive cytokine release; 2) the suppression of gastric H- secretion by Hp may contribute to hypergastrinemia observed during progression of gastric erosions into ulcers induced by I/R and 3) the I/R induced gastric ulcers may be a useful model of studying the role of Hp infection on gastric ulcerogenesis.
significantly larger (by about 30%) than that in animals inoculated with type II Hp strain. This marked prolongation of ulcer healing in Hp-inoculated mice was associated with increased apoptosis rate reaching in group A: 2.7 - 0.24, B: 2.5 -+ 0.2 and C: 1.6 -+ 0.18, while PCNA(%) was significantly reduced in group A: 34.80%+ 1.95% and B: group as compared to group C: 39.3 -+ 1.75). In type I unlike in type 1I Hp inoculated mice, there was a rise in expression of both apoptosis related proteins bax and bcl-2 (bax/bcl-2 ratio > 1). Conclusion: Infection with cagA+ Hp strains delays ulcer healing via decrease in cell proliferation at the ulcer margin and increased cell loss due to apoptosis and increased expression of apoptosis related proteins. • G0765 GASTRIC MUCOSA REPEATEDLY EXPOSED TO HELICOBACTER PYLORI (ItP) LIPOPOLYSACCHARIDE (LPS) SHOWS INCREASED TOLERANCE TO INJURY VIA INOS, COX-2 AND HSP 70 MECHANISMS. PC. Konturek*, T. Brzozowski #, SJ Konturek#, A. Taut*, P. Pierzchalski #, E.G. Hahn*.Dept. of Med. I, Univ. Erlangen, Germany*; Inst. Physiol. Univ. Sch. Med., Krakow, Poland #. Background: Hp-LPS is known to impair intestinal integrity but its influence on gastric mucosa has been little studied.Bacterial LPS stimulate the local production of nitric oxide (NO) and prostaglandins (PG), at least partly, through the induction of inducible form of cyclooxygenase (COX-2) and nitric oxide synthase (iNOS). Heat shock proteins (HSP) play a pivotal role in protecting mucosal cells from different types of injury including the Hp infection. Aim: This study was undertaken: 1) to determine the effects of repeatedly given Hp-LPS on the gastric damage induced by 100% ethanol or water immersion and restraint stress (WRS); 2) to assess the expression of iNOS, COX-2 and HSP 70 after Hp-LPS treatment and 3) to examine the role of gastric mucosal blood flow (GBF) in the LPS-induced gastroprotection. Methods: Rats received either lmg/kg of Hp-LPS (i.p.) or vehicle (saline) once daily for 5 days. After 5 days of repeated Hp-LPS administration, rats were challenged with strong irritants such as 100% ethanol or with WRS for 3,5h. The mRNA and protein expression of iNOS, COX-2 and HSP-70 in gastric mucosa were assessed by reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blot, respectively. Results: After 5 days of treatment with Hp-LPS no macroscopic lesions in the gastric mucosa were visible, but the subsequent challenge with 100% ethanol or WRS resulted in significant reduction in lesion area as compared with respective values obtained in vehicle-treated rats. This reduction in gastric lesions after Hp-LPS treatment was associated by a significant enhancement in GBF accompanied by a significant increase in protein and mRNA expression for iNOS, COX-2 and HSP 70. Conclusion: 1) Repeated administration of Hp-LPS enhances gastric mucosal defense against noxious factors (100% ethanol, stress) 2) this enhanced mucosal defense may be mediated, at least in part, by increased expression of iNOS, COX-2 and HSP 70.
• G0766 IMPACT OF HELICOBACTER PYLORI (HP)-INFECTION ON HEAT SHOCK PROTEIN RESPONSE IN GASTRIC MUCOSA. PCh Konturek*, H. Fischer #, EG Hahn*, W. Domschke #, J. Neumann##, W. Schmitz##, JW Konturke #. *Dept. Med. I, Univ. Erlangen-Nuremberg and #Dept. Med. B and ##Inst. of Pharmacol. Univ.Muenster, Germany. Background: Heat shock proteins (Hsp) play an important role in protecting mucosal cells from different types of injury. Although Hp is considered as a major cause of gastritis, there is little information about the heat shock protein response to Hp infection. The aim of the present study was to determine the mRNA and protein expression for Hsp 70, one of the member of Hsp-family, in Hp-positive patients before and after eradication of Hp as well as in mice infected with cagA and vacA positive Hp-strains. Material and Methods: In 15 patients with Hp-infection and chronic type B gastritis and in 8 normal controls with normal mucosa (Hp-), the expression of mRNA and protein for Hsp 70 was analyzed before and 4 weeks after eradication therapy (omeprazole 20 mg bid + clarithromycin 500 mg bid + metronidazole 500 mg bid for 7 days) using reverse transcriptase polymerase chain reaction (RT-PCR) and Western-blot, respectively. The activity and severity of gastritis in Hp-infected patients were assessed according to Sydney classification. Additionally, the mRNA expression of Hsp 70 was determined by RT-PCR in the gastric mucosa of mice inoculated three times (2x109 CFU) with Hp expressing cagA and vacA cytotoxins. The animals were sacrificed 14 days after first inoculation with Hp. The total RNA was isolated from gastric mucosa and then reverse transcribed to eDNA using reversetranscription kit (Stratagene). RT-generated eDNA was amplified by PCR using specific primers for Hsp70 and GAPDH as internal standard. PCR products were separated on 1,5% agarnse gel and their intensity was determined using video image analysis (Kodak Science, US). The Hsp70 signal was normalized to the GAPDH signal from the same RNA and expressed as Hsp70/GAPDH ratio. Results: The eradication of Hp resulted in a significant improvement of activity and severity of gastritis as well as in a significant increase in mRNA and protein expression (8.7 -+ 1.2x10'* phosphor imager units before eradication vs. 12 -+ 1.5x104 phosphor imager units after eradication) for Hsp70. The gastric infection of mice with Hp assessed by histology and the culture of gastric homogenates resulted in a significant decrease in expression of mRNA for Hsp 70 as compared to vehicle (saline) treated controls (HSP70/GAPDH ratio: 0.97 + 0.05 in infected mice vs. 1.62 -+ 0.07 in the control group). Conclusion: 1) Hp infection is associated
Esophageal, Gastric, and Duodenal Disorders A187
with a reduced protein expression and synthesis of Hsp 70 in gastric mucosal cells that in humans was partly reversed by Hp eradication. 2) The alteration in Hsp 70 expression in gastric mucosa infected by Hp may contribute to decreased cell resistance to the injury caused by Hp. G0767 PANCREATIC SECRETORY TRYPSIN INHIBITOR (PSTI) IN HEALING OF STRESS-INDUCED GASTRIC LESIONS IN RATS. PC. Konturek*, T. Brzozowski #, SJ. Konturek #, A. Taut*, G. Elia÷, S.Yagi**, E.G. Hahn*. Dept. Med. I, Univ. Erlangen, Germany*; Inst. Physiol. Univ. Sch. Med. Krakow, Poland#; ICRF, London+; Shionogi &Co, Japan**.
Background: PSTI is a serine protease inhibitor that prevents excessive digestion of the gastrointestinal mucus, but its role in the mechanism of mucosal defense has been little studied. The aim of this study was to determine the effect of exogenous PSTI on gastric H÷ secretion and healing of gastric lesions induced by stress and to examine the immunohistochemical and gene expression of PSTI in gastric mucosa during healing of these lesions. Material and Methods: Stress ulcerations were induced by exposure of rats to a 3.5 h of water immersion and restraint stress (WRS) with or without pretreatment with vehicle or PSTI (0.1 mg/kg s.c.). Rats were then sacrificed immediately (0 h) and at 6h or 12h after the termination of stress. The gastric blood flow (GBF) was measured by H2-gas clearance method at each time period and the gastric mucosal samples were taken for PSTI immunohistochemistry and expression of mRNA for PSTI using reverse transcriptase polymerase chain reaction (RT-PCR) and Southern hybrydization. Results: Stress produced numerous gastric lesions and decreased GBF by about 30% as compared to that in vehicle-treated mucosa in non-stressed rats. PSTI given s.c. in graded doses (0.01-1 mg/kg) inhibited dose dependently gastric H+ and pepsin outputs in conscious rats with gastric fistula and accelerated significantly healing of WRS-induced gastric lesions. The healing effect of PSTI (0.1 mg/kg s.c.) recorded at 6h and 12h after WRS was accompanied by a significant rise in the GBF. The expression of PSTI mRNA in the intact mucosa was weak, but following exposure to stress it increased significantly to reach maximum at 6 h after the termination of WRS. Conclusions: 1) exogenous PSTI accelerates healing of stress-induced gastric lesions, at least in part, due to its antisecretory activity and enhancement of gastric microcirculation. 2) mucosal healing results in the induction of gene expression for PSTI that might play an important role in the mechanism of mucosal recovery from lesions induced by stress. Supported in part by grant from IZKF-Erlangen G0768 HELICOBACTER PYLORI (HP) INFECTION DELAYS HEALING OF ISCHEMIA-REPERFUSION (I/R)-INDUCED GASTRIC ULCERATIONS: A NEW MODEL FOR STUDYING PATHOGENESIS OF HPINFECTION IN THE RAT STOMACH. S.J. Konturek, T. Brzozowski, S. Kwieciefi, R. Pajdo, Z. ~liwowski, P.C. Konturek, E.G. Hahn. Dept. Physiol. Univ. Med. Sch. Cracow, Poland, Dept. Med. I. Univ. Erlangen-Nuremberg, Erlangen, Germany. Background: Hp infection is considered as a major risk factor in of gastric ulcerations but studies on Hp pathogenecity are limited due to lack of adequate animal model. In this study we developed the new model with gastric Hp infection in rat gastric mucosa with acute gastric erosions induced by I/R that progressed to chronic ulcers. Methods: I/R lesions were produced in rats by clamping the celiac artery for 0.5 h followed by 1 h of reperfusion and gastric inoculation with type I Hp expressing cagA cytotoxin (cultured from flesh clinical isolates) (2x109 CFU, i.g.) was applied immediately after 1 h of reperfusion (R). In separate group of rats, equipped with gastric fistula the effect of Hp without of with I/R on gastric secretion was determined. Rats were sacrificed at 0, 3, 12 h and then after 1, 3, 7, 9, 12 and 14 days after I/R without or with Hp inoculation. Gastric lesion area was determined planimetrically and gastric blood flow (GBF) was measured by H2-gas clearance technique. The blood was withdrawn for measurement of plasma IL-113 by ELISA and gastrin by RIA and gastric biopsy were taken for histology, rapid urease test and Hp-culture. Results: Gastric I alone by celiac artery clamping resulted in almost complete suppression of GBF and gastric H+ secretion but no gastric lesions were observed. When I was followed by 1 h of R the area of erosions induced by I/R averaged 34 -+ 6 mm2 and this was further significantly increased at 3 h to reach maximum at 12 h after I/R. The erosions progressed to ulcers after 3-7 days but then after 7 days the area of these ulcers declined progressively. Viable Hp were found to gradually increase from 1 to 14 days and as confirmed by histology, Hp staining, CLOtest and culture. Hp inoculation significantly delayed healing of I/R ulcers at each time interval tested and this effect was accompanied by decreased GBF and increased plasma IL-113 and gastrin levels. Gastric H+ that was completely inhibited up to 12 h after I/R but returned to control value after 2 days upon I/R, this effect being significantly decreased in Hp-infected rats and accompanied by higher increment in plasma gastrin and IL-l~3. Conclusions: 1) Gastric infection with Hp expressing cagA cytotoxin delays healing of I/R gastric injury due to impairment of gastric microcirculation and excessive cytokine release; 2) the suppression of gastric H- secretion by Hp may contribute to hypergastrinemia observed during progression of gastric erosions into ulcers induced by I/R and 3) the I/R induced gastric ulcers may be a useful model of studying the role of Hp infection on gastric ulcerogenesis.