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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
Department of Physiology, Jagiellonian University School of Medicine, Cracow, Poland (T.B., R.P., S.K., S.J.K., Z.S., D.D., W.W.P.); Department of Medicine I, University of Erlangen-Nuremberg, Erlangen, Germany (P.C.K., E.G.H.); and Department of Microbiology, National University of Ireland, Galway, Ireland (A.P.M.)
Received January 8, 2004; accepted March 15, 2004.
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Abstract |
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, and tumor necrosis factor-
levels were examined. Single (once) or repeated (five times) i.p. injections of Hp-LPS (1 mg/kg) or intestinal LPSs failed to produce macroscopic gastric damage and did not affect the GBF when compared with vehicle. Hp-LPS injected repeatedly suppressed the gastric acid secretion, up-regulated leptin mRNA and protein, and increased plasma leptin and gastrin levels. Hp-LPS significantly reduced the ASA-induced gastric damage and the accompanying decline in the GBF, and these effects were significantly attenuated by capsaicin denervation and selective antagonism of cholecystokinin-B (CCK2) receptors by RPR-102681 [N-(metoxy-3 phenyl) N-(N-methyl N-phenyl-carbamylmethyl) carbamoylmethyl]-3 ureido}-3 phenyl}-2 propronique] but not by loxiglumide, an antagonist of CCK1 receptors. We conclude that 1) daily application of Hp-LPS enhances gastric mucosal resistance against ASA damage due to the increase of GBF and the expression and release of leptin and gastrin exerting trophic and gastroprotective effects, and 2) this enhanced resistance to ASA damage in Hp-LPS-adapted stomach is mediated by the sensory afferents and specific CCK2/gastrin receptors.
; Konturek et al., 1999), but it remains unknown whether the gastric mucosa is capable of adapting to repeated Hp insults and whether such Hp adaptation might alter the mucosal resistance to the injurious action of strong irritants.
Various pathogenic factors originating from Hp have been implicated in the damaging effect of this bacterium on the gastric mucosa, the most important in addition to ammonia being cytotoxins released by Hp strains expressing the vacuolating cytotoxin A and cytotoxin-associated gene A proteins, Hp-derived lipopolysaccharides (Hp-LPSs), and the enhanced generation of reactive oxygen species (Megraud et al., 1992; Crabtree, 1996; Figura and Tabaqchali, 1996; Moran, 2001a,b). Hp-LPS exhibits a low immunological activity, and this property has been assumed to play an important role in the persistency of Hp infection in the human stomach (Moran, 2001a,b). Nevertheless, the deleterious action of LPS derived from Hp in the stomach includes an interaction of this endotoxin with laminin (Valkonen et al., 1994), its influence on the gastric mucus formation and composition (Slomiany et al., 1992), and expression of proinflammatory cytokines (Crabtree et al., 1994; Moran, 2001a). Recent evidence suggests, however, that LPS derived from Escherichia coli applied parenterally also induces gastroprotective activity against lesions induced by strong topical irritants such as ethanol (Tepperman and Soper, 1994; Konturek et al., 1998a,b; Ng et al., 2002) and results in mucosal adaptation to topical irritants after prolonged administration (Ferraz et al., 1997).
Leptin is accepted as a protein product of the ob gene acting directly and through the sensory afferent on central leptin receptors (Ob-R) in the hypothalamus that controls food intake and energy expenditure (Friedman and Halaas, 1998). Recent studies documented the presence of leptin in the plasma of experimental animals, such as mice and rats, as well as in humans (Shalev et al., 1997; Barbier et al., 1998). Leptin is believed to be secreted mainly by adipocytes and the placenta, but recent studies have revealed that leptin messenger RNA (mRNA) and leptin protein can also be detected in the rat gastric oxyntic mucosa, suggesting that the gastric corpus may be another important source of leptin (Bado et al., 1998; Brzozowski et al., 1999).
The importance of leptin in the action of bacterial LPS has been supported by evidence that reduced levels of leptin during starvation increased animal susceptibility to endotoxic shock (Faggioni et al., 2000). Since parenteral LPS was shown to attenuate ethanol-induced gastric damage (Tepperman and Soper, 1994; Konturek et al., 1998a; Brzozowski et al., 2003), the question remains whether leptin, which exhibits gastroprotective activity in the stomach (Brzozowski et al., 1999), can also contribute to the LPS-induced protection against mucosal damage induced by aspirin (ASA). Finally, the physiological significance of gut hormones such as leptin and gastrin in the adaptation of gastric mucosa, developed by daily injections of endotoxins such as Hp-LPS, requires elucidation.
This study was designed to determine the effect of single or repeated parenteral applications of Hp-LPS on acute gastric lesions induced by intragastric (i.g.) administration of acidified ASA and accompanying changes in the gastric blood flow (GBF), gastric secretion, and the gene expression and release of leptin. An attempt was made to compare the effects of five daily injections with Hp-LPS with those exhibited by different LPSs isolated from enteric bacteria such as B. fragilis, Y. enterocolitica, and C. jejuni on gastric acid secretion and ASA-induced gastric damage. Furthermore, we attempted to compare the effects of Hp-LPS with those of exogenous leptin, CCK, and peptone meal, a potent releaser of both CCK and leptin, and to examine the involvement of specific CCK1 (for CCK) and CCK2 (for gastrin) receptors, sensory nerve activity, proinflammatory cytokines such as interleukin (IL)-1 and tumor necrosis factor (TNF)- in gastric mucosal integrity, and possible gastric mucosal adaptation afforded by Hp-LPS.
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Materials and Methods |
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). Series C was designed to determine the effect of single and repeated parenteral injections of LPS derived from the intestinal bacteria B. fragilis, Y. enterocolitica, and C. jejuni. B. fragilis NCTC 9343 was obtained from the National Collection of Type Cultures (London, UK), C. jejuni ATCC 43431 was purchased from the American Type Culture Collection (Manassas, Virginia), and Y. enterocolitica IY-9 was a clinical isolate originating from a human with diarrheal disease.
Induction of Gastric Adaptation to Hp-LPS. Gastric adaptation was achieved by daily parenteral administration of Hp-LPS or vehicle (saline) to normally fed rats for the entire period of the study. The parenteral route of LPS administration was chosen based on our previous observations (Konturek et al., 1998a; Brzozowski et al., 2003) that gastric mucosa directly exposed to LPS applied in a dose of 1 mg/kg (i.g.) failed to adapt to this endotoxin and that such LPS applied i.g. also failed to influence the mucosal lesions induced by strong irritants (e.g., ethanol, ASA, and stress). Since LPS produced by the bacteria contaminating the gastrointestinal lumen and adherent to mucosal cells under the mucus layer covering the surface epithelium may penetrate the mucosa and reach the general circulation, we decided to employ parenteral injection rather than the intragastric route as the route of bacterial LPS administration to mimic the fate of this systemic LPS. Our preliminary observation with intragastric daily application of Hp-LPS at a dose of 10 mg/kg also exerted gastroprotection against ASA-induced gastric damage, but such an investigation required large doses of Hp-LPS that were not available to us; therefore, only parenteral administration of this LPS was employed in the present study. The animals received Hp-LPS once and LPS isolated from intestinal bacteria (1 mg/kg) by i.p. route for comparison, or they were treated repeatedly by the same route with Hp-LPS and that of other intestinal bacteria for 4 consecutive days as described in detail in our previous studies with ASA-induced gastric adaptation (Konturek et al., 1994; Brzozowski et al., 1995). Rats with single or repeated daily (five times) injections of Hp-LPS were sacrificed, the stomach was quickly removed, opened along the greater curvature, and the gastric mucosa was photographed to subsequently measure the area of gastric lesions by two observers using planimetry (Morphomat; Carl Zeiss, Berlin, Germany). The gastric mucosa of separate overnight-fasted rats treated repeatedly with vehicle, Hp-LPS, or intestinal bacterial LPS was then challenged 120 min after the last dose of vehicle, Hp-LPS, or intestinal bacterial LPS with acidified ASA applied i.g. in a volume of 1.5 ml.
Three hours after ASA application, the animals were lightly anesthetized with ether, their abdomen was opened by the midline incision, and the stomach was exposed for the measurement of GBF by means of the H2 gas clearance technique (Konturek et al., 1994; Konturek et al., 2001a). The measurements were made in three areas of the oxyntic mucosa, and the mean values of the measurements were calculated and expressed as a percentage of changes of those recorded in the vehicle (saline)-treated animals.
The alterations of gastric secretions in rats treated with the vehicle (saline), Hp-LPS, or LPS derived from intestinal bacteria applied once or given repeatedly were tested in a separate group of 60 fasted rats surgically equipped with chronic gastric fistulas as described in our earlier studies (Brzozowski et al., 2000a). The rats that have been treated once with Hp-LPS or injected repeatedly (five times) with Hp-LPS or LPS isolated from intestinal bacteria were placed in individual Bollman cages (to which the animals were well conditioned) at day 4 to prevent coprophagy and to maintain the necessary restraint. In addition, the effect of five daily injections with Hp-LPS with or without loxiglumide, an inhibitor of CCK1 receptors, and RPR-102681, the selective CCK2 receptor antagonist, was determined (Konturek et al., 1995; Brzozowski et al., 2000b). Each fistula was then opened, and the stomach was rinsed gently with 5 to 8 ml of tap water at 37°C. Basal gastric secretion was collected for 120 min, during which time all animals received saline at a rate of 4 ml/h subcutaneously. The gastric juice was collected every 30 min, the volume was measured, and then the acid concentration and output were determined and expressed as the output per 30 min as described previously (Brzozowski et al., 2000a).
Experimental Groups and Treatments. Vehicle or Hp-LPS (dose of 1 mg/kg i.p.) was given once or administered at the same dose for 4 consecutive days. After five daily injections with Hp-LPS or vehicle, the gastric mucosa was challenged with acidified ASA. The protective effect of Hp-LPS applied i.p. 2 h prior to ASA was compared with known gastroprotective agents (Brzozowski et al., 1999), such as those of leptin and CCK administered i.p. (dose of 10 µg/kg) or 8% peptone meal applied i.g. in a volume of 1 ml per rat.
The following groups of rats were used: 1) vehicle (1 ml of saline i.p.) followed 120 min later by acidified ASA (150 mg/kg i.g.); 2) Hp-LPS (1 mg/kg i.p.) followed 120 min later by ASA; 3) B. fragilis-LPS, Y. enterocolitica-LPS, and C. jejuni-LPS (1 mg/kg i.p.) followed 120 min later by ASA; 4) leptin (10 µg/kg i.p.) and CCK-8 (10 µg/kg i.p.) followed 120 min later by ASA; 5) 8% peptone meal (1 ml per rat i.g.) followed 120 min later by ASA; 6) vehicle (saline) or Hp-LPS (1 mg/kg i.p.) administered daily for 4 days with or without the challenge with ASA applied at day 4; and 7) B. fragilis-LPS, Y. enterocolitica-LPS, and C. jejuni-LPS (1 mg/kg i.p.) administered daily for 4 days with or without the challenge with ASA applied at day 4.
Effect of Suppression of CCK1 and CCK2 Receptors and Sensory Nerves on Gastroprotection and Adaptation Induced by Hp-LPS. To check whether gastrin/CCK is involved in the action of bacterial LPS on mucosal integrity, separate subgroups of rats were used, and the effects of inhibition of CCK1 and CCK2 receptors with loxiglumide and RPR-102681, respectively, on the protection and adaptation induced by LPS were examined. RPR-102681 is a novel, nonpeptide selective antagonist of the CCK2/gastrin receptor, which has been shown to display nanomolar affinity of about 2000-fold greater selectivity for CCK2 than CCK1 receptors (Bohme et al., 1997). Loxiglumide was a generous gift of Dr. Rovati (University of Milan, Italy), and RPR-102681 was purchased from Aventis (Strasbourg, France). In subsequent studies, two series of experiments were carried out.
Series I was used to examine the effect of Hp-LPS applied (i.p.) against the mucosal lesions induced by ASA in rats with or without the blockade of CCK1 receptors with loxiglumide (30 mg/kg i.p.) or CCK2 receptors with RPR-102681 (30 mg/kg i.p.) (Brzozowski et al., 2000b). The dose of loxiglumide and RPR-102681 was selected on the basis of our previous studies in rats that showed that loxiglumide attenuated the gastroprotective and secretory effects of CCK by having no influence on gastroprotection and secretory activity of gastrin and leptin, whereas RPR-102681 reversed the leptin-induced gastroprotection without a significant effect on that induced by CCK (Konturek et al., 1995; Brzozowski et al., 2000b).
The involvement of sensory nerves (series II) in gastroprotection and adaptation by Hp-LPS was studied in rats with or without deactivation of afferent nerves with a neurotoxic dose of capsaicin as described previously (Brzozowski et al., 1996). For this purpose, the animals were pretreated with capsaicin (Sigma-Aldrich, St. Louis, MO) injected subcutaneously (s.c.) for 3 consecutive days at respective doses of 25, 50, and 50 mg/kg (total of 125 mg/kg) about 2 weeks before the experiment. All injections of capsaicin were performed under ether anesthesia to counteract the respiratory impairment associated with injection of this agent. Control rats received vehicle injections. All animals pretreated with capsaicin showed a negative wiping movement test, thereby confirming the functional denervation of the capsaicin-sensitive afferent fibers and the loss of corneal reflex.
The following groups of rats, each consisting of 6 to 8 animals, that had been exposed to Hp-LPS applied once or administered five times were used: 1) vehicle (saline) or Hp-LPS (1 mg/kg i.p.) applied once or given five times and followed 2 h later by acidified ASA with or without capsaicin denervation; 2) loxiglumide or RPR-102681 (30 mg/kg i.p.) followed 60 min later by vehicle applied once or given five times and followed 2 h later by acidified ASA; 3) loxiglumide and RPR-102681 (30 mg/kg i.p.) followed 60 min later by Hp-LPS (1 mg/kg i.p.) applied once or given five times and then finally followed 2 h later by acidified ASA. Subsequently, rats were anesthetized, the GBF was measured, and the area of gastric lesions was determined by planimetry in a similar manner to that mentioned above.
Determination of Plasma Leptin Levels by Radioimmunoassay (RIA) and Plasma IL-1 and TNF- by Enzyme-Linked Immunosorbent Assay. Upon the termination of some experiments after treatment with vehicle or Hp-LPS, leptin, CCK-8, and 8% peptone meal administration followed 2 h later by acidified ASA, the rats were anesthetized with ether, and the blood samples (about 3 ml) were taken from the vena cava for the measurement of plasma leptin by RIA (Brzozowski et al., 1999) and determination of plasma IL-1 and TNF- levels by enzyme-linked immunosorbent assay (Pierce Endogen, Rockford, IL) as described previously (Brzozowski et al., 2000a). For comparison, intact rats that had been fasted overnight and only given i.p. vehicle saline were also anesthetized with ether, and blood samples were collected for the determination of control values of leptin, IL-1, and TNF- in plasma. For determination of plasma leptin, blood samples collected in heparin-coated polypropylene tubes were centrifuged at 3000g for 20 min at 4°C, and the clear supernatant plasma was then stored at -80°C until analysis. Plasma leptin was measured by using an RIA kit for rat leptin from Linco Research, Inc. (St. Charles, MO). Briefly, this RIA involved the competition of a rat leptin sample with 125I-rat leptin tracer for binding to a specific rabbit anti-leptin polyclonal antibody. The limit of assay sensitivity was 0.5 ng/ml, the intra-assay variation was less than 7%, and the interassay variation was less than 9%.
Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) for Detection of mRNA of Leptin. Stomachs were removed from rats treated with vehicle (control) and those treated with Hp-LPS with or without i.g. application of ASA for the determination of leptin mRNA expression by RT-PCR with specific primers (Brzozowski et al., 2000b). Gastric mucosa was scraped off from the oxyntic gland area using a slide glass and immediately snap frozen in liquid nitrogen and stored at -80°C until analysis. Total RNA was extracted from mucosal samples by a guanidinium isothiocyanate/phenol chloroform method using a kit from Stratagene (Heidelberg, Germany). Aliquoted RNA samples were stored at -80°C until analysis.
Single-stranded cDNA was generated from 5 µg of total cellular RNA using StrataScript reverse-transcriptase and oligo(dT) primers (Stratagene). The nucleotide sequences of the primers for leptin and -actin were based on published cDNA-encoding leptin and -actin, respectively (Bado et al., 1998; Brzozowski et al., 1999). The sense primer for leptin was CTG CTC AAA GCC ACC ACC TCT G, and the antisense primer was CCT GTG GCT TTG GTC CTA TCT G. The sense primer for -actin was TTG TAA CCA ACT GGG ACG ATA TGG, and the antisense primer was GAT CTT GAT CTT CAT GGT GCT AGG. The primers were synthesized by Invitrogen (Carlsbad, CA).
Polymerase chain reaction products were detected by electrophoresis on a 1.5% agarose gel containing ethidium bromide. The location of predicted products was confirmed by using a DNA 100-base pair ladder (Invitrogen) as a standard size marker.
Protein Extraction and Analysis of Leptin Expression in the Gastric Mucosa by Western Blotting. Shock-frozen tissue from rat stomach was homogenized in lysis buffer (100 mmol Tris-HCl, pH 7.4, 15% glycerol, 2mmol EDTA, 2% SDS, 100 mmol DL-dithiothreitol) by the addition of 1:20 dilution of aprotinin and 1:50 dilution of 100 mmol phenylmethylsulfonyl fluoride. Insoluble material was removed by centrifugation at 12000g for 15 min. Approximately 100 µg of cellular protein extract were loaded into a well, separated electrophoretically through a 13.5% SDS-polyacrylamide gel, and transferred onto Sequi-Blot PVDF membrane (Bio-Rad, Hercules, CA) by electroblotting. Skim fast milk powder (5% w/v) in Tris-buffered saline/Tween 20 buffer (137 mmol NaCl, 20 mmol Tris-HCl, pH7.4, 0.1% Tween 20) was used to block filters for at least 1 h at room temperature. As a primary antibody, 1:500 dilution of specific goat polyclonal antiserum against leptin (Santa Cruz Biochemicals, Santa Cruz, CA) or 1:1000 dilution of rabbit polyclonal anti--actin (Sigma Aldrich) antiserum was added to the membrane, followed by an anti-goat or anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (1:2000; Santa Cruz). Incubation of the primary antibody was followed by three washes with Tris-buffered saline/Tween 20 buffer for 10 min. Incubation of the secondary antibody was followed by four washes for 10 min. Nonisotopic visualization of immunocomplexes was achieved by chemiluminescence using BM chemiluminescence blotting substrate (Boehringer Ingelheim GmbH, Ingelheim, Germany). Thereafter, the developed membrane was exposed to an X-ray film (Kodak, Wiesbaden, Germany).
Statistical Analysis. Results are expressed as means ± S.E.M. Statistical analysis was done using analysis of variance and a two way analysis of variance test with post hoc Tukey's honestly significant difference test. Differences of p < 0.05 were considered significant.
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Results |
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Effect of Hp-LPS and Bacterial LPSs Applied Once or Five Times Daily on ASA-Induced Gastric Lesions with Accompanying Alterations in GBF, Plasma Leptin, and Gastrin Levels. As shown in Fig. 1, the parenteral application of Hp-LPS (1 mg/kg i.p.) produced negligible macroscopic injury in the stomach when applied once or injected daily five times and failed to significantly alter the GBF when compared with that recorded in vehicle-control rats. Similarly, single or repeated injections of LPSs from B. fragilis, Y. enterocolitica, and C. jejuni produced only small gastric mucosal lesions and failed to influence GBF compared with vehicle treatment. In vehicle-pretreated rats, acidified ASA resulted in typical multiple gastric lesions and a significant decline in the GBF by about 30% compared with the respective value recorded in animals pretreated with vehicle alone without ASA (Fig. 1). Five daily injections with each endotoxin significantly reduced ASA-induced gastric damage and the accompanying decline in the GBF compared with those in vehicle-pretreated rats exposed to ASA. Representative gross macroscopic evidence of the ASA-induced gastric damage and the reduction in these lesions in the animal stomach with and without the daily injections of Hp-LPS is presented in Fig. 2A–D. Compared with the intact gastric mucosa, the exposure of gastric mucosa to ASA in the rat treated five times with vehicle resulted in multiple gastric lesions localized mainly in the oxyntic mucosa (Fig. 2, A and B). In contrast, the repeated treatment with Hp-LPS alone produced only a few gross gastric mucosal lesions (Fig. 2C). In rats injected daily five times with Hp-LPS and then subsequently exposed to ASA, there was a significant attenuation of the gastric mucosal injury compared with those treated five times with vehicle and then exposed to ASA (Fig. 2D).
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Hp-LPS applied in a single dose of 1 mg/kg i.p. significantly reduced the ASA-induced gastric lesions, and these protective effects were accompanied by a significant rise in GBF and an elevation of plasma immunoreactive leptin and gastrin levels (Fig. 3). In intact animals without ASA, the plasma gastrin concentration averaged 52 ± 5 pmol/l, and plasma leptin reached a value of 0.65 ± 0.04 ng/ml; these values remained relatively unchanged in animals treated with vehicle (Fig. 3). In rats injected once with Hp-LPS, both plasma leptin and gastrin concentrations showed a several-fold increase, being significantly higher than those in vehicle-treated animals. In rats injected daily with Hp-LPS, the plasma leptin and gastrin concentrations gave a further significant rise compared with that recorded in vehicle-treated animals or those exposed to single treatment with this endotoxin (Fig. 3). The ASA damage was significantly attenuated in the gastric mucosa of rats exposed to single or repeated (five times) injections of Hp-LPS, and these effects were accompanied by a significant elevation of plasma gastrin and leptin increments (Fig. 3).
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The single parenteral application of B. fragilis-LPS, Y. enterocolitica-LPS, and C. jejuni-LPS applied i.p. at a dose of 1 mg/kg also resulted in the attenuation of ASA-induced gastric damage and significantly raised the GBF (Table 2). As shown in Fig. 1, the repeated parenteral application of these LPSs also significantly reduced the lesions induced by acidified ASA. The protective effects against ASA-induced gastric lesions of these endotoxins injected repeatedly were accompanied by a significant rise in GBF compared with the respective values obtained in gastric mucosa exposed to ASA alone (Fig. 1).
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Figure 4 shows the results of parenteral administration of leptin, CCK-8, Hp-LPS, and 8% peptone meal on the mean area of ASA-induced gastric lesions and the accompanying changes in the GBF and plasma leptin levels. Exogenous leptin and CCK-8, both given in a single dose of 10 µg/kg i.p., or i.g. application of 8% peptone meal, which increased the plasma leptin levels by 2- to 3-fold and significantly raised GBF, resulted in a significant attenuation of gastric lesions induced by ASA, with an extent similar to those achieved with single parenteral injection of Hp-LPS.
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Effect of Pretreatment with Loxiglumide and RPR-102681 and Deactivation of Sensory Nerves on the ASA-Induced Gastric Lesions in Rats. As shown in Fig. 5, the i.g. application of acidified ASA produced similar gastric lesions and a similar decline in GBF as those presented in Figs. 1 and 2. The area of these lesions and the accompanying decline in GBF were significantly reduced in rats injected once with Hp-LPS or daily (five times) with this endotoxin. Suppression of CCK1 receptors with loxiglumide by itself failed to significantly influence the ASA-induced gastric damage and the accompanying decline in GBF. Loxiglumide also failed to affect the reduction in the area of ASA-induced gastric damage and the accompanying increase in GBF attained with Hp-LPS applied once or administered repeatedly. Pretreatment with RPR-102681 to suppress specific CCK2 receptors, which by itself also failed to influence the ASA-induced gastric damage, almost completely abolished the decrease in the area of these lesions and the accompanying rise in the GBF evoked by single or repeated treatment with this endotoxin.
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Hp-LPS applied once or injected daily (five times) significantly reduced the area of ASA-induced gastric lesions and significantly raised the GBF compared with those recorded in rats treated with ASA without endotoxin administration (Fig. 6). Capsaicin denervation failed to enhance the area of ASA-induced gastric damage and significantly influence GBF but resulted in almost complete elimination of the protective and hyperemic effects induced by Hp-LPS injected once or daily (five times) (Fig. 6).
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Effect of Single and Repetitive Treatment with Hp-LPS on Plasma IL-1 and TNF- Levels. As shown in Table 3, the plasma levels of both proinflammatory cytokines (IL-1 and TNF-) in the intact animals were negligible, but they were significantly increased in Hp-LPS-treated animals and further dramatically raised in rats exposed to acidified ASA that caused widespread acute gastric mucosal lesions. In rats injected once or daily with Hp-LPS and later exposed to ASA, a significant decrease in plasma IL-1 and TNF- levels was recorded, although the levels in plasma of these cytokines reached significantly higher values than those obtained in intact gastric mucosa.
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Determination of Leptin mRNA and Protein by RT-PCR and Western Blotting in the Gastric Mucosa of Rats Treated Once or Repeatedly with Hp-LPS. The internal control with the -actin mRNA and protein showed intense signals in all the samples tested, indicating a high integrity of RNA that was isolated from the gastric mucosa of vehicle-control rats as well as from those injected once or daily with Hp-LPS (Fig. 7, left and right panels). Expression of leptin mRNA was detectable in intact mucosa not exposed to Hp-LPS and in that treated with vehicle or Hp-LPS injected once or given daily (Fig. 7, left panel). No trace of leptin was recorded in rats serving as the negative control (saline), and this result has been omitted for clarity. In rats injected daily with Hp-LPS, the strong signal for leptin mRNA was greater than that in the vehicle-treated gastric mucosa. A weak signal for leptin protein was detected in the vehicle-treated gastric mucosa (Fig. 7, right panel). In contrast, an increased expression of leptin protein occurred in the gastric mucosa of rats treated with Hp-LPS injected once or daily five times.
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; Bado et al., 1998; Brzozowski et al., 2000b; Konturek et al., 2001a). The protective effects of Hp-LPS were accompanied by a rise in plasma gastrin and were significantly attenuated by pretreatment with RPR-102681, a highly specific inhibitor of mucosal gastrin receptors (CCK2), but not influenced by loxiglumide, an antagonist of receptors for CCK (CCK1), whose plasma level was not affected by Hp-LPS (unpublished observation). These results emphasize the importance of gastrin and CCK2 rather than CCK and CCK1 receptors in the protective and adaptive response of Hp-LPS. Both the protection and adaptation to Hp-LPS in ASA-injured mucosa were accompanied by up-regulation of leptin at the level of both mRNA and protein and subsequent release of leptin, indicating that this hormone indeed contributes to Hp-LPS-induced attenuation of ASA-induced gastric damage. Also, in rats with intact gastric mucosa injected with Hp-LPS, a marked increase in plasma leptin was observed. Thus, this study shows for the first time that an increase in the expression of leptin at the levels of mRNA and protein with subsequent plasma release of this peptide occurs in rats injected once or daily with Hp-LPS and thereby emphasizes that leptin, which has been shown previously to exert a protective effect against gastric mucosal injury by strong irritants (Bado et al., 1998; Brzozowski et al., 1999), might contribute to the enhanced resistance of gastric mucosa of rats treated with Hp-LPS against damage induced by acidified ASA. This notion agrees with the original finding of Tepperman and Soper (1994), who showed protection of the rat gastric mucosa against ethanol lesions after parenteral administration of E. coli-LPS. The present study is also consistent with the evidence of Ferraz et al. (1997) and our own recent observation (Brzozowski et al., 2003) that animals treated repeatedly with E. coli- or Hp-derived LPS developed mucosal tolerance to these endotoxins and that this adaptive response enhanced gastric mucosal resistance to ethanol-induced gastric damage. Our data also agrees with the observations by Sugiyama et al. (2001), who demonstrated that the extent of ethanol-induced damage of gastric mucosa was greatly limited in an experimental model of Hp-infection in the stomach of Mongolian gerbils. These authors concluded that Hp-infection, possibly due to the release of endotoxin and the mild irritant effects of these cytotoxins, exhibits an apparent paradoxical (protective) effect on gastric mucosal integrity by enhancing the resistance of this mucosa to damage induced by necrotizing irritants (Sugiyama et al., 2001). This "protective" action of Hp infection against ethanol lesions has been attributed to the increased generation of prostaglandin E2 derived from cyclooxygenase-2 overexpression in Hp-infected stomach and has been confirmed recently in a model using daily treatment with LPS (Konturek et al., 2001b).
It is known that LPS produces several neuroendocrine effects. Some of these effects are believed to be mediated through cytokines and hormones (for instance, leptin and prolactin), and this process involves the activation of peripheral autonomic nerves such as efferent and afferent vagal nerves (Mastronardi et al., 2001). This prompted our study to determine the role of leptin, neuropeptides released from sensory afferents, and gastric hormones such as gastrin in the mechanism of enhancing the resistance of gastric mucosa to ASA damage as induced by repetitive treatment with Hp-LPS. In a hamster model of Gram-negative bacterial infection, systemic leptin was increased after prolonged administration of LPS, and this was considered to enhance host response to endotoxemia (Grunfeld et al., 1996). Turrin et al. (2001) have shown that LPS applied i.p. activated cytokine production in the brain and at the periphery including in adipose tissue, liver, and spleen. In another study, LPS-induced leptin release was mediated through IL-1 because a soluble IL-1 receptor antagonist completely blocked the LPS-induced increase in the leptin levels (Francis et al., 1999). Thus, we can conclude that Hp-LPS-induced protection and adaptation resulting in the limitation of ASA damage may depend upon leptin expression and release and, as shown in this study, could also be mediated by neuropeptides released from sensory afferent nerves. The latter is supported by our present observation that the capsaicin-induced functional ablation of sensory nerves abolished the protective and hyperemic effects of single and repeated administration of Hp-LPS. It is suggested that endotoxins such as Hp-LPS can affect sensory afferent nerves that in turn may activate the brain-gut axis, resulting in limitation of ASA-induced gastric damage. This conclusion agrees with the observation by Hua et al. (1996) that endotoxin treatment enhanced the release of the vasoactive calcitonin gene-related peptide from the primary sensory afferents due to sensitizing their terminals. The mechanism by which enhancement in the plasma IL-1 and TNF- induced by ASA was reduced in rats treated repeatedly with Hp-derived endotoxin remains to be elucidated, but it could be due to the suppressive action on these cytokines of prostaglandins, nitric oxide, and heat shock proteins released via overexpression of cyclooxygenase-2, inducible nitric-oxide synthase, and heat shock protein 70 mRNA, as reported recently (Brzozowski et al., 2003).
The major finding of the present study is that Hp-LPS is capable of inhibiting gastric acid secretion while showing a significant rise in plasma gastrin level. The importance of gastrin in the observed protection and hyperemia seems to be particularly significant because the antagonism of receptors for gastrin (CCK2) with RPR-102681 completely reversed the inhibition of gastric secretion, gastroprotection and adaptation of gastric mucosa, and the rise in GBF afforded by Hp-LPS, whereas the blockade of CCK1 receptors by loxiglumide that eliminated the action of CCK was ineffective.
It is now becoming evident that nonsteroidal anti-inflammatory drugs such as ASA may influence the pathogenic effects of Hp as a result of possible direct interaction with this microorganism (Wang et al., 2003). However, this was not the case in our study, because the single or repeated parenteral injections with Hp-derived endotoxin actually increased mucosal resistance to the damaging effect of ASA in animals treated repeatedly with this endotoxin. The major drawback of this study is that only parenteral administration of LPS but not its intragastric administration was employed. Although our previous studies documented that LPS administered intragastrically in a relatively small dose (1 mg/kg) failed to influence the lesions provoked by acidified ASA, a large intragastric dose of LPS (10 mg/kg i.g.) was found to be effective in our preliminary observations. This difference requires further explanation, but it could be that small Hp-LPS doses given into the stomach lumen may not be able to penetrate the thick mucus covering of the surface epithelium to activate the mucosa protective mechanism. However, when Hp remains under the mucus layer and is in direct contact with the epithelial cells, it could activate the protective mechanism by local release of its endotoxins, such as LPS. Further studies are needed to determine whether Hp-LPS present in the gastric lumen can reach the gastric circulation sufficiently to mimic the changes observed after repeated parenteral Hp-LPS injections.
Our results with antisecretory effects of Hp-LPS and other non-Hp-related LPSs agree with those of Uehara et al. (1990) and Konturek et al. (2001) that show that peripheral and central applications of LPS derived from E. coli to rats produces profound dose-dependent inhibition of gastric acid output. Since ASA damage depends upon gastric acidity, it is reasonable to assume that suppression of gastric acid secretion by this endotoxin could contribute to the limitation of this damage. As shown in the present study, a marked increase in plasma gastrin in rats treated repeatedly with Hp-LPS, which is known to exert gastroprotective influence on the gastric mucosa, could result from hypochlorhydria and contribute to the greater tolerance of this mucosa to ASA-induced gastric damage. This was confirmed in the present study by blocking the receptors for gastrin (CCK2), which resulted in attenuation of the protective effects of Hp-LPS and the related rise in the plasma gastrin levels.
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ABBREVIATIONS: Hp, Helicobacter pylori; LPS, lipopolysaccharide; ASA, aspirin; i.g., intragastric; GBF, gastric blood flow; CCK, cholecystokinin; IL, interleukin; TNF, tumor necrosis factor; RPR-102681, N-(metoxy-3 phenyl) N-(N-methyl N-phenyl-carbamylmethyl) carbamoylmethyl]-3 ureido}-3 phenyl}-2 propronique; RIA, radioimmunoassay; RT-PCR, reverse-transcriptase polymerase chain reaction.
Address correspondence to: Dr. T. Brzozowski, Department of Physiology, Jagiellonian University School of Medicine, 16 Grzegorzecka Str., 31-531 Cracow, Poland. E-mail: mpbrzozo{at}cyf-kr.edu.pl
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