J. JAWOREK1, A. LEJA-SZPAK1, K. NAWROT-PORABKA1, J. BONIOR1, J. SZKLARCZYK1,M. KOT1, S.J. KONTUREK2, R. TOMASZEWSKA3, W.W. PAWLIK2

EFFECT OF NEONATAL ENDOTOXEMIAON THE PANCREAS OF ADULT RATS

1Department of Medical Physiology Faculty of Health Sciences, Jagiellonian University Schoolof Medicine, Krakow; 2Chair of Physiology, Medical Faculty, Jagiellonian University School of Medicine, Krakow, 3Chair of Cell Morphology, Medical Faculty, Jagiellonian University

School of Medicine, Krakow, Poland

Bacterial endotoxin (lipopolysaccharide, LPS), is the component of the cellular wallof Gram negative bacteria. Endotoxemia (sepsis) could produce multiorgan failureand could be particularly danger in the early period of life. The effects ofendotoxemia induced in the neonatal period of life on the pancreatic secretoryfunction and on pancreatic defense of adult organism have not been investigated yet.To induce endotoxemia suckling rats (30g) have been injected intraperitoneally withLPS from E. coli (5, 10 or 15 mg/kg-day) during 5 consecutive days. Three monthslater in these animals (300g) the studies on pancreatic secretion and acutepancreatitis were carried out. In the adult rats, which have been subjected in infancyto endotoxemia, basal pancreatic secretion was unaffected, whereas amylasesecretions stimulated by caerulein or by diversion of pancreatic-biliary juice to theexterior were significantly, and dose-dependently reduced as compared to theuntreated control. In the rats pretreated with LPS in the suckling period of lifecaerulein-induced amylase release from isolated pancreatic acini was significantlydecreased, and dose-dependent reduction of mRNA signal for CCK1 receptor onpancreatic acini have been observed. Caerulein infusion (25 µg/kg) producedcaerulein induced pancreatitis (AP) in all animals tested, that was confirmed byhistological examination. In the rats, which have been subjected in the neonatalperiod of life to LPS (10 or 15 mg/kg-day x 5 days) all manifestations of AP havebeen reduced. In these animals acute inflammatory changes of pancreatic tissue havebeen significantly diminished. Pancreatic weight and plasma lipase activity, havebeen markedly decreased in these animals as compared to the control rats, subjectedin the infancy to saline injection instead of LPS. Caerulein-induced fall in anantioxidative enzyme; SOD concentration was reversed and accompanied bysignificant reduction of lipid peroxidation products; MDA + 4 HNE in the pancreatictissue. Conclusions: 1/ neonatal endotoxemia reduces gene expression for CCK1receptor and could produce impairment of the exocrine pancreatic function at adultage; 2/ Prolonged exposition of suckling rats to bacterial endotoxin attenuated acute

JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2008, 59, Suppl 4, 87–102www.jpp.krakow.pl

pancreatitis induced in these animals at adult age and this effect could be related tothe increased concentration of antioxidative enzyme SOD in the pancreatic tissue.

K e y w o r d s : lipopolysaccharide (endotoxin, LPS), neonatal endotoxemia, pancreatic enzymesecretion, acute pancreatitis

INTRODUCTION

Lipopolysaccharide (LPS, endotoxin) is the constituent of outer leaflets ofGram negative bacteria, such as; E. coli or S typhi (1). LPS molecule constitutesof three elements; lipid A, the core and an antigen O. Lipid A represents the toxicpart of LPS, the core consists of sugars linked to lipid A and to the antigen O,which is highly immunogenic and composed of 20-40 units, each unit represents3 sugars (2).

Under normal conditions endotoxins are incorporated to the bacterial outermembrane, and not toxic, because they are covered with a polysaccharidecapsule. Following cell damage LPS are released into the environment to activateimmune cells (2, 3). These cells, such as macrophages, monocytes andpolymorphonuclear leukocytes, produce and secrete the inflammatory mediators;cytokines, reactive oxygen species (ROS), nitric oxide (NO), etc (4-7). Highconcentrations of LPS leads to the septic shock (8, 9). In acute pancreatitisendotoxins are responsible for the multiorgan damage (10, 11).

When LPS are released into the blood, lipid A binds to the specific protein -LPS binding protein (LBP) (12). This is an acute phase protein, produced in theliver, and also in the kidney, heart and lungs in response to interleukin 1 (IL-1)and to interleukin 6 (IL-6) (13, 14). The constitutive blood level of LPB is low,but it increases during infection (15). LBP transfers LPS molecule to the cell, andthis complex LPS-LBP binds to the CD14 receptor (16). CD14 is a membranereceptor lacking of the transmembrane domain and thus devoid of intracellularactivity (17). The additional molecule involved in LPS-induced signaltransduction is toll-like receptor 4 (TLR4), which initiates the signal pathwayleading finally to the activation of NF-κB (18-20).

TLR4 receptors have been detected in the pancreas. They have been located inthe ductal epithelium, vascular endothelium and in the islets (21, 22). Our recentstudy have shown that, TLR4 receptors are present on the normal pancreaticacinar cells isolated from the rat. Exposition of the animals to LPS resulted in thesignificant and dose-dependent increase of TLR4 on the pancreatic acinar cells.

Previous observations have revealed that chronic exposition of the rats to LPS(4 or 10 µg/kg during 5 days) resulted in mild pancreatic necrosis and in thereduction of pancreatic protein secretion (23, 24).

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Acute pancreatitis is a serious disease of still unclear pathogenesis. Severeacute pancreatitis, which constitutes of 20-30% of cases, could be fatal (25). Themortality in acute pancreatitis results from the complications such asendotoxemia (sepsis), infected pancreatic necrosis and multiple organ damage(26-28). Endotoxemia in acute pancreatitis appears in the early phase of severeform of this disease (29). It has been reported recently, that the injury of intestinalmucosa and the increase of mucosal cells apoptosis leads to the increase ofintestinal permeability (30). Translocation of Gram minus bacteria to thecirculation and high concentration of LPS resulted in the systemic endotoxemia(31). Inhibition of caspases prevented the dysfunction of intestinal mucosa andattenuates endotoxemia (32).

Experimental studies in the rats have shown that pretreatment of these animalswith low concentrations of LPS reduced tissue damage in acute pancreatitis anddecreased pro-inflammatory cytokine production (33-35). However it is notknown if endotoxemia, which take place in the early period of life, could affectpancreatic resistance to acute pancreatitis of the adult organism.

Endotoxemia is particularly danger for the young organisms, because it couldproduce tissue damage and sepsis with high mortality (36). It has been shown thatendotoxemia, which take place in the early period of life often resulted in the

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Fig. 1. The experimental protocol of the study on the effects of endotoxemia, induced in thesuckling period of life, on the pancreatic secretory function and on pancreatic defense against acutepancreatitis in the rats.

decreased protein synthesis (37). In newborn rats LPS administration enhancedbrain damage (38, 39). It is a question if endotoxemia in neonates could affectpancreatic function in adults? To answer this question we have investigated theinfluence of neonatal endotoxemia on the pancreatic enzyme secretion and onpancreatic defense of adult organism.

MATERIAL AND METHODS

The experimental protocol has been approved by the Jagiellonian University Ethical Committeefor Animals Experimentation.

In our study we have employed the suckling rats (2 weeks old, weighing 30 - 40 g). Rat pupshave been divided into two mains group (I and II). Rats from group I were injected with 0.5 ml ofvehicle saline intraperitonealy (i.p), once a day, during 5 consecutive days (control group). Ratfrom group II were injected with LPS from E. coli during 5 consecutive days. LPS has beenadministered at various doses: 5, 10 or 15 mg/kg-day (Fig. 1). Each group of rats received separatedose of LPS, so rats obtained total doses of 25, 50 or 75 mg/kg. Three months later the same rats,as adults, have been used for the studies on pancreatic exocrine secretory functions and on theexperiments on acute pancreatitis.

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Fig. 2. Pancreatic amylase outputs in response to caerulein in the control adult rats and in the adultanimals, which have been subjected to exposition to LPS from E. coli (25, 50 or 75 mg/kg) in thesuckling period of life. Basal = unstimulated amylase outputs. Results are means ± SEM from 4separate experiments, each performed on 8-10 rats. Asterisk indicates significant (p<0.05) decreasebelow the value obtained from control rats, without previous pretreatment with LPS.

RESULTS AND DISCUSSION

Effect of neonatal endotoxemia on the pancreatic enzyme secretion in adultsThe effect of neonatal endotoxemia on the pancreatic exocrine secretion in

the adult organism has not been previously investigated. In the study on themature rats Vaccaro et al. have demonstrated that 7-days treatment of these ratswith LPS resulted in the subsequent decrease of pancreatic secretory function(40). Above effect has been related to the inflammation of the pancreas producedby LPS, because prolonged administration of high doses of endotoxin to the ratsresulted in the acute inflammatory changes in the pancreatic tissue in theseanimals (23, 40).

It has been reported that LPS is able to affect directly pancreatic cells, toinduce apoptosis in AR42J cells exposed to endotoxin and to increase mRNAsignal for pancreatitis associated protein (PAP), and for pro-inflammatorycytokines in these (24). Chronic treatment of the adult rats with LPS resulted inthe pancreatic tissue damage (41).

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Fig. 3. Pancreatic amylase outputs evoked by diversion of pancreatic juice to the exterior (DPJ) inthe control adult rats and in the adult animals, which have been subjected to exposition to LPS fromE. coli (25, 50 or 75 mg/kg) in the suckling period of life. Basal = unstimulated amylase outputs.Results are means ± SEM from 4 separate experiments, each performed on 8-10 rats. Asteriskindicates significant (p<0.05) decrease below the value obtained from control rats without previouspretreatment with LPS.

In our study histological assessment of pancreatic tissue taken from adult rats,which have been subjected to endotoxin pretreatment in the suckling period of life,have not revealed any manifestations of pancreatic inflammation. In our study on theadult rats, which have been subjected to LPS in the early period of life, amylaseblood level was not significantly different from the enzyme blood concentrationobserved in the control rats, not pretreated with LPS. Also in the adult rats, pretreatedin infancy with LPS, unstimulated (basal) secretion of amylase was not affected byneonatal endotoxemia (Fig. 2). However enzyme secretion induced by caerulein (1µg/kg) was significantly diminished by pretreatment of the rats with LPS at totaldoses of 50 or 75 mg/kg in the suckling period of life (Fig. 2). The lowest responseto caerulein was observed in the rats, which have been pretreated with the highestused dose of LPS (75 mg/kg ). Also enzyme secretion stimulated by diversion ofpancreatic juice to the exterior (DPJ) was reduced in the animals, which have beensubjected to neonatal endotoxemia, comparing to the untreated controls (Fig. 3).

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Fig. 4. Amylase release from isolated pancreatic acini in response to caerulein (10-10 M). The aciniwere isolated from the pancreata of rats pretreated in the suckling period of life with LPS from E.coli (50 or 75 mg/kg). Control - animals pretreated with 0.9% NaCl instead of LPS. Results aremeans ± SEM from 3 separate experiments, each performed in duplicate. Asterisk indicatessignificant (P<0.05) decrease below the value obtained from control rats without previouspretreatment with LPS.

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Fig. 5. mRNA signal for CCK1receptor measured by RT-PCRin the pancreatic acini isolatedfrom the control rats (lane 1) orfrom the animals pretreated inthe suckling period of life withLPS from E. coli at dose of 75mg/kg (lane 2), at dose of 50mg/kg (lane 3) or at dose of 25mg/kg (lane 4). Control -animals pretreated with 0.9%NaCl instead of LPS.

Fig. 6. Histological picture of the pancreatic tissue taken from the adult rats, with caerulein-inducedpancreatitis, which have been subjected in the suckling period of life to i.p injections ofphysiological saline (control), or treated with LPS from E.coli at total dose of 75 mg/kg.

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Fig. 7. Plasma lipase activity measured in the adult rats with caerulein-induced pancreatitis, whichhave been pretreated with LPS from E. coli ( 25, 50 or 75 mg/kg) in the suckling period of life.Results are means ± SEM from 6 separate experiments, each performed on 8-10 rats. Asteriskindicates significant (P<0.05) decrease below the value obtained from rats subjected to caerulein-induced pancreatitis without previous pretreatment with LPS.

Fig. 8. Plasma interleukin 1β (IL-1β) concentration in the adult rats with caerulein-inducedpancreatitis, which have been pretreated with various doses of LPS from E. coli (25, 50 or 75mg/kg) in the suckling period of life. Results are means ± SEM from 6 separate experiments, eachperformed on 8-10 rats. Asterisk indicates significant (P<0.05) decrease below the value obtainedfrom rats subjected to caerulein-induced pancreatitis without previous pretreatment with LPS.

For the in vitro experiments, pancreatic acini, obtained from rats pretreated inthe suckling period of life with LPS have been used. These acini showeddecreased responsiveness to caerulein. Amylase release from pancreatic aciniisolated from the rats subjected to LPS (at total dose of 50 or 75 mg/kg) wassignificantly lower then those observed in the control acini originating fromuntreated with endotoxin young rats (Fig. 4).

What about the CCK receptors? We have observed that the signal for CCK1was significantly and dose dependently reduced in the pancreatic acini obtainedfrom rats injected with endotoxin in the early period of life (Fig 5). It is verylikely that this reduction of signal for CCK1 receptor could be responsible, atleast in part, for the some kind of down-regulation of this receptor and decreasedsecretory response of pancreatic acini to caerulein.

Our observations could be summarized as follows: exposition of suckling rats toLPS resulted in the impairment of pancreatic enzyme secretion in the adults. This effectcould be related, at least in part, to the changes of CCK1 receptor on pancreatic acini.

Effect of neonatal endotoxemia on the course of acute experimental pancreatitisin adults

In this part of the study rat pups were injected with various doses of LPSduring 5 consecutive days, in the same way as in the secretory studies. After 3

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Fig. 9. Plasma interleukin 10 (IL-10) concentration in the adult rats with caerulein-inducedpancreatitis, which have been pretreated with various doses of LPS from E. coli (25, 50 or 75mg/kg) in the suckling period of life. Results are means ± SEM from 6 separate experiments, eachperformed on 8-10 rats. Asterisk indicates significant (P<0.05) increase above the value obtainedfrom rats rats subjected to caerulein-induced pancreatitis without previous pretreatment with LPS.

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Fig. 10. The amount of lipid peroxidation products; malondialdehyde and 4-hydroxynonenal(MDA+4 HNE) in the pancreatic tissue taken from adult rats with caerulein-induced pancreatitis,which have been pretreated with various doses of LPS from E. coli (25, 50 or 75 mg/kg) in thesuckling period of life. Results are means ± SEM from 6 separate experiments, each performed on8-10 rats. Asterisk indicates significant (P<0.05) decrease below the value obtained from ratssubjected to caerulein-induced pancreatitis, without previous pretreatment with LPS.

Fig. 11. Concentration of superoxide dismutase (SOD) in the pancreatic tissue taken from rats withcaerulein-induced pancreatitis pretreated with from LPS E. coli (25, 50 or 75 mg/kg) in the sucklingperiod of life. Results are means ± SEM from 6 separate experiments, each performed on 8-10 rats.Asterisk indicates significant (P<0.05) decrease below the value obtained from rats subjected tocaerulein-induced pancreatitis, without previous pretreatment with LPS.

months animals were subjected to caerulein-induced pancreatitis (AP). In thegroups of rats which have been treated, in the early period of life, with LPS attotal doses of 50 or 75 mg/kg pancreatic inflammatory changes were significantlyless pronounced, as was shown by macroscopic observation supported byhistological assessment (Fig. 6). Lipase blood level is the indicator of the severityof acute pancreatitis. In these groups of rats plasma lipase levels, weresignificantly diminished, as compared with that measured in the control rats withacute panceatitis untreated with LPS in neonatal period of life (Fig. 7).

Attenuation of acute pancreatitis in the rats pretreated in the suckling periodof life with LPS at total doses of 50 or 75 mg/kg was expressed by significantdecrease of an pro-inflammatory IL-1β plasma concentration, as compared to thecontrol rats with acute pancreatitis without LPS pretreatment (Fig. 8). In spite ofabove reduction of pro-inflammatory IL-1β, exposition of the infant rats to LPSsignificantly increased production of an anti-inflammatory IL-10 in the course ofacute pancreatitis (Fig. 9).

Activation of immune response and production of cytokines is essential for theintensity of pancreatic necrosis and systemic complications in acute pancreatitis(42-45). Aggravation of inflammatory process is often associated with increasedproduction of pro-inflammatory interleukins such as tumor necrosis factor α(TNF α), interleukin 1 β (IL-1β), interleukin 6 (IL-6), or interleukin 8 (IL-8) (23,

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Fig. 12. HSP60 protein in thepancreatic acini isolated fromthe control rats (lane 1) orfrom the animals pretreated inthe suckling period of lifewith LPS from E. coli at doseof 75 mg/kg (lane 2), at doseof 50 mg/kg (lane 3) or at doseof 25 mg/kg (lane 4). Control- animals pretreated with0.9% NaCl instead of LPS.

46-49). Anti-inflammatory cytokines, such as interleukin 4 (IL-4), or interleukin10 (IL-10) alleviated the severity of this disease (42, 50).

In our study it was observed that exposition of the rats to endotoxin in theinfancy, reduced concentration of pro-inflammatory IL-1β plasma concentrationtogether with increased blood level of an anti-inflammatory IL-10 in theseanimals subjected to acute pancreatitis at adult age. This indicates that neonatalendotoxemia affects the ability of the immune cells to produce the cytokines andincreases the resistance of the organism to pancreatic inflammation.

Acute pancreatitis is characterized by the dramatic increase of reactive oxygenspecies (ROS) in the pancreatic tissue (52). ROS are the toxic compoundsresponsible for membrane cell damage (53). These substances are criticallyimplicated in the development of septic shock and pancreatitis-associatedmultiple organ dysfunction syndrome (MODS) (54-57). In acute pancreatitis highamounts of ROS are generated in the neutrophils infiltrating the pancreas and inthe pancreatic acinar cells (58). The level of lipid peroxidation products;malondialdehyde and 4-hydroxynonenal (MDA + 4 HNE) is commonly used asan indicator of ROS formation in acute pancreatitis (59). Pancreatic inflammationcaused sudden increase of MDA + 4 HNE (42-44). Above rise of lipidperoxidation products was significantly lower in the pancreatic tissue taken fromthe rats subjected in the suckling period to endotoxemia, than in the rats withacute pancreatitis without LPS pretreatment (Fig. 10). This indicates that in theanimals pretreated with endotoxins formation of ROS was reduced.

Under normal conditions nonenzymatic or enzymatic antioxidants (such assuperoxide dysmutase; SOD) protect the tissue against the noxious effects ofROS (58). Impairment of the scavenging system and increased production ofROS promoted leukocyte activation, cytokine production, and causeddysfunction of pancreatic microcirculation (54-57). Concentration of SOD, anantioxidative enzyme, in pancreatic tissue shows negative correlation with lipidperoxidation products in the gland of rats with acute pancreatitis (42, 60-62).

Looking for the mechanism of LPS-induced protection of the pancreas thelevel of SOD, in the pancreatic tissue have been measured. It was observed in ourstudy, that pancreatic SOD increased in the rats with AP, which have been treatedin the early period of life with LPS ( at total doses of 50 or 75 mg/kg), comparingto the untreated control (Fig. 11). This is consistent with the observation thatpancreatic level of lipid peroxidation products; such as MDA + 4 HNO, wasmarkedly diminished in the these rats (Fig. 10).

Among the other defense mechanisms activated by neonatal endotoxemia theincreased production of heat shock protein (HSP) in the pancreatic acini could betaken into consideration. HSP has been reported to protect the pancreatic tissueagainst the damage and to limit the severity of acute pancreatitis (63). Severalmechanisms, which have been shown to protect the pancreas against the damageact via activation of HSP's (64, 65). We have observed that in the pancreatic acini

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obtained from the rats pretreated in the early period of life with LPS the level ofHSP60 protein was significantly increased (66) (Fig. 12).

CONCLUSIONS

Above results of the study on the effects of neonatal endotoxemia on thepancreas of adult rats could be summarized as follows:

• Endotoxemia in the early period of life resulted in the impairment ofpancreatic enzyme secretion.

• Endotoxemia in the neonatal period of life improved pancreatic defenseagainst inflammation though the activation of SOD, stimulation of HSP60and modulation of cytokine production.

Conflict of interest statement: None declared.

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R e c e i v e d : May 15th, 2008A c c e p t e d : August 30th, 2008

Author’s address: Jolanta Jaworek MD PhD, Dept. Med. Physiology, Faculty of HealthSciences Jagiellonian University CM, Grzegorzecka Street 16, 31-531 Krakow, Poland; phone:(+48 12) 424-72-30; fax: +48 +12 421-15-78; e-mail: mpjawore@cyf-kr.edu.pl

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