Author Affiliations: Department of Gastroenterology (Yang LJ, Wan R, Shen JQ, Shen J and Wang XP), and Shanghai Key Laboratory of Pancreatic Diseases (Yang LJ, Wan R and Wang XP), Shanghai First People's Hospital, Shanghai Jiaotong University, Shanghai 200080, China; Department of Gastroenterology, Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai 200072, China (Wan R and Wang XP)

Corresponding Author: Xing-Peng Wang, MD, PhD, Department of Gastroenterology, Shanghai First People's Hospital, Shanghai Jiaotong University, Shanghai 200080, China (Tel/Fax: 86-21-63240090; Email: wangxingpeng@hotmail.com)

 

© 2013, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(13)60067-3

 

 

Contributors: WR and WXP proposed the study. YLJ and WR performed research and wrote the first draft. YLJ and WR contributed equally to this work. SJQ and SJ collected and analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts. WXP is the guarantor.

Funding: This work was supported by grants from the National Natural Science Foundation of China (30971359) and the Shanghai Key Laboratory of Pancreatic Diseases for open research project (P2012001).

Ethical approval: This study was approved by Science and Technology Commission of Shanghai Municipality (SYXK 2007- 0006).

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

 

 

BACKGROUND:Remote organ failure occurs in cases of acute pancreatitis (AP); however, the reports on AP induced by pancreatic duct obstruction are rare. In this study we determined the effect of L-cysteine on pancreaticobiliary inflammation and remote organ damage in rats after pancreaticobiliary duct ligation (PBDL).

 

METHODS: AP was induced by PBDL in rats with 5/0 silk. Sixty rats were randomly divided into 4 groups. Groups A and B were sham-operated groups that received injections of saline or L-cysteine (10 mg/kg) intraperitoneally (15 rats in each group). Groups C and D were PBDL groups that received injections of saline or L-cysteine (10 mg/kg) intraperitoneally (15 rats in each group). The tissue samples of the pancreas and remote organs such as the lung, liver, intestine and kidney were subsequently examined for pathological changes under a light microscope. The samples were also stored for the determination of malondialdehyde and glutathione levels. Blood urea nitrogen (BUN), plasma amylase, ALT and AST levels were determined spectrophotometrically using an automated analyzer. Also, we evaluated the effect of L-cysteine on remote organ injury in rats with AP induced by retrograde infusion of 3.5% sodium taurocholate (NaTc) into the bile-pancreatic duct.

 

RESULTS:Varying degrees of injury in the pancreas, lung, liver, intestine and kidney were observed in the rats 24 hours after PBDL. The severity of injury to the lung, liver and intestine was attenuated, while injury status was not changed significantly in the pancreas and kidney after L-cysteine treatment. Oxidative stress was also affected by L-cysteine in PBDL-treated rats. The concentration of tissue malondialdehyde decreased in the pancreas and remote organs of PBDL and L-cysteine administrated rats, and the concentration of glutathione increased more significantly than that of the model control group. However, L-cysteine administration reduced the severity of injury in remote organs but not in the pancreas in rats with NaTc-induced AP.

 

CONCLUSION: L-cysteine treatment attenuated multiple organ damage at an early stage of AP in rats and modulated the oxidant/antioxidant imbalance.

 

(Hepatobiliary Pancreat Dis Int 2013;12:428-435)

 

KEY WORDS: pancreatitis; oxidative stress; lung injury; liver failure; animal models; pathological changes

Acute pancreatitis (AP), mostly in severe forms causes high morbidity and mortality, because it is a systemic inflammatory condition.^[1] AP-induced pathological changes, which are not only limited to the pancreas but also extend to remote organs, increase the severity of the disease and mortality.^{[2, 3]} At present, the AP related mortality rate is approximately 10%, which increases to a mortality rate of 20%-30% related to multiple organ dysfunction syndromes,^[4] in spite of changes in therapeutic strategies.^[5] Oxidative stress plays an important role in the pathogenesis of AP and there is also a correlation between the degree of oxidative stress and the severity of AP. The detrimental effects of reactive oxygen and reactive nitrogen species are mediated by their direct actions on bio-molecules (lipids, proteins, and nucleic acids) and the activation of pro-inflammatory signal cascades, which subsequently lead to the activation of immune responses.^[6-8] In this study we observed the protective effect of L-cysteine as an antioxidant treatment on multiple organ injuries in AP rats and these results provide a theoretical basis for the clinical application of L-cysteine.

 

 

Male Sprague-Dawley rats weighing 250-300 g were purchased from Shanghai SLAC Laboratory Animal Co. Ltd., Shanghai, China. The rats were maintained on a 12-hour light/dark cycle at constant temperature (22±1 ��) and humidity (65%-70%). They were fed with standard rat chow and tap water ad libitum, but were fasted for 8 hours before surgery and 24 hours after surgery, and allowed to acclimatize for a minimum of 1 week. All the procedures for the rats were approved by the Animal Care and Use Committee of Shanghai Tenth People's Hospital (Permission number: 2011-RES1). This study was also approved by the Science and Technology Commission of Shanghai Municipality (SYXK 2007-0006).

 

Under anesthesia with 3% pentobarbital sodium, two experimental models of AP were prepared. Sixty rats were randomly assigned to four groups, 15 rats in each model. In one model, AP was induced by ligation of the common pancreaticobiliary duct (PBDL) of the rats with 5/0 silk.^[9] In another model, 3.5% sodium taurocholate (NaTc) was used to prepare the AP model via retrograde injection into the bile-pancreatic duct. In this study, groups A and B were sham-operated, indicating that the rats were surgically treated without PBDL or NaTc. Subsequently, the rats in group A received an intraperitoneal injection of normal saline (NS) 30 minutes and 12 hours respectively after the operation and the rats in group B received two doses of L-cysteine (10 mg/kg, Sigma-Aldrich, St. Louis, MO, USA) 30 minutes and 12 hours after the operation. Groups C and D were induced with AP by PBDL or NaTc. The rats in group C received an intraperitoneal injection of NS 30 minutes and 12 hours respectively after PBDL or NaTc and those in group D received L-cysteine. The survival rate was also studied in both models (10 rats in each group).

 

Twenty-four hours after the operation, the rats were sacrificed under anesthesia with 3% pentobarbital sodium, and samples of the pancreas, lung, liver, intestine and kidney were quickly removed, fixed in 4% para-formaldehyde buffered with PBS overnight at 4 ��, and embedded in paraffin wax or frozen immediately at -80 ��. Blood samples were collected and plasma was separated by centrifugation for 20 minutes at 2000×g, and was stored at -80 �� for further studies. Dead rats were replaced with new ones to maintain 15 rats in each group. The pathological changes of multiple organs were assessed under a light microscope.

 

For light microscopy, hematoxylin-eosin (HE) staining was done according to standard procedures. All specimens were scored by two pathologists who were unaware of the origin of the specimens. Three sections were randomly selected and scored from each rat. Median scores were calculated to morphologically assess tissue damage. The pancreas was evaluated according to Jahovic's pathological scores:^[10] acinar cell degeneration; interstitial edema; leukocyte infiltration; and vasocongestion. Scores were graded from 0 to 3 (0, none; 1, mild; 2, moderate; and 3, severe), with a maximum score of 12. Lung injury was evaluated according to Ozveri's pathological scores:^[11] vascular congestion; interstitial edema; alveolar structural disturbance; leukocyte infiltration; and general edema. The scores were graded from 0 to 3 (0, none; 1, mild; 2, moderate; and 3, severe), with a maximum score of 15. Liver injury was graded by the scores 0 to 3: hepatocyte degeneration; vascular congestion and sinusoidal dilation; and Kupffer cell enlargement, with a maximum score of 9.^[11] The intestinal mucosa was scored according to Chiu's pathological scoring:^[12] 0=normal mucosa; 1=development of subepithelial space at the tip of the villus; 2=extension of the space with epithelial lifting; 3=massive epithelial lifting; 4=denuded villi; and 5=disintegration of the lamina propria. Renal damage was graded from 0 to 3 by leukocyte infiltration, vasocongestion, glomerular degeneration, and tubular degeneration, with a total score of 12.^[13]

 

Plasma amylase, blood urea nitrogen (BUN), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured using a Kodak Ektachem DT60 analyzer (Rochester, NY, USA).

 

The levels of malondialdehyde (MDA) in the tissues of the pancreas, lung, liver, intestine and kidney were measured to assess lipid peroxidation, using a LPO-586 commercial kit (Enzolife, BML-AK170), according to the manufacturer's protocol. Briefly, samples of tissues weighing about 100 mg were homogenized with ice-cold PBS. Prior to homogenization, 10 µL of 0.5 mol/L butylated hydroxytoluene in acetonitrile was added to every 1 mL of tissue homogenate for each sample to prevent tissue oxidation. Tissue samples were collected and centrifuged, and the supernatant was frozen at -70 �� until assay. MDA levels were measured spectrophotometrically in duplicate and expressed as nmol/g of tissue. Glutathione (GSH) concentrations were estimated in the tissue samples using a total GSH quantification kit (Dojindo Molecular Technologies Inc, Kunamoto, Japan) according to the manufacturer's protocol. Briefly, samples of tissues weighing about 100 mg were homogenized in 5% 5-sulfosalicylic acid. The homogenates were centrifuged for 10 minutes at 8000×g at 4 ��. The supernatant was used to determine GSH content via a standard enzymatic recycling procedure. The concentration of GSH was calculated against a standard curve which was measured at 405 nm at 20 minutes. Results were expressed as µmol/g tissue.

 

Data are presented as mean±SD. Statistical analysis was performed using one-way analysis of variance followed by SNK tests as post-hoc test. Survival curves were analyzed by the Kaplan-Meier method and the log-rank test. Mean pathological scores were compared using the Kruskal-Wallis test and the post-hoc Wilcoxon's rank-sum test. P<0.05 was considered statistically significant.

 

 

Cumulative survival rates from 0 to 7 days after induction of AP in the two rat models (n=10 in each group) were expressed as horizontal step lines (Fig. 1). All rats in sham-operated groups survived. L-cysteine reduced the mortality of rats in both models. The 7-day mortality rate was 70% in the PBDL+NS group and 40% in the PBDL+L-cysteine group (P>0.05). The 7-day survival rates were 40% and 70% in the NaTc+NS and NaTc+L-cysteine groups, respectively (P>0.05).

 

Severe pancreatic injury was induced after PBDL in rats in accordance with histological changes and the increases of plasma amylase. Histological observation showed extensive acinar necrosis and inflammatory cell infiltration, interstitial edema and hemorrhage in the pancreas 24 hours after PBDL, which were typical characteristics of AP (Fig. 2A). At the same time, the application of L-cysteine could not palliate the severity of acute pancreatic lesions, indicated by plasma amylase and histological scores (Tables 1 and 2). Meanwhile, L-cysteine could not attenuate pancreatic injury in NaTc-induced AP rats either, which can be attested by histological observation and plasma amylase (Tables 1 and 2, Fig. 3A).

 

Alveolar-capillary membrane thickening and inflammatory cell infiltration were the main histological changes in PBDL- and NaTc-launched lung injury, while the L-cysteine administration attenuated these tissue alterations in accordance with histological findings i.e., less thick capillary-alveolar membrane and less pronounced inflammation in group D (Figs. 2B and 3B). Moreover, L-cysteine treatment significantly decreased histological scores (Table 1).

 

Plasma ALT and AST showed a significant increase in both AP models; however, lower enzyme activities (ALT and AST) were detected in the PBDL or NaTc+L-cysteine group (Table 2). The amelioration of hepatic injury was also confirmed histologically in tissue sections (Table 1). Hepatocyte necrosis, vascular congestion, sinusoidal dilatation and inflammatory infiltration were relieved after L-cysteine administration, suggesting a protective effect of L-cysteine in PBDL- and NaTc-induced liver lesions (Figs. 2C and 3C).

 

A protective effect of L-cysteine was observed on intestinal injury caused by PBDL and NaTc. The length of the villi in PBDL- and NaTc-induced AP was increased in the L-cysteine-treated group compared with the NS-treated group, and inflammatory cell infiltration was improved (Figs. 2D and 3D). The same tendency was seen in the evaluation of pathological severity scores (Table 1).

 

Histological changes of kidney were also observed after PBDL or NaTc treatment. There were unclear margins of renal tubular epithelial cells, stenosis and atresia of the lumens, congestion of the renal glomerulus, and interstitial edema in both AP models (Figs. 2E and 3E). L-cysteine administration, in our study, failed to affect the severity of renal injury in rats with PBDL- induced AP according to plasma BUN tests and tissue section observation (Tables 1 and 2, Fig. 2E). However, histological analysis and plasma BUN tests showed that L-cysteine may alleviate the renal injury of NaTc-induced AP rats (Tables 1 and 2, Fig. 3E).

 

MDA levels were increased when the pancreaticobiliary duct was ligated. This elevation was reduced after L-cysteine treatment (P<0.05). The early depletion of GSH activity after obstruction was recovered after L-cysteine treatment (P<0.05). A similar tendency was observed in the lung, liver, intestinal mucosa and kidney in NaTc-induced AP rats (Fig. 4). Oxidative damage was involved in multiple organ injury to the pancreas, lung, liver, intestinal mucosa and kidney after ligation of the pancreaticobiliary duct.

 

 

Multiple organ failure is one of the leading causes of death in patients with AP.^[14] The lung is considered to be the major target in patients with pancreatitis. Pulmonary complications are frequent and serious as systemic complications.^[15] The intestinal barrier appears to play a pivotal role in the development of multiple organ failure.^{[16, 17]} Breakdown of this barrier may lead to sepsis because of the absorption of viable bacteria and their antigens, initiation of cytokines mediated systemic inflammatory response, multiple organ dysfunction and death. Acute renal and hepatic failure also increases the mortality of patients with AP.^{[2, 3, 18]}

 

As a useful model, PBDL-induced AP can be used to mimic gallstone-induced pancreatitis, which is a common etiological factor in humans.^[19] Current studies^{[20, 21]} have confirmed that multiple organ injury (the lung, liver, intestine and kidney) is induced after PBDL. In the present study L-cysteine administration palliated extra-pancreatic organ (lung, liver, and intestinal mucosa) injury assessed by reduced pathological scores, biochemical indexes and lipid peroxidation. The protective effect was not specific to the PBDL-induced AP model. In the NaTc-induced AP model, L-cysteine also reduced the severity of injury to remote organs but not to the pancreas.

 

Oxidative stress is thought to play an essential role in multisystem organ failure during AP.^{[22, 23]} Reactive oxygen species are also associated with inflammatory response in the early phase of obstructive pancreatitis through cytokine production by several signaling pathways.^{[24, 25]} Studies have shown that therapy targeting oxidative stress may protect rats against AP-induced tissue injury in the pancreas and extra-pancreatic organs such as the lung,^[26-29] liver,^{[18, 30]} kidney and intestine.^[31] Thus antioxidant treatment may be effective in preventing extra-pancreatic complications in AP patients.

 

L-cysteine reduces body metabolism. It is naturally utilized in the synthesis of both protein and non-protein compounds including taurine, reduced inorganic sulfur, sulfate, and GSH.^[32] The major factor regulating GSH biosynthesis is the availability of cysteine. The present study showed that L-cysteine reduced the levels of MDA and enhanced the content of GSH in pancreatic tissues and remote organs, indicating that its antioxidant effect would attenuate remote organ injury. These findings suggest protective effects of antioxidant against AP associated tissue injury.

 

In addition, oxidative stress and cytokines are critical factors in the outcome of AP.^{[22, 33]} There is a positive feedback between them. The results of our previous study showed that L-cysteine could modulate nuclear erythroid-related factor 2 (Nrf2), an antioxidant response element signal pathway, through which it enhances the antioxidant effect and modulates inflammatory cytokines.^[34] The study further elucidated the mechanism by which L-cysteine enhances antioxidant activity.

 

In our study, L-cysteine treatment did not show any effect on AP injury in both AP models. As a progressive and irreversible pathological process, PBDL-induced pancreatitis was thought to be more severe than that in other AP models.^[35] Therefore, the PBDL model is not suitable for the study of L-cysteine for the treatment of AP. Reproducible acute injury to the pancreas makes the study in pancreatitis-associated remote organ injuries possible. As to the NaTc-induced AP model, the antioxidant effect of L-cysteine was overwhelmed by an intense generation of reactive oxygen species, and thus L-cysteine treatment failed to relieve pancreatic injury.

 

L-cysteine treatment was not effective for the pancreatitis-induced renal lesion in rats with PBDL-induced AP. We postulate that in the PBDL-induced AP model oxidative stress may not be a pivotal regulator in the kidney although the levels of MDA and GSH in the kidney are changed significantly after L-cysteine treatment.

 

Our results are in agreement with a previous study by Manso et al,^[21] in which remote organ injury during AP induced by PBDL was palliated by N-acetylcysteine (NAC) treatment. Some reports have indicated that L-cysteine is more effective than NAC in some aspects. A study^[36] found that the initial rate of NAC uptake in hepatocytes was lower than that of L-cysteine after the assay of ³⁵S-labeled substrates. Rougee et al^[37] reported that more effective deactivation of singlet oxygen was seen on L-cysteine treatment than NAC. In summary, these findings suggest that L-cysteine may act as an efficient antioxidant in tissues.

 

In conclusion, L-cysteine has a protective effect against multiple organ injury induced by PBDL and NaTc and this effect may be mediated by its antioxidant properties.

 

 

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