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Pharmacological blockade of the MaxiK channel attenuates experimental acute pancreatitis and associated lung injury in rats
International Immunopharmacology 21 (2014) 220–224
Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Pharmacological blockade of the MaxiK channel attenuates experimental acute pancreatitis and associated lung injury in rats Jian-Dong Ren a,⁎, Yong-Jun Xing b, Kai-Hua Fan a,⁎⁎, Bo-Tao Yu a, Wei-Hua Jin a, Yan Jiang a, Li Jing a, Xue-Chai Wu a, Shi-Hua Wang a, Juan Wu a, Hua Chen a a b
Department of Pharmacy, Chengdu Military General Hospital, Chengdu, China Department of Stomatology, Chengdu Military General Hospital, Chengdu, China
a r t i c l e
i n f o
Article history: Received 24 December 2013 Received in revised form 13 March 2014 Accepted 1 April 2014 Available online 15 May 2014 Keywords: Acute pancreatitis Lung injury Toll like receptor 4 Heparan sulfate MaxiK Paxilline
a b s t r a c t Increasing evidence has recently demonstrated that soluble heparan sulfate (HS), a degradation product of extracellular matrix produced by elastase, plays a key role in the aggravation of acute pancreatitis (AP) and associated lung injury. However little is known about the detailed mechanism underlying HS-induced inflammatory cascade. Our previous work has provided a valuable clue that a large-conductance K+ channel (MaxiK) was involved in the HS-stimulated activation of murine macrophages. Here we attempted to ask whether pharmacological inhibition of the MaxiK channel will exert beneficial effects on the treatment of AP and secondary lung injury. The protective effects of paxilline, a specific blocker of MaxiK, on rats against sodium taurocholate induced AP were evaluated. Our data showed that paxilline substantially attenuated AP and resultant lung injury, mainly by limiting the burst of inflammatory responses, as proven by decreased plasma concentrations of tumor necrosis factor-α and macrophage inflammatory protein-2, together with unimpaired pancreatic enzyme activities in rats suffering from AP. Compared with the therapeutic administration, pre-treatment of paxilline showed superior potential to slow down the progress of AP. Furthermore, AP rats received paxilline exhibited improved histopathologic alterations both in the pancreas and the lungs, and even lower lung MPO activity. Taken together, our study provides evidence that MaxiK is involved in the spread of inflammatory responses and the following lung injury during the attack of AP, indicating that this ion channel is a promising candidate as a therapeutic target for AP. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Acute pancreatitis (AP) is a sudden inflammatory disease triggered by inappropriate activation of pancreatic enzymes in the pancreas. Most cases of AP are mild and self-limiting and resolve in a few days with supportive treatment. However, approximately 15% of patients with AP will develop into severe acute pancreatitis that usually results in a life-threatening condition with multiple organ dysfunction, and in particular the following acute lung injury, often contributes to the majority of AP-associated deaths [1,2]. So it is urgently needed to reveal the detailed mechanism and explore new therapeutic strategies to prevent the progression of AP.
⁎ Correspondence to: J.-D. Ren, Department of Pharmacy, Chengdu Military General Hospital, No. 270, Rongdu Avenue, Jinniu District, Chengdu, Sichuan 610083, China. Tel.: +86 28 86570825. ⁎⁎ Correspondence to: K.-H. Fan, Department of Pharmacy, Chengdu Military General Hospital, No. 270, Rongdu Avenue, Jinniu District, Chengdu, Sichuan 610083, China. Tel.: +86 28 86570425. E-mail addresses: [email protected] (J.-D. Ren), [email protected] (K.-H. Fan).
http://dx.doi.org/10.1016/j.intimp.2014.04.003 1567-5769/© 2014 Elsevier B.V. All rights reserved.
Lately, increasing research evidence has been found that elastase is implicated in the aggravation of AP and the resultant lung injury [3–6]. In detail, after the initiation of AP, massively activated pancreatic elastase can cleave extracellular matrix and lead to the release of soluble degradation products, especially heparan sulfate (HS), a negatively charged glycosaminoglycan, which can be recognized by Toll like receptor 4 (TLR4). Macrophages, dendritic cells, and endothelial cells that abundantly express TLR4, trigger intracellular inflammatory cascades and the burst of inflammatory mediators after activated by HS [7,8]. Moreover, the high systemic level of elastase usually induces pulmonary inflammatory responses and accumulation of polymorphonuclear leukocytes, which secrete neutrophil elastase and further strengthen HS/TLR4 signaling pathway, resulting in exacerbation of inflammation and tissue damage [9,10]. Therefore, blockade of HS-caused transmembrane signal transduction will probably bring potential beneficial effects in controlling the severity of AP and secondary lung injury. Although much effort has been devoted to disclose the mechanism underlying HS-mediated inflammatory cascade and the resultant aggravation of AP, little is known about the molecules involved in the HS/TLR4 interaction and consequent signaling process. In recent years,
J.-D. Ren et al. / International Immunopharmacology 21 (2014) 220–224
researchers have found that cellular potassium ion (K+) channels participate in the transmembrane signaling pathway of lipopolysaccharide (LPS), another known TLR4 ligand, indicating the potential roles of K+ channels in HS/TLR4 mediated intracellular inflammatory cascade [11–14]. Moreover, the findings from our previous work revealed that a large-conductance K+ channel (MaxiK or BK channel) was involved in the activation of murine macrophages in response to HS. Pharmacological blockade of MaxiK by its specific blocker paxilline substantially abolished HS-stimulated activation of transcription factors and the following considerable production of cytokines in macrophages [15]. Therefore, the herein study aims to ask whether pharmacological blockade of the MaxiK channel may confer beneficial effects on attenuating the severity of AP and associated lung injury in rats. 2. Materials and methods 2.1. Reagents Paxilline and sodium taurocholate were purchased from Sigma (St. Louis, MO, USA). Enzyme linked immunosorbent assay (ELISA) kits for tumor necrosis factor-α (TNF-α) and macrophage inflammatory protein-2 (MIP-2) were obtained from R&D system (Minneapolis, MN, USA). The myeloperoxidase (MPO) assay kits were purchased from Jian Cheng Corporation (Nanjing, Jiangsu, China). Paxilline was suspended in 0.05% dimethyl sulfoxide (DMSO) and diluted in phosphate buffered saline (PBS) when used. 2.2. Animals Male Wistar rats weighing 205–240 g were obtained from the Experimental Animal Center of Sichuan University (Chengdu, Sichuan, China). All animals were housed at a constant temperature of 23 °C and under 12 h light–dark cycle conditions with free access to standard laboratory chow and water. Animals were acclimatized for one week prior to any experimentation. All procedures involving the rats were carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee of Sichuan University. 2.3. Experimental AP model in rats Sodium taurocholate induced AP model was produced according to the method described by Aho et al. [16]. Briefly, under pentobarbital anesthesia (50 mg/kg body weight), a laparotomy was performed and 5% sodium taurocholate (1 ml/kg body weight) was injected into the biliopancreatic duct of the rat in one minute. Then the rat recovered from the anesthesia and was allowed access to water. Paxilline (2.2 μg/kg) was intravenously injected to rats 1 h before or after the injection of sodium taurocholate, respectively. Rats received intravenous injection of only normal saline (1 ml/kg) or vehicle solution (solvent for paxilline) composed of 0.05% DMSO in PBS to AP rats served as the control. The sham operation control group only underwent laparotomy. Then all rats were sacrificed by CO2 asphyxiation at indicated time points and immediately the blood and tissue samples were collected. 2.4. Enzyme activity and cytokine concentrations in plasma Plasma samples were obtained from harvested blood specimens by centrifugation (1800 ×g for 15 min at 4 °C) 6 h after the operation and the amylase or lipase activity of was detected by an enzymebased colorimetric assay on a fully automated Hitachi 7170 biochemistry analyzer (Hitachi, Tokyo, Japan). To evaluate the anti-inflammatory effects of paxilline on AP rats, the concentrations of TNF-α and MIP-2 in plasma were measured using quantitative ELISA kits (R&D, Minneapolis, MN, USA) according to the manufacturer's instructions.
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2.5. Histological examination Pancreas and lung tissue samples were removed from sacrificed rats 12 h after the initiation of AP. Paraffin-embedded samples were sectioned and stained with hematoxylin and eosin, using a standard staining procedure. Histopathological evaluation was performed under a light microscope by an experienced laboratory pathologist who was blinded to the group identity. 2.6. Water content and MPO activity in pancreas and lung tissues A removed sample of the pancreas or the lungs was weighed then dried for 72 h at 60 °C and weighed again. Then water content (ratio of wet/dry weight) was calculated to evaluate the consequence of pancreatic or pulmonary tissue edema. For MPO activity determination, pancreas or lung tissues were collected from sacrificed animals and homogenized immediately in 10 volumes of ice-cold potassium phosphate buffer (20 mM, pH 7.4) containing 30 mM KCl. The homogenate was centrifuged at 14,000 ×g for 10 min at 4 °C. The pellet was then rehomogenized with an equivalent volume of 50 mM acetic acid containing 5% hexadecyltrimethylammonium bromide. Subsequently the MPO activity in homogenate was determined using a commercial MPO assay kit following the manufacturer's recommendations. One unit of MPO activity is defined as degrading 1 μmol of hydrogen peroxide at 37 °C, and MPO activity of tissue was expressed as unit per gram (U/g) of protein. 2.7. Statistical and presentation of data All experimental values are expressed as mean ± SE. Statistical comparisons were examined by the one-way ANOVA. Differences in values were considered significant if P values b0.05. 3. Results 3.1. MaxiK blockade inhibited the release of inflammatory mediators in rats during AP Plasma amylase and lipase activities markedly increased in rats 6 h after the injection of sodium taurocholate, indicating the development of pancreatitis (Fig. 1A and B). Likewise, significantly higher concentrations of TNF-α and MIP-2 in plasma were also found which are capable of contributing to the amplification of inflammatory process during AP (Fig. 1C and D). In contrast, either pre- or post-administration of paxilline effectively decreased plasma TNF-α and MIP-2 levels, but failed to attenuate amylase and lipase activities. Moreover, compared to therapeutic administration, prophylactic treatment with paxilline showed better inhibitory effects on the production of both TNF-α and MIP-2. 3.2. Paxilline attenuated pancreatic and pulmonary tissue injury Edema in the pancreas and the lungs occurred in rats after the initiation of pancreatitis, as assessed gravimetrically by measuring tissue wet/dry weight ratio. We found that either pre- or post-administration of paxilline substantially improved the pulmonary edema, but had no beneficial effect on the pancreas (Fig. 2), which was in agreement with the findings in histopathological examination. Prophylactic or therapeutic administration of paxilline resulted in less extensive histopathological changes of hemorrhage, interstitial leukocyte infiltration and even necrosis in the pancreas and the lungs, except the edema (Fig. 3). 3.3. Paxilline decreased MPO activity in the pancreas and the lungs Generally, MPO activity in pancreas or lung homogenates was detected as an indicator for neutrophil infiltration or inflammation in the organ [17]. Rats suffering from AP showed profound increase in
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Fig. 1. Plasma pancreatic enzyme activities and cytokine concentrations in rats suffering from AP. Experimental AP model was induced after intraductal sodium taurocholate injection in rats. Animals received intravenous injection of paxilline (Px, 2.2 μg/kg) 1 h prior to or after the operation, respectively. Animals were sacrificed by CO2 asphyxiation 6 h after the operation and immediately the plasma samples were collected. Then the amylase (A) and lipase (B) activities in plasma were determined by a fully automated biochemistry analyzer. The plasma concentrations of TNF-α (C) and MIP-2 (D) were measured using quantitative ELISA kits according to the manufacturer's instructions. Treatment with only saline or PBS containing 0.05% DMSO in AP rats served as control. Animals in sham operation control group underwent only laparotomy. *P b 0.05, compared with sham group; and #P b 0.05, compared between preand post-treatments of Px.
pancreatic and pulmonary MPO activities, implying potential tissue damage in both organs. Paxilline exerted effective inhibition on pancreatic MPO activity only when administered 1 h before the initiation of AP. In contrast, either prophylactic or therapeutic treatment of paxilline diminished the increase in pulmonary MPO activity. Compared to therapeutic administration of paxilline, significantly more potent inhibition of MPO activity in lung homogenates was observed in animals preinjected with paxilline (Fig. 4A and B).
4. Discussion
Fig. 2. The water content of pancreatic and pulmonary tissues in AP rats. Experimental AP model was developed by a retrograde injection of sodium taurocholate into the pancreatic duct of rats. Animals received intravenous injection of paxilline (Px, 2.2 μg/kg) 1 h prior to or after the operation, respectively. Pancreas and lung tissue samples were obtained from sacrificed rats 12 h after the initiation of AP. A removed sample of the pancreas or the lungs was weighed then dried for 72 h at 60 °C and weighed again. Then water content (ratio of wet/dry weight) was calculated. *P b 0.05, pancreatic ratio of W/D weight compared with AP rats treated with normal saline alone; and #P b 0.05, pulmonary ratio of W/D weight compared with AP rats treated with normal saline alone.
It is well-known that LPS, the principal component of the outer membrane of Gram-negative bacteria, is capable of triggering systemic inflammatory responses and resultant sepsis via TLR4 [18,19]. Similarly, some endogenous molecules, known as damage associated molecular patterns (DAMPs), also act as the inducer of inflammatory responses during tissue damage and inflammation independent of infectious state and finally lead to different complications [20–22]. During the process of AP, the extracellular matrix is degraded by activated pancreatic elastase and then soluble HS, an important member of DAMPs, can be recognized by TLR4, consequently provoking considerable release of inflammatory cytokines, resulting in the aggravation of AP and associated organ injury [7,8]. Currently, LPS neutralization by cationic compounds or peptides is regarded as a potential therapeutic candidate for sepsis. However, it is obviously impractical that direct HS neutralization is applied as a therapeutic strategy for AP due to its ubiquitous distribution in mammalian tissues. Therefore, much effort should be focused on
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Fig. 3. Histological sections from rats that underwent various treatments. Experimental AP model was induced after intraductal sodium taurocholate injection in rats. Animals received intravenous injection of paxilline (Px, 2.2 μg/kg) 1 h prior to or after the operation, respectively. Pancreas and lung tissue samples were obtained from sacrificed rats 12 h after the initiation of AP. Paraffin-embedded samples were sectioned and stained with hematoxylin and eosin. Histopathological evaluation was performed under light microscope. Row 1 indicates the pancreas histological sections; and row 2 shows the lung histological sections. Scale bars = 50 μm.
blocking HS/TLR4 mediated signal transduction so as to slow down the progress of AP. Growing evidence has accumulated in recent years indicating that activated elastase is mainly responsible for acute lung injury during AP [3–6]. Elastase from the pancreas is released to the blood and then induces pulmonary damage with following neutrophil infiltration, which subsequently secretes neutrophil elastase, further amplifying and worsening the inflammatory state. That is why treatment with elastase inhibitors conferred significant protection against AP and secondary lung injury in animal models [4,23]. It is encouraging that similar results were obtained in our previous work that blockade of the MaxiK channel produced significant inhibition on HS/TLR4 mediated
inflammatory cascade in vitro, which sheds a new light on preventing the aggravation of AP and associated organ injury, especially lung injury, one of the most life-threatening complications following AP [15]. In the present study, the finding that prophylactic blockade of MaxiK produced superior activity to attenuate the development of AP and lung injury than therapeutic treatment, manifested the significance of early control of inflammation in the management of AP. As for lung injury, it often occurs after the onset of an attack of AP, maximal between 48 and 96 h [24]. Accordingly, treatment of paxilline either 1 h before or after the initiation of AP offered early inhibition on the spread of inflammation from the pancreas into the lung, which just provided the explanation why the protective effects of paxilline against lung injury in AP
Fig. 4. Alterations in pancreatic and pulmonary MPO activity in AP rats. Experimental AP model was developed by a retrograde injection of sodium taurocholate into the pancreatic duct of rats. Animals received intravenous injection of paxilline (Px, 2.2 μg/kg) 1 h prior to or after the operation, respectively. Pancreas and lung tissue samples were obtained from sacrificed rats 12 h after the initiation of AP. After tissues were homogenized and centrifuged at 14,000 ×g for 10 min at 4 °C, the pellets were homogenized again with an equivalent volume of 50 mM acetic acid containing 5% hexadecyltrimethylammonium bromide. Then the MPO activity in homogenate was determined using a commercial MPO assay kit. AP rats treated with only saline or PBS containing 0.05% DMSO served as control. Animals in sham operation control group underwent only laparotomy. The MPO activity of tissue was expressed as unit per gram (U/g) of protein. *P b 0.05, compared with AP rats treated with normal saline alone; and #P b 0.05, compared between pre- and post-treatments of Px.
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rats were more potent than pancreatic injury. In addition, paxilline failed to inhibit the overwhelming activation of pancreatic enzymes, suggesting that paxilline attenuated AP and resultant lung injury mainly by limiting the release of inflammatory mediators, and it further provides a valuable clue that combined inhibition of MaxiK and pancreatic enzyme activity might probably bring more significant beneficial effects on alleviating the severity of AP. Although our work has presented the evidence that MaxiK channel blockade could be a promising candidate in the management of AP and associated lung injury, there are still some questions that need to be addressed by future research. For example, it is worth to be noted that the MaxiK channel is widely distributed in various mammalian tissues, function in modulating neurotransmitter release and regulating vascular, urinary bladder and respiratory tone [25–28], implying the risk that non-selected blockade of MaxiK might bring unwanted side effects. In our preliminary experiment, we found that although paxilline had no influence on plasma cytokine levels when injected alone to healthy rats, slight behavioral abnormalities such as involuntary movements and abnormal gait were observed (data not shown). Therefore, it should be noted that extensive blockade of MaxiK will likely affect its normal physiological roles in spite of the potential beneficial effects in the treatment of AP. In this case, further studies, especially the development of new inhibitors of the MaxiK channel specifically against HS/ TLR4-mediated inflammatory cascade are warranted. Acknowledgments We are grateful for the support and funding of the National Natural Science Foundation of China (NSFC) granted to Jian-Dong Ren (Grant No. 81000185). References [1] Bradley EL, Dexter ND. Management of severe acute pancreatitis: a surgical odyssey. Ann Surg 2010;251:6–17. [2] Elder AS, Saccone GT, Dixon DL. Lung injury in acute pancreatitis: mechanisms underlying augmented secondary injury. Pancreatology 2012;12:49–56. [3] Millson CE, Charles K, Poon P, Macfie J, Mitchell CJ. A prospective study of serum pancreatic elastase-1 in the diagnosis and assessment of acute pancreatitis. Scand J Gastroenterol 1998;33:664–8. [4] Fric P, Slabý J, Kasafírek E, Kocna P, Marek J. Effective peritoneal therapy of acute pancreatitis in the rat with glutaryl-trialanin-ethylamide: a novel inhibitor of pancreatic elastase. Gut 1992;33:701–6. [5] Jaffray C, Yang J, Carter G, Mendez C, Norman J. Pancreatic elastase activates pulmonary nuclear factor kappa B and inhibitory kappa B, mimicking pancreatitisassociated adult respiratory distress syndrome. Surgery 2000;128:225–31. [6] Lungarella G, Gardi C, de Santi MM, Luzi P. Pulmonary vascular injury in pancreatitis: evidence for a major role played by pancreatic elastase. Exp Mol Pathol 1985; 42:44–59.
[7] Liu H, Li Y, Wang L, Chen H, Guan J, Zhou Z. Aggravation of acute pancreatitis by heparan sulfate in mice. Scand J Gastroenterol 2009;44:626–32. [8] Sawa H, Ueda T, Takeyama Y, Yasuda T, Shinzeki M, Nakajima T, et al. Role of Tolllike receptor 4 in the pathophysiology of severe acute pancreatitis in mice. Surg Today 2007;37:867–73. [9] Kawabata K, Hagio T, Matsuoka S. The role of neutrophil elastase in acute lung injury. Eur J Pharmacol 2002;451:1–10. [10] Hashimoto S, Okayama Y, Shime N, Kimura A, Funakoshi Y, Kawabata K, et al. Neutrophil elastase activity in acute lung injury and respiratory distress syndrome. Respirology 2008;13:581–4. [11] Blunck R, Scheel O, Müller M, Brandenburg K, Seitzer U, Seydel U. New insights into endotoxin-induced activation of macrophages: involvement of a K+ channel in transmembrane signaling. J Immunol 2001;166:1009–15. [12] Papavlassopoulos M, Stamme C, Thon L, Adam D, Hillemann D, Seydel U, et al. MaxiK blockade selectively inhibits the lipopolysaccharide-induced I kappa B-alpha/NFkappa B signaling pathway in macrophages. J Immunol 2006;177:4086–93. [13] Jo HY, Kim SY, Lee S, Jeong S, Kim SJ, Kang TM, et al. Kir3.1 channel is functionally involved in TLR4-mediated signaling. Biochem Biophys Res Commun 2011;407:687–91. [14] Qiu MR, Campbell TJ, Breit SN. A potassium ion channel is involved in cytokine production by activated human macrophages. Clin Exp Immunol 2002;130:67–74. [15] Ren JD, Fan L, Tian FZ, Fan KH, Yu BT, Jin WH, et al. Involvement of a membrane potassium channel in heparan sulfate induced activation of macrophages. Immunology 2014;141:345–52. [16] Aho Hj, Koskensalo SM-L, Nevalainen TJ. Experimental pancreatitis in the rat. Sodium taurocholate-induced acute haemorrhagic pancreatitis. Scand J Gastroenterol 1980;15:411–6. [17] Allan G, Bhattacherjee P, Brook CD, Read NG, Parke AJ. Myeloperoxidase activity as a quantitative marker of polymorphonuclear leukocyte accumulation into an experimental myocardial infarct—the effect of ibuprofen on infarct size and polymorphonuclear leukocyte accumulation. J Cardiovasc Pharmacol 1985;7:1154–60. [18] Alves-Filho JC, de Freitas A, Russo M, Cunha FQ. Toll-like receptor 4 signaling leads to neutrophil migration impairment in polymicrobial sepsis. Crit Care Med 2006; 34:461–70. [19] Roger T, Froidevaux C, Le Roy D, Reymond MK, Chanson AL, Mauri D, et al. Protection from lethal Gram-negative bacterial sepsis by targeting Toll-like receptor 4. Proc Natl Acad Sci U S A 2009;106:2348–52. [20] Johnson GB, Brunn GJ, Platt JL. Cutting edge: an endogenous pathway to systemic inflammatory response syndrome (SIRS)-like reactions through Toll-like receptor-4. J Immunol 2004;172:20–4. [21] Tang AH, Brunn GJ, Cascalho M, Platt JL. Pivotal advance: endogenous pathway to SIRS, sepsis, and related conditions. J Leukoc Biol 2007;82:282–5. [22] Lee KM, Seong SY. Partial role of TLR4 as a receptor responding to damageassociated molecular pattern. Immunol Lett 2009;125:31–9. [23] Wang HH, Tang AM, Chen L, Zhou MT. Potential of sivelestat in protection against severe acute pancreatitis-associated lung injury in rats. Exp Lung Res 2012;38:445–52. [24] Bhatia M, Brady M, Zagorski J, Christmas SE, Campbell F, Neoptolemos JP, et al. Treatment with neutralising antibody against cytokine induced neutrophil chemoattractant (CINC) protects rats against acute pancreatitis associated lung injury. Gut 2000; 47:838–44. [25] Robitaille R, Charlton MP. Presynaptic calcium signals and transmitter release are modulated by calcium-activated potassium channels. J Neurosci 1992;12:297–305. [26] Brayden JE, Nelson MT. Regulation of arterial tone by activation of calciumdependent potassium channels. Science 1992;256:532–5. [27] Herrera GM, Etherton B, Nausch B, Nelson MT. Negative feedback regulation of nerve-mediated contractions by KCa channels in mouse urinary bladder smooth muscle. Am J Physiol Regul Integr Comp Physiol 2005;289:R402–9. [28] Kume H, Takai A, Tokuno H, Tomita T. Regulation of Ca2+-dependent K+-channel activity in tracheal myocytes by phosphorylation. Nature 1989;341:152–4.
Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Pharmacological blockade of the MaxiK channel attenuates experimental acute pancreatitis and associated lung injury in rats Jian-Dong Ren a,⁎, Yong-Jun Xing b, Kai-Hua Fan a,⁎⁎, Bo-Tao Yu a, Wei-Hua Jin a, Yan Jiang a, Li Jing a, Xue-Chai Wu a, Shi-Hua Wang a, Juan Wu a, Hua Chen a a b
Department of Pharmacy, Chengdu Military General Hospital, Chengdu, China Department of Stomatology, Chengdu Military General Hospital, Chengdu, China
a r t i c l e
i n f o
Article history: Received 24 December 2013 Received in revised form 13 March 2014 Accepted 1 April 2014 Available online 15 May 2014 Keywords: Acute pancreatitis Lung injury Toll like receptor 4 Heparan sulfate MaxiK Paxilline
a b s t r a c t Increasing evidence has recently demonstrated that soluble heparan sulfate (HS), a degradation product of extracellular matrix produced by elastase, plays a key role in the aggravation of acute pancreatitis (AP) and associated lung injury. However little is known about the detailed mechanism underlying HS-induced inflammatory cascade. Our previous work has provided a valuable clue that a large-conductance K+ channel (MaxiK) was involved in the HS-stimulated activation of murine macrophages. Here we attempted to ask whether pharmacological inhibition of the MaxiK channel will exert beneficial effects on the treatment of AP and secondary lung injury. The protective effects of paxilline, a specific blocker of MaxiK, on rats against sodium taurocholate induced AP were evaluated. Our data showed that paxilline substantially attenuated AP and resultant lung injury, mainly by limiting the burst of inflammatory responses, as proven by decreased plasma concentrations of tumor necrosis factor-α and macrophage inflammatory protein-2, together with unimpaired pancreatic enzyme activities in rats suffering from AP. Compared with the therapeutic administration, pre-treatment of paxilline showed superior potential to slow down the progress of AP. Furthermore, AP rats received paxilline exhibited improved histopathologic alterations both in the pancreas and the lungs, and even lower lung MPO activity. Taken together, our study provides evidence that MaxiK is involved in the spread of inflammatory responses and the following lung injury during the attack of AP, indicating that this ion channel is a promising candidate as a therapeutic target for AP. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Acute pancreatitis (AP) is a sudden inflammatory disease triggered by inappropriate activation of pancreatic enzymes in the pancreas. Most cases of AP are mild and self-limiting and resolve in a few days with supportive treatment. However, approximately 15% of patients with AP will develop into severe acute pancreatitis that usually results in a life-threatening condition with multiple organ dysfunction, and in particular the following acute lung injury, often contributes to the majority of AP-associated deaths [1,2]. So it is urgently needed to reveal the detailed mechanism and explore new therapeutic strategies to prevent the progression of AP.
⁎ Correspondence to: J.-D. Ren, Department of Pharmacy, Chengdu Military General Hospital, No. 270, Rongdu Avenue, Jinniu District, Chengdu, Sichuan 610083, China. Tel.: +86 28 86570825. ⁎⁎ Correspondence to: K.-H. Fan, Department of Pharmacy, Chengdu Military General Hospital, No. 270, Rongdu Avenue, Jinniu District, Chengdu, Sichuan 610083, China. Tel.: +86 28 86570425. E-mail addresses: [email protected] (J.-D. Ren), [email protected] (K.-H. Fan).
http://dx.doi.org/10.1016/j.intimp.2014.04.003 1567-5769/© 2014 Elsevier B.V. All rights reserved.
Lately, increasing research evidence has been found that elastase is implicated in the aggravation of AP and the resultant lung injury [3–6]. In detail, after the initiation of AP, massively activated pancreatic elastase can cleave extracellular matrix and lead to the release of soluble degradation products, especially heparan sulfate (HS), a negatively charged glycosaminoglycan, which can be recognized by Toll like receptor 4 (TLR4). Macrophages, dendritic cells, and endothelial cells that abundantly express TLR4, trigger intracellular inflammatory cascades and the burst of inflammatory mediators after activated by HS [7,8]. Moreover, the high systemic level of elastase usually induces pulmonary inflammatory responses and accumulation of polymorphonuclear leukocytes, which secrete neutrophil elastase and further strengthen HS/TLR4 signaling pathway, resulting in exacerbation of inflammation and tissue damage [9,10]. Therefore, blockade of HS-caused transmembrane signal transduction will probably bring potential beneficial effects in controlling the severity of AP and secondary lung injury. Although much effort has been devoted to disclose the mechanism underlying HS-mediated inflammatory cascade and the resultant aggravation of AP, little is known about the molecules involved in the HS/TLR4 interaction and consequent signaling process. In recent years,
J.-D. Ren et al. / International Immunopharmacology 21 (2014) 220–224
researchers have found that cellular potassium ion (K+) channels participate in the transmembrane signaling pathway of lipopolysaccharide (LPS), another known TLR4 ligand, indicating the potential roles of K+ channels in HS/TLR4 mediated intracellular inflammatory cascade [11–14]. Moreover, the findings from our previous work revealed that a large-conductance K+ channel (MaxiK or BK channel) was involved in the activation of murine macrophages in response to HS. Pharmacological blockade of MaxiK by its specific blocker paxilline substantially abolished HS-stimulated activation of transcription factors and the following considerable production of cytokines in macrophages [15]. Therefore, the herein study aims to ask whether pharmacological blockade of the MaxiK channel may confer beneficial effects on attenuating the severity of AP and associated lung injury in rats. 2. Materials and methods 2.1. Reagents Paxilline and sodium taurocholate were purchased from Sigma (St. Louis, MO, USA). Enzyme linked immunosorbent assay (ELISA) kits for tumor necrosis factor-α (TNF-α) and macrophage inflammatory protein-2 (MIP-2) were obtained from R&D system (Minneapolis, MN, USA). The myeloperoxidase (MPO) assay kits were purchased from Jian Cheng Corporation (Nanjing, Jiangsu, China). Paxilline was suspended in 0.05% dimethyl sulfoxide (DMSO) and diluted in phosphate buffered saline (PBS) when used. 2.2. Animals Male Wistar rats weighing 205–240 g were obtained from the Experimental Animal Center of Sichuan University (Chengdu, Sichuan, China). All animals were housed at a constant temperature of 23 °C and under 12 h light–dark cycle conditions with free access to standard laboratory chow and water. Animals were acclimatized for one week prior to any experimentation. All procedures involving the rats were carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee of Sichuan University. 2.3. Experimental AP model in rats Sodium taurocholate induced AP model was produced according to the method described by Aho et al. [16]. Briefly, under pentobarbital anesthesia (50 mg/kg body weight), a laparotomy was performed and 5% sodium taurocholate (1 ml/kg body weight) was injected into the biliopancreatic duct of the rat in one minute. Then the rat recovered from the anesthesia and was allowed access to water. Paxilline (2.2 μg/kg) was intravenously injected to rats 1 h before or after the injection of sodium taurocholate, respectively. Rats received intravenous injection of only normal saline (1 ml/kg) or vehicle solution (solvent for paxilline) composed of 0.05% DMSO in PBS to AP rats served as the control. The sham operation control group only underwent laparotomy. Then all rats were sacrificed by CO2 asphyxiation at indicated time points and immediately the blood and tissue samples were collected. 2.4. Enzyme activity and cytokine concentrations in plasma Plasma samples were obtained from harvested blood specimens by centrifugation (1800 ×g for 15 min at 4 °C) 6 h after the operation and the amylase or lipase activity of was detected by an enzymebased colorimetric assay on a fully automated Hitachi 7170 biochemistry analyzer (Hitachi, Tokyo, Japan). To evaluate the anti-inflammatory effects of paxilline on AP rats, the concentrations of TNF-α and MIP-2 in plasma were measured using quantitative ELISA kits (R&D, Minneapolis, MN, USA) according to the manufacturer's instructions.
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2.5. Histological examination Pancreas and lung tissue samples were removed from sacrificed rats 12 h after the initiation of AP. Paraffin-embedded samples were sectioned and stained with hematoxylin and eosin, using a standard staining procedure. Histopathological evaluation was performed under a light microscope by an experienced laboratory pathologist who was blinded to the group identity. 2.6. Water content and MPO activity in pancreas and lung tissues A removed sample of the pancreas or the lungs was weighed then dried for 72 h at 60 °C and weighed again. Then water content (ratio of wet/dry weight) was calculated to evaluate the consequence of pancreatic or pulmonary tissue edema. For MPO activity determination, pancreas or lung tissues were collected from sacrificed animals and homogenized immediately in 10 volumes of ice-cold potassium phosphate buffer (20 mM, pH 7.4) containing 30 mM KCl. The homogenate was centrifuged at 14,000 ×g for 10 min at 4 °C. The pellet was then rehomogenized with an equivalent volume of 50 mM acetic acid containing 5% hexadecyltrimethylammonium bromide. Subsequently the MPO activity in homogenate was determined using a commercial MPO assay kit following the manufacturer's recommendations. One unit of MPO activity is defined as degrading 1 μmol of hydrogen peroxide at 37 °C, and MPO activity of tissue was expressed as unit per gram (U/g) of protein. 2.7. Statistical and presentation of data All experimental values are expressed as mean ± SE. Statistical comparisons were examined by the one-way ANOVA. Differences in values were considered significant if P values b0.05. 3. Results 3.1. MaxiK blockade inhibited the release of inflammatory mediators in rats during AP Plasma amylase and lipase activities markedly increased in rats 6 h after the injection of sodium taurocholate, indicating the development of pancreatitis (Fig. 1A and B). Likewise, significantly higher concentrations of TNF-α and MIP-2 in plasma were also found which are capable of contributing to the amplification of inflammatory process during AP (Fig. 1C and D). In contrast, either pre- or post-administration of paxilline effectively decreased plasma TNF-α and MIP-2 levels, but failed to attenuate amylase and lipase activities. Moreover, compared to therapeutic administration, prophylactic treatment with paxilline showed better inhibitory effects on the production of both TNF-α and MIP-2. 3.2. Paxilline attenuated pancreatic and pulmonary tissue injury Edema in the pancreas and the lungs occurred in rats after the initiation of pancreatitis, as assessed gravimetrically by measuring tissue wet/dry weight ratio. We found that either pre- or post-administration of paxilline substantially improved the pulmonary edema, but had no beneficial effect on the pancreas (Fig. 2), which was in agreement with the findings in histopathological examination. Prophylactic or therapeutic administration of paxilline resulted in less extensive histopathological changes of hemorrhage, interstitial leukocyte infiltration and even necrosis in the pancreas and the lungs, except the edema (Fig. 3). 3.3. Paxilline decreased MPO activity in the pancreas and the lungs Generally, MPO activity in pancreas or lung homogenates was detected as an indicator for neutrophil infiltration or inflammation in the organ [17]. Rats suffering from AP showed profound increase in
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Fig. 1. Plasma pancreatic enzyme activities and cytokine concentrations in rats suffering from AP. Experimental AP model was induced after intraductal sodium taurocholate injection in rats. Animals received intravenous injection of paxilline (Px, 2.2 μg/kg) 1 h prior to or after the operation, respectively. Animals were sacrificed by CO2 asphyxiation 6 h after the operation and immediately the plasma samples were collected. Then the amylase (A) and lipase (B) activities in plasma were determined by a fully automated biochemistry analyzer. The plasma concentrations of TNF-α (C) and MIP-2 (D) were measured using quantitative ELISA kits according to the manufacturer's instructions. Treatment with only saline or PBS containing 0.05% DMSO in AP rats served as control. Animals in sham operation control group underwent only laparotomy. *P b 0.05, compared with sham group; and #P b 0.05, compared between preand post-treatments of Px.
pancreatic and pulmonary MPO activities, implying potential tissue damage in both organs. Paxilline exerted effective inhibition on pancreatic MPO activity only when administered 1 h before the initiation of AP. In contrast, either prophylactic or therapeutic treatment of paxilline diminished the increase in pulmonary MPO activity. Compared to therapeutic administration of paxilline, significantly more potent inhibition of MPO activity in lung homogenates was observed in animals preinjected with paxilline (Fig. 4A and B).
4. Discussion
Fig. 2. The water content of pancreatic and pulmonary tissues in AP rats. Experimental AP model was developed by a retrograde injection of sodium taurocholate into the pancreatic duct of rats. Animals received intravenous injection of paxilline (Px, 2.2 μg/kg) 1 h prior to or after the operation, respectively. Pancreas and lung tissue samples were obtained from sacrificed rats 12 h after the initiation of AP. A removed sample of the pancreas or the lungs was weighed then dried for 72 h at 60 °C and weighed again. Then water content (ratio of wet/dry weight) was calculated. *P b 0.05, pancreatic ratio of W/D weight compared with AP rats treated with normal saline alone; and #P b 0.05, pulmonary ratio of W/D weight compared with AP rats treated with normal saline alone.
It is well-known that LPS, the principal component of the outer membrane of Gram-negative bacteria, is capable of triggering systemic inflammatory responses and resultant sepsis via TLR4 [18,19]. Similarly, some endogenous molecules, known as damage associated molecular patterns (DAMPs), also act as the inducer of inflammatory responses during tissue damage and inflammation independent of infectious state and finally lead to different complications [20–22]. During the process of AP, the extracellular matrix is degraded by activated pancreatic elastase and then soluble HS, an important member of DAMPs, can be recognized by TLR4, consequently provoking considerable release of inflammatory cytokines, resulting in the aggravation of AP and associated organ injury [7,8]. Currently, LPS neutralization by cationic compounds or peptides is regarded as a potential therapeutic candidate for sepsis. However, it is obviously impractical that direct HS neutralization is applied as a therapeutic strategy for AP due to its ubiquitous distribution in mammalian tissues. Therefore, much effort should be focused on
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Fig. 3. Histological sections from rats that underwent various treatments. Experimental AP model was induced after intraductal sodium taurocholate injection in rats. Animals received intravenous injection of paxilline (Px, 2.2 μg/kg) 1 h prior to or after the operation, respectively. Pancreas and lung tissue samples were obtained from sacrificed rats 12 h after the initiation of AP. Paraffin-embedded samples were sectioned and stained with hematoxylin and eosin. Histopathological evaluation was performed under light microscope. Row 1 indicates the pancreas histological sections; and row 2 shows the lung histological sections. Scale bars = 50 μm.
blocking HS/TLR4 mediated signal transduction so as to slow down the progress of AP. Growing evidence has accumulated in recent years indicating that activated elastase is mainly responsible for acute lung injury during AP [3–6]. Elastase from the pancreas is released to the blood and then induces pulmonary damage with following neutrophil infiltration, which subsequently secretes neutrophil elastase, further amplifying and worsening the inflammatory state. That is why treatment with elastase inhibitors conferred significant protection against AP and secondary lung injury in animal models [4,23]. It is encouraging that similar results were obtained in our previous work that blockade of the MaxiK channel produced significant inhibition on HS/TLR4 mediated
inflammatory cascade in vitro, which sheds a new light on preventing the aggravation of AP and associated organ injury, especially lung injury, one of the most life-threatening complications following AP [15]. In the present study, the finding that prophylactic blockade of MaxiK produced superior activity to attenuate the development of AP and lung injury than therapeutic treatment, manifested the significance of early control of inflammation in the management of AP. As for lung injury, it often occurs after the onset of an attack of AP, maximal between 48 and 96 h [24]. Accordingly, treatment of paxilline either 1 h before or after the initiation of AP offered early inhibition on the spread of inflammation from the pancreas into the lung, which just provided the explanation why the protective effects of paxilline against lung injury in AP
Fig. 4. Alterations in pancreatic and pulmonary MPO activity in AP rats. Experimental AP model was developed by a retrograde injection of sodium taurocholate into the pancreatic duct of rats. Animals received intravenous injection of paxilline (Px, 2.2 μg/kg) 1 h prior to or after the operation, respectively. Pancreas and lung tissue samples were obtained from sacrificed rats 12 h after the initiation of AP. After tissues were homogenized and centrifuged at 14,000 ×g for 10 min at 4 °C, the pellets were homogenized again with an equivalent volume of 50 mM acetic acid containing 5% hexadecyltrimethylammonium bromide. Then the MPO activity in homogenate was determined using a commercial MPO assay kit. AP rats treated with only saline or PBS containing 0.05% DMSO served as control. Animals in sham operation control group underwent only laparotomy. The MPO activity of tissue was expressed as unit per gram (U/g) of protein. *P b 0.05, compared with AP rats treated with normal saline alone; and #P b 0.05, compared between pre- and post-treatments of Px.
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rats were more potent than pancreatic injury. In addition, paxilline failed to inhibit the overwhelming activation of pancreatic enzymes, suggesting that paxilline attenuated AP and resultant lung injury mainly by limiting the release of inflammatory mediators, and it further provides a valuable clue that combined inhibition of MaxiK and pancreatic enzyme activity might probably bring more significant beneficial effects on alleviating the severity of AP. Although our work has presented the evidence that MaxiK channel blockade could be a promising candidate in the management of AP and associated lung injury, there are still some questions that need to be addressed by future research. For example, it is worth to be noted that the MaxiK channel is widely distributed in various mammalian tissues, function in modulating neurotransmitter release and regulating vascular, urinary bladder and respiratory tone [25–28], implying the risk that non-selected blockade of MaxiK might bring unwanted side effects. In our preliminary experiment, we found that although paxilline had no influence on plasma cytokine levels when injected alone to healthy rats, slight behavioral abnormalities such as involuntary movements and abnormal gait were observed (data not shown). Therefore, it should be noted that extensive blockade of MaxiK will likely affect its normal physiological roles in spite of the potential beneficial effects in the treatment of AP. In this case, further studies, especially the development of new inhibitors of the MaxiK channel specifically against HS/ TLR4-mediated inflammatory cascade are warranted. Acknowledgments We are grateful for the support and funding of the National Natural Science Foundation of China (NSFC) granted to Jian-Dong Ren (Grant No. 81000185). References [1] Bradley EL, Dexter ND. Management of severe acute pancreatitis: a surgical odyssey. Ann Surg 2010;251:6–17. [2] Elder AS, Saccone GT, Dixon DL. Lung injury in acute pancreatitis: mechanisms underlying augmented secondary injury. Pancreatology 2012;12:49–56. [3] Millson CE, Charles K, Poon P, Macfie J, Mitchell CJ. A prospective study of serum pancreatic elastase-1 in the diagnosis and assessment of acute pancreatitis. Scand J Gastroenterol 1998;33:664–8. [4] Fric P, Slabý J, Kasafírek E, Kocna P, Marek J. Effective peritoneal therapy of acute pancreatitis in the rat with glutaryl-trialanin-ethylamide: a novel inhibitor of pancreatic elastase. Gut 1992;33:701–6. [5] Jaffray C, Yang J, Carter G, Mendez C, Norman J. Pancreatic elastase activates pulmonary nuclear factor kappa B and inhibitory kappa B, mimicking pancreatitisassociated adult respiratory distress syndrome. Surgery 2000;128:225–31. [6] Lungarella G, Gardi C, de Santi MM, Luzi P. Pulmonary vascular injury in pancreatitis: evidence for a major role played by pancreatic elastase. Exp Mol Pathol 1985; 42:44–59.
[7] Liu H, Li Y, Wang L, Chen H, Guan J, Zhou Z. Aggravation of acute pancreatitis by heparan sulfate in mice. Scand J Gastroenterol 2009;44:626–32. [8] Sawa H, Ueda T, Takeyama Y, Yasuda T, Shinzeki M, Nakajima T, et al. Role of Tolllike receptor 4 in the pathophysiology of severe acute pancreatitis in mice. Surg Today 2007;37:867–73. [9] Kawabata K, Hagio T, Matsuoka S. The role of neutrophil elastase in acute lung injury. Eur J Pharmacol 2002;451:1–10. [10] Hashimoto S, Okayama Y, Shime N, Kimura A, Funakoshi Y, Kawabata K, et al. Neutrophil elastase activity in acute lung injury and respiratory distress syndrome. Respirology 2008;13:581–4. [11] Blunck R, Scheel O, Müller M, Brandenburg K, Seitzer U, Seydel U. New insights into endotoxin-induced activation of macrophages: involvement of a K+ channel in transmembrane signaling. J Immunol 2001;166:1009–15. [12] Papavlassopoulos M, Stamme C, Thon L, Adam D, Hillemann D, Seydel U, et al. MaxiK blockade selectively inhibits the lipopolysaccharide-induced I kappa B-alpha/NFkappa B signaling pathway in macrophages. J Immunol 2006;177:4086–93. [13] Jo HY, Kim SY, Lee S, Jeong S, Kim SJ, Kang TM, et al. Kir3.1 channel is functionally involved in TLR4-mediated signaling. Biochem Biophys Res Commun 2011;407:687–91. [14] Qiu MR, Campbell TJ, Breit SN. A potassium ion channel is involved in cytokine production by activated human macrophages. Clin Exp Immunol 2002;130:67–74. [15] Ren JD, Fan L, Tian FZ, Fan KH, Yu BT, Jin WH, et al. Involvement of a membrane potassium channel in heparan sulfate induced activation of macrophages. Immunology 2014;141:345–52. [16] Aho Hj, Koskensalo SM-L, Nevalainen TJ. Experimental pancreatitis in the rat. Sodium taurocholate-induced acute haemorrhagic pancreatitis. Scand J Gastroenterol 1980;15:411–6. [17] Allan G, Bhattacherjee P, Brook CD, Read NG, Parke AJ. Myeloperoxidase activity as a quantitative marker of polymorphonuclear leukocyte accumulation into an experimental myocardial infarct—the effect of ibuprofen on infarct size and polymorphonuclear leukocyte accumulation. J Cardiovasc Pharmacol 1985;7:1154–60. [18] Alves-Filho JC, de Freitas A, Russo M, Cunha FQ. Toll-like receptor 4 signaling leads to neutrophil migration impairment in polymicrobial sepsis. Crit Care Med 2006; 34:461–70. [19] Roger T, Froidevaux C, Le Roy D, Reymond MK, Chanson AL, Mauri D, et al. Protection from lethal Gram-negative bacterial sepsis by targeting Toll-like receptor 4. Proc Natl Acad Sci U S A 2009;106:2348–52. [20] Johnson GB, Brunn GJ, Platt JL. Cutting edge: an endogenous pathway to systemic inflammatory response syndrome (SIRS)-like reactions through Toll-like receptor-4. J Immunol 2004;172:20–4. [21] Tang AH, Brunn GJ, Cascalho M, Platt JL. Pivotal advance: endogenous pathway to SIRS, sepsis, and related conditions. J Leukoc Biol 2007;82:282–5. [22] Lee KM, Seong SY. Partial role of TLR4 as a receptor responding to damageassociated molecular pattern. Immunol Lett 2009;125:31–9. [23] Wang HH, Tang AM, Chen L, Zhou MT. Potential of sivelestat in protection against severe acute pancreatitis-associated lung injury in rats. Exp Lung Res 2012;38:445–52. [24] Bhatia M, Brady M, Zagorski J, Christmas SE, Campbell F, Neoptolemos JP, et al. Treatment with neutralising antibody against cytokine induced neutrophil chemoattractant (CINC) protects rats against acute pancreatitis associated lung injury. Gut 2000; 47:838–44. [25] Robitaille R, Charlton MP. Presynaptic calcium signals and transmitter release are modulated by calcium-activated potassium channels. J Neurosci 1992;12:297–305. [26] Brayden JE, Nelson MT. Regulation of arterial tone by activation of calciumdependent potassium channels. Science 1992;256:532–5. [27] Herrera GM, Etherton B, Nausch B, Nelson MT. Negative feedback regulation of nerve-mediated contractions by KCa channels in mouse urinary bladder smooth muscle. Am J Physiol Regul Integr Comp Physiol 2005;289:R402–9. [28] Kume H, Takai A, Tokuno H, Tomita T. Regulation of Ca2+-dependent K+-channel activity in tracheal myocytes by phosphorylation. Nature 1989;341:152–4.