Protective effects of MCP-1 inhibitor on a rat model of severe acute pancreatitis

Guo-Xiong Zhou, Xue-Juan Zhu, Xiao-Ling Ding, Hong Zhang, Jian-Ping Chen,Hui Qiang, Hai-Feng Zhang and Qun Wei

Nantong, China

Protective effects of MCP-1 inhibitor on a rat model of severe acute pancreatitis

Guo-Xiong Zhou, Xue-Juan Zhu, Xiao-Ling Ding, Hong Zhang, Jian-Ping Chen,Hui Qiang, Hai-Feng Zhang and Qun Wei

Nantong, China

(Hepatobiliary Pancreat Dis Int 2010; 9: 201-207)

severe acute pancreatitis;monocyte chemotactic protein-1;Bindarit;pathogenesis

Introduction

Acute pancreatitis (AP), especially severe acute pancreatitis (SAP), is a serious disease with a high morbidity of 10%-20%. Most cases are secondary to gallstones or excessive alcohol consumption.Irrespective of the cause, activation of digestive enzymes within pancreatic acinar cells is thought to be a critical initiating event. Pancreatic damage then leads to a localized and a subsequent systemic in fl ammatory response. If the latter is marked it may result in leukocytemediated distant organ damage and the development of multiple organ dysfunction syndrome, which is responsible for the majority of deaths in this condition.Thus, in fl ammatory mediators play a key role in the pathogenesis of AP.[1,2]

Chemokines are a family of small (8-10 kDa)inducible secreted cytokines with chemotactic and activating effects on leukocyte subsets. They can be divided into major subgroups on the basis of the orientation of the fi rst two cysteines. In CXC chemokines the fi rst two cysteine residues (C) are separated by a single amino acid (X), while in the CC subfamily the fi rst two cysteine residues are adjacent.[1,2]CXC and CCchemokines have different patterns of tissue distribution and are reported to have different effects. It is suggested that the CC chemokines, such as monocyte chemotactic protein-1 (MCP-1), MIP-1α, and regulation upon activation normal T-cell expressed and secreted(RANTES), primarily activate monocytes, whereas the CXC chemokines, such as IL-8 and GRO-α/CINC, tend to preferentially stimulate neutrophils. MCP-1, a potent chemokine released by in fl ammatory mononuclear cells, attracts lymphocytes, monocytes, mast cells,and eosinophils during in fl ammation. MCP-1 not only provides chemotactic cues for the recruitment of monocytes from the bloodstream to the tissues but also is responsible for monocyte activation and induction of the respiratory burst.[3]In fact, upregulated MCP-1 expression has been found during acute and chronic pancreatitis in both animal models and human tissues,suggesting a contribution of this chemokine in the pathogenesis of mononuclear in fi ltration.[4-6]MCP-1 is one of several chemokines upregulated in pancreatitis,but evidence for its pathogenic role is still lacking.

Bindarit (2-methyl-2-[[1-(phenylmethyl)-1H-indazol-3yl]methoxy] propanoic acid) is a novel molecule that has been shown to preferentially inhibit MCP-1 production in monocytes in vitro and in vivo without affecting the production of the cytokines IL-1 and IL-6,or the chemokines IL-8, MIP-1α, and RANTES.[7]In this study, we investigated the effect of Bindarit on AP in rats.

MethodsReagents

Bindarit was purchased from ACRAF, Aprilia (Italy),and multi-clonal antibody against MCP-1 from Abcam Chemicals (USA). Primers were synthesized by Shanghai Biotech (Shanghai, China). The reverse transcription system was purchased from QIAGEN Biotech (Germany),and the total RNA isolation kit from Invitrogen Biotech(Shanghai, China).

Experimental animals and grouping

Seventy-two male Sprague-Dawley rats weighing 250±30 g were purchased from the Experimental Animal Center, Nantong University, China. They were randomly divided into three groups (n=24 for each group): saline control group (group S), SAP group(group P), and Bindarit treatment group (group T). SAP models in groups P and T were induced by retrograde injection of 4% sodium taurocholate (Sigma Co., USA)into the bilio-pancreatic duct. Bindarit (100 mg/kg) was injected intraperitoneally in group T, while 0.5% methyl cellulose was injected intraperitoneally in groups S and P. In group S, saline was retrogradely infused into the bilio-pancreatic duct. Rats were sacri fi ced at 1, 3, 6 and 12 hours after operation (6 rats at each time point).

Establishment of the SAP rat model

Rats were fasted for 12 hours before operation. After intraperitoneal injection of 2% chlorine aldehyde, a 2-cm cut was made into the abdominal cavity, the duodenum was elevated and the bilio-pancreatic duct unfolded. The bile duct was blocked at the hepatic hilum using a nontraumatic clamp. The side wall of the duodenum was punctured by a fi ne needle with a one-milliliter syringe to the bile-pancreatic duct. Taurocholate (4%, 1 ml/kg)was injected at a pressure of 20 cm H2O at 0.1 ml/min.The puncture point was compressed with a cotton stick for 2 minutes to avoid re fl ux of the solution. Five minutes of observation con fi rmed no bleeding from pancreatic tissues at the spleen-stomach portion, and the SAP rat model was established successfully. Afterward,the blockage at the hepatic hilum was released and the wound was closed.

Amylase estimation

After the models were established, the rats were sacri fi ced at different time points. Two milliliters of blood was drawn from the inferior vena cava, centrifuged, and kept at -80 ℃. Later, it was used to assess the levels of amylase with an auto-biochemical analysis instrument.

Morphological examination

Paraf fi n-embedded pancreas samples were cut at 4 μm,stained with hematoxylin-eosin, and examined under a light microscope. Ten microscopic fi elds in each specimen were randomly selected and examined. The degree of injury was evaluated on the basis of the destruction of tissue architecture and cellular vacuolization and swelling associated with an in fl ammatory reaction. Pancreatic damage was scored according to edema, infection,hemorrhage, and necrosis on a scale of 0-4 (0, least severe and 4, most severe for each parameter).

Enzyme-linked immunoadsorbent assay (ELISA)

Plasma was assayed for MCP-1 using a sandwich ELISA kit according to the manufacturer's instructions.Brie fl y, anti-MCP-1 primary antibody was coated onto ELISA plates and incubated for 120 minutes at room temperature. Samples and standards were added to the wells and incubated for 1 hour. Then the wells were washed and a biotinylated goat anti-rat MCP-1 antibody was added for 1 hour. The plates were washed again, and streptavidin conjugated to horseradishperoxidase was added for 10 minutes. After washing,tetramethylbenzidine was added for color development and the reaction was terminated with 1 mol/L H2SO4.Absorbance was measured at 490 nm.

Immunohistochemistry

Immunohistochemistry was performed to examine MCP-1 protein expression and localization in the tissues.Brie fl y, pancreas samples were isolated and immediately fi xed in 10% pH-neutral phosphate-buffered formalin.The fi xed tissues were then embedded in paraf fi n and kept until use. Paraf fi n sections (4 μm) were cut,deparaf fi nized and hydrated. Antigens were retrieved in 10 mmol/L sodium citrate buffer (pH 6.0) preheated to 95 ℃for 10 minutes. Immunohistochemistry was also used to examine chemokine protein expression and localization in the tissues. Immunohistochemical staining of MCP-1 protein was performed using the streptavidin-peroxidase method with anti-MCP-1 antibody at a dilution of 1∶100.Negative control sections were processed in the same manner by replacing the primary antibody with buffered saline. The stained sections were reviewed and scored using an Olympus microscope. Positive staining was de fi ned as 5% or more of the cells stained.

Reverse transcriptase-polymerase chain reaction(RT-PCR)

Total RNA was extracted from pancreatic tissues.After reverse transcription into cDNA, 1 μl of RT product was used as a template for PCR. The primer sequences of the MCP-1 gene were 5'-TAG CAT CCA CGT GCT GTC TC-3' (forward) and 5'-CAT TCA AAG GTG CTG AAG TCC-3' (reverse). The primers for the GAPDH gene were 5'-ATG GGA AGC TGG TCA TCA AC-3' (forward) and 5'-TTC AGC TCT GGG ATG ACC TT-3' (reverse). The expected ampli fi cation fragment lengths of MCP-1 and GAPDH were 299bp and 484bp respectively. The reaction parameters of PCR were:94 ℃ for 2 minutes, 40 ampli fi cation cycles including 94 ℃ for 40 seconds, 55 ℃ for 55 seconds and 72 ℃ for 1 minute, and a fi nal extension at 72 ℃ for 7 minutes.Ampli fi cation was performed in a Perkin Elmer 2400 thermocycler (Applied Biosystems, Foster City, CA,USA). The PCR products were run on electrophoresis using 1.5% agarose gel, and visualized after ethidium bromide staining and ultraviolet irradiation. The relative level of MCP-1 mRNA expression was analyzed by normalizing the band intensity of MCP-1 to that of GAPDH. The detection was repeated six times.

Western blotting

MCP-1 protein expression was detected by Western blotting analysis. Total protein extract was separated by 10% SDS/PAGE before electrophoretic transfer (120 V, 1 hour) to hybond PVDF membrane. The membranes were probed with 10 ml anti-MCP-1 monoclonal antibody (Abcam, USA) in TBS-T (1∶500), and the immunocomplexed membranes were re-probed at room temperature for 1 hour with horseradish peroxidaseconjugated anti-rat secondary antibody in TBS-T (1∶4000),with 5% blocking reagent. Finally, the immunoreactive proteins were visualized using the ECL Western blotting analysis system (Santa Cruz, USA).

Statistical analysis

Data are presented as mean±standard error (SEM)and analyzed with STATA 7.0. A P value less than 0.05 was considered signi fi cantly signi fi cant.

ResultsLevels of serum amylase

The levels of amylase increased with the development of SAP at different time points in group P. Serum amylase levels in group P and T were higher than those in group S. The amylase levels were signi fi cantly lower at 6 and 12 hours in group T than in group P (Fig. 1).

Effects of Bindarit treatment on MCP-1 expression in serum

The levels of MCP-1 in serum did not change at different time points in group S. The levels of MCP-1 in serum at 6 and 12 hours after operation were signi fi cantly higher in group P than in group S, and were signi fi cantly lower in group T than in group P at 6 and 12 hours after operation (Fig. 2).

Morphological changes in the pancreas after Bindarit treatment

In group P, obvious edema, focal hemorrhage,leukocyte in fi ltration, and disrupted acinar cells were seen at 1 and 3 hours after the establishment of the model.Large regions of gland cell necrosis were shown at 6 and 12 hours (Fig. 3A), while milder injury occurred in group S (Fig. 3B). These pathological changes were greatly ameliorated by treatment with MCP-1 inhibitors.Compared with group P, pancreatic histopathological injury was signi fi cantly reduced in group T (Fig. 3C).As summarized in the Table, treatment with MCP-1 inhibitors markedly reduced the severity of sodium taurocholate-induced pancreatitis. Scores for pancreatic in fl ammation and necrosis, including the total pancreatic histological score were all decreased.

Effects of Bindarit treatment on immunohistochemical localization of MCP-1 in the pancreas

Immunohistochemical staining was made to further con fi rm the attenuating effect of Bindarit on the expression levels of MCP-1 in the pancreas and to localize MCP-1 to its cellular sources in the tissues.MCP-1 expression was found mostly in the pancreasof rats given sodium taurocholate but without Bindarit treatment (Fig. 4A). There was an apparent reduction of MCP-1 expression in tissues from rats treated with Bindarit (Fig. 4B). The pancreas from rats administered with saline showed no signi fi cant staining for MCP-1(Fig. 4C). In the pancreas, MCP-1 expression was found mostly in pancreatic acinar cells and occasionally in in fi ltrating leukocytes.

Table. Histopathological scores of pancreatic lesions

Fig. 3. Morphological changes in sodium taurocholate-induced rat pancreatitis. Representative light micrographs of pancreas sections stained by HE (A-C, original magni fi cation ×20). A: group P; B: group S; C: group T.

Fig. 4. Bindarit treatment affected immunohistochemical localization of MCP-1 protein in the pancreas. Representative immunohistochemical staining of MCP-1 in the pancreas (A-C, original magni fi cation ×20) at 12 hours after operation. A: group P; B:group T; C: group S.

Fig. 5. MCP-1 mRNA expression detected by RT-PCR in the pancreas. Lanes 1-4: group S at 1, 3, 6, and 12 hours; lanes 5-8:group P at 1, 3, 6, and 12 hours; lane 9: marker; lanes 10-13: group T at 1, 3, 6, and 12 hours.

Fig. 6. MCP-1 protein expression detected by Western blotting in the pancreas. A: lanes 1-4: group S at 1, 3, 6, and 12 hours; lanes 5-8:group P at 1, 3, 6, and 12 hours; B: lanes 1-4, group P at 1, 3, 6, and 12 hours; lanes 5-8: group T at 1, 3, 6, and 12 hours.

Effects of Bindarit treatment on MCP-1 mRNA expression in the pancreas

In the pancreas, the expression of MCP-1 mRNA showed a time-related increase in group P and was markedly higher at 6 and 12 hours than in group S(P＜0.05). The expression level of MCP-1 mRNA was signi fi cantly lower at 6 and 12 hours in group T than in group P (Fig. 5). The expression of MCP-1 mRNA were higher in groups P and T than in group S.

Effects of Bindarit treatment on MCP-1 protein expression in the pancreas

The expression levels of MCP-1 protein as assessed by Western blotting were markedly higher at 6 and 12 hours in group P than in group S. And the expression levels of pancreatic MCP-1 were signi fi cantly lower at 6 and 12 hours in group T than in group P (Fig. 6).

Discussion

AP, an in fl ammatory disease involving the production of different cytokines and chemokines, is characterized by leukocyte in fi ltration. Activated leukocytes are important in the pathogenesis of AP. Chemokines are key in fl ammatory mediators by directing leukocytes to the areas of injury. They in fl uence leukocyte migration into tissues in two principal ways. Firstly, chemokine ligand binding to a speci fi c leukocyte receptor leads to activation of cell surface integrins and allows strong adhesion to the endothelium. Secondly, chemokines promote migration of adherent leukocytes across the endothelium and through the extracellular matrix. Chemokines are also important regulators of leukocyte activation in situ.[8-11]

Most studies evaluating the role of chemokines in AP focused on CXC chemokines, whereas others on the critical role of CC chemokines.[4,11,12]CC chemokines such as MCP-1 have been shown to play a role in sepsis and other in fl ammatory conditions.[13,14]MCP-1 has a direct homologue in the rat as a potent monocyte chemoattractant and activator. Recently, it has been demonstrated that the levels of MCP-1 protein increase in the rat pancreas after AP is induced by caerulein hyperstimulation or bile salt infusion.[15]Thus, MCP-1 may be an early mediator of the in fl ammatory response in AP.

Bindarit is an original indazolic derivative devoid of systemic immunosuppressive effects and of activity on arachidonic acid metabolism, which has been shown to be effective in a number of animal models involving chronic in fl ammation such as arthritis[16-18]and lupus.[19-21]Bindarit, in fact, selectively inhibits chronic in fl ammation and ameliorates joint damage in rat adjuvant arthritis[16-18]and is able to limit glomerular injury and prolong the survival of NZB/W lupus mice.[19,21]Several studies have demonstrated that Bindarit is a preferential inhibitor of MCP-1 production in vitro and in vivo and have suggested that its bene fi cial effects in models of joints and kidney in fl ammation are related to its anti-MCP-1 activity.[7,17,21]In this study, we investigated the role of MCP-1 in AP by using Bindarit, an inhibitor of MCP-1.

In our study, Bindarit treatment in rats reduced the severity of pancreatitis. It also signi fi cantly attenuated the hyperamylasemia and diminished pancreatic injury(acinar cell injury/necrosis as well as edema). Another interesting fi nding is that MCP-1 serum levels were markedly elevated in experimental AP. We demonstrated that increased serum levels of MCP-1 play a pathological role during the course of sodium taurocholate-induced AP. The increased level of MCP-1 was signi fi cantly attenuated by treatment with Bindarit, as demonstrated by a signi fi cant reduction in serum amylase and decreased histopathological lesions. It is interesting to note that MCP-1 production was completely inhibited by Bindarit treatment, while the pancreatic levels of MCP-1 were only partially reduced in vivo. The reasons for this difference are not clear but may be explained by the fact that the in vivo system is much more complex than isolated pancreatic acini in vitro.

Several papers reported that a chemokine receptor antagonist can in fl uence the course of an attack of AP, including the complications, suggestive of a novel method of therapy for this condition.[1,2,11,22,23]The levels of the in fl ammation-related genes MCP-1, PAI, TF, IL-6,and ICAM-1 and the extent of lung in fl ammation can be reduced during the initiation of caerulein-induced AP in EGR-1-de fi cient mice. In this study, MCP-1 was identi fi ed as likely important in the development and severity of AP.[24]Sun et al[2]reported that caerulein induces early upregulation of MCP-1 in the pancreas.CP-96345 administration suppresses caerulein-induced increase in MCP-1 mRNA and protein expression. In the present study, we investigated the expression of MCP-1 mRNA and protein in the pancreas treated with Bindarit. The results showed that MCP-1 mRNA and protein expression showed a time-related increase in the AP group. The expression levels of MCP-1 mRNA and protein were markedly higher at 6 and 12 hours than in the control group. After Bindarit treatment, the mRNA and protein levels of MCP-1 were signi fi cantly lower at 6 and 12 hours than in the untreated group. These results suggested that MCP-1 as an early proin fl ammatory mediator probably plays a more important role in local than in systemic in fl ammatory responses.

We suggest inhibition of MCP-1 production is the mechanism by which Bindarit treatment protects against AP. The drug, however, has been reported to affect other in fl ammatory mediators such as TNF-α[17]but not IL-1,IL-6, or the chemokines IL-8, MIP-1α, and RANTES.[7]It is possible that these actions may contribute to its protective action. The precise mechanism by which Bindarit protects against AP will be the subject of future studies.

In conclusion, our results showed for the fi rst time that MCP-1 inhibitor treatment in established AP reduced the severity of AP and the mortality rate in an animal model of SAP. These fi ndings suggest that anti-MCP-1 strategies are of potential therapeutic value in the treatment of AP.

Funding: This study was supported by grants from the social Burteall Foundation of Nantong (S5054).

Ethical approval: Not needed.

Contributors: ZXJ, DXL and QH proposed the study. ZGX wrote the fi rst draft. ZLM analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts.ZGX is the guarantor.

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

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BACKGROUND: Chemokines and their receptors play key roles in the pathogenesis of acute pancreatitis. This study aimed to establish a rat model of severe acute pancreatitis (SAP)for investigating monocyte chemotactic protein-1 (MCP-1)expression in the pathogenesis of the disease. We assessed the effects of the inhibitor of MCP-1, Bindarit, on SAP and explored the mechanisms underlying SAP.

METHODS: Seventy-two Sprague-Dawley rats were randomly divided into a saline control group (group S), an SAP group(group P), and a Bindarit group (group T). The SAP model was induced by retrograde infusion of 4% sodium taurocholate into the bilio-pancreatic duct. Based on the SAP model,Bindarit was injected intraperitoneally in group T, and 0.5%methyl cellulose was injected intraperitoneally in groups S and P. In group S, saline was retrogradely infused into the bilipancreatic duct. Serum amylase levels and the histological changes in the pancreas were assessed at different timepoints in each group. Expression of MCP-1 in serum was measured by enzyme-linked immunoadsorbent assay (ELISA).MCP-1 protein and mRNA expression levels were detected by immunohistochemistry, Western blotting, and semiquantitative reverse transcriptase-polymerase chain reaction(RT-PCR).

RESULTS: Serum amylase levels in groups P and T were higher than those in group S. Serum amylase levels were signi fi cantly lower in group T than in group P at 6 and 12 hours after operation. The levels of MCP-1 in serum at 6 and 12 hours after operation in group P were signi fi cantly higher than in group S, and signi fi cantly lower in group T than in group P at 6 and 12 hours after operation. The pathological damage in the pancreas was milder in group T than in group P. MCP-1 protein and mRNA expression levels in the pancreas were higher in groups P and T than in group S. These expression levels were positively correlated with the pathological damage of pancreatic tissues. The activity of MCP-1 in group T was signi fi cantly lower than in group P.

CONCLUSION: MCP-1 may play important roles in the pathogenesis of SAP. The data suggest that Bindarit ameliorates SAP by inhibiting the activity of MCP-1 in vivo.

Author Af fi liations: Department of Gastroenterology, Af fi liated Hospital of Nantong University, Nantong 226001, China (Zhou GX, Zhu XJ, Ding XL,Zhang H, Chen JP, Qiang H, Zhang HF and Wei Q)

Guo-Xiong Zhou, MD, Department of Gastroenterology, Af fi liated Hospital of Nantong University, Nantong 226001,China (Tel: 86-513-81161826; Fax: 86-513-85519820; Email: zhouguoxiong@medmail.com.cn)

? 2010, Hepatobiliary Pancreat Dis Int. All rights reserved.

May 27, 2009

Accepted after revision February 12, 2010