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    Toll-like receptor 4-mediated apoptosis of pancreatic cells in cerulein-induced acute pancreatitis in mice

    2010-07-07 00:59:37SiQinDingYuanLiZongGuangZhouCunWangLanZhanandBinZhou

    Si-Qin Ding, Yuan Li, Zong-Guang Zhou, Cun Wang, Lan Zhan and Bin Zhou

    Chengdu, China

    Toll-like receptor 4-mediated apoptosis of pancreatic cells in cerulein-induced acute pancreatitis in mice

    Si-Qin Ding, Yuan Li, Zong-Guang Zhou, Cun Wang, Lan Zhan and Bin Zhou

    Chengdu, China

    BACKGROUND:Toll-like receptor 4 (TLR4) plays an important role in the occurrence and development of acute pancreatitis (AP). Apoptosis of pancreatic cells is closely related to the severity of AP. TLR4 is known to induce apoptosis in some cell types and therefore it is of importance to investigate potential associations between TLR4 activity and apoptosis in the setting of AP.

    METHODS:A total of 50 wild-type (C57BL/10J) and TLR4-deficient (C57BL/10ScNJ) mice were divided into three groups: 2-hour, 4-hour, and control groups. Each group was divided into two equal subgroups: TLR4-wild-type mice and TLR4-deficient mice. AP was experimentally induced by 7 intraperitoneal injections of 50 μg/kg cerulein at hourly intervals. Control mice received 7 injections of equal volumes of saline. The severity of pancreatic injury during AP was assessed by serum amylase concentration and histopathology. The level of apoptosis of pancreatic cells in response to AP was evaluated by calculating the apoptotic index (AI) and comparing the expression levels of cytochrome C and Fasassociated protein with death domain (FADD) between TLR4-wild-type and TLR4-deficient mice at 2 time points.

    RESULTS:The AI was found to be significantly lower in the pancreas of TLR4-deficient mice with AP compared to TLR4-wild-type mice at two hours after the last treatment injection. Enzyme-linked immunosorbent assay and realtime reverse transcription-polymerase chain reaction also revealed significantly lower expression of cytochrome C and FADD in the pancreas of TLR4-deficient mice than in TLR4-wild-type animals at the same time point. Serum amylase concentration and morphological severity of AP in pancreatic tissue were found to be similar in the two strains of mice at both time points.

    CONCLUSION:We postulate that TLR4 can mediate apoptosis of pancreatic cells during the early stages of AP, via the activation of both intrinsic and extrinsic apoptotic signaling pathways.

    (Hepatobiliary Pancreat Dis Int 2010; 9: 645-650)

    acute pancreatitis; toll-like receptor 4; apoptosis; cytochrome C; Fas-associated protein with death domain

    Introduction

    Acute pancreatitis (AP) is a common clinical pancreatic disorder, and its incidence has been increasing in the last two decades.[1]The pathogenesis of AP has not yet been elucidated,[2]thus stimulating research into the underlying mechanisms of its development and activity. Severe acute pancreatitis (SAP) which is the most critical form of AP has a high mortality rate. Recent studies have suggested that pancreatic acinar cell apoptosis is the predominant cause of cell death in AP, and that pancreatic acinar cell necrosis is the main cause of cell death in SAP.[3-5]Apoptosis is the process of programmed cell death, during which no inflammatory reaction occurs. In contrast, necrosis is a form of premature cell death, which can lead to the release of a variety of inflammatory agents. The reported elevation of pancreatic acinar cell apoptosis may indicate a beneficial response following the onset of pancreatitis.[6-8]

    Toll-like receptors (TLRs) play a fundamental role in the inflammatory response and host defense against invading microorganisms. Studies found that toll-like receptor 4 (TLR4) is the key receptor to bind bacterial lipopolysaccharide (LPS),[9-11]and it also recognizes some endogenous ligands.[12,13]Activation of TLR4 triggers intracellular signal-transduction cascades leading to the activation of NF-κB, regulatingthe release of inflammatory cytokines. For example, systemic inflammatory response syndrome is due to TLR4 activity,[13]and AP in the rat has been successfully induced by heparan sulphate, which is also an endogenous ligand of TLR4.[14]TLR4 is reported to be rapidly upregulated during the early stage of rat cerulein-induced pancreatitis[15]and downregulated in the late phase of SAP in mice.[16]TLR4 is implicated in regulating mechanisms underlying organ dysfunction and bacterial translocation in the setting of SAP,[16]with silencing of TLR4 alleviating the progression of AP.[17]The upregulation of TLR4 in the early phase of AP is suggested to trigger an inflammatory response, resulting in damage to the organ(s). The downregulation of TLR4 in the late phase of SAP may promote bacterial translocation and subsequent infection. Moreover, in addition to mediating the inflammatory response, TLR4 activity is also associated with the increased apoptosis.[18]LPS-stimulated apoptosis is demonstrated to be dependent on TLR4[10,19,20]in macrophages,[21]endothelial[20]and microglial cells.[22]Furthermore, chlamydia heat shock protein 60 (HSP60) induces trophoblast cell apoptosis through TLR4.[23]

    In conclusion, both apoptosis and TLR4 are strongly associated with AP and activation of TLR4 alone can induce apoptosis in some cell types. However, few studies have found the correlation of TLR4 expression and activity with apoptosis in the setting of AP. The present study investigated the hypothesis that TLR4 mediates pancreatic cell apoptosis in the setting of AP.

    Methods

    Animal groups

    Nine-week-old male and female TLR4-deficient C57BL/ 10ScNJ and wild-type C57BL/10J mice (The Jackson Laboratory, Bar Harbor, Maine, USA), weighing 20-22 g, were used for the current study. The mice were divided into three groups based on the time of sampling: 2 hours and 4 hours (n=20 for each group), and control (n=10). Each group was divided into two equal subgroups: TLR4-wild-type mice and TLR4-deficient mice. The mice were maintained at 23 ℃ on a 12-hour light/dark cycle and fasted for 12 hours prior to experimentation with free access to water. All animal experiments were conducted according to the guidelines of the local Animal Use and Care Committee, and executed according to the National Animal Welfare Law of China.

    Induction of AP

    All of the mice were fasted throughout experiments. AP was induced by intraperitoneal injections of cerulein (Sigma, USA) at 50 μg/kg.bw at 1-hour intervals for 7 hours. Control mice received 7 injections of equal volumes of saline.

    Preparation of serum and tissue samples

    The animals were euthanized by cervical dislocation 2 hours (control and 2-hour group) or 4 hours (4-hour group) after receiving their last injection. The pancreas was rapidly removed and blood samples were obtained by cardiac puncture. All tissues collected were rinsed in Trizol (Gibco, USA) for subsequent RNA isolation, frozen at -80 ℃ for protein extraction, or fixed with 10% formaldehyde before paraffin sectioning for histological examination. Blood samples were immediately centrifuged and serum was stored at -80 ℃ until use.

    Histological examination

    For morphological evaluation of the pathological severity of AP in the pancreatic tissue, 4-mm sections of 10% formalin-fixed, paraffin-embedded tissue were prepared and stained with hematoxylin and eosin (HE). The severity of AP in pancreatic tissue was determined by histological scoring according to the Schmidt score standard.[24]All microscopic sections were analyzed blind. Edema, inflammatory infiltration, parenchymal necrosis and hemorrhage were also scored, ranking from 0 (normal) to 3 (severe).

    Serum amylase determination

    Serum amylase was determined using a commercial kit produced by Jiancheng Institute of Biotechnology (Nanjing, China). The test was performed according to the manufacturer's instructions.

    Apoptosis TUNEL assay

    The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was performed using anin situcell death detection kit (Roche Applied Science, Germany), according to the manufacturer's instructions. Dewaxed, rehydrated paraffin sections (4 μm) of pancreatic tissue were washed in phosphatebuffered saline (PBS) and digested for 20 minutes at 37 ℃ with 20 μg/ml proteinase K (Boehringer, Mannheim, Germany). After washing in PBS, the sections were incubated with TUNEL reaction mixture containing TdT and fluorescein-dUTP, at 37 ℃ for 60 minutes, followed by extensive washing. Finally, a drop of PBS was added to the sections before they were analyzed under a fluorescence microscope (Olympus, Tokyo, Japan) at an objective magnification of 40×, at450-500 nm. Images of the tissue were obtained using an Olympus DD70 BX51 image acquisition system.

    TUNEL-positive cells displayed brilliant green fluorescence. The apoptotic index (AI) was determined as the percentage of TUNEL-positive cells in a highpowered field (×400). The average percentage of TUNEL-positive cells was calculated as the number of TUNEL-positive cells divided by the total number of cells. The number of positive cells was evaluated on 10 randomly-selected high-power fields (×400) by averaging 10 counts per tissue section.

    Enzyme-linked immunosorbent assay (ELISA)

    Pancreatic tissue was frozen to enable ELISA to be performed at a later date. RIPA lysis buffer (Beyotime, Beijing, China) was used to extract total protein from pancreatic tissue. After the frozen pancreatic tissue was cut into small fragments, RIPA lysis buffer containing 1 mmol/L phenylmethylsulfonyl fluoride was added at a ratio of 100 μl RIPA lysis buffer/20 mg of tissue. Samples were then homogenized sufficiently using a glass tissue homogenizer and the homogenates centrifuged at 1400 g for 5 minutes at 4 ℃. The supernatant containing the cytoplasmic protein fractions were collected for analysis of cytochrome C and FADD (Fas-associated protein with death domain). Cytochrome C and FADD were detected using an ELISA (R&D, Minneapolis, MN, USA) according to the manufacturer's instructions. Total protein concentration of pancreatic homogenate was assayed using a BCA protein assay kit (Beyotime) and measured using a microplate reader (Bio-Rad, M550, Hercules, CA, USA) at 595 nm.

    Real-time reverse transcription-polymerase chain reaction (RT-PCR)

    Total RNA from pancreatic tissue was extracted with TRIzol (Gibco, Gaithersburg, MD, USA) following the TRIzol kit protocol. Primers were designed and synthesized by Shanghai Sangon Co. (Shanghai, China).

    Primers for RT cytochrome C and FADD were as follows: Cytochrome C F: 5'-CAA ATC TCC ACG GTC TGT T-3'; Cytochrome C R: 5'-CCC TTT CTC CCT TCT TCT TA-3'; TaqMan probe for cytochrome C: 5'-FAMCAG GCT GCT GGA TTC TCT TAC ACA-TAMRA-3'; FADD F: 5'-CGC CGA CAC GAT CTA CT-3'; FADD R: 5'-CCT CAA TCC CAT CCA TCT T-3'; TaqMan probe for FADD: 5'-FAM-TGC AGG TGG CAT TTG ACA TTG TG-TAMRA-3'.

    Total RNA (5 μg) was reverse transcribed (RT) into cDNA using FTC-2000 (Fengling, Shanghai, China). Five microliters of the RT products were amplified using real-time PCR (iCycler iQ; Bio-Rad Corp., Hercules, CA, USA) over 40 cycles as follows: 94 ℃ for 30 seconds, 55 ℃ for 30 seconds, and 60 ℃ for 1 minute (30 μl total reaction mix). The amplified products were resolved by electrophoresis in 2.0% agarose gel, and visualized by ethidium bromide staining. β-actin was performed simultaneously as a standard internal control. The specific pairs of oligonucleotide primers were as follows: β-actin F: 5'-CGT GAA AAG ATG ACC CAG AT-3'; β-actin R: 5'-ACC CTC ATA GAT GGG CAC A-3'; TaqMan probe for β-actin: 5'-FAM-TCA ACA CCC CAG CCA TGT ACG T-TAMRA-3'.

    The relative expression ratio and real-time RTPCR efficiencies (E) of samples were determined by the following equations:

    The relative expression levels of amplified PCR products were estimated by the relative expression ratio (R). A ratio of <1 indicates downregulation of mRNA expression of the target gene.

    Statistical analysis

    Data were expressed as mean±SD. Real time RTPCR data were analyzed using REST (Relative Expression Software Tool) software.[27]Other experimental data from C57BL/10ScNJ and C57BL/10J mice were compared using unpaired Student'sttests, performed using SPSS 11.5 (SPSS, Chicago, IL., USA). APvalue of less than 0.05 (two-tailed) was considered statistically significant.

    Results

    Serum amylase

    Serum amylase concentration was found not to differ between the TLR4-deficient and TLR4-wild-type mice in the control (t=-0.59,P=0.57), 2-hour (t=0.25,P=0.80) or 4-hour (t=-0.15,P=0.88) groups (Fig. 1).

    Histological examination

    Pancreatic tissue samples taken from all groups were stained with HE, showing varying degrees of inflammation in pancreatic tissue collected at each time point. However, the total histopathological scores between TLR4-wild-type and TLR4-deficient mice did not significantly differ (2 hours,t=0.85,P=0.42; 4 hours,t=0.93,P=0.36; data not shown).

    ELISA analysis of cytochrome C and FADD

    Fig. 1. Serum amylase concentration in control and cerulein-induced pancreatic animals. No significant difference was detected between TLR4-wild-type and TLR4-deficient mice at all time points (P>0.05).

    Fig. 2. TUNEL staining of pancreatic tissue sections from a TLR4-wild-type mouse (A) and a TLR4-deficient mouse (B) from the 2-hour group (original magnification ×400).

    Table 1. ELISA results of cytochrome C and FADD assays

    Table 2. mRNA expression of cytochrome C and FADD (2-hour group)

    Cytochrome C and FADD protein concentrations were determined by ELISA, and normalized to those of total pancreatic protein. The concentrations of both cytochrome C and FADD were significantly elevated in TLR4-wild-type mice of the 2-hour group compared to TLR4-deficient mice of the same group; however, no significant difference was found between the two strains of mice in the 4-hour group (Table 1).

    Apoptosis TUNEL assay

    Results from the TUNEL assay (Fig. 2) were consistent with those of the ELISA. The AI of TLR4-wild-type mice was 8.82±0.46%, compared to 6.74± 0.52% of TLR4-deficient mice in the 2-hour group (t=2.35,P<0.01). No significant difference in AI was found between the two strains of mice comprising the 4-hour group (t=1.26,P>0.05, data not shown).

    Real-time RT-PCR

    Amplified fragments of the expected base pairs were 186 for cytochrome C, 181 for FADD and 180 for β-actin. Real-time PCR amplification efficiencies were calculated from the slopes using FTC-2000 software (Fengling, Shanghai, China) and REST analysis. REST analysis revealed that the mRNA expressions of cytochrome C and FADD were significantly elevated in the 2-hour group of TLR4-wild-type mice when compared to TLR4-deficient mice (Table 2). No significant difference wasobserved between the TLR4-wild-type mice and TLR4-deficient mice in the 4-hour group (data not shown).

    Discussion

    TLR is recognized to activate both innate immune response and adaptive immune response.[28,29]Recent reports have suggested that activation of TLR can also induce apoptosis.[30,31]TLR4 was identified in 1997 as the first mammalian TLR.[10]Through the identification of its exogenous and endogenous ligands, TLR4 is now known to trigger a series of intracellular responses including inflammatory response.[13,14]Some evidence suggests a link between TLR4 activity and apoptosis. Equils et al[23]have demonstrated that chlamydia HSP60 induces apoptosis through TLR4, and that LPS-stimulated apoptosis is dependent on TLR4 in C3H/HeJ mice that lack functional TLR4.[10,19,20]

    Previously we reported TLR4 expression to be rapidly upregulated in the pancreas during the early stages of AP.[15]We hypothesized that the upregulation of TLR4 is associated with the elevated induction of apoptosis in AP. Thus, the present study investigated potential links between TLR4 and apoptosis in AP using a TLR-deficient mouse model. The level of pancreatic cell apoptosis in response to AP was evaluated by calculating AI and comparing the expression levels of cytochrome C and FADD between TLR4-wild-type and TLR4-deficient mice. Cytochrome C is an important member of the intrinsic apoptotic pathway, which functions in the oxidative respiration chain triggering caspase 3 cleavage by activation of caspase 9 during the formation of the apoptosome. FADD is an adaptor molecule involved in the recruitment and activation of caspases, which plays a key role in apoptosis by mediating apoptotic signals from cell membrane receptors into the cells (the extrinsic apoptotic pathway).

    The current study determined the 2-hour group as the only group with significant differences between wild-type and TLR4-deficient mice. The AI of TLR4-wild-type mice was significantly higher than that of TLR4-deficient mice, and the expressions of cytochrome C and FADD in the TLR4-wild-type mice were significantly elevated when compared to the TLR4-deficient mice. These results suggest that TLR4-mediated apoptosis in pancreatic cells occurs early in the onset of AP. This finding is consistent with the observed rapid upregulation of TLR4 and rapid increase in the generation of TLR4 ligands during AP. The significant upregulation of cytochrome C and FADD in the TLR4-wild-type mice 2 hours following the last treatment injection indicated that TLR4-mediated apoptosis may be dependent on both intrinsic and extrinsic pathways. No significant differences were found between the TLR4-wild-type and TLR4-deficient mice in the 4-hour group, potentially due to the low expression of TLR4 in pancreatic tissue.

    In all groups the severity of pancreatic inflammation was assessed by serum amylase concentration, pancreatic edema, inflammatory infiltration, parenchymal necrosis and hemorrhage. The severity of cerulein-induced pancreatitis was found to be similar in the TLR4-wildtype and TLR4-deficient mice in our study, in contrast to the report[17]that the severity of AP is ameliorated in mice that lack TLR4, and TLR4 plays a significant proinflammatory role in the progression of AP.[17]We found no significant difference between the two strains of mice by analysis of histopathology and serum amylase concentration possibly because of the short experimental time. Apoptosis is considered a favorable response in AP as it is suggested to reduce the severity of AP.[6-8]In our study the level of apoptosis differed significantly between the TLR4-wild-type and TLR4-deficient mice in the 2-hour group, suggesting TLR4-mediated apoptosis of pancreatic cells in AP may be a significant mechanism regulating its severity. We believe that while TLR4 induces a pathogenic pro-inflammatory response in AP, it also promotes apoptosis of pancreatic cells. These two reciprocal mechanisms mediated by TLR4 may limit pancreatic inflammation to some extent.

    In summary, our results indicate that when compared with the TLR4-deficient mice, the AI and the expression of cytochrome C and FADD were significantly higher in the pancreas of the TLR4-wild-type mice with AP at two hours after injection. These results suggest that TLR4 mediates apoptosis during the early stage of AP, likely via activation of the intrinsic and extrinsic apoptotic signaling pathways. Further studies are required to clarify the role of TLR4 in SAP.

    Funding:None.

    Ethical approval:All animal experiments were conducted in accordance with the guidelines approved by the Chinese Association of Laboratory Animal Care.

    Contributors:DSQ proposed the study and wrote the first draft. WC analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts. ZZG is the guarantor.

    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.

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    May 4, 2010

    Accepted after revision July 24, 2010

    Author Affiliations: Institute of Digestive Surgery (Ding SQ, Li Y, Zhou ZG, Wang C, Zhanland Zhou B), Department of Gastrointestinal Surgery (Ding SQ, Zhou ZG and Wang C), and Department of Pediatric Surgery (Li Y), West China Hospital, Sichuan University, Chengdu 610041, China

    Zong-Guang Zhou, MD, Institute of Digestive Surgery, West China Hospital, Sichuan University, Chengdu 610041, China (Tel: 86-28-85164035; Fax: 86-28-85164036; Email: Zhou767@163.com)

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

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