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Effect of Chaiqinchengqi decoction on inositol requiring enzyme 1α in alveolar macrophages of dogs with acute necrotising pancreatitis induced by sodium taurocholate
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J Tradit Chin Med 2015 August 15; 35(4): 434-439 ISSN 0255-2922 © 2015 JTCM. All rights reserved.
EXPERIMENTAL STUDY TOPIC
Effect of Chaiqinchengqi decoction on inositol requiring enzyme 1α in alveolar macrophages of dogs with acute necrotising pancreatitis induced by sodium taurocholate
Guo Jia, Wang Xiaoxiang, Luo Ruijie, Zhang Xiaoxin, Yang Xiaonan, Xia Qing, Xue Ping aa Guo Jia, Wang Xiaoxiang, Luo Ruijie, Zhang Xiaoxin, Yang Xiaonan, Xia Qing, Xue Ping, Department of Integrated Traditional Chinese and Western Medicine, West China Hospital, Sichuan University, Chengdu 610041, China Supported by the National Natural Science Foundation of China (the Mechanism of Protein Molecular Brake of Suppression the Releasing of Pro-Inflammatory Cytokines of Macrophage in Acute Necrotizing Pancreatitis by Chaiqinchengqi Decoction, No. 81072910); Science and Technology Support Program of Sichuan (Effect of Chaiqincheng Decoction on the Gastrointestinal Dysfunction in Severe Acute Pancreatitis and the Establishment of Evaluation System, No. 2014SZ0211) Correspondence to: Associate Prof. Xue Ping, Department of Integrated Traditional Chinese and Western Medicine, West China Hospital, Sichuan University, Chengdu 610041, China. [email protected] Telephone: +86-28-85423373 Accepted: September 12, 2014
CQCQD every 2 h for 6 h. Two hours after the last administration of either CQCQD or saline, they were sacrificed by anaesthesia. AMs were collected to determine the IRE1α and Interleukin-1β (IL-1β) mRNA and protein expression, and pancreatic tissues were collected for histopathology analysis. RESULTS: Compared with the ANP group, the mRNA and protein expression of IRE1α and the protein expression of IL-1β of AMs in the CQCQD group were significantly down-regulated, and the pancreatic histopathology score of the CQCQD group also was lower. There was no significant difference in the mRNA expression of IL-1β of AMs between the two groups. CONCLUSION: The CQCQD-induced down-regulation of the IL-1β protein expression may involve the down-regulation of the mRNA and protein expression of IRE1α in AMs.
Abstract
© 2015 JTCM. All rights reserved.
OBJECTIVE: To investigate the effect of Chaiqinchengqi decoction (CQCQD) on inositol requiring enzyme 1α (IRE1α) in alveolar macrophages (AMs) of the dog model of acute necrotising pancreatitis (ANP) induced by sodium taurocholate.
Key words: IRE1alpha protein, dog; Pancreatitis; Macrophages, Alveolar; Chaiqinchengqi decoction
INTRODUCTION
METHODS: Fifteen beagle dogs were randomised into a control group, ANP group and CQCQD group (n = 5 per group). ANP was induced by a retrograde duct injection of 50 mg/kg of 5% sodium taurocholate. The dogs in the control group received injections of the same volume of saline as the sodium taurocholate. After the models were induced, the dogs in the CQCQD group were administered 10 mL/kg JTCM | www. journaltcm. com
Multiple organ dysfunction syndrome (MODS) as a result of systemic inflammatory response syndrome (SIRS) is the major cause of death of patients suffering from severe acute pancreatitis (SAP) in its acute phase.1-3 Timely and effective mitigation of SIRS can reduce the incidence of organ failure.4 It has been confirmed that SIRS is caused by the imbalance of pro-inflammatory cytokines and anti-inflammatory cyto434
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kines, which is induced by the amplifying inflammatory cascade through excessive release of inflammatory cytokines.5 Therefore, reducing the release of the inflammatory cytokines has become the key to preventing the incidence of MODS in SAP patients. Our previous studies have shown that Chaiqinchengqi decoction (CQCQD), a Chinese medication modified from Dachengqi decoction, was effective in treating acute pancreatitis (AP) both in vivo and in vitro.6-9 We found that CQCQD may have multiple activities, including protection against pancreatic acinar cell Ca2 + overload and reduction in the serum levels of interleukin-6 (IL-6), tumour necrosis factor-alpha (TNF-α) and nuclear factor-kappa B,10,11 in the treatment of AP. Notably, in an experimental AP induced by ductal infusion of taurocholate into the pancreas, CQCQD treatment significantly reduced choline esterase, increased cholineacetyl transferase, up-regulated neuronal acetylcholine receptor alpha 7 (nAChRα7) in peritoneal macrophages and simultaneously reduced IL-6 and TNF-α. The interactions of Ach and nAChRα7 inhibited the release of macrophage pro-inflammatory cytokines, such as IL-1, IL-6 and IL-18, at the post-transcriptional level of protein synthesis, but did not influence the release of anti-inflammatory cytokines, such as IL-10.12-14 However, the exact mechanism underlying these effects and the targets are unclear. Several studies have found that inositol requiring enzyme 1α (IRE1α), a protein possessing protein kinase and endoribonuclease functions, is one of the important components of the unfolded protein response (UPR) signalling pathways of the endoplasmic reticulum (ER).15-17 The UPR signalling pathways play a key role in increasing the ER protein-folding capacity when the mRNA translation rate increases. The UPR signalling pathways include IRE1α, which activates transcription factor 6 (ATF6) and is important in the UPR, and PKR-like ER kinase (PERK).18,19 However, it is unclear whether CQCQD affects the IRE1α expression to regulate the protein processing functions of the ER in macrophages and reduce the release of inflammatory cytokines in SAP. In the present study, we used an acute necrotising pancreatitis (ANP) model induced by sodium taurocholate in beagle dogs to investigate the effect of CQCQD on the mRNA and protein expression of IRE1α in alveolar macrophages (AMs).
(Carlsbad, CA, USA). dNTPs and the RNAase inhibitor were from Takara Co. (Katsushika-Ku, Tokyo, Japan). Taq DNA polymerase was from Promega (Madison, WI, USA). Dulbecco's modified Eagle medium (DMEM) is a product of Life Technologies (GIBCO BRL, Grand Island, NY, USA), which was pre-oxygenated for 30 min before use. All other reagents were purchased from Sigma. Preparation of CQCQD The Chinese medicinal herbs in CQCQD, including Chaihu (Radix Bupleuri Chinensis) 15 g, Huangqin (Radix Scutellariae Baicalensis) 15 g, Dahuang (Radix Et Rhizoma Rhei Palmati) 20 g, Mangxiao (Nalrii Sulfas) 20 g, Houpu (Cortex Magnoliae Officinalis) 15 g, Zhishi (Fructus Aurantii Immaturus) 15 g, Yinchen (Herba Artemisia Capillaris) 15 g, Zhizi (Fructus Gardeniae) 20 g, were purchased from the pharmacy of West Chinese Hospital of Sichuan University (Chengdu, China). These herbs were first made into a 200 mL decoction and were then lyophilised into powder. Before administration to the animals, the lyophilised CQCQD powder was prepared at a concentration of 2 g/mL of crude herbs. Animals and protocol Fifteen 8-month-old beagle dogs of ordinary grade, weighing (10.0 ± 0.5) kg were purchased from the Laboratory Animal Centre of Huaxi, Sichuan University (certificate of quality No. SCXK[Chuan] 2008-01). The animals were kept at constant room temperature with a 12-h light/dark cycle and were allowed free access to water and standard laboratory chow. The animal experiments performed in this study were approved by the Animal Care Committee of Sichuan University. The beagle dogs were randomised into a control group, AP group and CQCQD group (n = 5 in each group). ANP was induced by retrograde duct injection of 50 mg/kg of 5% sodium taurocholate with a micropump (60 mL/h).20 The dogs in the control group were injected the same volume of saline as that of sodium taurocholate. After the model was induced, the dogs in the CQCQD group were administered 10 mL/kg CQCQD by a jejuna tube every 2 h for 6 h, while the dogs in the other two groups received an equivalent volume of saline. Dogs were sacrificed by anaesthesia 2 h after the last administration of either CQCQD or saline. AMs were collected for reverse transcription and polymerase chain reaction (RT-PCR), and western blot analysis, to determine the IRE1α and IL-1β mRNA and protein expression. Pancreatic tissues were collected for histopathology analysis.
METHODS Reagents Sodium taurocholate and bovine serum albumin (BSA) were purchased from Sigma (St. Louis, MO, USA). Trizol and diethyl pyrocarbonate (DEPC) were from Invitrogen (Waltham, MA, USA). Hexanucleotide random primers and Superscript II RNase H-reverse transcriptase (RNase H-RT) were from Life Technologies JTCM | www. journaltcm. com
Collection of Dog AMs The AMs were collected as follows.21-23 After the dogs were sacrificed, the chest skin was cut after being disinfected with Eric for 3 min. The right lower lobe was 435
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ing the Novex X-Cell Ⅱ system. The membranes were blocked overnight in 5% BSA, followed by a 3-h incubation with primary antibodies (0.5 μg/mL) at room temperature. Then, the membranes were washed and incubated with secondary antibodies for 1 h. β-Actin was used as an internal housekeeping protein. Immunoreactivity was detected using a chemiluminescence system and quantified by Image J software.
clipped along the hilum, and then 40 mL of cold (4 ℃) PBS were injected into the main bronchus alveola five times to collect the rinse fluid. Then, AMs were collected with differential adhesion methods as follows. The samples were centrifuged at 400 × g for 10 min, and the supernatant was discarded. The cells were resuspended twice with 100 mL PBS, and the settling cells were separated, collected and reserved. Detection of IRE1α and IL-1β mRNA with RT-PCR After the AMs of each group were collected, the total RNA was extracted using Trizol. The integrity of the RNA was confirmed by agarose gel electrophoresis. The quantity and purity of the RNA were determined by an ultraviolet spectrophotometer. The RNA was reverse-transcribed using hexanucleotide random primers with RNase H-RT. The cDNA was amplified as a template for subsequent PCR, using Taq DNA polymerase in a Perkin Elmer/Cetus DNA Thermal Cycler. The PCR reaction system included 5 μL of cDNA-RT product, 3 μL of 10 × PCR buffer, 3 μL of 25 mmol/L MgCl2, 0.36 μL of 25 mmol/L dNTP, 1 μL of 10 μmol/L upstream primer, 1 μL of 10 μmol/L downstream primer, 0.6 μL of 10 μmol/L probe, 0.3 μL of 5 U/μL Taq polymerase, and 15.74 μL of deionised double-distilled water. The PCR conditions were as follows: initial denaturation at 94 ℃ for 2 min, followed by 45 cycles of 94 ℃ for 20 s, 55 ℃ for 30 s and 60 ℃ for 40 s. The negative control contained the same volume of deionised double-distilled water instead of cDNA, and was amplified to check for DNA contamination. The β-actin gene was used as an internal housekeeping gene. The primers for IRE1α and IL-1β were designed according to a method reported previously.24 Primers for IRE1α, IL-1β and β-actin genes were synthesised by Shanghai Biotechnology Co. Ltd. (Shanghai, China; Table 1). The amplified DNA was resolved by agarose gel electrophoresis and stained with ethidium bromide. The bands were visualised and photographed under ultraviolet light.
Histopathological analysis of pancreatic tissues After the removal of pancreatic tissues, the tissue samples were fixed in 4% neutral buffer formaldehyde, embedded with paraffin wax, cut into slices, stained with haematoxylin and eosin (HE), and then examined under a light microscope. For each HE-stained slide, 10 visual fields under a high-power microscope (magnification × 400) were randomly selected and scored by one specific pathologist who was blinded to the experimental design.25,26 The mean score of the 10 visual fields of each slide was calculated as the histology score. Statistical analysis All data were analysed with SPSS 14.0 (SPSS Inc., Chicago, IL, USA). Data were expressed as the mean ± standard deviation ( xˉ ± s). Variables with normal distribution were analysed by LSD test. Variables with abnormal distribution were analysed by Kruskal-Wallis H test. The Relative Expression Software Tool was used for statistical analysis of gene expression. P < 0.05 was considered statistically significant.
RESULTS Histopathological scores of the pancreas Photographs of the pancreatic pathology are shown in Figure 1A-C. The histopathological mean score of the pancreatic tissues in the ANP group (7.2 ± 1.3) was the highest among the three groups, followed by the CQCQD group (5.1 ± 0.9) and then the control group (1.0 ± 0.6, P < 0.05, Figure 1D).
Detection of IRE1α and IL-1β protein expression with western blotting AMs were lysed using the Sonics Vibracell sonicator with a 0.4-mm diameter probe. The amount of protein in each sample was determined by the Bradford assay using BSA as a standard. The samples were denatured at 95 ℃ for 3 min in 1× Nu Page SDS sample buffer. Equal amounts of protein were loaded, electrophoresed, and transferred to Immobilon-P membranes us-
Expression of IRE1α and IL-1β mRNA In the RT-PCR analysis, β-actin, IRE1α and IL-1β mRNA were confirmed to be 111, 99 and 178 bp, respectively (Figure 2). There were no significant differences in the cycle threshold (Ct) value of β-actin
Table 1 Probes, Primers and Products (bp) in RT-PCR Gene
Probe
Upstream primer
Downstream primer
Product (bp)
5'-ACCACCAGCCCATCACCA 5'-CAAGATGTCCCACTTTGTGT 5'-CAAGATGTCCCACT 99 TTGG -3' TTGTGTCC -3' CC -3' 5'-TTCCATCTTCCTCTTTGG 5'-CAAACAAGTGGTGT 5'-GATACCTGTGGTCTTGGGC 178 IL-1β GTAG -3' TCCACATG -3' ATC -3' 5'-CTGTCCACCTTCCAGCAGA - 5'-GAAGATCAAGATCAT 5'-TACTCCTGCTTGCTGATC 111 β-actin CA -3' TGCTCCT 3' 3' Notes: RT-PCR: reverse transcriptase-polymerase chain reaction; IRE1α: inositol requiring enzyme 1α; IL-1β: Interleukin-1β. IRE1α
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10 Histopathological score
C
B
A
D
a
8 6 4 2 0
Control
ANP CQCQD Groups Figure 1 Histopathological photographs and histopathological scores of the pancreas in the three groups A-C: histopathological photographs of the pancreas (HE staining, ×400); A: control group; B: ANP group; C: CQCQD group. D: histopathological score of the pancreas (n = 5). control group: dogs injected intraductally with normal saline; ANP group: AP dogs induced by sodium taurocholate; CQCQD group: the AP dogs were administered 10 mL/kg CQCQD by jejuna tube every 2 h for 6 h. The dogs in the control group and ANP group received an equivalent volume of saline. ANP: acute necrotising pancreatitis; AP: acute pancreatitis; CQCQD: Chaiqinchengqi decoction, HE: Hematoxylin-Eosin. aP < 0.05, compared with the control group and the CQCQD group. 1
2
3
4
IRE1α
99
β-actin
111
IL-1β
Table 2 Ct values of β-actin, IRE1α and IL-1β in RT-PCR (n = 5, xˉ ± s)
bp
178
Figure 2 Agar gel electrophoresis images of PCR products of β-actin, IRE1α and IL-1β mRNA 1: marker; 2: control group; 3: ANP group; 4: CQCQD group. control group: dogs injected intraductally with normal saline; ANP group: AP dogs induced by sodium taurocholate; CQCQD group: the AP dogs were administered 10 mL/kg CQCQD by jejuna tube every 2 h for 6 h. The dogs in the control group and ANP group received an equivalent volume of saline. ANP: acute necrotising pancreatitis; AP: acute pancreatitis; CQCQD: Chaiqinchengqi decoction; IRE1α: inositol requiring enzyme 1α; IL-1β: interleukin-1β; PCR: polymerase chain reaction.
IRE1α
IL-1β
Control group
25.0±1.5
26.0±1.3
27.9±1.6b
ANP group
24.3±1.3
24.5±1.2
25.1±1.2
CQCQD group
24.0±1.6
28.0±1.1
24.9±1.4
a
Notes: control group: dogs injected intraductally with normal saline; ANP group: AP dogs induced by sodium taurocholate; CQCQD group: the AP dogs were administered 10 mL/kg CQCQD by jejuna tube every 2 h for 6 h. The dogs in the control group and ANP group received an equivalent volume of saline. ANP: acute necrotising pancreatitis; AP: acute pancreatitis; CQCQD: Chaiqinchengqi decoction; IRE1α: inositol requiring enzyme 1α; IL-1β: interleukin-1β.aP < 0.05, compared with the ANP group; bP < 0.05, compared with the ANP group and the CQCQD group.
group was significantly higher than that of the control group.
mRNA among the three groups (P > 0.05, Table 2), but the Ct of IRE1α mRNA in the CQCQD treatment group was significantly higher than that of the other two groups (P < 0.05). These findings suggest that the expression of IRE1α mRNA in the CQCQD group was significantly lower than that of the other two groups. The Ct of IL-1β mRNA in the CQCQD group and the ANP group was significantly lower than that of the control group (P < 0.05). There were no significant differences in the Ct values of IL-1β mRNA between the CQCQD group and the ANP group (P > 0.05, Table 2). These findings suggest that the expression of IL-1β mRNA in the CQCQD group and ANP JTCM | www. journaltcm. com
β-actin
Groups
IRE1α and IL-1β protein expression The images of the western blot analysis of IRE1α, IL-1β and β-actin are shown in Figure 3A. The band intensity ratio of IRE1α and β-actin in the control group (0.8 ± 0.3) was smaller than that of the ANP group (0.9 ± 0.4), but was higher than that of the CQCQD group (0.4 ± 0.1, P < 0.05, Figure 3B). The band intensity ratio of IL-1β and β-actin in the ANP group (1.4 ± 0.5) was higher than those of the CQCQD group (0.8 ± 0.4) and the control group (0.6 ± 0.3, P < 0.05, Figure 3C). 437
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Guo J et al. / Experimental Study A IRE1β
1
2
3
4
5
6
7
8
9
QD affects the expression of IRE1α to reduce the synthesis of IL-1β in AMs of ANP dogs. Our data showed that the mRNA and protein expression of IRE1α in AMs was significantly down-regulated after CQCQD treatment (P < 0.05, Figures 2B, 3A and 3B). The mRNA expression of IL-1β in AMs in the ANP group and the CQCQD group was significantly up-regulated compared with that in the control group (P < 0.05, Figure 2C), but the protein expression of IL-1β was significantly down-regulated after treatment with CQCQD (P < 0.05, Figures 2C, 3A and 3C). . These findings suggest that the down-regulated IRE1α in AMs may play a key role in the anti-inflammatory effect of CQCQD on AP. IRE1α is widely expressed in various types of inflammatory cells including lymphocytes, monocytes and macrophages.28 The current study only explored IRE1α in AMs to investigate the anti-inflammatory effect of CQCQD on ANP. Arguably IRE1α in other cell types may also contribute to the anti-inflammatory effect on ANP. Therefore, it is of interest to examine whether CQCQD also affects IRE1α expression in other cell types. In summary, down-regulating the expression of IRE1α in AMs may be the central mechanism behind the anti-inflammatory effect of CQCQD treatment on ANP.
kDa 120
β- actin
43
IL-1β
31
IRE1β / β-actin
B 1.5
1.0
a
0.5
0
Control
C 2.0
ANP
CQCQD
IL-1β / β-actin
b
1.5 1.0 0.5 0
REFERENCES
Control
ANP CQCQD Groups Figure 3 Western blot analysis of IRE1α and IL-1β (n = 5) A: agar gel electrophoresis photographs of western blots of IRE1α, IL-1β and β-actin. 1-3: control group; 4-6: ANP group; 7-9: CQCQD group. B: the ratio of the band intensity of IRE1α and β-actin. aP < 0.05, compared with the control group and ANP group. C: the ratio of the band intensity of IL-1β and β-actin. aP < 0.05, compared with the ANP group; b P < 0.01, compared with the control group. control group: dogs injected intraductally with normal saline; ANP group: AP dogs induced by sodium taurocholate; CQCQD group: the AP dogs were administered 10 mL/kg CQCQD by jejuna tube every 2 h for 6 h. The dogs in the control group and ANP group received an equivalent volume of saline. ANP: acute necrotising pancreatitis; AP: acute pancreatitis; CQCQD: Chaiqinchengqi decoction.
1
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DISCUSSION The CQCQD modified from the Dachengqi decoction is an effective herbal prescription for the treatment of AP.7 It has been shown to reduce SIRS through the cholinergic anti-inflammatory pathway in rats with acute necrotising pancreatitis.10 Our previous studies have confirmed that CQCQD could up-regulate the nAChRα7 expression of peritoneal macrophages and reduce the serum IL-6 and TNF-α levels in mice with AP, but the specific mechanism was unclear. Study had confirmed that the IRE1α plays a key role in the UPR signalling pathways to increase the ER protein-folding capacity when mRNA translation is increased.27 In this study, we investigated whether CQCJTCM | www. journaltcm. com
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J Tradit Chin Med 2015 August 15; 35(4): 434-439 ISSN 0255-2922 © 2015 JTCM. All rights reserved.
EXPERIMENTAL STUDY TOPIC
Effect of Chaiqinchengqi decoction on inositol requiring enzyme 1α in alveolar macrophages of dogs with acute necrotising pancreatitis induced by sodium taurocholate
Guo Jia, Wang Xiaoxiang, Luo Ruijie, Zhang Xiaoxin, Yang Xiaonan, Xia Qing, Xue Ping aa Guo Jia, Wang Xiaoxiang, Luo Ruijie, Zhang Xiaoxin, Yang Xiaonan, Xia Qing, Xue Ping, Department of Integrated Traditional Chinese and Western Medicine, West China Hospital, Sichuan University, Chengdu 610041, China Supported by the National Natural Science Foundation of China (the Mechanism of Protein Molecular Brake of Suppression the Releasing of Pro-Inflammatory Cytokines of Macrophage in Acute Necrotizing Pancreatitis by Chaiqinchengqi Decoction, No. 81072910); Science and Technology Support Program of Sichuan (Effect of Chaiqincheng Decoction on the Gastrointestinal Dysfunction in Severe Acute Pancreatitis and the Establishment of Evaluation System, No. 2014SZ0211) Correspondence to: Associate Prof. Xue Ping, Department of Integrated Traditional Chinese and Western Medicine, West China Hospital, Sichuan University, Chengdu 610041, China. [email protected] Telephone: +86-28-85423373 Accepted: September 12, 2014
CQCQD every 2 h for 6 h. Two hours after the last administration of either CQCQD or saline, they were sacrificed by anaesthesia. AMs were collected to determine the IRE1α and Interleukin-1β (IL-1β) mRNA and protein expression, and pancreatic tissues were collected for histopathology analysis. RESULTS: Compared with the ANP group, the mRNA and protein expression of IRE1α and the protein expression of IL-1β of AMs in the CQCQD group were significantly down-regulated, and the pancreatic histopathology score of the CQCQD group also was lower. There was no significant difference in the mRNA expression of IL-1β of AMs between the two groups. CONCLUSION: The CQCQD-induced down-regulation of the IL-1β protein expression may involve the down-regulation of the mRNA and protein expression of IRE1α in AMs.
Abstract
© 2015 JTCM. All rights reserved.
OBJECTIVE: To investigate the effect of Chaiqinchengqi decoction (CQCQD) on inositol requiring enzyme 1α (IRE1α) in alveolar macrophages (AMs) of the dog model of acute necrotising pancreatitis (ANP) induced by sodium taurocholate.
Key words: IRE1alpha protein, dog; Pancreatitis; Macrophages, Alveolar; Chaiqinchengqi decoction
INTRODUCTION
METHODS: Fifteen beagle dogs were randomised into a control group, ANP group and CQCQD group (n = 5 per group). ANP was induced by a retrograde duct injection of 50 mg/kg of 5% sodium taurocholate. The dogs in the control group received injections of the same volume of saline as the sodium taurocholate. After the models were induced, the dogs in the CQCQD group were administered 10 mL/kg JTCM | www. journaltcm. com
Multiple organ dysfunction syndrome (MODS) as a result of systemic inflammatory response syndrome (SIRS) is the major cause of death of patients suffering from severe acute pancreatitis (SAP) in its acute phase.1-3 Timely and effective mitigation of SIRS can reduce the incidence of organ failure.4 It has been confirmed that SIRS is caused by the imbalance of pro-inflammatory cytokines and anti-inflammatory cyto434
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kines, which is induced by the amplifying inflammatory cascade through excessive release of inflammatory cytokines.5 Therefore, reducing the release of the inflammatory cytokines has become the key to preventing the incidence of MODS in SAP patients. Our previous studies have shown that Chaiqinchengqi decoction (CQCQD), a Chinese medication modified from Dachengqi decoction, was effective in treating acute pancreatitis (AP) both in vivo and in vitro.6-9 We found that CQCQD may have multiple activities, including protection against pancreatic acinar cell Ca2 + overload and reduction in the serum levels of interleukin-6 (IL-6), tumour necrosis factor-alpha (TNF-α) and nuclear factor-kappa B,10,11 in the treatment of AP. Notably, in an experimental AP induced by ductal infusion of taurocholate into the pancreas, CQCQD treatment significantly reduced choline esterase, increased cholineacetyl transferase, up-regulated neuronal acetylcholine receptor alpha 7 (nAChRα7) in peritoneal macrophages and simultaneously reduced IL-6 and TNF-α. The interactions of Ach and nAChRα7 inhibited the release of macrophage pro-inflammatory cytokines, such as IL-1, IL-6 and IL-18, at the post-transcriptional level of protein synthesis, but did not influence the release of anti-inflammatory cytokines, such as IL-10.12-14 However, the exact mechanism underlying these effects and the targets are unclear. Several studies have found that inositol requiring enzyme 1α (IRE1α), a protein possessing protein kinase and endoribonuclease functions, is one of the important components of the unfolded protein response (UPR) signalling pathways of the endoplasmic reticulum (ER).15-17 The UPR signalling pathways play a key role in increasing the ER protein-folding capacity when the mRNA translation rate increases. The UPR signalling pathways include IRE1α, which activates transcription factor 6 (ATF6) and is important in the UPR, and PKR-like ER kinase (PERK).18,19 However, it is unclear whether CQCQD affects the IRE1α expression to regulate the protein processing functions of the ER in macrophages and reduce the release of inflammatory cytokines in SAP. In the present study, we used an acute necrotising pancreatitis (ANP) model induced by sodium taurocholate in beagle dogs to investigate the effect of CQCQD on the mRNA and protein expression of IRE1α in alveolar macrophages (AMs).
(Carlsbad, CA, USA). dNTPs and the RNAase inhibitor were from Takara Co. (Katsushika-Ku, Tokyo, Japan). Taq DNA polymerase was from Promega (Madison, WI, USA). Dulbecco's modified Eagle medium (DMEM) is a product of Life Technologies (GIBCO BRL, Grand Island, NY, USA), which was pre-oxygenated for 30 min before use. All other reagents were purchased from Sigma. Preparation of CQCQD The Chinese medicinal herbs in CQCQD, including Chaihu (Radix Bupleuri Chinensis) 15 g, Huangqin (Radix Scutellariae Baicalensis) 15 g, Dahuang (Radix Et Rhizoma Rhei Palmati) 20 g, Mangxiao (Nalrii Sulfas) 20 g, Houpu (Cortex Magnoliae Officinalis) 15 g, Zhishi (Fructus Aurantii Immaturus) 15 g, Yinchen (Herba Artemisia Capillaris) 15 g, Zhizi (Fructus Gardeniae) 20 g, were purchased from the pharmacy of West Chinese Hospital of Sichuan University (Chengdu, China). These herbs were first made into a 200 mL decoction and were then lyophilised into powder. Before administration to the animals, the lyophilised CQCQD powder was prepared at a concentration of 2 g/mL of crude herbs. Animals and protocol Fifteen 8-month-old beagle dogs of ordinary grade, weighing (10.0 ± 0.5) kg were purchased from the Laboratory Animal Centre of Huaxi, Sichuan University (certificate of quality No. SCXK[Chuan] 2008-01). The animals were kept at constant room temperature with a 12-h light/dark cycle and were allowed free access to water and standard laboratory chow. The animal experiments performed in this study were approved by the Animal Care Committee of Sichuan University. The beagle dogs were randomised into a control group, AP group and CQCQD group (n = 5 in each group). ANP was induced by retrograde duct injection of 50 mg/kg of 5% sodium taurocholate with a micropump (60 mL/h).20 The dogs in the control group were injected the same volume of saline as that of sodium taurocholate. After the model was induced, the dogs in the CQCQD group were administered 10 mL/kg CQCQD by a jejuna tube every 2 h for 6 h, while the dogs in the other two groups received an equivalent volume of saline. Dogs were sacrificed by anaesthesia 2 h after the last administration of either CQCQD or saline. AMs were collected for reverse transcription and polymerase chain reaction (RT-PCR), and western blot analysis, to determine the IRE1α and IL-1β mRNA and protein expression. Pancreatic tissues were collected for histopathology analysis.
METHODS Reagents Sodium taurocholate and bovine serum albumin (BSA) were purchased from Sigma (St. Louis, MO, USA). Trizol and diethyl pyrocarbonate (DEPC) were from Invitrogen (Waltham, MA, USA). Hexanucleotide random primers and Superscript II RNase H-reverse transcriptase (RNase H-RT) were from Life Technologies JTCM | www. journaltcm. com
Collection of Dog AMs The AMs were collected as follows.21-23 After the dogs were sacrificed, the chest skin was cut after being disinfected with Eric for 3 min. The right lower lobe was 435
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ing the Novex X-Cell Ⅱ system. The membranes were blocked overnight in 5% BSA, followed by a 3-h incubation with primary antibodies (0.5 μg/mL) at room temperature. Then, the membranes were washed and incubated with secondary antibodies for 1 h. β-Actin was used as an internal housekeeping protein. Immunoreactivity was detected using a chemiluminescence system and quantified by Image J software.
clipped along the hilum, and then 40 mL of cold (4 ℃) PBS were injected into the main bronchus alveola five times to collect the rinse fluid. Then, AMs were collected with differential adhesion methods as follows. The samples were centrifuged at 400 × g for 10 min, and the supernatant was discarded. The cells were resuspended twice with 100 mL PBS, and the settling cells were separated, collected and reserved. Detection of IRE1α and IL-1β mRNA with RT-PCR After the AMs of each group were collected, the total RNA was extracted using Trizol. The integrity of the RNA was confirmed by agarose gel electrophoresis. The quantity and purity of the RNA were determined by an ultraviolet spectrophotometer. The RNA was reverse-transcribed using hexanucleotide random primers with RNase H-RT. The cDNA was amplified as a template for subsequent PCR, using Taq DNA polymerase in a Perkin Elmer/Cetus DNA Thermal Cycler. The PCR reaction system included 5 μL of cDNA-RT product, 3 μL of 10 × PCR buffer, 3 μL of 25 mmol/L MgCl2, 0.36 μL of 25 mmol/L dNTP, 1 μL of 10 μmol/L upstream primer, 1 μL of 10 μmol/L downstream primer, 0.6 μL of 10 μmol/L probe, 0.3 μL of 5 U/μL Taq polymerase, and 15.74 μL of deionised double-distilled water. The PCR conditions were as follows: initial denaturation at 94 ℃ for 2 min, followed by 45 cycles of 94 ℃ for 20 s, 55 ℃ for 30 s and 60 ℃ for 40 s. The negative control contained the same volume of deionised double-distilled water instead of cDNA, and was amplified to check for DNA contamination. The β-actin gene was used as an internal housekeeping gene. The primers for IRE1α and IL-1β were designed according to a method reported previously.24 Primers for IRE1α, IL-1β and β-actin genes were synthesised by Shanghai Biotechnology Co. Ltd. (Shanghai, China; Table 1). The amplified DNA was resolved by agarose gel electrophoresis and stained with ethidium bromide. The bands were visualised and photographed under ultraviolet light.
Histopathological analysis of pancreatic tissues After the removal of pancreatic tissues, the tissue samples were fixed in 4% neutral buffer formaldehyde, embedded with paraffin wax, cut into slices, stained with haematoxylin and eosin (HE), and then examined under a light microscope. For each HE-stained slide, 10 visual fields under a high-power microscope (magnification × 400) were randomly selected and scored by one specific pathologist who was blinded to the experimental design.25,26 The mean score of the 10 visual fields of each slide was calculated as the histology score. Statistical analysis All data were analysed with SPSS 14.0 (SPSS Inc., Chicago, IL, USA). Data were expressed as the mean ± standard deviation ( xˉ ± s). Variables with normal distribution were analysed by LSD test. Variables with abnormal distribution were analysed by Kruskal-Wallis H test. The Relative Expression Software Tool was used for statistical analysis of gene expression. P < 0.05 was considered statistically significant.
RESULTS Histopathological scores of the pancreas Photographs of the pancreatic pathology are shown in Figure 1A-C. The histopathological mean score of the pancreatic tissues in the ANP group (7.2 ± 1.3) was the highest among the three groups, followed by the CQCQD group (5.1 ± 0.9) and then the control group (1.0 ± 0.6, P < 0.05, Figure 1D).
Detection of IRE1α and IL-1β protein expression with western blotting AMs were lysed using the Sonics Vibracell sonicator with a 0.4-mm diameter probe. The amount of protein in each sample was determined by the Bradford assay using BSA as a standard. The samples were denatured at 95 ℃ for 3 min in 1× Nu Page SDS sample buffer. Equal amounts of protein were loaded, electrophoresed, and transferred to Immobilon-P membranes us-
Expression of IRE1α and IL-1β mRNA In the RT-PCR analysis, β-actin, IRE1α and IL-1β mRNA were confirmed to be 111, 99 and 178 bp, respectively (Figure 2). There were no significant differences in the cycle threshold (Ct) value of β-actin
Table 1 Probes, Primers and Products (bp) in RT-PCR Gene
Probe
Upstream primer
Downstream primer
Product (bp)
5'-ACCACCAGCCCATCACCA 5'-CAAGATGTCCCACTTTGTGT 5'-CAAGATGTCCCACT 99 TTGG -3' TTGTGTCC -3' CC -3' 5'-TTCCATCTTCCTCTTTGG 5'-CAAACAAGTGGTGT 5'-GATACCTGTGGTCTTGGGC 178 IL-1β GTAG -3' TCCACATG -3' ATC -3' 5'-CTGTCCACCTTCCAGCAGA - 5'-GAAGATCAAGATCAT 5'-TACTCCTGCTTGCTGATC 111 β-actin CA -3' TGCTCCT 3' 3' Notes: RT-PCR: reverse transcriptase-polymerase chain reaction; IRE1α: inositol requiring enzyme 1α; IL-1β: Interleukin-1β. IRE1α
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10 Histopathological score
C
B
A
D
a
8 6 4 2 0
Control
ANP CQCQD Groups Figure 1 Histopathological photographs and histopathological scores of the pancreas in the three groups A-C: histopathological photographs of the pancreas (HE staining, ×400); A: control group; B: ANP group; C: CQCQD group. D: histopathological score of the pancreas (n = 5). control group: dogs injected intraductally with normal saline; ANP group: AP dogs induced by sodium taurocholate; CQCQD group: the AP dogs were administered 10 mL/kg CQCQD by jejuna tube every 2 h for 6 h. The dogs in the control group and ANP group received an equivalent volume of saline. ANP: acute necrotising pancreatitis; AP: acute pancreatitis; CQCQD: Chaiqinchengqi decoction, HE: Hematoxylin-Eosin. aP < 0.05, compared with the control group and the CQCQD group. 1
2
3
4
IRE1α
99
β-actin
111
IL-1β
Table 2 Ct values of β-actin, IRE1α and IL-1β in RT-PCR (n = 5, xˉ ± s)
bp
178
Figure 2 Agar gel electrophoresis images of PCR products of β-actin, IRE1α and IL-1β mRNA 1: marker; 2: control group; 3: ANP group; 4: CQCQD group. control group: dogs injected intraductally with normal saline; ANP group: AP dogs induced by sodium taurocholate; CQCQD group: the AP dogs were administered 10 mL/kg CQCQD by jejuna tube every 2 h for 6 h. The dogs in the control group and ANP group received an equivalent volume of saline. ANP: acute necrotising pancreatitis; AP: acute pancreatitis; CQCQD: Chaiqinchengqi decoction; IRE1α: inositol requiring enzyme 1α; IL-1β: interleukin-1β; PCR: polymerase chain reaction.
IRE1α
IL-1β
Control group
25.0±1.5
26.0±1.3
27.9±1.6b
ANP group
24.3±1.3
24.5±1.2
25.1±1.2
CQCQD group
24.0±1.6
28.0±1.1
24.9±1.4
a
Notes: control group: dogs injected intraductally with normal saline; ANP group: AP dogs induced by sodium taurocholate; CQCQD group: the AP dogs were administered 10 mL/kg CQCQD by jejuna tube every 2 h for 6 h. The dogs in the control group and ANP group received an equivalent volume of saline. ANP: acute necrotising pancreatitis; AP: acute pancreatitis; CQCQD: Chaiqinchengqi decoction; IRE1α: inositol requiring enzyme 1α; IL-1β: interleukin-1β.aP < 0.05, compared with the ANP group; bP < 0.05, compared with the ANP group and the CQCQD group.
group was significantly higher than that of the control group.
mRNA among the three groups (P > 0.05, Table 2), but the Ct of IRE1α mRNA in the CQCQD treatment group was significantly higher than that of the other two groups (P < 0.05). These findings suggest that the expression of IRE1α mRNA in the CQCQD group was significantly lower than that of the other two groups. The Ct of IL-1β mRNA in the CQCQD group and the ANP group was significantly lower than that of the control group (P < 0.05). There were no significant differences in the Ct values of IL-1β mRNA between the CQCQD group and the ANP group (P > 0.05, Table 2). These findings suggest that the expression of IL-1β mRNA in the CQCQD group and ANP JTCM | www. journaltcm. com
β-actin
Groups
IRE1α and IL-1β protein expression The images of the western blot analysis of IRE1α, IL-1β and β-actin are shown in Figure 3A. The band intensity ratio of IRE1α and β-actin in the control group (0.8 ± 0.3) was smaller than that of the ANP group (0.9 ± 0.4), but was higher than that of the CQCQD group (0.4 ± 0.1, P < 0.05, Figure 3B). The band intensity ratio of IL-1β and β-actin in the ANP group (1.4 ± 0.5) was higher than those of the CQCQD group (0.8 ± 0.4) and the control group (0.6 ± 0.3, P < 0.05, Figure 3C). 437
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Guo J et al. / Experimental Study A IRE1β
1
2
3
4
5
6
7
8
9
QD affects the expression of IRE1α to reduce the synthesis of IL-1β in AMs of ANP dogs. Our data showed that the mRNA and protein expression of IRE1α in AMs was significantly down-regulated after CQCQD treatment (P < 0.05, Figures 2B, 3A and 3B). The mRNA expression of IL-1β in AMs in the ANP group and the CQCQD group was significantly up-regulated compared with that in the control group (P < 0.05, Figure 2C), but the protein expression of IL-1β was significantly down-regulated after treatment with CQCQD (P < 0.05, Figures 2C, 3A and 3C). . These findings suggest that the down-regulated IRE1α in AMs may play a key role in the anti-inflammatory effect of CQCQD on AP. IRE1α is widely expressed in various types of inflammatory cells including lymphocytes, monocytes and macrophages.28 The current study only explored IRE1α in AMs to investigate the anti-inflammatory effect of CQCQD on ANP. Arguably IRE1α in other cell types may also contribute to the anti-inflammatory effect on ANP. Therefore, it is of interest to examine whether CQCQD also affects IRE1α expression in other cell types. In summary, down-regulating the expression of IRE1α in AMs may be the central mechanism behind the anti-inflammatory effect of CQCQD treatment on ANP.
kDa 120
β- actin
43
IL-1β
31
IRE1β / β-actin
B 1.5
1.0
a
0.5
0
Control
C 2.0
ANP
CQCQD
IL-1β / β-actin
b
1.5 1.0 0.5 0
REFERENCES
Control
ANP CQCQD Groups Figure 3 Western blot analysis of IRE1α and IL-1β (n = 5) A: agar gel electrophoresis photographs of western blots of IRE1α, IL-1β and β-actin. 1-3: control group; 4-6: ANP group; 7-9: CQCQD group. B: the ratio of the band intensity of IRE1α and β-actin. aP < 0.05, compared with the control group and ANP group. C: the ratio of the band intensity of IL-1β and β-actin. aP < 0.05, compared with the ANP group; b P < 0.01, compared with the control group. control group: dogs injected intraductally with normal saline; ANP group: AP dogs induced by sodium taurocholate; CQCQD group: the AP dogs were administered 10 mL/kg CQCQD by jejuna tube every 2 h for 6 h. The dogs in the control group and ANP group received an equivalent volume of saline. ANP: acute necrotising pancreatitis; AP: acute pancreatitis; CQCQD: Chaiqinchengqi decoction.
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DISCUSSION The CQCQD modified from the Dachengqi decoction is an effective herbal prescription for the treatment of AP.7 It has been shown to reduce SIRS through the cholinergic anti-inflammatory pathway in rats with acute necrotising pancreatitis.10 Our previous studies have confirmed that CQCQD could up-regulate the nAChRα7 expression of peritoneal macrophages and reduce the serum IL-6 and TNF-α levels in mice with AP, but the specific mechanism was unclear. Study had confirmed that the IRE1α plays a key role in the UPR signalling pathways to increase the ER protein-folding capacity when mRNA translation is increased.27 In this study, we investigated whether CQCJTCM | www. journaltcm. com
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