ORIGINAL ARTICLE
Cartilage Oligomeric Matrix Protein-Angiopoietin-1 Has a Protective Effect of Vascular Endothelial Barrier in Rat With Acute Necrotizing Pancreatitis Ya-Feng Chen, MD,* Ping-Ting Kong, MD,* Hong-Chang Li, MD,* Xin-Juan Fan, MD,* Jia-Min Tu, MD,* Jin-Kun Xie, MD,* Ji-Yun Tian, MD,* Li-Yun Pan, MD,* Teng Chen, MD, PhD,* Yi-Jun Cao, MD,* Pei-Hao Yin, MD, PhD,* Wen Peng, MD, PhD,† and Dian-Xu Feng, MD, PhD* Objective: To investigate the protective effect of angiopoietin-1 (Ang-1) from capillary endothelial damage in rats with acute necrotizing pancreatitis (ANP). Methods: 96 male Sprague-Dawley rats were randomly averaged and divided into control group, ANP group, Si-Ang-1 group, and COMP (cartilage oligomeric matrix protein)-Ang-1 group. Animals were killed at 6, 12, and 24 hours after molding. Levels of serum amylase, porcine endothelin 1, C-reactive protein, and Ang-1 were detected; histopathological changes in the pancreas were observed; capillary permeability and Ang-1 expression of the pancreatic tissue were detected by Evans Blue extravasation assay, immunohistochemistry, Western blot, and quantitative polymerase chain reaction. Results: (1) Levels of serum amylase, C-reactive protein, and porcine endothelin-1 increased and level of Ang-1 decrease in the ANP group and Si-Ang-1 group compared with the control group, whereas COMPAng-1 group could improve the changes. (2) The order of pancreas pathological changes (mild to severe) is: control group, COMP-Ang-1 group, ANP group, and Si-Ang-1 group. (3) Capillary permeability of the pancreatic tissue in the COMP-Ang-1 group was lower than that in the ANP group. (4) Ang-1 mRNA and protein expression in the COMP-Ang-1 group was significantly higher than in the ANP group. Conclusions: COMP-Ang-1 can upregulate the expression of Ang-1 protein to promote angiogenesis and improve early inflammatory and pathological damage in ANP group. Key Words: acute necrotizing pancreatitis, angiopoietin-1, COMP-Ang-1, capillary leak (Pancreas 2016;45: 142–147)
S
evere acute pancreatitis (SAP) is a common and severe clinical emergency with a mortality rate as high as 20%.1 Inflammatory capillary leak caused by vascular barrier dysfunction is one of the key steps in early pathogenesis of SAP. Therefore, protection of endothelial function to alleviate capillary permeability is essential for the prevention of leakage in later developed SAP. Therapeutic vascular regeneration has been used in experimental and clinical studies investigating microvascular lesions in recent
From the *Department of General Surgery and †Laboratory Center, Putuo Hospital, Shanghai University of Traditional Chinese Medicine Shanghai, China. Received for publication September 1, 2014; accepted March 2, 2015. Reprints: Dian-Xu Feng, MD, PhD, Department of General Surgery, Putuo Hospital, Shanghai University of Traditional Chinese Medicine. 164 Lanxi Rd, Shanghai, 200062 China. (e‐mail: [email protected]). This study was supported by the Shanghai Municipal Education Commission program (No. 2011JW62), the Putuo District Health System Innovation Research Fund (No. 2012PTKW003), Predominant Disease of Shanghai Integrative Medicine (pancreatitis), the Innovation Team of Shanghai Traditional Chinese Medicine, and the Key Medical Discipline Project of Shanghai Putuo Distinct, Training Scheme of Back-up Experts of Shanghai University of Traditional Chinese Medicine. The authors declare no conflict of interest. Ya-Feng Chen and Ping-Ting Kong contributed to the study equally. Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
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years.2–4 Angiopoietin-1 (Ang-1), an important member of the vascular endothelial cell-specific angiogenic factor family, plays a vital role in keeping the stability and integrity of the vascular wall and prevention of vascular leakage in face of inflammatory mediators,5,6 Therefore, it is expected to a potential therapeutic target in prevention of SAP early onset inflammatory leakage. This study used a rat model of acute necrotizing pancreatitis (ANP) to investigate the effects of recombinant cartilage oligomeric matrix protein Ang-1 (COMP-Ang-1) on vascular permeability in the pathogenesis of ANP.
MATERIALS AND METHODS Animal Model and Specimen Collection A total of 120 male Sprague-Dawley rats (purchased from Sino-British Sippr/BK Lab, Animal Ltd., Co. Shanghai, China) weighing 220 (30) g were randomly divided into control group, ANP group, silent angiopoietin-1 (Si-Ang-1) interference plasmid group, and COMP-Ang-1 group. Preoperative fasting, as well as free drinking water and ventilation, was given to the animals. No modeling operations were performed in the control group. Cerulein (Sigma) was given (20 μg/kg intraperitoneal injection for 4 times with interval of 1 hour) to rats in the ANP group, intraperitoneal injection of lipopolysaccharide (Sigma) was given (2 mg/kg) within 30 minutes after the last injection of cerulein.7 The Si-Ang-1 group had intravenous injection of Ang-1 SiRNA (100 nM/UL, Genechem, Shanghai, China) within 30 minutes after the first injection of cerulein. The Si-Ang-1 model was prepared jointly with the ANP group.8 In the COMPAng-1 group, 2 shots intravenous COMP-Ang-1 (200 ng/mL, purchased from Shanghai Propack Ltd.) were given within 30 minutes after the first injection of cerulein and 30 minutes after lipopolysaccharide injection, respectively, before the COMPAng-1 model was jointly prepared with the ANP group.9 At 6, 12, and 24 hours after molding, respectively, 8 rats randomly selected from each group were killed 1 hour after the injection of 2% Evans Blue (EB) via the penile vein. Abdominal aortic blood was drawn and immediately centrifuged (3000 r/min for 5 minutes). The supernatant was extracted and frozen at lower than 80°C.
Serum Amylase Test Serum amylase was carried out in the automatic biochemistry analyzer (Roche Ltd, Basel, Switzerland). Detections of serum C-reactive protein (CRP), endothelin (porcine endothelin-1, ET-1), and Ang-1 were performed with enzyme-linked immunosorbent assay, in accordance with the manufacturer's instructions (HuShang Biological Technology, ShangHai, China).
Pathological Changes of Pancreas A small piece of tissue from the pancreatic head was taken for histological examination. The sample was fixed with 4% Pancreas • Volume 45, Number 1, January 2016
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Pancreas • Volume 45, Number 1, January 2016
formalin, dehydrated, and embedded in paraffin wax machine before slicing and Hematoxylin Eosin staining. Pathological injury severity score was determined under light microscope according to Schmidt method.10
Capillary Permeability Test Tissue described previously (300 mg) was extracted using the EB extravasation method11 and placed in 2 mL of 50% trichloroacetic acid solution. The extraction was then incubated and centrifuged (3000 r/min for 10 minutes), and the supernatant of 150 uL was used to measured absorbance values at 620 nm (Bio-Rad, Hercules, Calif ) in formamide solution. Absorbance values measured were then compared with the standard curve of known EB tissue. The content of EB in the tissues was calculated, which can represent the permeability of the capillary.
Immunohistochemistry Instant StreptAvidin Biotin Complex immunohistochemical staining kit (Maixin BIO, Fuzhou, China) was used according to manufacturer's instruction. The positive film provided by the manufacturer was used as positive control. Phosphate buffer, instead of primary antibody, was used as negative control. The Ang-1 antibody (Santa Cruz) was diluted into 1:50 with phosphate buffer solution. Angiopoietin-1 is mainly expressed in the cytoplasma of capillary endothelial cell. Therefore, positive results would primarily present as brown or tan particles on the capillary endothelial cell cytoplasma.
Pancreatic Tissue Ang-1 mRNA Quantitative polymerase chain reaction (PCR) was used for the detection of pancreatic tissue Ang-1 mRNA. Tissue RNA was extracted according to the Trizol kit operation instruction and then reverse transcribed into cDNA. Ang-1 primer sequences: Upstream 5′-CCAAGGGTCGGGATGCCAGATA-3′, Downstream 5′-CTCCGTAAGGGCTTCCATTCGC-3′, Probe 5′-GACCCCTTCAACTCTGGCTC-CCAGGTACACGAAGTTC CTGACTCAGC-CAGTATCTGGCATCCCGACC-3′.
COMP-Angiopoietin-1 in Acute Pancreatitis
GAPDH primer sequences: Upstream 5′-CCTCAAGATTGTCAGCAAT-3′, Downstream 5′-CCATCCAGCAGTCTTCTGAGT-3′.
Expression of Ang-1 in the Pancreatic Tissue It was detected by Western blot. The sample tissue was cut into small pieces, lysates were added in a proportion of 1 mL/250 mg for homogenization, and total protein was extracted. Detection of tissue protein concentration was performed using bicinchoninic acid protein assay kit (Sangon Biotech, Shanghai, China). A total of 50-μg protein was loaded onto the preprepared gel, and sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed before film transfer. The sample was then incubated in 5% Bovine Serum Albumin blocking solution at room temperature for 2 hours. The membrane was washed 3 times by Tris Buffered Saline with Tween (10 minutes for each time), Ang-1 antibody (1:300) was added, and the sample was incubated at 4°C overnight. Horseradish peroxidase–coupled secondary antibody was added the next day after washing the membrane. After incubation at room temperature for 1 hour, the sample underwent enhanced chemiluminescence color and x-ray exposure. The results of the scan film were analyzed with the ImageJ software.
Statistical Methods SPSS15.0 (SPSS Inc, Chicago, Ill) statistical software was used for statistical analysis. Analysis of variance and t test were used for comparison of the experimental results. A P value of less than 0.05 was regarded statistically significant.
RESULTS Serum Amylase, CRP, ET-1, and Ang-1 Levels Compared with those at the same time point in the control group, serum amylase, CRP, and ET-1 levels were all elevated in the ANP group, Si-Ang-1 group, and COMP-Ang-1 group, whereas Ang-1 level was lower than the control group (P < 0.05). Most significant change in the ANP group and Si-Ang-1 group was observed at 12 hours. Serum amylase, CRP, and ET-1 levels at 24 hours in the Si-Ang-1 group are higher than those in the
FIGURE 1. Comparison of levels of serum amylase, CRP, ET-1, and Ang-1 in different groups. Serum amylase (A), serum CRP (B), serum ET-1 (C), and serum Ang-1 (D). © 2015 Wolters Kluwer Health, Inc. All rights reserved.
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ANP group. The level of Ang-1, on the other hand, was lower in the Si-Ang-1 group than the ANP group (P < 0.05), with no significant difference both at 6 and 12 hours (Fig. 1).
Pathological Changes No abnormal pathology appeared in the control group, whereas the ANP group showed mild leukocyte infiltration, moderate interstitial edema, and hemorrhage in the pancreatic tissue at 6 hours. Pancreatic tissue in the ANP group at 24 hours showed large number of leukocyte infiltration, interstitial edema, and necrosis surrounding tissue. Injury in the Si-Ang-1 group was found to be more severe than the ANP group with massive pancreatic necrosis, vascular congestion, and prominent pancreatic interstitial edema. Pathological damage in the treatment group COMP-Ang-1 was progressively alleviated with time, compared with the model groups (Fig. 2).
Capillary Permeability Compared with those at the same time point in the control group, pancreatic EB levels in both ANP group and Si-Ang-1 group were elevated (P < 0.05) and peaked at 12 hours, where EB level at 24 hours in the Si-Ang-1 group was higher than the ANP group (P < 0.05). The EB permeability decreased with time in the COMP-Ang-1 group with time (Fig. 3).
Fluorescent Quantitative-Polymerase Chain Reaction Result of Pancreatic Tissue mRNA Compared with those at the same time point in the control group, pancreatic Ang-1 mRNA expressions in both ANP group and Si-Ang-1 group were found to be lower (P < 0.05). The Ang-1 mRNA expression at 24 hours was lower in the Si-Ang-1 group than the ANP group (P < 0.05), with no significant difference both at 6 hours and 12 hours (Fig. 3).
FIGURE 2. Pathologic changes in the pancreatic tissue at 12 hours (Hematoxylin Eosin, 200). Control, normal pancreatic tissues (A); ANP group, widened pancreatic lobules with edema, patchy hemorrhage, necrosis, and inflammatory infiltration (B); Si-Ang-1 group (Hematoxylin Eosin, 200), severe hemorrhage and necrosis with more widened intersititium and massive inflammatory infiltration (C); COMP-Ang-1 group, less space among pancreatic lobules, patchy necrosis, and mild inflammatory infiltration (D); and pathologic scores of the pancreatic tissue (E).
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© 2015 Wolters Kluwer Health, Inc. All rights reserved.
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Pancreas • Volume 45, Number 1, January 2016
COMP-Angiopoietin-1 in Acute Pancreatitis
FIGURE 3. Pancreatic vessel permeability and Ang-1 mRNA levels. Pancreatic vessel permeability (A) and pancreatic Ang-1 mRNA level (B).
Angiopoietin-1 Protein Expression in Immunohistochemistry Angiopoietin-1 is mainly expressed in the cytoplasma of pancreatic capillary endothelial cells. Compared with those at the same
time point in the control group, pancreatic Ang-1 expressions in both ANP group and Si-Ang-1 group were found to be lower (P
Cartilage Oligomeric Matrix Protein-Angiopoietin-1 Has a Protective Effect of Vascular Endothelial Barrier in Rat With Acute Necrotizing Pancreatitis Ya-Feng Chen, MD,* Ping-Ting Kong, MD,* Hong-Chang Li, MD,* Xin-Juan Fan, MD,* Jia-Min Tu, MD,* Jin-Kun Xie, MD,* Ji-Yun Tian, MD,* Li-Yun Pan, MD,* Teng Chen, MD, PhD,* Yi-Jun Cao, MD,* Pei-Hao Yin, MD, PhD,* Wen Peng, MD, PhD,† and Dian-Xu Feng, MD, PhD* Objective: To investigate the protective effect of angiopoietin-1 (Ang-1) from capillary endothelial damage in rats with acute necrotizing pancreatitis (ANP). Methods: 96 male Sprague-Dawley rats were randomly averaged and divided into control group, ANP group, Si-Ang-1 group, and COMP (cartilage oligomeric matrix protein)-Ang-1 group. Animals were killed at 6, 12, and 24 hours after molding. Levels of serum amylase, porcine endothelin 1, C-reactive protein, and Ang-1 were detected; histopathological changes in the pancreas were observed; capillary permeability and Ang-1 expression of the pancreatic tissue were detected by Evans Blue extravasation assay, immunohistochemistry, Western blot, and quantitative polymerase chain reaction. Results: (1) Levels of serum amylase, C-reactive protein, and porcine endothelin-1 increased and level of Ang-1 decrease in the ANP group and Si-Ang-1 group compared with the control group, whereas COMPAng-1 group could improve the changes. (2) The order of pancreas pathological changes (mild to severe) is: control group, COMP-Ang-1 group, ANP group, and Si-Ang-1 group. (3) Capillary permeability of the pancreatic tissue in the COMP-Ang-1 group was lower than that in the ANP group. (4) Ang-1 mRNA and protein expression in the COMP-Ang-1 group was significantly higher than in the ANP group. Conclusions: COMP-Ang-1 can upregulate the expression of Ang-1 protein to promote angiogenesis and improve early inflammatory and pathological damage in ANP group. Key Words: acute necrotizing pancreatitis, angiopoietin-1, COMP-Ang-1, capillary leak (Pancreas 2016;45: 142–147)
S
evere acute pancreatitis (SAP) is a common and severe clinical emergency with a mortality rate as high as 20%.1 Inflammatory capillary leak caused by vascular barrier dysfunction is one of the key steps in early pathogenesis of SAP. Therefore, protection of endothelial function to alleviate capillary permeability is essential for the prevention of leakage in later developed SAP. Therapeutic vascular regeneration has been used in experimental and clinical studies investigating microvascular lesions in recent
From the *Department of General Surgery and †Laboratory Center, Putuo Hospital, Shanghai University of Traditional Chinese Medicine Shanghai, China. Received for publication September 1, 2014; accepted March 2, 2015. Reprints: Dian-Xu Feng, MD, PhD, Department of General Surgery, Putuo Hospital, Shanghai University of Traditional Chinese Medicine. 164 Lanxi Rd, Shanghai, 200062 China. (e‐mail: [email protected]). This study was supported by the Shanghai Municipal Education Commission program (No. 2011JW62), the Putuo District Health System Innovation Research Fund (No. 2012PTKW003), Predominant Disease of Shanghai Integrative Medicine (pancreatitis), the Innovation Team of Shanghai Traditional Chinese Medicine, and the Key Medical Discipline Project of Shanghai Putuo Distinct, Training Scheme of Back-up Experts of Shanghai University of Traditional Chinese Medicine. The authors declare no conflict of interest. Ya-Feng Chen and Ping-Ting Kong contributed to the study equally. Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
142
www.pancreasjournal.com
years.2–4 Angiopoietin-1 (Ang-1), an important member of the vascular endothelial cell-specific angiogenic factor family, plays a vital role in keeping the stability and integrity of the vascular wall and prevention of vascular leakage in face of inflammatory mediators,5,6 Therefore, it is expected to a potential therapeutic target in prevention of SAP early onset inflammatory leakage. This study used a rat model of acute necrotizing pancreatitis (ANP) to investigate the effects of recombinant cartilage oligomeric matrix protein Ang-1 (COMP-Ang-1) on vascular permeability in the pathogenesis of ANP.
MATERIALS AND METHODS Animal Model and Specimen Collection A total of 120 male Sprague-Dawley rats (purchased from Sino-British Sippr/BK Lab, Animal Ltd., Co. Shanghai, China) weighing 220 (30) g were randomly divided into control group, ANP group, silent angiopoietin-1 (Si-Ang-1) interference plasmid group, and COMP-Ang-1 group. Preoperative fasting, as well as free drinking water and ventilation, was given to the animals. No modeling operations were performed in the control group. Cerulein (Sigma) was given (20 μg/kg intraperitoneal injection for 4 times with interval of 1 hour) to rats in the ANP group, intraperitoneal injection of lipopolysaccharide (Sigma) was given (2 mg/kg) within 30 minutes after the last injection of cerulein.7 The Si-Ang-1 group had intravenous injection of Ang-1 SiRNA (100 nM/UL, Genechem, Shanghai, China) within 30 minutes after the first injection of cerulein. The Si-Ang-1 model was prepared jointly with the ANP group.8 In the COMPAng-1 group, 2 shots intravenous COMP-Ang-1 (200 ng/mL, purchased from Shanghai Propack Ltd.) were given within 30 minutes after the first injection of cerulein and 30 minutes after lipopolysaccharide injection, respectively, before the COMPAng-1 model was jointly prepared with the ANP group.9 At 6, 12, and 24 hours after molding, respectively, 8 rats randomly selected from each group were killed 1 hour after the injection of 2% Evans Blue (EB) via the penile vein. Abdominal aortic blood was drawn and immediately centrifuged (3000 r/min for 5 minutes). The supernatant was extracted and frozen at lower than 80°C.
Serum Amylase Test Serum amylase was carried out in the automatic biochemistry analyzer (Roche Ltd, Basel, Switzerland). Detections of serum C-reactive protein (CRP), endothelin (porcine endothelin-1, ET-1), and Ang-1 were performed with enzyme-linked immunosorbent assay, in accordance with the manufacturer's instructions (HuShang Biological Technology, ShangHai, China).
Pathological Changes of Pancreas A small piece of tissue from the pancreatic head was taken for histological examination. The sample was fixed with 4% Pancreas • Volume 45, Number 1, January 2016
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Pancreas • Volume 45, Number 1, January 2016
formalin, dehydrated, and embedded in paraffin wax machine before slicing and Hematoxylin Eosin staining. Pathological injury severity score was determined under light microscope according to Schmidt method.10
Capillary Permeability Test Tissue described previously (300 mg) was extracted using the EB extravasation method11 and placed in 2 mL of 50% trichloroacetic acid solution. The extraction was then incubated and centrifuged (3000 r/min for 10 minutes), and the supernatant of 150 uL was used to measured absorbance values at 620 nm (Bio-Rad, Hercules, Calif ) in formamide solution. Absorbance values measured were then compared with the standard curve of known EB tissue. The content of EB in the tissues was calculated, which can represent the permeability of the capillary.
Immunohistochemistry Instant StreptAvidin Biotin Complex immunohistochemical staining kit (Maixin BIO, Fuzhou, China) was used according to manufacturer's instruction. The positive film provided by the manufacturer was used as positive control. Phosphate buffer, instead of primary antibody, was used as negative control. The Ang-1 antibody (Santa Cruz) was diluted into 1:50 with phosphate buffer solution. Angiopoietin-1 is mainly expressed in the cytoplasma of capillary endothelial cell. Therefore, positive results would primarily present as brown or tan particles on the capillary endothelial cell cytoplasma.
Pancreatic Tissue Ang-1 mRNA Quantitative polymerase chain reaction (PCR) was used for the detection of pancreatic tissue Ang-1 mRNA. Tissue RNA was extracted according to the Trizol kit operation instruction and then reverse transcribed into cDNA. Ang-1 primer sequences: Upstream 5′-CCAAGGGTCGGGATGCCAGATA-3′, Downstream 5′-CTCCGTAAGGGCTTCCATTCGC-3′, Probe 5′-GACCCCTTCAACTCTGGCTC-CCAGGTACACGAAGTTC CTGACTCAGC-CAGTATCTGGCATCCCGACC-3′.
COMP-Angiopoietin-1 in Acute Pancreatitis
GAPDH primer sequences: Upstream 5′-CCTCAAGATTGTCAGCAAT-3′, Downstream 5′-CCATCCAGCAGTCTTCTGAGT-3′.
Expression of Ang-1 in the Pancreatic Tissue It was detected by Western blot. The sample tissue was cut into small pieces, lysates were added in a proportion of 1 mL/250 mg for homogenization, and total protein was extracted. Detection of tissue protein concentration was performed using bicinchoninic acid protein assay kit (Sangon Biotech, Shanghai, China). A total of 50-μg protein was loaded onto the preprepared gel, and sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed before film transfer. The sample was then incubated in 5% Bovine Serum Albumin blocking solution at room temperature for 2 hours. The membrane was washed 3 times by Tris Buffered Saline with Tween (10 minutes for each time), Ang-1 antibody (1:300) was added, and the sample was incubated at 4°C overnight. Horseradish peroxidase–coupled secondary antibody was added the next day after washing the membrane. After incubation at room temperature for 1 hour, the sample underwent enhanced chemiluminescence color and x-ray exposure. The results of the scan film were analyzed with the ImageJ software.
Statistical Methods SPSS15.0 (SPSS Inc, Chicago, Ill) statistical software was used for statistical analysis. Analysis of variance and t test were used for comparison of the experimental results. A P value of less than 0.05 was regarded statistically significant.
RESULTS Serum Amylase, CRP, ET-1, and Ang-1 Levels Compared with those at the same time point in the control group, serum amylase, CRP, and ET-1 levels were all elevated in the ANP group, Si-Ang-1 group, and COMP-Ang-1 group, whereas Ang-1 level was lower than the control group (P < 0.05). Most significant change in the ANP group and Si-Ang-1 group was observed at 12 hours. Serum amylase, CRP, and ET-1 levels at 24 hours in the Si-Ang-1 group are higher than those in the
FIGURE 1. Comparison of levels of serum amylase, CRP, ET-1, and Ang-1 in different groups. Serum amylase (A), serum CRP (B), serum ET-1 (C), and serum Ang-1 (D). © 2015 Wolters Kluwer Health, Inc. All rights reserved.
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Chen et al
ANP group. The level of Ang-1, on the other hand, was lower in the Si-Ang-1 group than the ANP group (P < 0.05), with no significant difference both at 6 and 12 hours (Fig. 1).
Pathological Changes No abnormal pathology appeared in the control group, whereas the ANP group showed mild leukocyte infiltration, moderate interstitial edema, and hemorrhage in the pancreatic tissue at 6 hours. Pancreatic tissue in the ANP group at 24 hours showed large number of leukocyte infiltration, interstitial edema, and necrosis surrounding tissue. Injury in the Si-Ang-1 group was found to be more severe than the ANP group with massive pancreatic necrosis, vascular congestion, and prominent pancreatic interstitial edema. Pathological damage in the treatment group COMP-Ang-1 was progressively alleviated with time, compared with the model groups (Fig. 2).
Capillary Permeability Compared with those at the same time point in the control group, pancreatic EB levels in both ANP group and Si-Ang-1 group were elevated (P < 0.05) and peaked at 12 hours, where EB level at 24 hours in the Si-Ang-1 group was higher than the ANP group (P < 0.05). The EB permeability decreased with time in the COMP-Ang-1 group with time (Fig. 3).
Fluorescent Quantitative-Polymerase Chain Reaction Result of Pancreatic Tissue mRNA Compared with those at the same time point in the control group, pancreatic Ang-1 mRNA expressions in both ANP group and Si-Ang-1 group were found to be lower (P < 0.05). The Ang-1 mRNA expression at 24 hours was lower in the Si-Ang-1 group than the ANP group (P < 0.05), with no significant difference both at 6 hours and 12 hours (Fig. 3).
FIGURE 2. Pathologic changes in the pancreatic tissue at 12 hours (Hematoxylin Eosin, 200). Control, normal pancreatic tissues (A); ANP group, widened pancreatic lobules with edema, patchy hemorrhage, necrosis, and inflammatory infiltration (B); Si-Ang-1 group (Hematoxylin Eosin, 200), severe hemorrhage and necrosis with more widened intersititium and massive inflammatory infiltration (C); COMP-Ang-1 group, less space among pancreatic lobules, patchy necrosis, and mild inflammatory infiltration (D); and pathologic scores of the pancreatic tissue (E).
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© 2015 Wolters Kluwer Health, Inc. All rights reserved.
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Pancreas • Volume 45, Number 1, January 2016
COMP-Angiopoietin-1 in Acute Pancreatitis
FIGURE 3. Pancreatic vessel permeability and Ang-1 mRNA levels. Pancreatic vessel permeability (A) and pancreatic Ang-1 mRNA level (B).
Angiopoietin-1 Protein Expression in Immunohistochemistry Angiopoietin-1 is mainly expressed in the cytoplasma of pancreatic capillary endothelial cells. Compared with those at the same
time point in the control group, pancreatic Ang-1 expressions in both ANP group and Si-Ang-1 group were found to be lower (P
