Influence of calcitonin gene-related peptide on model mice with acute pancreatitis

Reagents

Caerulein (#HY-A0190), CGRP (α-CGRP, #HY-P1462) and CGRP_(8–37) (CGRP receptor antagonist, #HY-P1014) were purchased from MedChemexpress Co. (New Jersey, USA). Lipopolysaccharide (LPS, from Escherichia coli (O111:B4)) was purchased from Sigma-Aldrich Co. (St. Louis, Missouri, USA). Pentobarbital sodium (#096956-001, for animal experiments only) was purchased from Beijing Huaye Huanyu Chemical Co. (Beijing, China). Anhydrous ethanol (#10009218), xylene (#10023418) and paraffin wax (#69018961) were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Hematoxylin-Eosin Staining Kit (#PH0516), DAB Horseradish Peroxidase Color Development Kit (#PH0417), 5% BSA Blocking Buffer (#PH0420), Phosphate Buffered Saline Powder (#PH1403) and 4% Paraformaldehyde Fixation (#PH0427) were purchased from Phygene Life Sciences Co., Ltd (Fuzhou, China). Anti-CGRP antibody (#ab47027), Goat Anti-Rabbit IgG H&L (HRP, #ab205718), Recombinant Anti-CD20 antibody (#ab64088) and PE/R-Phycoerythrin Conjugation Kit (#ab102918) were purchased from Abcom Co. (Cambridge, England).

Preparation of stock solution

The 1 mg of caerulein was dissolved into 1 mL of sterilized normal saline to obtain 1 mg/mL of clarified stock solution. If caerulein could not be fully dissolved, the ultrasonic treatment was performed briefly for 10 min with less than 30 °C. The stock solution was kept at −20 °C after separately packed in enzyme-free sterile centrifuge tube to avoid repeated freezing-thawing. The 10 mg of LPS was dissolved into 10 mL of sterilized normal saline with lightly swirl to obtain 1 mg/mL of clarified stock solution. The stock solution was kept in enzyme-free sterile centrifuge tube at −20 °C when not in use. The 1 mg of CGRP/CGRP_(8–37) was dissolved into 1 mL of sterilized normal saline to obtain 1 mg/mL of clarified stock solution, which was separately packed in enzyme-free sterile centrifuge tube and stored at −80 °C.

Grouping of experimental animals and establishment of acute pancreatitis model

Animal experiments were performed in accordance with the Guideline for Animal Experimentation with the approval of the Animal Care and Use Committee of Putian University (No. PUACUC 2019-03011). In this study, the male C57/6 mice with healthy clean grade and 18–22 g of weight were purchased from Shanghai Shrek Laboratory Animal Company. All the mice were fed with a standard diet and fasted for 18 h before the induction of acute pancreatitis, and accessed to water ad libitum during the experiments. After weighing 40 mice separately, they were completely randomly divided into five groups, namely control group (Con), acute pancreatitis group (AP), CGRP pretreatment group (preCGRP+AP), CGRP treatment group (CGRP+AP) and CGRP_(8–37) pretreatment group (preCGRP_(8–37)+AP). There were no less than eight mice in each group, and the number of mice in the corresponding groups was increased appropriately according to the experimental needs. In the experiment, the administration volume of 20 mL/kg was used for intraperitoneal injection in mice [16]. All the injection doses mentioned herein represent a single dose, including 100 μg/kg, 10 mg/kg and 50 mg/kg. Details of each group were as follows [17]:

Con: the mice were intraperitoneally injected with sterile normal saline at a dose of 20 mL/kg at a 1 h interval six times.

AP: the mice were intraperitoneally injected with caerulein at a dose of 100 μg/kg at a 1 h interval six times, and an intraperitoneal injection of LPS was administered at a dose of 10 mg/kg after the last intraperitoneal injection of caerulein.

preCGRP+AP: CGRP was intraperitoneally injected into the mice at a dose of 100 μg/kg 30 min before the first dose of caerulein.

CGRP+AP: CGRP was intraperitoneally injected into mice at a dose of 100 μg/kg 12 h after successive six doses of caerulein and one dose of LPS.

preCGRP_(8–37)+AP: CGRP_(8–37) (CGRP receptor antagonist) was intraperitoneally injected into mice at a dose of 100 μg/kg 30 min before the first dose of caerulein.

Treatment of CGRP on acute pancreatitis

The mice were intraperitoneally injected with CGRP at a dose of 100 μg/kg in preCGRP+AP, CGRP+AP and preCGRP_(8–37)+AP group, and the same dose of sterile normal saline in Con and AP group, 12 h after successive six doses of caerulein and one dose of LPS.

Collection of serum and pancreatic tissue in mice

According to the preparation method of acute pancreatitis model, six doses of caerulein and one doses of LPS were intraperitoneally injected successively, which successfully induced acute pancreatitis in mice. The blood in mice was collected in AP group 12 and 24 h after successive six doses of caerulein and one dose of LPS, respectively, and in preCGRP+AP, CGRP+AP and preCGRP_(8–37)+AP group 12 h after treatment of CGRP. In Con group, the blood was collected 12 and 24 h after the mice were intraperitoneally injected with seven doses of sterile normal saline equal to six doses of caerulein and one dose of LPS. The collected blood method were as follows: the mice were anesthetized by intraperitoneal injection of pentobarbital sodium at a dose of 50 mg/kg, and then the blood in the mice was harvested via retrobulbar plexus without anticoagulation [16]. The harvested blood samples were placed at 4 °C for 1 h, and then centrifuged at 3,500 rpm/min for 10 min, and the upper serums were collected to detect various biochemical indicators. The obtained serums were kept in the refrigerator at −80 °C when not in use. After blood collection, the experimental mice were euthanized and laparotomized, and then the mice were dissected to observe and record the changes in the pancreas. Meanwhile, the pancreas and surrounding tissues were observed to record edema, congestion, necrosis and other phenomena. The head tissue of pancreas was cut and fixed with 4% paraformaldehyde for preparation of pathological sections.

Determination of activity of amylase and trypsin

The activities of amylase and trypsin were determined by colorimetric method using amylase assay kit and trypsin assay kit, respectively. Assay kits of amylase (C016-1-1) and trypsin (A080-2-2) for mice were purchased from Nanjing Jiancheng Biological Products Institute (Nanjing, China). All materials and prepared reagents were equilibrated to room temperature prior to use. Each well was repeated three times in the experiment. The detailed experimental procedure is as follows:

Standard wells: The 50 µL of standard solutions with different concentrations were added into different wells, which was used to draw standard curves.

Positive control wells: A 5 µL of positive substrate was added into well and adjusted to 50 µL/well with assay buffer.

Sample wells: A 10 µL of serum samples was add into well and adjusted to 50 µL/well with assay buffer.

A 100 µL of reaction mixture was added into each well and mixed thoroughly. The reaction mixture consists of assay buffer and substrate in a proportion of 1:1 for amylase and 14:1 for trypsin. The absorbance at 405 nm was immediately determined by a Microplate Reader after incubation for 30 min at 37 °C in dark. Finally, the standard curves were drawn to calculate the activity of amylase and trypsin, respectively.

Evaluation of pancreatic lesions

After observing the appearance characteristics of the pancreas in the experimental mice, the head tissues of the pancreas, such as the size of green beans, were fixed with formaldehyde, embedded, sectioning and stained with HE, and the pathological changes were observed under an optical microscope. The total scores of pathological findings were derived from five items, including edema, hemorrhage, acinar necrosis, pancreatic duct lesion and inflammatory infiltration. Each item was rated on five scales, namely 0, 1, 20, 30, and 40.

Preparation of histological sections

After fixed for more than 24 h, the pancreatic tissues were removed from the fixing solution and trimmed to make the target site of the tissues smooth. And then, the tissues were dehydrated in the different dehydration boxes, which successively were 75% ethanol for 4 h →85% ethanol for 2 h →95% ethanol for 1 h →anhydrous ethanol for 30 min →xylene for 10 min →paraffin wax for 1 h. Next, the waxed tissue was embedded and processed on the slicer to obtain 4 µm of thick sections. The sections were flatted on a glass slide and baked at 60 °C for 30 min. After the wax melted, the sections were successively placed in xylene for 20 min →anhydrous ethanol for 5 min →75% ethanol for 5 min →tap water for washing. Subsequently, hematoxylin-eosin (H&E) staining and immunohistochemical staining were performed on the obtained sections.

Hematoxylin-eosin (H&E) staining

The sections were stained with hematoxylin solution for 5 min, and then rinsed with tap water. After the sections were placed into 1% hydrochloric acid for a few seconds and rinsed with tap water, 0.6% ammonia water was added to turn blue and tap water rinsed the sections. After that, the sections were stained with eosin solution for 5 min, and then dehydrated, successively in anhydrous ethanol for 5 min →xylene for 5 min. Finally, the sections were sealed with neutral resin.

Immunohistochemical staining

The sections were immersed into 0.01 mol/L citrate buffer solution and heated with microwave for 10 min to repair antigen. After cooling for 10 min, the sections were heated for another 3 min. Next, the sections were placed in 3% H₂O₂ solution for 10 min to inactivate endogenous peroxidase activity, and then were sealed with 5% BSA blocking buffer at room temperature to block the non-specific adsorption of antibodies and reduce the background staining. After the excess BSA solution was discarded, 30 µL of primary antibody solution was added onto the sections and incubated in a humidified box at 4 °C overnight, followed by reheating at 37 °C for 20 min and soaking in PBS buffer for 5 min repeatedly three times. After that, 30 µL of biotinylated secondary antibody solution was added onto the sections and incubated at 37 °C for 60 min, followed by soaking in PBS buffer for 5 min repeatedly three times. The sections were developed by DAB Immunohistochemistry Color Development Kit at room temperature for 10 min, rinsed with tap water, and then redyed with hematoxylin for 1 min. After washing, the sections were dehydrated, transparent and sealed with neutral resin.

Image acquisition

The images of the sections of H&E staining and immunohistochemical staining were acquired on an optical microscope (Nikon Eclipse E100, Japan).

Statistical analysis

The statistical analyses were carried out by GraphPad Prism 6.0 (GraphPad Software, San Diego, CA). One-way ANOVA was used for multiple comparisons and was followed by Bonferroni’s test. Statistical differences with p values less than 0.05 were considered significant. All data were represented as the mean ± standard deviation (SD) from eight parallel independent experimental results.

Morphological characteristics of pancreas and activity of trypsin and amylase in mice with AP

After the mice were successively intraperitoneally injected with six doses of caerulein and one dose of LPS for 12 h, the volume of pancreas increased, the texture of pancreas was slightly hard, the boundary structure was blurred, and there was obvious swelling and hyperemia in pancreas. The head and tail of pancreas showed the most prominent edema and dark red color. Saponification spots occurred on the pancreas surface. There was a small amount of fluid in the abdominal cavity of mice with AP, obvious saponification spots occurred in the mesentery and omentum, and the pancreas had a severe adhesion to the surrounding tissues.

The experimental results in Figure 1A showed that the activity of trypsin in AP group increased significantly compared with Con group. The activity of trypsin in Con group was 5,550 ± 860 U/L, and the activity of trypsin in AP group for 12 and 24 h were 7,810 ± 1,021 and 9,396 ± 1354 U/L, respectively. The activity of trypsin in AP group was significantly stronger than that in Con group (**p<0.01). In addition, the activity of trypsin in AP group for 24 h was significantly higher than AP group for 12 h (*p<0.05). The experimental results in Figure 1B showed that the activity of amylase in AP group for 12 and 24 h was obviously higher than that in Con group (**p<0.01). The activity of amylase in AP group for 12 and 24 h were 14,263 ± 1,491 and 16,393 ± 1180 U/L, respectively, and the activity of amylase in Con group was 4,549 ± 670 U/L. And the activity of amylase in AP group for 24 h was also significantly higher than that in AP group for 12 h (*p<0.05).

Figure 1:

Comparison of activity of trypsin and amylase in serum between Con group and AP group. The activity of trypsin and amylase in Con group and AP group was measured at 12 and 24 h, respectively. In Con group, there was no significant difference in the activity of trypsin and amylase during the whole experiment process, so it was shown as a set of data in the figure. One-way ANOVA was performed with Bonferroni’s test to identify the significant difference between multiple groups. The error bars represents standard deviation and reflects the dispersion of the data. *p<0.05, **p<0.01, and ***p<0.001.

The above results suggested that the mice model with AP induced by caerulein combining with lipopolysaccharide were successfully established.

Pancreatic histopathology in mice with AP

The typical pathologic feature of pancreatic tissue in Con group was shown in Figure 2A. The boundaries of pancreatic tissue were clear and uniform in color. The lobules were complete in structure and normal in shape, and there was no infiltration of neutrophils and erythrocytes and no obvious edema. The pancreatic acini was also well defined and had a complete structure, and the staining of cytoplasm and nucleoli was clearly visible, without bleeding or necrosis. The pancreatic tissues of mice in AP group were observed under light microscope. As can be seen from Figure 2B, the total volume of the pancreas was larger than that of Con group, and the structure of the glands was more disordered, there was marked edema and rupture of the acinus in pancreas, and the outline was not clear. The nucleus is partially pyknotic (green arrow), and inflammatory cells infiltrated (yellow arrow), and there was the exudation of red blood cells around the blood vessels and between the glands (blue arrow).

Figure 2:

Representative micrographs of H&E-stained pancreatic histopathological sections in mice (×400). A and B were Con group and AP group, respectively. Green, yellow and blue arrow indicated nucleus-pyknotic cell, inflammatory cell and red blood cells, respectively.

Evaluation results of pancreatic lesions

In AP group, the pancreatic duct was swollen, the color was deepened, the pancreas was hyperemic, and there were scattered brown necrotic foci and a small amount of bloody ascites, and mesenteric edema was obvious, 12 h after intraperitoneal injection of successive six doses of caerulein and one dose of LPS. In preCGRP+AP and CGRP+AP group, the pancreas of the mice showed acini necrosis, but the degree of necrosis was significantly less severe than AP group. The pathological scores of the pancreas in each group were shown in Table 1. In preCGRP_(8–37)+AP group, both each score and total scores were the highest in all the groups, and the pathological changes were significantly worse than those in preCGRP+AP and CGRP+AP group. The total scores were close to each other in preCGRP+AP and CGRP+AP group, and the difference between preCGRP+AP and CGRP+AP group was not significant. Whether it’s preCGRP+AP/CGRP+AP group vs. AP group, or preCGRP+AP/CGRP+AP group vs. pre-CGRP_(8–37)+AP group, there were significant differences among the total scores (p<0.05), as shown in Table 1. The score of inflammatory infiltration in Table 1 showed that the pancreatic inflammation of mice in pre-CGRP_(8–37)+AP group was more severe than AP group. Thus, the experiment confirmed the protective effect of CGRP on pancreatic injury from positive and negative aspects.

Amylase of mice in different groups

As shown in Figure 3, the activity of amylase in AP group was 16,393 ± 1180 U/L and significantly higher than that in Con group (4,549 ± 670 U/L, **p<0.01). In the CGRP intervention study, we found that the activity of amylase was 5,012 ± 788 and 5,610 ± 866 U/L in preCGRP+AP and CGRP+AP group, respectively, and had no significant difference between preCGRP+AP and CGRP+AP group (p=0.1103). And there was no significant difference of amylase activity between the two groups and Con group (preCGRP+AP vs. Con: p=0.3033; CGRP+AP vs. Con: p=0.0517). The activity of amylase in preCGRP_(8–37)+AP group was 15,902 ± 1685 U/L and significantly higher than that in Con, preCGRP+AP and CGRP+AP (***p<0.001). However, there was no significant difference for the activity of amylase between preCGRP_(8–37)+AP and AP group (p=0.3764).

Figure 3:

Comparison of amylase activity in Con, AP, preCGRP+AP, CGRP+AP, and preCGRP_(8–37)+AP group. The activity of amylase was measured in Con and AP group 24 h after induction of AP, and in preCGRP+AP, CGRP+AP and preCGRP_(8–37)+AP group 12 h after treatment of CGRP. One-way ANOVA was performed with Bonferroni’s test to identify the significant difference between multiple groups. The error bars represents standard deviation and reflects the dispersion of the data. ***p<0.001; n.s., no significance; p>0.05.

Expression of CGRP in different groups

We analyzed the expression of CGRP in pancreatic tissues of mice in different groups by immunohistochemistry. Figure 4A showed that CGRP was low expressed in pancreatic tissue of Con group. However, pancreatic tissue of AP group showed significant expression of CGRP, which was the dark brown within the green dotted line in Figure 4B. In preCGRP+AP and CGRP+AP group, CGRP was strongly high expressed in pancreatic tissue and distributed in clusters, as shown the dark brown within the green dotted line in Figure 4C, D. Unexpectedly, after the intervention of CGRP receptor blocker (CGRP_(8–37)) in advance, the expression of CGRP was low or weak in preCGRP_(8–37)+AP group (Figure 4E), which was similar to the expression of CGRP in Con group.

Figure 4:

Representative micrographs of immunohistochemistry-stained pancreatic histopathological sections in mice (×400). A, B, C, D and E were Con group, AP group, preCGRP+AP group, CGRP+AP group, and preCGRP_(8–37)+AP group, respectively. The dark brown within the dotted green line represented the high expression of CGRP.

Influence of CGRP on inflammatory cells in different groups

Figure 5A showed that there was no CD20⁺ B lymphocytes in Con group. In stark contrast, a lot of CD20⁺ B lymphocytes were present in the pancreatic tissue of AP group (Figure 5B), indicating that humoral immunity was involved in the pathological process of AP. Figures 5C, D showed that CD20⁺ B lymphocytes were significantly reduced in preCGRP+AP and CGRP+AP group, respectively. However, many CD20⁺ B lymphocytes appeared in the pancreatic tissue of preCGRP_(8–37)+AP group (Figure 5E), which was similar to AP group.

Figure 5:

Representative micrographs of immunohistochemistry-stained pancreatic histopathological sections in mice (×400). A, B, C, D, and E were Con group, AP group, preCGRP+AP group, CGRP+AP group and preCGRP_(8–37)+AP group, respectively. The dark brown represented the infiltration of CD20⁺ B lymphocytes in pancreatic tissue.