| Pancreatitis is one of the essential diseases that threaten the health of human being. It has been commonly accepted that besides the abnormal enzyme secretion or ectopic activation mechanism, there still exist complex inflammatory responses mediated by complex immune or nerve secretion, which may be key determinants of patients’ prognosis and mortality. Therefore, neuro-endocrin-immunological regulation network attract tremendous attention in recent years. Calcitonin gene-related peptide(CGRP), as an important neurogenous negative immune regulatory peptide, can prevent the damage caused by excessive activation of immune function and participate in the maintenance of homeostasis in vivo environment.Besides involved in the pain and inflammation of pancreatitis, CGRP has the protective effect on neurons as well.⑴ ClinicallyThis paper tests the expression of CGRP in serum and other neurotransmitters or serum trypsin in order to verify whether it is related to the disease on the basis of large sample. Also, the paper detect the serum trypsin, CGRP and other indicators of biliary pancreatitis(n=72), hyperlipdemic pancreatitis(n=60), idiopathic pancreatitis(n=8) and Normal control(n=60) by ELISA so as to analyze the correlation and the association with disease. Besides, we collect the general clinical data of patients at the same time for statistic.⑵ Animal levelIn this paper the mouse model of acute necrotizing pancreatitis was induced by intraperitoneal injection of caerulein. The expression changes of calcitonin gene related peptide in pancreatic tissues of mice were analyzed by using tissue pathology and immunohistochemistry. To verify the protective effect of CGRP in pancreatitis from two opposite aspects: on one hand, we inject he pancreatitis model mouse with CGRP to investigate whether the symptoms of acute pancreatitis relieve; On the other hand, the pancreatitis model mouse accept hypodermic injection with commercial CGRP receptor antagonist(CGRP8-37) which was meant to verify whether the symptoms of acute pancreatitis aggravated.Method:The experiment is divided into 2 steps:1.To investigate the connection between patients’ serum CGRP level and pancreatitis; to analyze the general clinical data of different types of pancreatitis.(1)To determine of serum trypsin and CGRP of pancreatitic patients and concurrent normal control;( 2) To analyze the reliability of serum trypsin and CGRP as the biomarker for pancreatitis screening;(3)To investigate the change of pancreatitis patients’ peripheral blood lymphocyte subsets;(4) To explore the differences of clinical indicators among the different types of pancreatitis(biliary pancreatitis, hyperlipidemia pancreatitis and idiopathic pancreatitis);(5) To analyze the concentration correlation between CGRP and trypsin.2.To verify the role of the role of CGRP in the development of acute pancreatitis using the C57BL/6 mouse model.(1) The mouse model of pancreatitis was induced by intraperitoneal injection of caerulein. In this study mice were divided into four groups: normal control group, pancreatitis group, treatment group with CGRP and treatment group with CGRP receptor antaonist(CGRP8–37)(2) To detect and analyze the the expression changes of CGRP and lipase of the normal control group and pancreatitis group in the time point of 12 h after injecting.(3) To detect and analyze the the expression changes of CGRP and lipase of the normal control group and pancreatitis group in the time point of 24 h after injecting.(4) To set up 4 groups: normal control group, pancreatitis group, treatment group with CGRP and treatment group with CGRP receptor antaonist(CGRP8–37),thus to detect and analyze the expression changes of both serum CGRP and lipase, and the pathological changes of pancreatic tissue 24 hours after the pancreatitis happened.(5) To investigate the expression of CGRP in pancreatic tissues of mice of various models(including control group, model group, CGRP treatment and CGRP8-37 treatment group) and their relationship with inflammatory infiltration cells.Results:1. The data was performed by one-way analysis of variance suggested that the three groups(biliary, hyperlipidemic, idiopathic) of pancreatitic patients showed differences in index of blood lipids and diabetes mellitus(Hb A1 C,GLU,TG), and indicated no significant differences in general clinical examination index as ages,gender, et al.2. while the serum trypsin level of male patients(n=94,75.25 ± 33.62pg/m L) was significantly higher than that in female patients(n=46,55.23 ± 22.32 pg/m L),p=0.023; the serum levels of trypsin were significantly lower in patients with idiopathic pancreatitis( 42.36 ± 18.25 pg/m L) than that of other patients with pancreatitis. Besides, the serum concentrations of trypsin were positively correlated to the courses and severity of pathogenetic conditions.3. The expression of serum CGRP in every group(biliary, hyperlipidemic, idiopathic pancreatitis) was significantly different(p<0.01), which was shown as the following: serum CGRP in male patients( n=94, 98.63±25.45pg/m L) was significantly higherthan that in female patients( n=46, 70.12±30.56 pg/m L),p=0.029; and serum CGRP in patients with inflammatory exudation(125.32±58.45 pg/m L) was significantly highert han that in patients without inflammatory exudation( 65.23 ±33.20 pg/m L), p< 0.01. In addition, the concentration of serum CGRP was positively related to the severity of pancreatitis.4. Pancreatic protease activity of pancreatitis model mice have increased significantly compared to that of normal control group. The activity of trypsin of control group is 5600±1500U/mgprot, which in pancreatitis model(12h) group is7800 ±1800U/mgprot and that of pancreatic model(24h) is 9200 ±650U/mgprot.Pancreatic protease activity in pancreatic model mice was significantly higher than that in normal control group(p=0.0152), and the pancreatic protease activity in pancreatic model mice(24h) was significantly higher than that in pancreatic model mice(12h)(p=0.0215). Both of them show a statistical difference, which suggest that mouse model of acute pancreatitis induced by caerulein plus lipopolysaccharide was established successfully.5. The study confirmed the protective effect of CGRP on pancreatic injury from two aspects. In the evaluation of pancreatic lesions, the CGRP intervention group(pre- or at the same time) had the appearance of pancreatic congestion and swelling,but the degree was significantly lower than that of the model group. Meanwhile,the inflammatory infiltration in CGRP8-37 group was more severe than that in the model group.6. The serum amylase(AMY) of the model group( 18580±1952 U/ml) was significantly higher than that in the normal control group(4985±545 U/ml,p<0.001); and in the study of drug intervention, we found that there was no significant difference between pre-and post treatment(5012 + 630U/ml and 6203 + 596U/ml).Interestingly, after the intervention of CGRP, there was no significant difference between the serum amylase of the intervention group and normal control group.However, the serum amylase(16590 + 780 U/ml) in the CGRP8-37 receptor blocker group was significantly higher than that in the normal control group and CGRP intervention group, which showed no significant difference when compared with the model group.7. The serum levels of CGRP in CGRP8-37 receptor blocking group(median 13.92pg/ml) were significantly lower than those in the CGRP intervention group(median, 54.71 and 73.60 pg/ml) and model group(median 30.27 pg/ml), but not significantly different from the normal control group(median 14.37). Time points of intervention had little effect on serum CGRP, and the serum concentrations of CGRP in CGRP treated mice were significantly higher than those in model group.8. CGRP was low expression or weak expression in mice pancreatic tissue which pretreated with CGRP receptor blocker, which had little difference with that of normal control group. And in the CGRP treatment group, it could be seen that the CGRP is in high expression around portal area, which was shown as cluster distribution.Conclution:1. Serum CGRP was significantly increased in acute pancreatitis, which indicated that CGRP plays an important role in the development of acute pancreatitis;2. Exogenous CGRP can improve the microcirculation of pancreatitis and reduce the pathological damage of pancreatitis.3. CGRP receptor antagonist CGRP8- 37 can aggravate the pathological damage of pancreatitis. |
PDF Full Text Request