An Analytic Study On Metabolic Features Of Acute Pancreatitis And Early Stage Of Chronic Pancreatitis

Part one The establishment of the animal model of acute pancreatitis and early chronic pancreatitis in ratsObjective:To establish a animal model as similar as clinical aspects of acute necrotizing pancreatitis based on etiology, pathogenesis, and pathology. The models will be used to study the metabolic characteristics and biological characteristics in rats. To establish a chronic pancreatitis model in rats which is very similar to human chronic pancreatitis. The models are used to investigate the processes leading to chronic pancreatitis and metabolic features.Methods:(1) Forty male Wistar rats, weighing about 180-200g, were randomly divided into experimental and control groups, each group was subdivided into 6,12,24,48h,four observation time points,5 rats in each time point. The rats in experimental group received a high-dose intraperitoneal injection of L-arginine (2 X 2.5mg/g) two times. The interval time is 1 hour. The Control group is injected the same dose of saline. After modeling 1,12,24,48 h, obtain the blood from the heart of rats, then the serum amylase, lipase, hyaluronic acid, and pancreas wet-dry weight ratio will be detected. To spin-off the pancreatic tissue carefully, after generally observed, a small part used to pathological study, the large part of the pancreatic tissue will be fixed in liquid nitrogen. (2) Sixty male Wistar rats, weighing about 180-200g, were randomly divided into experimental and control groups, each group was subdivided into four observation time points 1,8,15,21,28,60d. Dibutyltin dichloride (DBTC) first is dissolved into 100% alcohol, and then mixed with glycerol (the ratio of the three is 1:2:3). The experiential group is injected DBTC solution 8mg/kg body wt via the tail vein. The control group only received an injection of the same dose solvent. After modeling 1,8,15,21,28,60 days, obtain the blood from the heart of rats, then the serum amylase, lipase, hyaluronic acid will be detected. Peeling the pancreatic tissue carefully, some of the pancreatic tissue will be sent to the pathology department, the tissue will receive HE stain and the pathological changes in different periods also be observed. Two experienced pathologists will assess the inflammation degree using double-blind method. Modified Van Gieson staining also be used to the fibrosis assessment.Results:After injection of L-arginine, the time point of 6,12,24,48h the number of deaths were 1,0,1,1 in AP group of rats.The total mortality was 7.5%. After killing the survival rats of every time point, we found that 6h, pancreatic tissue swelling associated with punctate bleeding points; 12h, the pancreas is more significant swelling, bleeding points, with mild increase in liquefied change;after 24 and 48h, the structure of pancreas is blurred, a large number of fatty tissue can be seen around the pancreas. Light microscope, we can see after modeling 6h, congestion and edema in the pancreas is obviously, also we found amount of pancreatic necrosis and interstitial inflammatory cell infiltration. we can found pancreatic edema associated with congestive hemorrhage, coagulation necrosis of large areas of pancreatic parenchyma during 12h. After 24 and 48h, the structure of pancreas is fuzzy with amount of fatty tissue around it.6h after modeling the serum amylase level was significantly higher,12h increased even more pronounced,24h rise reached a peak,48h post-began to decline. After 1day of DBTC injection, we can see an acute edematous pancreatitis. The pancreatic tissue was more edema in day 8; The pancreatic duct Can be seen dilated in day 15, especially in the head and body of the pancreas. After 21 days, we can see the pancreatic tissue contraction, surface irregularities and rough. At day 21 to day 28, level of pancreatic fibrosis was aggravatingly.The pancreatic tissue was characterized by an extended interstitial fibrosis at the end of 2 months. Blood biochemistry showed mold one days later, serum amylase, fats, enzymes and hyaluronic acid that is significantly higher.8 days later the serum amylase returned to normal levels,15 days later the lipase return to normal levels, hyaluronic acid concentrations were have been higher, but no clear correlation with time.Conclusions:By high-dose intraperitoneal injection of L-arginine could obtain an acute pancreatitis model of which bionomics similar to human, it is suitable to study on the model. DBTC can induced a chronic pancreatitis model characterized by dynamically increase of fibrosis, which is very similar to human. The model is appropriate for studying the development of fibrosis in chronic pancreatitis patients. Part Two Comparison of Metabolic Changes between Acute Pancreatitis and Early Chronic Pancreatitis in ratsObjective:To compare the metabolic characteristics of normal pancreas, acute pancreatitis, and early chronic pancreatitis in rats, to analyze the relevance between metabolic changes and pathological changes. By comparing the different metabolic changes of acute pancreatitis and early chronic pancreatitis, we provide a good animal model for clinical research in such disease.Methods:High-resolution Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy, HRMAS-NMR) and Principal Component Analysis(PCA) were combined to study the the different metabolic changes of acute pancreatitis and early chronic pancreatitis, meanwhile study the relationship between some metabolic changes and histopathology. HRMAS-NMR and PCA have performed to Wistar rats. The pancreatic tissues were divided into normal group(n=5), acute pancreatitis (n=6) and early chronic pancreatitis(n=6). All in vitro samples HRMAS-NMR experiments were carried out on BRUKER AVANCE-600 spectrometer, magic angle speed is 5k Hz, test temperature is 300K,the peak of 1H spectrometry was worked out though pre-saturation method and the CPMG method. The J-resolved and JRES, HSQC experiments were used to dimension reduction. The total spin-echo time is 320ms. PCA was executed by using AMix soft ware, we can obtain PC1 (first principal component) and PC2 (second principal component) and Scores map and Loadings map.Results:The PCA scores plot of data from 1H CPMG spectra presents great different metabolic changes among the three group of samples. The loading plot shows the difference can be identified by some principal components. Compared with the 1H CPMG spectra of the normal group, the signal intensity of taurine(Tau), acetic acid(Ace), alanine(Ala) of the acute pancreatitis group were increased. Oppositely the signal intensities of phosphocholine(Pc), glycerophosphocholine(GPc) and betine(Bet) were decreased. The signal intensities of choline(Cho), glutamic acid (Glu), lactate(Lac) were no change observed. Compared with the normal group, the signal intensity of taurine(Tau), lactate(Lac),phosphocholine(Pc),glycerophosphocholine(GPc) of early stage of chronic pancreatitis were increased, the signal intensities of acetic acid(Ace), alanine(Ala), betine(Bet), choline(Cho), glutamic acid (Glu) were no changes observed. Compared to the early chronic pancreatitis group, the signal intensities of acetic acid(Ace), alanine(Ala), isoleucine (Ile), leucine (Leu), valine (Val) of acute pancreatitis were increased; the intensititis of betine(Bet), phosphocholine(PC),glycerophosphate choline (GPC) were decreased. The signal intensities of taurine(Tau), choline(Cho), lactate(Lac), glutamic acid (Glu) no such changes were observed.Conclusions:The HRMAS-NMR technique can extract many other factors which can increase the errors in the experiment. HRMAS-NMR coupled with PCA were suitable to study on metabolic changes of biology. These important results from NMR experiments and PCA method show that the normal pancreas, acute pancreatitis and early chronic pancreatitis could be differentiated by their metabolic characters. This must be a effective assistance to clinical research for such kind diseases.