| BackgroundAcute pancreatitis(AP) is an inflammatory condition of the pancreas. AP will spontaneously resolve in most cases, but it can develop into severe AP(SAP), which can be fatal. The overall mortality rate of SAP is approximately 20%. Several risk factors for SAP have been reported including alcohol, obesity, opioid drugs, and non-alcoholic fatty liver disease(NAFLD), also termed hepatic steatosis(HS). Rather than representing a specific disease, NAFLD is a clinical phenomenon characterized by inflammation, necrosis, and degeneration of liver cells. NAFLD affects 10 to 24% of the general population in many countries.The mechanisms by which fatty liver disease(FLD) aggravates pancreatitis remain to be elucidated; however, several potential processes have been proposed. FLD is commonly accompanied by hyperlipidemia, which has been reported to cause microcirculatory disturbances, oxidative stress, free radical accumulation, and/or acinar necrosis. Hyperlipidemia may also reduce red blood cell velocity, and thus increase hemoglobin-oxygen affinity in the microcirculation, which can induce tissue hypoxia. In addition, increasing free radical accumulation and oxidative stress may promote AP, and interstitial release of triglyceride degradation products may exacerbate cellular disruption. FLD may indeed exacerbate pancreatitis through a variety of mechanisms.Alpha-1antitrypsin(AAT) is a tissue-diffusible and water-soluble glycoprotein. Over 80% of AAT is synthesized and secreted by hepatocytes, macrophages, and some cancer cells. As serum levels of AAT increase in response to tissue injury, this glycoprotein is considered an acute-phasereactant. In addition to inhibiting neutrophil elastase, chymase, and trypsin, AAT also inhibits proteolytic enzymes such as cathepsin-G and proteinase-3(PR3). However, under inflammatory conditions, AAT activity has been reported to be reduced. For example, hydrogen peroxide, a component of cigarette smoke, can inactivate AAT. Severe AAT deficiency leads to an imbalance between proteinases and inhibitors, promoting the development of diseases such as(chronic obstructive pulmonary diseases) COPD, cirrhosis, and hepatocellular carcinoma. However, the mechanism of AAT in acute pancreatitis has not been fully elucidated.In the present study, we successfully established the rat model of fatty liver pancreatitis(HSAP) and no-fatty liver pancreatitis model(NHSAP). First, we used proteomic methods to screen the differentially expressed proteins between HSAP and NHSAP, the results showed that AAT was one of differently expressed proteins between two groups, and then we used clinical serum samples to verify the results. In order to further explore the molecular mechanism of fatty liver aggravating pancreatitis, we analyzed the differentially expressed genes and related molecular pathways between HSAP and NHSAP by RNA sequencing high throughput method, which laid a foundation for clinical application. Study one AimTo explore the differentially expressed proteins between HSAP and NHSAP. MethodsWe use the method of sodium taurocholate retrograde pancreatic duct injection to establish a rat model of acute pancreatitis. Screening differentially expressed proteins in the serum of rats with fatty liver pancreatitis and non-fatty liver pancreatitis by two dimensional gel electrophoresis and mass spectrometry. In order to verify the results of proteomics, we used ELISA to detect the serum AAT levels in normal diet group(ND), high fat diet group(HFD), non fatty liver pancreatitis group(NHSAP), fatty liver pancreatitis group(HSAP) four groups, followed by blot Western and immunohistochemistry to do further validation. ResultsWe successfully established HSAP and NHSAP model, two-dimensional gel electrophoresis showed that there were 14 down regulated proteins, 4 up-regulated proteins, and AAT was one of the differentially expressed proteins between two groups identified by mass spectrometry. The results of ELISA showed that the serum AAT levels in HSAP group were significantly decreased compared with the NHSAP group, and the results were consistent with the proteomics. Immunohistochemistry also showed that the expression of AAT in HSAP was significantly decreased compared with that in NHSAP group. ConclusionsIn summary, we have demonstrated that the serum AAT levels in HSAP rats is in the state of decompensation. Study two AimTo explore the relationship between AAT changes and the severity of acute pancreatitis. MethodsELISA was used to detect the content of AAT in the serum of the following groups: healthy control group(NC), fatty liver group(FLD), non fatty liver pancreatitis group(NHSAP) and fatty liver pancreatitis group(HSAP). The venous blood meeting the inclusion criteria was collected to obtain serum after centrifugation at 860 g, 15 min, and then it is stored in-80°C. Analysis of the relationship between serum AAT and APACHE-II scores in HSAP group. ResultsAAT level in HSAP group: 1.63±0.34 mg/ml(range: 0.74-2.59 mg/ml); the normal group 2.54±0.21 mg/ml(range: 2.16-2.90 mg/ml); NHSAP group is 3.92±0.37 mg/ml(range: 3.1-4.78 mg/ml); FLD group is 2.30±0.27 mg/ml(range: 1.81-2.69 mg/ml). APACHE-II score was negatively correlated with serum AAT levels in HSAP patients(r=-0.85, P<0.01). ConclusionsIn HSAP patients, the decompensation of AAT caused by fatty liver can aggravates the condition of pancreatitis. Study three AimComparison of the difference between fatty liver pancreatitis(APFL) and non-fatty liver pancreatitis(AP) by RNA-seq technique. MethodsHigh throughput sequencing(RNA-seq) technique was used to analyze the differentially expressed genes between APFL and AP, then they were subjected to GO function analysis and KEGG pathway enrichment analysis. Finally, the differentially expressed genes were verified by RT-PCR. ResultsKEGG results showed that the PPAR pathway and its regulated downstream pathways such as the fatty acid metabolism and JAK/SATA inflammatory pathways were involved in the pathological process of AP and APFL, and the degree of enrichment in the APFL group was more obvious. q RT-PCR validated the genes participating in the PPAR signaling pathway and the fatty acid metabolic pathway, which were consistent with the results of RNA-seq analysis. ConclusionsFatty liver may affect the lipid metabolism and inflammatory response through the PPAR gamma pathway, which would aggravate pancreatitis processes. |
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