| Part1. The study of intra-hepatic bile duct injury in severe acute pancreatitis in ratsObjective:To explore the changes of pathology features of intrahepatic bile duct, liver function and the apoptosis rate of intrahepatic bile duct cells in the rats with severe acute pancreatitis (SAP). And to investigate the expression and roles of macrophage migration inhibitory factor (MIF) in intra-hepatic bile duct cells injury progress after severe acute pancreatitis.Methods:48SPF male Wistar rats were randomly divided into4groups (N=12). Sham operation group (SO group) and severe acute pancreatitis model groups (SAP3h,6h,12h three subgroups). Pancreatitis model was established by retrograde infusion of5%sodium taurocholate into the bilio-pancreatic duct. In the sham operation group, pancreas and duodenum was reversed after a midline incision. Serum amylase (AMY), lipase (LIPA), Alanine aminotransferase(ALT), Alkaline phosphatase (ALP), Total bile acid(TBA) and Total bilirubin (TBiL) levels were detected. Pathological changes of histology in pancreas,liver and intra-hepatic bile duct were analyzed by stained with HE (hematoxylin and eosin stain) for light microscopy. Immunohistochemistry was used to detect the expression of MIF in intrahepatic bile duct cells, and TUNEL methods to analyze apoptosis rate of intra-hepatic bile duct cells in rats.Results:With the progress of pancreatitis, pancreatic enzyme indicators AMY, LIPA, and pancreatic histology scores were elevated significantly. Liver function indicators ALT,ALP,TBA and TBiL were also that. Liver and intra-hepatic bile duct pathological grading in SAP3h,6h,12h group showed increased gradually. There was significantly statistical difference between group(P<0.05). It was the same comparing with SO group. Immunohistochemistry showed that the expression of MIF in intrahepatic bile duct in SAP group was significantly increased than that in SO group. It was expressed mainly in the nucleus, and appeared increased gradually with the pancreatitis progress. The difference was statistically significant (P<0.05). TUNEL assay showed that the apoptosis rates in intrahepatic bile duct cells were significantly increased in SAP group than that in SO group in rats (P<0.05), the difference was significant.Conclusion:The different pathological injury degree were found in intrahepatic bile duct cells in SAP rats. And expression of MIF in intrahepatic duct cells was increased gradually with SAP progress. Apoptosis rates of intrahepatic bile duct cell showed the same as MIF. It indicates that MIF may play an important role in the process of SAP complicated with intrahepatic bile duct cells injury in rats.Part2. To explore the mechanism of macrophage migration inhibitory factor in SAP associated intra-hepatic bile duct injury in ratsObjective:To explore the changes of MIF expression, and the protein expression changes of P38-MAPK signal pathway related inflammatory factors in intrahepatic bile duct in SAP rats,.And to study the mechanisms of MIF plays in SAP complicated with intrahepatic bile duct cells damage.Methods:48SPF male Wistar rats were randomly divided into4groups (N=12). Sham operation group (SO group) and severe acute pancreatitis model groups (SAP3h,6h,12h three subgroups). SAP model was established according to the Part1. Immunohistochemistry was used to detect the expression of P-38in intrahepatic bile duct cells. Expression of MIF mRNA and TNF-a mRNA in intrahepatic bile duct cells were analyzed by Real time fluorescent quantitative polymerase chain reaction (Real-time PCR). In the same time, Western-blot was used to detect the protein expression of P38, P-P38, nuclear factor-?B(NF-?B p65) and tumor necrosis factor (TNF-a). And enzyme linked immunosorbent assay (ELISA) kits was used to analyze the contents of TNF-alpha (TNF-a), IL-1beta (IL-1?) and IL-6in intrahepatic bile duct in rats.Results:Immunohistochemistry showed that the expression of P-38in intrahepatic bile duct in SAP group was significantly increased than that in SO group. It was expressed mainly in the nucleus. The difference was statistically significant (P<0.05). Real-time PCR assay showed that the expression of MIF mRNA and TNF-a mRNA in intrahepatic bile duct cells were significantly increased in SAP group than that in SO group in rats (P<0.05), the difference was significant. As the same, the results of Western-blot showed that the protein expression of P38,P-P38,NF-?B p65and TNF-a in intrahepatic bile duct increased in SAP group than that in SO group. The difference was significant (P<0.05). ELISA indicated that the contents of TNF-a, IL-1? and IL-6in intrahepatic bile duct in rats were significantly increased in SAP group than that in SO group, and those were increased gradually with acute pancreatitis progress. There was significant difference between SAP group and SO group (P<0.05).Conclusion:We can concluded that MIF could activate the activity of P38mitogen activated protein kinases (MAPK,P38-MAPK) in intrahepatic bile duct cells of rats in SAP, and further to arouse the NF-?B inflammatory signal pathway, and lead to the downstream mediators of inflammation of TNF-a, IL-1?, IL-6release a large amounts. In the end, it aggravates the inflammatory reaction and pathological injury of intrahepatic bile duct cells in SAP, and promotes the progress of SAP in rats.Part3. Protection mechanism of macrophage migration inhibitory factor inhibitor ISO-1in SAP associated intra-hepatic bile duct injury in ratsObjective:The aim of this study is to explore the effects of MIF inhibitor ISO-1on pathological injury and ultrastructure of intrahepatic bile duct of rats in SAP. To analyze the activation of NF-?B and protein expression changes of downstream inflammatory factors in intrahepatic bile duct, and to investigate the protection mechanism of ISO-1on severe acute pancreatitis-associated intrahepatic bile duct injury in rats.Methods:48SPF male Wistar rats were randomly divided into4groups (N=12). Sham operation group (SO group), severe acute pancreatitis model group (SAP group), MIF inhibitor ISO-1treatment group(ISO-1+SAP group) and ISO-1drug control group (ISO-1+SO group). ISO-1treatment group rats were injected MIF inhibitor ISO-1(0.1ml/100g) solution by intraperitoneal injection before30minutes of making SAP model. ISO-1drug control group rats were injected intraperitoneally with ISO-1(0.1ml/100g) solution before30min of sham operation. Each group rats were killed after12hours of being made model. SAP model was established according to the Part1. Serum AMY, LIPA,and ALT,ALP, TBA and TBiL levels were detected respectively.Pathological changes of histology in pancreas and intra-hepatic bile duct were analyzed by stained with HE for light microscopy. Electron microscopic was used to observe the ultrastructural changes of intrahepatic bile duct cells. Immunohistochemistry was used to detect the expression of MIF and P-38in intrahepatic bile duct cells. Expression of MIF mRNA and TNF-a mRNA in intrahepatic bile duct cells were analyzed by Real time fluorescent quantitative polymerase chain reaction (Real-time PCR). In the same time, Western-blot was used to detect the protein expression of P38, P-P38, NF-?B p65and TNF-a. And ELISA kits was used to analyze the contents of TNF-a, IL-1? and IL-6in intrahepatic bile duct in rats.Results:L Comparine with SAP, after treatment with ISO-1, pancreatic enzyme indicators AMY, LIPA, and pancreatic histology scores were reduced significantly. Liver function indicators ALT, ALP, TBA and TBiL were also that. The pathological grading of liver and intra-hepatic bile duct in ISO-1group showed decreased than SAP group. There was significantly statistical difference between group(P<0.05). It was the same comparing with SO group. Immunohistochemistry showed that the expression of MIF and P-38in intrahepatic bile duct in SAP group was significantly higher than that in ISO-1group. It was expressed mainly in the nucleus. The difference was statistically significant (P<0.05). Real-time PCR assay showed that the expression of MIF mRNA and TNF-?mRNA in intrahepatic bile duct cells were significantly decreased in ISO-1group than that in SAP group in rats (P<0.05), the difference was significant. As the same, the results of Western-blot showed that the protein expression of P-P38, NF-?B p65and TNF-a in intrahepatic bile duct droped in ISO-1group than that in SAP group. The difference was significant (P<0.05). ELISA indicated that the contents of TNF-a, IL-1? and IL-6in intrahepatic bile duct in rats were significantly decreased in ISO-1group than that in SAP group. There was no statistical difference between ISO-1control group and SO group (P>0.05).Conclusion:MIF inhibitor ISO-1could inhibit the expression of MIF in intrahepatic bile duct cells of rats in SAP, and inhibit the activity of P38-MAPK, prevent the activation of NF-?B inflammatory signal transduction pathways, and reduce the release of inflammatory factors such as TNF-a, IL-1?, IL-6,and so on. So, ISO-1plays a role of protection for inflammatory reaction and pathological injury of intrahepatic bile duct cells in SAP, and decreases the histological injury degree of SAP in rats. |
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