| Acute pancreatitis(AP) is a common clinical acute abdomen with the characteristic of acute onset, severe and rapid progression. Besides the pancreatic lesions, AP can also causes the injury of other organs or tissues, even induces death because of the systemic inflammatory response syndrome(SIRS) or multiple-organ dysfunction syndrome(MODS). There are many researches on the mechanism of AP at present, including the intracellular signaling pathway. A great deal of studies have showed that the Hedgehog(Hh) signaling pathway, a new hot spot of intracellular signaling pathway, plays an important role not only in the animal embryonic development, cell differentiation and proliferation, organ development regulation, but also in a direct relationship with the occurrence and development of many diseases. However, the current studies of the relationship between Hh signaling pathway and diseases mostly focused on the malignant diseases caused by abnormal activation of Hh signaling pathway, such as carcinoma of pancreas, gastric cancer and oophoroma. Moreover, there were relatively few discussions about the correlation between Hh and inflammatory diseases. Therefore, this study was designed to investigate the role of Hh signaling pathway in AP and its possible mechanism.Objective:The aim of this study is to investigate the serum levels of amylase(AMY), tumor necrosis factor-alpha(TNF-α) and interleukin-10(IL-10) in AP rats by administrating purmorphamine, which is the agonist of Hh signaling pathway, and to explore the effect of activated Hh signaling pathway on AP rats and discuss its pathogenesis.Method:1 Experimental groups: 54 adult SD rats were randomly divided into 3 groups: N group(normal control group, n=18), AP group(acute pancreatitis group, n=18), Pur group(acute pancreatitis plus Purmorphamine, n=18). Then the rats in each group were divided into three subgroups according to time points: 6hs group, 12 hs group and 24 hs group, 6 rats in each group.2 Experimental models: The rats were given L-arginine(L-Arg) intraperitoneal injection to form AP. In AP groups, the rats were given 20% L-Arg intraperitoneal injection by 2.5 g/kg, twice at one-hour interval. In N groups, the rats received the same amount saline at the same time. Purmorphamine was administered to Pur groups intraperitoneally with the dosage of 0.69 mg/kg 1 hour before the model was made. Other groups were given the same amount DMSO intraperitoneal injection once before the first injection of L-Arg. All rats were killed at the time of 6hs, 12 hs and 24 hs respectively after the last injection of L-Arg to collect blood and pancreas tissue.3 Experimental methods: The serum levels of AMY were measured by Amylase Assay Kit; ELISA(enzyme linked immunosorbent assay) was used to detect the expression of serum TNF-α and IL-10. The expression of Ptch1 m RNA of pancreas tissue was evaluated by RT-PCR(real-time polymerase chain reaction, Real-time PCR).4 Statistical Methods: All statistical analysis was performed by SPSS 22.0 for Windows. The significance was assumed at P < 0.05.Results:1 The serum levels of AMY: At different time points, there was no significant difference in N group. Compared with N group, the serum levels of AMY were increased in AP group and Pur group(P < 0.05). The serum levels of AMY were significantly decreased in Pur group in comparison with AP group(P < 0.05).2 The expression of serum TNF-α: There were no obvious changes in N group at different time points. The expression of serum TNF-α rose in AP group and Pur group comparing with N group(P < 0.05). In Pur group, the expression of serum TNF-α were dramatically reduced when compared with that of the AP group(P < 0.05).3 The expression of serum IL-10: The expression of serum IL-10 was low at each time point in N group. This indicator in AP group and Pur group were evidently higher than those in N group(P < 0.05). Besides, compared with AP group, IL-10 were clearly increased at all-time points in Pur group(P < 0.05).4 The expression of pancreatic Ptch1 m RNA: At every time points, the expressions of Ptch1 m RNA in N group were in a relatively low level and have no obvious changes. When making a Comparison with N group, the Ptch1 m RNA in AP group and Pur group were dramatically jumped(P < 0.05); In Pur group, Ptch1 m RNA were higher than that in AP group(P < 0.05).5 Correlation tests of each indicators in AP group and Pur group: The serum levels of TNF-α and AMY were positively correlated(r=0.704, P < 0.01); The serum levels of IL-10 and AMY were negative correlated(r=-0.429, P < 0.01); The serum levels of IL-10 and TNF-α were negative correlated(r=-0.463, P < 0.01); The expression of pancreatic Ptch1 m RNA and the serum levels of IL-10 were positively correlated(rs=0.700, P < 0.01); There was no correlation between the expression of pancreatic Ptch1 m RNA and the serum levels of TNF-α(rs=-0.129, P=0.454 > 0.01).Conclusion:1 The application of L-Arg intraperitoneal injection method can successfully replicate rat AP models and also provide the condition for the research on the effect of Hh signaling pathway in AP rats.2 AMY is an important indicator of the inflammatory response in acute pancreatitis. After treated with Purmorphamine, the agonist of Hh signaling pathway, the serum levels of AMY were significantly decreased, which would result in reducing pancreatic damage and promoting the recovery of acute pancreatitis.3 As a critical proinflammatory factor, TNF-α plays an important role in AP occurrence and development. The level of inflammation severity can be directly reflected by the serum levels of TNF-α; Anti-inflammatory factor IL-10 play a protective role in the development of AP by reducing the serum level of AMY and inhibiting inflammatory factors, for example TNF-α.4 The expression of Ptch1 grew significantly after using the the agonist of Hh signaling pathway-Purmorphamine, so the expression of Ptch1 could be used as a marker of activation of Hh signaling pathway. And the activation of Hh signaling pathway in AP led to the increase of IL-10 then the decrease of TNF-α, indicating that Hh signal pathway is involved in the development process of AP and may play a protective role via up-regulating of IL-10. |
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