The Preventive And Therapeutic Effect Of Regulating The IL-6/STAT3 Signaling Pathway With Entrapement Scattered DaChaiHu On CP Pancreatic Fibrosis Induced By L-Arginine

[Objective]Using the mice model of chronic pancreatitis induced by intraperitoneal injection of L-Arginine, to observe the changes of IL-6/STAT3 signal pathway in the progression of chronic pancreatitis and the intervention of different doses of Entrapement scattered DaChaillu on it, to further study the molecular pharmacology of Entrapement scattered DaChaiHu in prevention and treatment of chronic pancreatitis.[Methods]108 healthy mice (half male and half female) were randomly divided into five groups:control group (12), chronic pancreatitis (CP) model group(24), Entrapement scattered DaChaiHu large, medium and low-dose treatment group (72). Model group and treatment group were given intraperitoneal injection of L-Arginine at a concentration of 20% once a week (3.0g/kg) for 6 weeks, two each time interval of 1 hour. From the second week of modeling, the treatment group were given large, medium and small doses of Entrapement scattered DaChaiHu,and the model group were given the same volume of normal saline. Control group were injected and treated by the same volume normal saline as the treatment group. Materials were taken at 2 week,4 week and 6 week three time points. Take blood from the inferior vena cava, detect the serum amylase activity by biochemical assay, and the serum content of IL-6 by Elisa. Take fresh pancreas tissue, embed and section, observe the histopathological changes of the pancreas through HE and Masson staining. The expression of alpha-SMA changes in pancreas were observed by immunohistochemistry. The total RNA was extracted from the pancreas and was detected with PCR, to detect the changes of expression in IL-6, MMP-1 and TIMP-1 mRNA; and extraction of total protein in pancreatic homogenates. Western-blot to detect p-STAT3, FN protein change.[Results]1. Effect of pathological changes of Entrapement scattered DaChaiHu on mice with chronic pancreatitis.HE staining showed:After L-Arginine intraperitoneal injection 2 weeks, pancreatic tissue was observed significant acute inflammation, edema in pancreas lobular, necrosis in acinar cells by point and inflammatory cell infiltration. And with repeated intraperitoneal injection, the structure of the pancreas destroyed more obvious after 4 weeks and visible deposition of fibrous tissue, after 6 weeks pancreatic fibrosis has replaced the normal pancreatic tissue. Compared with mice in model group, the pancreatic of Entrapement scattered DaChaiHu treatment group injury significantly lighter, and only have a small amount of fibrous tissue deposition at 6 weeks. Effect of pathological changes of Entrapement scattered DaChaiHu on mice with chronic pancreatitis.Masson staining showed:After L-Arginine intraperitoneal injection by 2 weeks, a very small amount of blue dye collagen fibers appear; large amounts of collagen fibers hyperplasia in the pancreas can be widely visible until 6 weeks, and the pancreatic tissue is separated by mesh grid. The degree of fibrosis of Entrapement scattered DaChaiHu treatment group were significantly less than model group.2. Effect of Entrapement scattered DaChaiHu on serum AMS activity in chronic pancreatitisAfter modeling with intraperitoneal injection of L-Arginine, pancreas of the mice occurred inflammation and fibrosis, activity of serum amylase increased significantly in 2W, and remained at a high level by the fourth week, decreased until 6 week. Compared with control group it had significant difference. The serum AMS levels of Entrapement scattered DaChaiHu treatment group were significantly decreased compared with the model group.3. Effect of Entrapement scattered DaChaiHu on serum IL-6 levels in the pancreas at different time points.After L-Arginine intraperitoneal injection by 2 weeks, detect the serum IL-6 level by ELISA, it increased significantly at the second, fouth and sixth week. It had significant differences compared with the control group, and the IL-6 levels of Entrapement scattered DaChaiHu group were lower than in the model group, meanwhile, the IL-6 expression level in the treatment groups: low-dose group> middle-dose group> high-dose group.4. Effects of Entrapement scattered DaChaiHu on mRNA levels of IL-6, MMP-1, TIMP-1 in the pancreatic tissue at different time pointsAfter L-Arginine intraperitoneal injection by 2 weeks, the MMP-1mRNA expression in mice pancreas tissue at the model group decreased, there is difference compared with normal group. After the treatment of Entrapement scattered DaChaiHu Decoction gavage, expression is markedly increased, it is different from the model group (P< 0.05). The mRNA expression of pancreatic tissue IL-6, TIMP-1 was significantly increased in the model of 2W. It declined in 4W, but remained at a high level. It reduced that the expression of the each dose Entrapement scattered DaChaiHu treatment group in the 2W,4W. Compared with the model group, it is very difference (P<0.05). But there is no significant difference between the expression of model group and treatment group at 6W.5. The effect of Entrapement scattered DaChaiHu on the expression of p-STAT3 and FN protein in pancreatic tissue at different time pointsAfter L-Arginine intraperitoneal injection, the expression of p-STAT3 protein detected by Western-blot in the model increased obviously. While the expression of the Entrapement scattered DaChaiHu treatment group was significantly reduced at different time points. There was no expression of FN in the blank group, the expression of FN in the model group was significantly enhanced at each time point, and the expression was the most obvious at 6W. After the treatment of Entrapement scattered DaChaiHu, the expression of FN in pancreatic tissue was significantly less than that in model group.[Conclusion]1. Taking intraperitoneal injection of L-Arginine can successfully replicate the CP model of mice. IL-6/STAT3 signaling pathway involved in the development of chronic pancreatitis, is one of the therapeutic targets.2. Entrapement scattered DaChaiHu can effectively prevent chronic pancreatitis, and the molecular pharmacological mechanisms is associated with the regulation of IL-6/STAT3 signaling pathway.