Effect Of The Treatment Of Smoothing Liver,Tonifying Spleen And Dredging Collaterals On Smad Signal Pathway Of Chronic Pancreatitis And Pancreatic Fibrosis

Objective By establishing a model of chronic pancreatitis and pancreatic fibrosis rats by tail intravenous single injection of dibutyltin dichloride(DBTC),the anti-fibrosis effect of treatment of smoothing liver tonifying spleen and dredging collaterals on rats model with chronic pancreatitis and pancreatic fibrosis is observed to further study the regulatory effect on chronic pancreatitis fibrosis rats’ related proteins Smad2,Smad3 and Smad7 of treatment of smoothing liver tonifying spleen and dredging collaterals.It is aimed to explore the the possible mechanism of the prevention and treatment of chronic pancreatitis and pancreatic fibrosis.Methods Sixty healthy and clean male Sprague-Dawley rats were randomly divided into normal group(n = 10)and model group(n = 50).The model group was set by tail intravenous single injection of DBTC(8mg / kg)for preparation of pancreatic fibrosis.The same volume of saline was injected by tail intravenous single injection to the normal group.In the next day after modeling,the rats in model preparation group were randomly divided into model group,pentoxifylline(PTX)group,low-dose Chinese medicine group,middle-dose traditional Chinese medicine group and high-dose Chinese medicine group,with 10 rats in each group.After grouping,drug intervention was carried out.Rats in low,medium and high dose Chinese medicine groups were intragastrically administrated with 18.5g / kg,37 g / kg and 55.5g / kg of traditional Chinese medicine every day while rats in PTX group were given daily Intraperitoneal injection of PTX 6mg /(kg.d),and the normal group was fed the same volume of distilled water every day.After continuous administration for 5 weeks,abdominal aorta blood were taken for serological test from anesthetized rats.Pancreatic tissues were isolated,and a part of them were fixed with 10% neutral formalin solution,dehydrated by gradual alcohol,embedded in paraffin and sliced for preparing the Hematoxylin and eosin(HE)staining,collagen staining(Masson)staining and Smad2,Smad3 and Smad7 protein immunohistochemistry and other histological observation and diagnosis;the other part was stored in liquid nitrogen and then stored in-80℃ refrigerator after 3h for extraction of RNA and protein.The expression of Smad2,Smad3 and Smad7 mRNA was detected by RT-PCR and Smad2 / 3 and Smad7 protein expression was detected by Western Blot.Results(1)The general situation of rats and the general structure of pancreas: The rats in normal group were generally good,and no pancreatic injury was observed.The rats in model group were generally in poor situation and their pancreas tissues were damaged severely.Compared with the model group,the general conditions of each dose TCM group and PTX group were better,and pancreatic tissue damage was also significantly less severe.(2)Serum amylase and hyaluronic acid determination: Compared with the normal group,serum amylase and hyaluronic acid in the model group,the traditional Chinese medicine group and the PTX group were significantly increased(P <0.05);compared with the model group,Serum amylase and hyaluronic acid were decreased in TCM group and PTX group(P <0.05).Serum amylase and hyaluronic acid in middle and high dose TCM group and PTX group were lower than those in low dose TCM group(P <0.05).The middle dose TCM group,high-dose group and PTX group have no significant difference between each other(P> 0.05).(3)Pathological score of HE staining and area determination of Masson-stained collagen fibers: The rats’ pancreas tissue structure in the normal group was normal and a small amount of collagen fibers were distributed in the interstitium.The pancreas tissue of the model group had severe pathological changes and extensive collagen fiber tissue deposition was observed.Compared with model group,pancreas injury and collagen fiber formation of rats in TCM group and PTX group were significantly reduced.The pathological score of the model group and the percentage of collagen fiber area were significantly higher than those in the normal group(P<0.01).The lesion degree of TCM group and PTX group was less severe than that of the model group(P <0.01).Compared to TCM middle、high-dose group and PTX group,the pancreatic pathological damage and collagen fiber formation of low-dose group are more severe(P <0.05).However,there was no significant statistical difference in pathological score and collagen fiber area between the groups of high and middle dose TCM group and PTX group(P> 0.05).(4)Immunohistochemical expression of Smad2,Smad3 and Smad7 protein:The expression of Smad2 and Smad3 protein in model group,low,medium and high dose TCM group and PTX group were significantly higher than those in normal group(P <0.01),and immunohistochemical expression of Smad7 were lower than normal group(P <0.01).Compared with the model group,the expressions of Smad2 and Smad3 were significantly decreased(P <0.01)and the expression of Smad7 protein was significantly increased in different dose TCM groups and PTX group(P <0.01).The expression of Smad2 and Smad3 protein decreased and the expression of Smad7 protein increased(P <0.05)in middle and high dose group compared with PTX group compared with low dose TCM group.There was no significant difference between the middle dose TCM group,high dose TCM group and PTX group(P> 0.05).(5)Western blot assay Smad2/3,Smad7 protein expression: Compared with the normal group,the model group Smad2/3 protein increased significantly,Smad7 protein expression decreased significantly.Compared with the model group,the expression of Smad2/3 protein decreased significantly and the expression of Smad7 increased significantly in each treatment group.(6)The expression of Smad2,Smad3 and Smad7 mRNA in the model group was significantly higher than that in the normal group(P <0.05).The mRNA expression of Smad2 and Smad3 in the model group were significantly higher than that in the normal group.Compared with the model group,the expression of Smad2 and Smad3 mRNA in each treatment group decreased significantly,and the expression of Smad7 mRNA increased in the treatment group(P <0.01).The expression of Smad2 mRNA and Smad3 mRNA in low dose TCM group were significantly higher than the middle and high dose group and PTX group(P <0.05),and the expression of Smad7 mRNA is in contrast.There was no significant difference between the medium and high dose groups and the PTX group.Conclusion(1)Rat model of pancreatic fibrosis induced by chronic pancreatitis can be successfully established by single tail intravenous injection of dibutyltin dichloride(DBTC).(2)Treatment of smoothing liver tonifying spleen and dredging collaterals can improve pathological changes of pancreatic fibrosis rats with chronic pancreatitis,reduce serum amylase and hyaluronic acid content,have obvious anti-fibrosis on pancreatic fibrosis in rats with chronic pancreatitis effect.(3)Treatment of smoothing liver tonifying spleen and dredging collaterals can inhibit the expression of Smad2 / 3 and promote the expression of Smad7,thus play a role in anti-fibrosis,which is one of the mechanisms of its treatment of pancreatic fibrosis.