Expression Of Fractalkine/CX3CR1 In The Pancreas Of Chronic Pancreatitis Rats And Therapeutic Effect Of α-Tocopherol And Tocotrienol-Rich Fraction

Chronic pancreatitis(CP)is characterized by progressive pancreatic damage that eventually results in significant impairment of exocrine as well as endocrine functions of the gland. Most patients currently diagnosed with CP have pain, steatorrhea, diabetes mellitus, pancreatic calcification and pseudocyst. The key histopathologic features of CP are impressive infiltration of various subsets of inflammatory cells, pancreatic fibrosis, acinar atrophy, and distorted ducts, of which severe fibrosis with distinct accumulation of extracellular matrix is its main pathlogical feature. The pathogenesis of pancreatic fibrosis has not yet been fully elucidated and there is no effective treatment for it.Part 1 Establishment of CP rat model by DBTC injection into tail veinObjective: To establish a rat model of CP induced by dibutyltin dichloride(DBTC)injection into the tail vein,and observe the pathological changes, pancreatic fibrosis degress and evaluate the oxidative stress of CP rat model.Methods: Twenty male Wistar rats(weighting 180-200g)were randomly divided into CP and VC(Vehicle control)groups. CP was induced by DBTC(8mg/kg)infusion into the tail vein once. As vehicle controls, rats were infused with the same volume of solvent. The body weight was measured once a week at the same time after the induction of CP. The rats were sacrificed by exsanguination under pentobarbital anesthesia(50mg/kg)4 weeks after the induction of CP. Histological examination, Sirius-Red staining, and measurement of the contents of hydroxyproline of the pancreas were performed to evaluate pancreatic damage and fibrosis. Pancreatic malondialdehyde(MDA)was tested to evaluate the oxidative stress level of the CP rats.Results: By the end of experiment(4 weeks after DBTC induction), the survival rate was 80% in the CP group, and 100% in the VC group. Significant body weight reduction was observed in rats with DBTC induction from the second week, compared with the vehicle controls(all P<0.05). Rats in CP group displayed histopathological signs of CP at the time of sacrifice, as reflected by abnormal architecture and fibrosis and inflammatory cell infiltrate. However, all marks in the VC group were almost normal. The pancreatic tissue content of MDA(1.18±0.37 nmol/mgprot vs. 0.22±0.27nmol/mgprot, P<0.05)and hydroxyproline content(650.58±52.27ug/mg vs. 167.88±11.20ug/mg, P<0.05) significantly increased in rats with DBTC induction comparing with the vehicle controls.Conclusions: DBTC induced CP rat model is reliable with good reproducibility and reproducibility. Because of its characteristic pathological damage in the pancreas, this rat model can be used for mechanism and therapy research.PartПExpression of Fractalkine(FKN)/CX3CR1 in the pancreas of CP rat modelObjective: To detect the expression of FKN and CX3CR1 in the pancreas of vehicle control rats and DBTC induced CP rats model.Methods: Sixteen male Wistar rats(weighting 180-200g)were randomly divided into CP and VC groups. CP was induced by DBTC(8mg/kg body weight)infusion into the tail vein once. As vehicle controls, rats were infused with the same volume of solvent. The rats were sacrificed by at the end of the forth week after induction of CP. Histological scores, Sirius-Red staining, and measurement of the contents of hydroxyproline of the pancreas were performed to evaluate pancreatic damage in two groups. Immunohistochemical analysis and real-time reverse transcription polymerase chain reaction(real-time RT-PCR)were used to detect the expression of FKN and CX3CR1in the pancreas of CP and VC group.Results: Histological scores of abnormal architecture, glandular atrophy pseudotubular complexes, fibrosis and inflammatory cell infiltrate were significantly higher in CP group. And the pancreatic tissue content of hydroxyproline content(690.19±49.49μg/mg vs. 139.75±13.39μg/mg, P<0.05)was significantly increased rats with DBTC induction comparing with the vehicle controls. The gene expression of FKN and CX3CR1 significantly increased in the pancreas of CP(P<0.05). The immunohistochemistry analysis showed that expression of FKN and CX3CR1 were up-regulated in inflammatory cells, damaged acini, pancreatic duct endothelial cells and vascular endothelial cells at different levels in CP. The gene expression of FKN and CX3CR1 significantly increased in the pancreas of CP.Conclusions: We revealed that FKN and CX3CR1 was up-regulated both at the mRNA and protein levels in the pancreas of CP rats. FKN-CX3CR1 axis may play an important role in the recruitment and adhesion of inflammatory cells and fibrogenesis of CP.PartШComparison of therapeutic effects ofα-tocopherol(Toc)and tocotrienol-rich fraction(TRF)in a at model of CPObjective: This study aimed to compare the therapeutic effects and to investigate the mechanisam ofα-Toc and TFR in DBTC-induced CP rats. Methods: Thirty-two rats were randomly divided into 4 groups, and rats of 3 groups after induction of CP selected to receive orogastric gavage of 0.6ml soybean oil , or soybean oil containingα-Toc(800 mg/kg) or TRF(800 mg/kg), once daily for 4 weeks. The control rats received orogastric gavage of 0.6 ml soybean oil once daily for 4 weeks. The body weight was measured weekly after the gavage. The rats were sacrificed by exsanguination under pentobarbital anesthesia(50 mg/kg)4 weeks after the gavage. Histological examination, Sirius-Red staining, and measurement of the contents of hydroxyproline and MDA of the pancreas were performed to evaluate pancreatic damage and fibrosis. Immunohistochemical analysis ofα-smooth muscle actin(SMA)and real time RT-PCR for transforming growth factor(TGF)-β1 and collagen(col)-α1(I)were performed to evaluate the activation of pancreatic stellate cells and the mRNA levels of fibrosis-related genes, respectively.Results: Bothα-Toc and TRF reduced MDA level, ameliorated pathlogical scores of inflammation and fibrosis and down-regulated the mRNA expression of TGF-β1, col-α1(I) in DBTC-induced CP. TRF was superior toα-tocopherol in alleviating inflammation and fibrosis and down-regulating TGF-β1 mRNA expression.Conclusions: Oral administration ofα-Toc and TRF improves pancreatic inflammation and fibrosis in DBTC induced CP rats, with TRF being more effective thanα-Toc. In summary:1.The CP rat model could be established successfully by DBTC injection into the tail vein.2.We revealed that FKN and CX3CR1 was up-regulated both at the mRNA and protein levels in the pancreas of CP rats. Fractalkine-CX3CR1 axis may play a important role in the recruitment and adhesion of inflammatory cells and fibrogenesis of CP.3. Oral administration ofα-Toc and TRF improves pancreatic inflammation and fibrosis in DBTC-induced CP rats, with TRF being more effective thanα-Toc.