Role Of ATP Citrate Lyase On The Repair Of Injured Pancreas After Acute Pancreatitis

Part 1 Pancreatic injury and repair after acute pancreatitis in ratsObjective: To investigate the process of pancreatic injury and repair after acute necrotizing pancreatitis in a rat model.Methods: 8-week-old Sprague-Dawley male rats,weighing 250-300 g,were divided into 6 groups according to random number table,sham-operation(SO)group,3d after acute pancreatitis(AP3d)group,AP7 d group,AP14 d group,AP28 d group,and AP56 d group,eight rats in each group.Rats in AP group were injected with 5% sodium taurocholate saline solution(50mg/kg)to establish the animal model of acute necrotizing pancreatitis.Continuous monitoring of rats’ weight changes was conducted after modelling.According to the presupposed time point in experimental group,at 3d,7d,14 d,28d and 56 d after surgery,rats were sacrificed,and blood samples were collected by inferior vena cava puncture to measure serum amylase activity.The head of the pancreas tissue was immediately excised for histopathological analysis by HE staining and immunofluorescent detection of α-Amylase expression in the acinar cells.Results: From 1 week to 8 weeks after modeling,the weight growth rate of rats in SO group was much higher than that in AP group at all time points(P<0.05).Level of serum amylase activity in AP3 d group was significantly higher than that in SO group(P<0.05);Level of serum amylase activity in AP7 d group was significantly lower than that in AP3 d group(P<0.05);Level of serum amylase activity was decreased further in AP14 d and AP28 d,and slightly increased in AP56 d,but there was no significant statistical difference between AP56 d and AP28d(P>0.05);and no significant difference was observed between AP56 d and SO group(P>0.05).In AP3 d group rats,obvious damage to the pancreatic structure was observed,with massive areas of coagulation necrosis,remarkable edema in residual acini,accompanied by a large number of inflammatory cell infiltration in the interlobular septa and acinar spaces,extensive proliferation of fibroblast-like cells in necrotic areas and around the residual acini,as well as acinar-to-ductal metaplasia(ADM)structure formation.As a result,pancreatic pathological score was significantly higher than that in SO group(P< 0.05).As progressed to AP28 d,a shrinking number of pancreatic acini necrosis,reduced tissue edema degree and inflammatory cells infiltration were detected,while the total number of ADM structure further increased and formed tubular complexes.Moreover,the interlobular septa and acinar spaces were surrounded by plenty of fibroblast-like cells,with abundant stromal fibrosis formation,and part of the acinar cells were replaced by fat.In AP56 d group,the pancreatic lobule structure was complete,and no obvious ADM structure or acinar necrosis was observed.A small number of inflammatory cells and fibroblast-like cells were observed in the interlobular space,while no inflammatory cell infiltration and fibroblast-like cell proliferation were observed around the acini,and the pancreatic pathological score was significantly lower than that of AP28d(P<0.05).Theα-Amylase staining showed that the number of acinar cells in AP3 d group was decreased significantly compared with that in SO group.As the disease progressed after modeling,the percentage of acinar cells was decreased gradually,while the percentage of acinar cells in the AP56 d group was increased significantly.Conclusion: After acute necrotizing pancreatitis,the weight gain of rats was limited,and the pancreas was significantly damaged,accompanied by the process of tissue repair.Inflammatory cells,fibroid cells and ADM structures play an important role in the repair process.Part 2 Dynamic changes of inflammatory cells and ACLY expression in rat pancreatic tissue after acute pancreatitisObjective: To investigate the expression location and changes of ATP citrate lyase(ACLY)in pancreatic tissue after acute necrotizing pancreatitis in a rat model.Methods: 8-week-old Sprague-Dawley male rats,weighing 250-300 g,were divided into 6 groups according to random number table,sham-operation(SO)group,3d after acute pancreatitis(AP3d)group,AP7 d group,AP14 d group,AP28 d group,and AP56 d group,eight rats in each group.Rats in AP group were injected with 5% sodium taurocholate saline solution(50mg/kg)to establish the animal model of acute necrotizing pancreatitis.According to the presupposed time point in experimental group,at 3d,7d,14 d,28d and 56 d after surgery,rats were sacrificed,the head of the pancreas tissue was immediately excised for assessing fibrosis level by Masson staining and Van Gieson staining.The m RNA level of ACLY,α-SMA and Collagen I were evaluated by real-time fluorescent quantitative PCR.The expression location and changes of ACLY in pancreatic tissue,and the expression of activated pancreatic stellate cell(PSC)marker,α-SMA,were detected by immunohistochemistry.The deposition of collagen type I and the expression of cell proliferation index,Ki67,were detected by immunofluorescence.Moreover,the infiltration of neutrophil(marked by MPO),macrophage(CD68),plasma cell(PC-1),and T lymphocyte(CD3)were assessed.Results: As shown by immunofluorescence,dynamic changes of the infiltration of inflammatory cells were observed in pancreatic tissue after AP.In AP3 d group,a large number of MPO-positive neutrophils and CD68-positive macrophages infiltration,a small amount of PC-1-positive plasma cells and CD3-positive T lymphocytes infiltration were detected.As time going,the number of neutrophils gradually reduced,but a large number of infiltrating macrophages was sustained until AP28 d.Little change of plasma cells occurred from AP3 d to AP14 d,and peaked at AP28 d,along with the gradual increase of T lymphocytes from AP3 d to AP28 d.The infiltration status of these inflammatory cells was significantly reduced at AP56 d group.The expression of α-SMA in the pancreatic tissues of rats increased gradually as the progression of AP,and reached the peak in the AP28 d group,both of the protein and m RNA levels.In the pancreas of rats in the AP56 d group,only a small number of α-SMA positive cells were detected,and the expression level of α-SMA m RNA was significantly decreased,except for blood vessels(P<0.05).The expression levels of Collagen type I protein and m RNA in rat pancreatic tissue also were increased as the progression of AP,and peaked in AP28 d group,which decreased significantly in AP56 d group(P<0.05).Masson staining and Van Gieson staining showed that the percentage of area covered by collagen fibers in rat pancreatic tissue increased gradually with the progression of AP,peaking in AP28 d group and decreasing significantly in AP56 d group(P<0.05).As shown by immunohistochemistry,ACLY was highly expressed in the islets of the rats in the SO group,showing a brown color;it was also expressed in the acinar cells and ductal epithelial cell,showing a pale brown color.ACLY expression was increased in residual acinar cells of AP3 d group and AP7 d group,and high expression of ACLY was also observed in ADM structure.The expression of ACLY increased in innate ductal epithelial cells,and increased in fibroblast-like cells and inflammatory cells.In the abovementioned cells,the expression level of ACLY in AP14 d group and AP28 d group higher than that in AP7 d group.The expression of ACLY protein in AP56 d group was similar to that in SO group.The expression level of ACLY m RNA in pancreatic tissues of rats was significantly lower in AP3 d group and AP7 d group than that in SO group,and it was significantly higher in AP14 d group than that in AP7 d group,while in AP28 d group and AP56 d group,it was further increased(P<0.05).The increased expression of Ki67 was observed in residual acinar cells and inflammatory cells of the AP3 d group.The high expression of Ki67 was maintained in residual acinar cells and infiltrating inflammatory cells of the AP7 d group.In AP14 d group,a higher expression state of Ki67 was still maintained in residual acinar cells and infiltrating inflammatory cells,which was decreased compared with AP7 d group.The expression level of Ki67 in residual acinar cells and infiltrating inflammatory cells in the pancreas of the AP28 d group was significantly lower than that of AP14 d group.The expression of Ki67 was mainly concentrated in acinar cells in AP56 d group.Conclusion: The infiltration of inflammatory cells in pancreatic tissue of AP rats were changed dynamically with the progression of the disease.High expression of ACLY was supposed to be involved in the repair of the pancreas after AP,presumably by modulating the proliferation of pancreatic acinar cells,infiltration of inflammatory cells,and fibrosis of pancreatic tissues.Part 3 Inhibition of ACLY promotes pancreatic repair after acute pancreatitis in ratsObjective: To investigate the protective effect of inhibiting ACLY by BMS-303141 on the pancreatic repair after acute necrotizing pancreatitis in a rat model.Methods: 8-week-old Sprague-Dawley male rats,weighing 250-300 g,were divided into 2 groups according to random number table,AP28 d group,and acute pancreatitis plus BMS-303141 intervention 28d(APB28d)group,eight rats in each group.Rats in APB28 d group were given the ACLY inhibitor BMS-303141 solution prepared with 5%DMSO through intraperitoneal injection after modeling of acute necrotizing pancreatitis,at the dose of 1.0mg/kg body weight once a day until 1 day before sacrificed.Rats in the AP28 d group were injected with 5%DMSO saline solution intraperitoneally after AP modeling,at a dose of 0.35 ml per rat once a day until 1d before sacrificed.According to the presupposed time point in experimental group,at 28 d after surgery,rats were sacrificed,and blood samples were collected by inferior vena cava puncture to measure serum amylase activity.The head of the pancreas tissue was immediately excised for histopathological analysis by HE staining,and for assessing fibrosis level by Masson staining and Van Gieson staining.The m RNA level of ACLY,α-SMA and Collagen I were evaluated by real-time fluorescent quantitative PCR.The expression location and changes of ACLY in pancreatic tissue,and the expression of activated pancreatic stellate cell(PSC)marker,α-SMA,were detected by immunohistochemistry.In addition,α-Amylase expression in the acinar cells,the deposition of collagen type I and the expression of cell proliferation index,Ki67,were detected by immunofluorescence.Moreover,the infiltration of neutrophil(marked by MPO),macrophage(CD68),plasma cell(PC-1),and T lymphocyte(CD3)were assessed by immunofluorescence.The expressions of NF-κB p65,IκBα,IL-6 and TNF-α in rat pancreas were detected by Western blot.Results: Level of serum amylase activity was slightly increased in APB28 d group than AP28 d group,but there was no significantly statistical difference between groups(P>0.05).As for morphology,in APB28 d group,the pancreatic lobule structure was complete,and no obvious acinar necrosis was observed.A small number of ADM structure,inflammatory cells and fibroblast-like cells were observed in the interlobular space,and the pancreatic pathological score was significantly lower than that of AP28d(P<0.05).As shown by immunofluorescence,the α-Amylase-positive acinar cells in APB28 d group were significantly increased when compared with that in AP28 d group(P<0.05),while the percentage of area occupied by acinar cells in APB28 d group was nearly 80%.In addition,as shown by immunohistochemistry,ACLY was highly expressed in the islets of the rats in the APB28 d group,showing a brown color.However,the expression level of ACLY protein in the acinar cells,ductal epithelial cell,and fibroblast-like cells of APB28 d group was lower than that of AP28 d group.The expression level of ACLY m RNA in pancreatic tissues of rats was significantly lower in APB28 d group than that in AP28 d group(P<0.05).In the pancreas of rats in APB28 d group,except for blood vessels,only a small number of α-SMA positive cells were detected,and the expression level of α-SMA m RNA was significantly lower than that in AP28 d group(P<0.05).Compared with AP28 d group,collagen type I protein and m RNA in rat pancreatic tissues were decreased significantly in APB28 d group(P<0.05).Masson staining and Van Gieson staining showed that the percentage of area covered by collagen fibers in rat pancreatic tissues of APB28 d group were significantly lower than that of AP28 d group(P<0.05).Besides,the number of MPO-positive neutrophils and CD68-positive macrophages,PC-1-positive plasma cells and CD3-positive T lymphocytes infiltrated in the rat pancreas of APB28 d group were significantly reduced,as compared with AP28 d group.The expression of Ki67 was mainly concentrated in acinar cells in APB28 d group,and the proliferation index was higher expression than that in AP28 d group(P<0.05).The expression of nuclear NF-κB p65,total IL-6 and TNF-α protein in rat pancreas of APB28 d group was lower than that of AP28 d group,while total IκBα protein was higher(P<0.05).Conclusion: ACLY was involved in the process of pancreatic repair after AP.Treatment with BMS-303141 could inhibit the expression of ACLY in pancreatic tissue of AP rats,and further reduce the chemogenic aggregation of inflammatory cells,suppress the activation and proliferation of PSC,improve the inflammatory microenvironment of pancreas,promote the proliferation of acinar cells,and consequently facilitate the repair of pancreatic structure.