| Objective:Acute pancreatitis(AP)is a common inflammatory disease,and its incidence has increased year by year in recent years.The main pathological manifestations were edema,acinar cell necrosis,inflammatory cell infiltration and hemorrhage.Most of them are clinically mild,but a few will also progress to severe acute pancreatitis,with a high mortality rate.At present,the pathogenesis of acute pancreatitis has not been elucidated,so the treatment is mostly symptomatic.Immune response is an important part of the development of acute pancreatitis,and cytotoxic T lymphocyte antigen 4(CTLA-4)is a common immune checkpoint on the surface of T cells.We want to explore the expression level of CTLA-4 in acute pancreatitis and its related mechanism,so as to explore its possibility as a potential therapeutic target and provide new ideas for the treatment of acute pancreatitis.Methods:Part Ⅰ:Thirty-two C57BL/6J mice were randomly divided into control group,acute pancreatitis 6 hours(AP-6h)group,acute pancreatitis 12 hours(AP-12h)group and acute pancreatitis 18 hours(AP-18h)group.A mouse model of acute pancreatitis was established by retrograde pancreatic duct injection of sodium taurocholate.In the acute pancreatitis 6hours group,acute pancreatitis 12 hours group and acute pancreatitis 18 hours group,10μL/10g body weight of 2.5%sodium taurocholate solution was retrogradely injected into the pancreaticobiliary duct.The saline control(Control)group was intraperitoneally injected with an equal volume of saline.The mice were anesthetized at the specified time point after modeling,and the eyeballs were removed to take blood and separate the serum,pancreas and spleen.The levels of serum amylase and lipase were detected by automatic biochemical analyzer.Paraffin sections of pancreatic tissue were prepared for HE staining and histopathological scoring.The expression of CTLA-4 and MPO in pancreatic tissue was detected by immunohistochemical staining.The expression levels of CTLA-4 and GAPDH mRNA in pancreatic tissue were detected by qRT-PCR.The expression of CTLA-4 on CD3~+T cells in spleen was detected by flow cytometry.Part Ⅱ:Twenty-four C57BL/6J mice were randomly divided into control group,AP group and Anti-CTLA-4 group,with 8 mice in each group.Mice in the Anti-CTLA-4 group were injected with 200μg of ipilimumab solution 12 hours before modeling,and the other two groups were injected with equal volume of normal saline.In AP group and Anti-CTLA-4group,2.5%sodium taurocholate solution of 10μL/10 g body weight was injected into the pancreatic duct.The Control group was injected with an equal volume of normal saline.The mice were anesthetized at the specified time point after modeling,and the eyeballs were removed to take blood and separate the serum,pancreas and spleen.The levels of serum amylase and lipase were detected by animal automatic biochemical analyzer.The pancreatic tissue and lung tissue were prepared into paraffin sections for HE staining and histopathological scoring.The expression of CTLA-4 and MPO in pancreatic tissue was detected by immunohistochemical staining.The mRNA expression levels of CTLA-4,IL-6,IL-10,IL-1β,TNF-α,CD80 and CD86 in pancreatic tissue were detected by qRT-PCR.The expression of CTLA-4 on spleen CD3~+T cells was detected by flow cytometry.The contents of IL-6 and IL-10 in serum of mice were detected by enzyme-linked immunosorbent assay.Immunofluorescence technique was used to detect the localization and expression of CD80 and CD86 on the surface of immune cells in pancreatic tissue.Results:Ⅰ.The high expression of CTLA-4 in acute pancreatitis mouse model1.Retrograde pancreatic duct injection of sodium taurocholate successfully constructed a mouse model of acute pancreatitis(1)The mice in the acute pancreatitis group were curled up in the trunk,occasionally twitched in the limbs,and had less food and water after modeling.HE staining showed that compared with the control group,typical pathological inflammation(such as edema,gap widening,inflammatory cell infiltration)could be seen in the pancreatic tissues of AP-6h,AP-12h and AP-18h groups.(2)The pathological scores of pancreatic tissue showed that the pathological scores of AP-6h,AP-12h and AP-18h groups were higher than those of the control group(P<0.05).(3)The contents of serum amylase and lipase in AP-6h,AP-12h and AP-18h groups were increased compared with the control group(P<0.05).(4)Immunohistochemical results of MPO showed that compared with the control group,MPO was highly expressed in pancreatic tissue of AP-6h,AP-12h and AP-18h groups,indicating an increase in neutrophil infiltration.2.The high expression of CTLA-4 in acute pancreatitis mouse model(1)The results of qRT-PCR showed that the mRNA level of CTLA-4 in pancreatic tissue of mice with acute pancreatitis was significantly higher than that of the control group(P<0.05).(2)The results of flow cytometry showed that the proportion of CD3~+CTLA-4~+T cells in spleen increased in AP-6h,AP-12h and AP-18h groups compared with the control group.(3)Immunohistochemistry showed that CTLA-4 was highly expressed in pancreatic tissue and spleen immune cells of mice with acute pancreatitis compared with the control group.Ⅱ.Inhibiting the expression of CTLA-4,aggravating pancreatic injury and increasing the level of inflammatory factors and its mechanism1.Inhibit the expression of CTLA-4,aggravate pancreatic injury and increase the level of inflammatory factors.(1)HE staining and pathological score of pancreatic tissue sections of mice showed that after 12 hours of modeling,the inflammatory pathological manifestations of pancreatic tissue were significantly aggravated compared with the normal modeling group(P<0.05).(2)The results of MPO immunohistochemical staining of pancreatic tissue in mice showed that after 12 hours of modeling,the infiltration of inflammatory cells in pancreatic tissue was increased in the model group compared with the normal model group(P<0.05).(3)The results showed that compared with AP group,the levels of amylase and lipase in Anti-CTLA-4 group were increased(P<0.05).(4)The pathological score of lung tissue in mice showed that the pathology of acute lung injury in Anti-CTLA-4 group was significantly worse than that in AP group(P<0.05).(5)The results of qRT-PCR showed that the expression levels of IL-6,IL-10 and IL-1βmRNA in pancreatic tissue of Anti-CTL-4 group were higher than those in AP group(P<0.05).(6)The levels of serum inflammatory factors in each group were detected by Elisa.The results showed that the levels of IL-6 and IL-10 in serum of Anti-CTLA-4 group were higher than those in AP group(P<0.05).2.CTLA-4 may play a regulatory role by binding to CD86(1)The results of qRT-PCR and immunofluorescence showed that there was no significant difference in the expression of CD80 mRNA and protein in Control group,AP group and Anti-CTLA-4 group.(2)QRT-PCR results of CD86 showed that compared with the control group,the expression level of CD86 mRNA in pancreatic tissue of AP group was significantly increased(P<0.05).Compared with AP group,the expression of CD86 mRNA in pancreatic tissue of Anti-CTLA-4 group was decreased(P<0.05).(3)Immunofluorescence results showed that CD86 was expressed on the surface of immune cell membrane in pancreatic tissue of AP group,while there was almost no CD86expression on the surface of immune cell membrane in pancreatic tissue of Anti-CTLA-4group.Conclusions:Ⅰ.CTLA-4 is highly expressed in the mouse model of acute pancreatitis.Ⅱ.Inhibition of CTLA-4 expression can aggravate pancreatic injury and increase the level of inflammatory factors,which may play a protective role in acute pancreatitis in mice by regulating inflammatory response. |
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