c-FLIP不同单体沉默对重症急性胰腺炎大鼠中性粒细胞凋亡的影响研究

Effects of Silencing of Different c-FLIP Monomers on Neutrophil Apoptosis in Rats with Severe Acute Pancreatitis

目的探讨细胞型Fas相关死亡域样白细胞介素-1β转换酶抑制蛋白(c-FLIP)不同单体沉默对重症急性胰腺炎(SAP)大鼠中性粒细胞凋亡的影响。方法选取50只SD雄性大鼠建立SAP模型,将造模成功的40只大鼠随机分为对照组、c-FLIP siRNA-NC组、c-FLIPS siRNA组、c-FLIPL siRNA组,每组10只;另取10只大鼠为假手术组。c-FLIP siRNA-NC组、c-FLIPS siRNA组、c-FLIPL siRNA组大鼠尾静脉注射1 nmol/ml的对应siRNA溶液,假手术组、对照组大鼠尾静脉注射等量生理盐水;转染后72 h,测定各组大鼠腹水量、血清淀粉酶及血清中性粒细胞凋亡水平,并观察胰腺组织病理变化。测定各组大鼠中性粒细胞中c-FLIPS、c-FLIPL、受体相互作用蛋白1(RIP1)、半胱氨酸天冬氨酸蛋白酶-8(Caspase-8)mRNA和蛋白表达水平。结果对照组、c-FLIP siRNA-NC组胰腺组织坏死严重、崩解,胰腺细胞排列紊乱,可见大量炎性细胞浸润;c-FLIPS siRNA组、c-FLIPL siRNA组胰腺细胞坏死减少,炎性细胞浸润减少,但胰腺组织疏松紊乱。与假手术组比较,对照组和c-FLIP siRNA-NC组腹水量、血清淀粉酶、中性粒细胞c-FLIPS mRNA和蛋白表达水平、c-FLIPL mRNA和蛋白表达水平升高,中性粒细胞凋亡率、RIP1 mRNA和蛋白表达水平、Caspase-8 mRNA和蛋白表达水平降低,差异有统计学意义(P<0.05);但对照组与c-FLIP siRNA-NC组上述指标比较差异无统计学意义(P>0.05)。与c-FLIP siRNA-NC组比较,c-FLIPS siRNA组和c-FLIPL siRNA组腹水量、血清淀粉酶、中性粒细胞c-FLIPS、c-FLIPL mRNA和蛋白表达水平降低,中性粒细胞凋亡率、RIP1、Caspase-8 mRNA和蛋白表达水平升高,差异有统计学意义(P<0.05);但c-FLIPS siRNA组比较与c-FLIPL siRNA组上述指标比较差异无统计学意义(P>0.05)。结论沉默c-FLIP的两种单体c-FLIPS、c-FLIPL可明显促进SAP大鼠血液中性粒细胞凋亡,且对SAP具有明显缓解作用;其机制与c-FLIPS、c-FLIPL能明显抑制SAP大鼠血液中性粒细胞c-FLIPS、c-FLIPL的mRNA和蛋白表达,促进RIP1、Caspase-8 mRNA和蛋白的表达,进而激活坏死性凋亡途径有关。

Objective To investigate effects of silencing of different cellular Fas-associated death domain-like interleukin-1β-converting enzyme inhibitory protein(c-FLIP)monomers on neutrophil apoptosis in rats with severe acute pancreatitis(SAP).Methods A total of 50 SD male rats were established SAP models.After successful modeling,40 rats were randomly divided into blank control group,c-FLIP siRNA-NC group,short c-FLIP(c-FLIPS)siRNA group and long c-FLIP(c-FLIPL)siRNA group(n=10 in each group),and another 10 rats were selected as sham operation group.Rats in c-FLIP siRNA-NC,c-FLIPS siRNA and c-FLIPL siRNA groups were injected with 1 nmol/ml corresponding siRNA solutions through caudal veins,while sham operation and blank control groups were injected with the same volumes of normal saline through caudal veins.After transfection for 72 h,values of ascites volume,serum amylase and neutrophil apoptosis were measured,and pathological changes of pancreatic tissues in rats were observed in all groups.Levels of neutrophil apoptosis,and mRNA and protein levels of c-FLIPS,c-FLIPL,receptor interacting protein 1(RIP1)and Caspase-8 in neutrophils were determined in all groups.Results In blank control and c-FLIP siRNA-NC groups,pancreatic tissues were serious necrosis and disintegration,and pancreatic cells were arranged disorderly with a large number of inflammatory cells infiltration.In c-FLIPS siRNA and c-FLIPL siRNA groups,the necrotic pancreatic cells and inflammatory cells infiltration were reduced,but the pancreatic tissue was loose and disordered.Compared with those in sham operation group,values of ascites volume,serum amylase,mRNA and protein levels of c-FLIPS and c-FLIPL in neutrophils were significantly increased,while values of neutrophil apoptosis rate,and mRNA and protein levels of RIP1 and Caspase-8 were significantly decreased in blank control and c-FLIP siRNA-NC groups(P<0.05);but there were no statistically significant differences in the above indicators between blank control group and c-FLIP siRNA-NC group(P>0.05).In c-FLIPS siRNA and c-FLIPL siRNA groups,values of ascites volume,serum amylase,and mRNA and protein levels of c-FLIPS and c-FLIPL in neutrophils were significantly decreased,while neutrophil apoptosis rate,and mRNA and protein levels of RIP1 and Caspase-8 were significantly increased compared with those in c-FLIP siRNA-NC group(P<0.05);but there were no statistically significant differences in the above indicators between c-FLIPS siRNA group and c-FLIPL siRNA group(P>0.05).Conclusion The silencing of c-FLIPS and c-FLIPL,which are the two monomers of c-FLIP,may significantly promote blood apoptosis of neutrophils and obviously alleviate the SAP in rats.The mechanism is related that the c-FLIPS and c-FLIPL may significantly inhibit the mRNA and protein expressions of c-FLIPS and c-FLIPL in blood neutrophils,and promote mRNA and protein expressions of RIP1 and Caspase-8,thereby it may activate the necrotic apoptotic pathway.