摘要
目的观察自噬抑制剂spautin-1干预雨蛙素诱导的急性胰腺炎(AP)AR42J细胞后对腺泡细胞自噬、凋亡率及坏死率的影响,探讨其在AP发病中的作用。方法分别给予AR42J细胞生理盐水(对照组)、100 nmol/L的雨蛙素(雨蛙素组)、100 nmol/L的雨蛙素联合10 μmol/L spautin-1(雨蛙素+spautin-1组)处理24 h,雨蛙素作用AR42J细胞0、6、12、24 h,采用蛋白质印迹法检测细胞自噬相关的LC3、p62、Beclin1蛋白表达,BGAM法检测细胞胰蛋白酶活性,流式细胞仪检测细胞凋亡率,乳酸脱氢酶细胞毒性测定细胞坏死率。结果0、6、12、24 h雨蛙素组LC3Ⅰ表达量分别为4.32±0.46、4.68±0.41、5.22±0.38、5.88±0.63,LC3Ⅱ表达量为0.36±0.02、0.64±0.05、0.93±0.08、2.43±0.23,p62表达量为0.24±0.01、0.22±0.02、0.84±0.09、1.25±0.13,随时间延长,表达量均逐渐增加,差异均有统计学意义(P值均<0.05),而各时间点Beclin1表达差异无统计学意义。与雨蛙素组比较,雨蛙素+spautin-1组LC3Ⅰ(0.24±0.01比0.65±0.06)、LC3Ⅱ(0.71±0.08比1.26±0.15)和p62(0.56±0.06比1.06±0.09)表达量均显著降低,同时细胞胰蛋白酶活性降低(1.13±0.14比1.65±0.18),细胞凋亡率增加[(23.64±2.12)%比(6.58±4.01)%],细胞坏死率下降[(7.64±2.12)%比(27.58±3.46)%],差异均有统计学意义(P值均<0.05)。结论spautin-1可通过阻止雨蛙素诱导的AP AR42J细胞自噬障碍,抑制胰蛋白酶激活,进而促进细胞凋亡,抑制细胞坏死。
ObjectiveTo investigate the potential of Spautin-1 to treat acute pancreatitis by inhibiting impaired autophagy and promoting apoptosis using an acute pancreatitis cell model induced by cerulein in vitro.Methods Pancreatic acinar cells AR42J were treated with sterile saline,100 nmol/L cerulein,and 100 nmol/L cerulein combined with 10 μmol/L spautin-1 for 24 h,respectively,and then western blot was used to detect the expression of LC3,p62 and Beclin1 in the cells to reflect autophagy;the trypsin activity in cells was detected by BGAM.Flow cytometry was used to detect the apoptosis;LDH cytotoxicity assay kit was used to detect cell necrosis.Results The expression of LC3Ⅰ(0 h-24 h: 4.32±0.46,4.68±0.41,5.22±0.38,5.88±0.63),LC3Ⅱ(0 h-24 h: 0.36±0.02,0.64±0.05,0.93±0.08,2.43±0.23) and p62 (0 h-24 h: 0.24±0.01,0.22±0.02,0.84±0.09,1.25±0.13) was increased in AR42J cell model treated with cerulein (P<0.05),indicated that cells undergo autophagy,but autophagy flux is blocked.The expression of LC3Ⅰ(0.65±0.06 vs 0.24±0.01),LC3Ⅱ(1.26±0.15 vs 0.71±0.08) and p62 (1.06±0.09 vs 0.56±0.06) were decreased after incubated with spautin-1,with the decreased trypsin activity (1.65±0.18 vs 1.13±0.14),increased apoptosis (6.58±4.01 vs 23.64±2.12)(P<0.05) and reduced cell necrosis(27.58±3.46 vs 7.64±2.12)(P<0.05).Conclusions In the AR42J cell model of acute pancreatitis induced by cerulein in vitro,spautin-1 can inhibit impaired autophagy induced by cerulein,decrease trypsin activity,increase apoptosis and reduce cell necrosis to protect pancreatic cells from damage,which has a certain value for remissing and even curing acute pancreatitis.
作者
张雪良
徐子琴
熊建华
Zhang Xueliang;Xu Ziqin;Xiong Jianhua(Department of Critical Care Medicine,Wenzhou People's Hospital,Wenzhou 325000,China)
出处
《中华胰腺病杂志》
CAS
2018年第4期247-250,共4页
Chinese Journal of Pancreatology
