内质网应激在高脂血症相关性大鼠急性胰腺炎发病中的作用

The effect of endoplasmic reticulum stress in hyperlipidemia rats with acute pancreatitis

目的探讨内质网应激在高脂血症大鼠合并急性胰腺炎(AP)发病中的作用。方法48只大鼠分为单纯AP组(n=18,正常大鼠腹腔内注射雨蛙肽40μg/kg×2次,间隔2 h)、正常组(n=6)、高脂血症组(n=6,普通饲料+3%胆固醇+20%猪油)和高脂血症性AP组(n=18,高脂血症大鼠腹腔内注射雨蛙肽40μg/kg×2次,间隔2 h)。单纯AP组和高脂血症性AP组大鼠均于造模后9、12、24 h分批处死(n=6),测定血淀粉酶活性.评价胰腺病理变化;TUNEL法评价胰腺组织凋亡指数;免疫组化法检测胰腺内葡萄糖调节蛋白(GRP)78/Bip的蛋白表达;PT-PCR法检测胰腺组织内内质网应激过程中的莺要分子GRP78/Bip、X结合蛋白(XBP)-1、CHOP/GADD153(C/EBP-homolo-gous protein,or growth arrest and DNA-damage-inducible gene 153)、caspase-12的基因动态表达;Western印迹法检测GRP78、caspase-12蛋白的动态表达。结果大鼠喂饲高脂饮食8周后,三酰甘油[(0.99±0.38)mmol/ L]和胆圃醇[(3.17±0.18)mmol/L]明显升高,且较正常组高281%和96%(P<0.05);高脂血症性AP组血清淀粉酶活性较单纯AP组高,胰腺病理变化亦较单纯AP组严重(P<0.05),尤其在造模后9 h最为显著。高脂血症AP大鼠造模后9、12、24 h,胰腺腺泡细胞凋亡均较正常组明显(P<0.05);GRP78/Bip表达明显;CHOP/GADD153在造膜(?)2、24 h时表达与正常对照组相比差异有统计学意义(P<0.05);在造模后12、24 h时胰腺组织发生XBP-1和caspase-12分裂,而正常组则无。结论内质网应激参与了高脂血症性AP的发病,并可通过caspase-12通路诱导胰腺腺泡细胞发生凋亡。加重AP病程。

Objective To investigate the effect of endoplasmic reticulum stress in hyperlipidemia rats with acute pancreatitis（AP）. Methods Forty-eight SD rats were divided into AP group（n = 18, induced by injection of 40μg/kg caerulein in normal rats twice with 2 hrs interval）, normal control（n=6）, hyperlipidemia group（n = 6, fed with a high-fat diet for 8 weeks） and hyperlipidemia AP group（n = 18, induced by injection of 40 μg/kg caerulein in hyperlipidemia rats twice with 2 hrs interval）. The rats were sacrificed at 9, 12, 24 hrs（6 of each）, respectively. cal changes of the pancreatic tissues were observed. Serum level of amylase was tested and the pathologi cal changes of the pancreatic tissues were observed. The index of pancreatic apoptosis was assessed by TUNEL method. The expression of GRP78/Bip（glucose regulated protein） protein was determined by immunochemistry, the endoplasmic reticulum stress related molecules of XBP-1 splicing（X box binding protein）, CHOP/GADD153 （C/EBP-homologous protein or growth arrest and DNA-damage-inducible gene 153）, caspase-12 were analyzed by RT-PCR. The dynamic expressions of GRP78/Bip and caspase 12 were determined by Western blot. Results After 8 weeks of fat diet in hyperlipidemia rats, the ser- um levels of triglyeeride[（0.99± 0.38） mmol/L] and cholesterol[（3. 17 ±0, 18） mmol/L] were significantly increased（P 〈0.05）. The serum level of amylase was higher in hyperlipidemia AP group than that in AP group and the pancreatic tissue injury was severe in hyperlipidemia AP group than that in AP group. The apoptotic indexes were higher in hyperlipidemia AP group than that in normal control（P〈 0.05）. The expression of GRP78 and CHOP/GADD153 were significantly increased, but XBP-1 and caspase-12 were splicing in hyperlipidemia AP group. Conclusions The endoplasmic retieulum stress is involved in pathogenesis of hyperlipidemia AP, and it may exacerbate the disease by inducing apoptosis of pancreatic acinar cells through caspase-12 pathway.