高三酰甘油血症对急性坏死性胰腺炎大鼠腺泡细胞自噬的影响

The effects of hypertriglyceridemia on autophagy of acinar cells in acute necrotic pancreatitis rats

目的观察高三酰甘油血症（HTG）性急性坏死性胰腺炎（ANP）大鼠腺泡细胞自噬的变化，探讨高脂因素对自噬及AP病情的影响。方法40只SD大鼠按数字表法随机分为对照组、ANP组、高脂血症组（HL组）、高脂血症性ANP组（HANP组）。对照组及ANP组大鼠先用正常饲料喂养2周，HL组及HANP组大鼠先用高脂饲料喂养2周，取血后，ANP组及HANP组大鼠采用胰胆管逆行注射3.5%牛磺胆酸钠方法制备ANP模型，对照组及HL组大鼠腹腔内注射等容积生理盐水。造模后48 h处死大鼠，取血测三酰甘油（TG）、总胆固醇（TC）及淀粉酶水平；取胰腺组织常规行病理检查并评分；透射电镜下观察细胞内超微结构改变，计算自噬体及自噬溶酶体数量；蛋白质印迹法检测胰腺组织自噬相关蛋白LC3Ⅱ、LC3Ⅰ、p62、LAMP-2表达。结果高脂饮食2周后HL组、HANP组大鼠血清TG和TC水平显著高于对照组和ANP组；HANP组和ANP组血淀粉酶水平显著高于对照组和HL组，差异均有统计学意义（P值均〈0.05）。透射电镜下见ANP组自噬小体数量显著高于其他3组，差异均有统计学意义（P值均〈0.05）；相对于ANP组HANP组腺泡细胞自噬活性被抑制。与对照组比较，ANP组胰腺组织LC3Ⅱ/Ⅰ比值及p62蛋白表达显著增加（2.11±0.03比1.70±0.08，9 420±624比4 973±577），LAMP-2蛋白表达下降（20 533±578比23 231±1 155）；与ANP组比较，HANP组LC3Ⅱ/Ⅰ比值、p62蛋白表达显著下降（1.22±0.07比2.11±0.03，7 882±83比9 420±624），差异均有统计学意义（P值均〈0.05）。结论高脂血症能加重AP的病理损伤，其机制可能与自噬相关蛋白LC3Ⅱ/Ⅰ、p62表达下降，自噬体数量减少有关。

Objectives To observe the changes of autophagy of acinar cells in hypertriglyceridemia （HTG） associated acute necrotic pancreatitis（ANP） rats, and investigate the effects of HTG on autophagy and AP.MethodsForty SD rats were randomly divided into 4 groups using random number method, including control group, ANP group, hyperlipemia （HL） group, hyperlipidemia associated acute necrotizing pancreatitis （HANP） group. Control group and ANP group were fed with normal diet for 2 weeks, while the rats in HL and HANP group were fed with a high-fat diet for 2 weeks, and after the blood collection ANP and HANP rats were induced by 3.5% sodium taurocholate retrograde injection into the pancreaticobiliary duct to establish the ANP model. Rats in control group and HL group were intraperitoneally injected with equal volume of normal saline solution. ALL rats were sacrificed at 48 h after the model establishment. Serum triglyceride （TG）, total cholesterol （TC） and serum amylase levels were measured; pancreatic tissue was histologically examined and scored; Transmission electron microscope were used to observe the intracellular ultrastructure changes and calculate the number of autophagosome and autophagic lysosome; the expressions of autophagy related proteins such as LC3, LC3Ⅱ , LC3Ⅰ, p62 and Lamp-2 were determined by Western-blot.ResultsAfter 2-week high fat diet, serum levels of TG and TC in HANP and HL groups were obviously higher than those in the control and ANP group; Serum amylase levels in HANP group and ANP group were higher than the control and HL group, and the differences were statistically significant （all P〈0.05） . Transmission electron microscope showed that the number of autophagosomes in ANP group was significantly higher than those in other three groups and the difference was statistically different （all P〈0.05）; and the autophagy activity in HANP group was impaired compared with ANP group. Compared with the control group, in ANP group the protein expressions of LC3 Ⅱ/Ⅰ ratio and p62 in pancreatic tissue were up-regulated （2.11±0.03 vs 1.70±0.08, 9 420±624 vs 4 973±577）, but LAMP-2 was down-regulated （20 533±578 vs 23 231±1 155）. Compared with ANP group, the proteins expression levels of LC3Ⅱ/Ⅰ and p62 in HANP group was obviously decreased（20 533±578 vs 23 231±1 155）, and the difference was statistically significant （all P〈0.05）.ConclusionsHypertriglyceridemia can worsen the pathology injury of AP, and the mechanism may be related to the decreased autophagy related proteins LC3Ⅱ/Ⅰ and p62 and decreased number of autophagosome.