大黄素对急性胰腺炎大鼠胰腺核因子-kB活化的影响

Effects of emodin on nuclear factor-kB activation in pancreatic lesions in rats with acute pancreatitis

目的观察大黄素对急性胰腺炎(AP)大鼠胰腺组织核因子-kB(NF-kB)活化的影响,初步阐明大黄素治疗 AP 的分子作用机制。方法雄性 SD 大鼠采用持续输注雨蛙素(5 μg·kg^(-1)·h^(-1))法建立急性水肿性胰腺炎模型,分为注射后5、30min 及1、2、4、6 h 组,停止输注后6h 组;造模前2h 大鼠腹腔注射大黄素(10、30、100mg/kg)组及生理盐水对照组。采用电泳迁移率改变分析实验检测 NF-kB DNA 结合活性,Western blot 检测 NF-kB 抑制蛋白(ⅠkBs)的ⅠkBα和ⅠkBβ降解水平,Northern blot 检测 NF-kB 信号下游效应分子单核细胞趋化蛋白-1(MCP-1)和细胞间粘附分子-1(ICAM-1)基因表达。结果超生理剂量的雨蛙素可诱导大鼠胰腺 NF-kB 发生双相活化,活化高峰出现在雨蛙素注射30min 和4h 后,其相对面积灰度值分别为正常对照的(3.9±2.0)和(7.7±3.2)倍(n=4,P 值均<0.01)。大黄素10、30和100mg/kg剂量治疗组大鼠胰腺 NF-kB 活性较 AP 模型组分别下降45%,62%和84%(30min)和28%,74%和80%(4h,n=4,P 值均<0.01)。同时,大黄素(100mg/kg)可明显抑制雨蛙素诱导的胰腺ⅠkBα和ⅠkBβ蛋白降解(n=4,P 值均<0.01)。与其对 NF-kB 活化的抑制作用一致,大黄素剂量依赖性地抑制雨蛙素诱导的胰腺 MCP-1和 ICAM-1基因表达。结论大黄素可抑制胰腺内ⅠkBs/NF-kB 信号转导通路活化,从而抑制炎性细胞因子和粘附分子基因表达,发挥其对 AP 的治疗作用。

Objective To investigate whether the action of emodin on acute pancreatitis is mediated through inhibiting nuclear factor-κB （NF-κB） signaling pathway in pancreatic lesions. Methods Acute pancreatitis was induced by continuous intravenous infusion of 5 μg·kg^-1·h^-1 cerulein for a period of up to 6 h in male Sprague-Dawley rats？ Different doses of emodin （ 10, 30,100 mg/kg） were injected 120 min prior to cerulein infusion. At indicted times, rats were killed and the cytoplasmic and nuclear protein extracts and the total RNA were prepared from the pancreatic tissues close to the spleen. DNA-binding activity of NF-κB was assessed as an index of NF-κB activation by using electrophoretic mobility shift assay. Pancreatic expression of IκcBα and IκBβ, the inhibitory proteins of NF-κB, were measured by using Western blot analysis. Gene expression of monocyte chemoattractant protein-1 （MCP-1）and intercllular adhesion molecular-1 （ICAM-1） were detected using Northern blot analysis. Results During cerulein infusion, NF-κB activation was induced in a biphasic manner, which peaked at 30 min and 4 h （3.9 ± 2.0 fold） and （7.7 ± 3.2 fold） vs. baseline, for 30 min and 4 h, respectively （ n = 4, both P 〈 0.01 ）. This activation was significantly blocked by emodin pretreatment in a dose-dependent manner（ decreased by 45 %, 62 %, and 84 % for 30 min, and by 28%, 74%, and 80% for 4 h. respectively by 10, 30, and 100 mg/kg emodin （ n = 4, each P 〈 0.01 vs. cerulein alone）. Western blot analysis revealed that the inhibition of NF-κB activation was linked to emodininduced preservation of IκBα and IκBβ proteins. In line with the inhibition of NF-κB activation, the levels of MCP-1 and ICAM-1 mRNA in pancreas were markedly and dose-dependently reduced by emodin. Conclusions These findings indicate emodin act as a potent inhibitor of NF-κB signaling pathway in pancreas induced by acute injuries, suggesting that emodin, at least in part, through this mechanism, exerts the protective effects against acute pancreatitis.