丹参多酚酸盐通过抑制内质网应激减轻小肠缺血再灌注损伤的研究

Salvia polyphenolate reduces the intestinal ischemia-reperfusion injury by inhibiting endoplasmic reticulum stress

关键词:  丹参多酚酸盐  小肠缺血再灌注  内质网应激  炎症  细胞凋亡

Key words:  Salvianolate  intestinal ischemia-reperfusion  endoplasmic reticulum stress  inflammation  apoptosis

摘要:

[目的] 探讨丹参多酚酸盐（SAL）对大鼠小肠缺血再灌注（I/R）后肠组织的保护作用并探索其机制。[方法] 将144只大鼠按照随机数字表法平均分为假手术组（Sham组），I/R组，SAL低（10 mg/kg）、中（20 mg/kg）、高（40 mg/kg）剂量组和银杏叶提取物（EGB）组（3.15 mg/kg）；造模前30 min腹腔注射给药；采用夹闭肠系膜上动脉60 min的方法复制小肠I/R大鼠模型。再灌注120 min后，采用干湿比重法计算肠组织含水量；分别通过苏木精-伊红（HE）染色法、原位末端转移酶标记（TUNEL）法行肠组织病理学检查和细胞凋亡观察，计算损伤评分（Chiu’s氏评分）和凋亡指数（AI）；酶联免疫吸附（ELISA）法检测肠组织炎症因子[C反应蛋白（CRP）、肿瘤坏死因子-α（TNF-α）、白介素-1β（IL-1β）]水平；蛋白免疫印迹（Western blot）法检测内质网应激相关蛋白[糖调节蛋白78（GRP78）、c-Jun氨基端激酶（p-JNK）、增强子结合蛋白同源蛋白（CHOP）]、核因子-κB（NF-κB）、激活型半胱氨酸蛋白酶-3（Cleved Caspase-3）蛋白表达。[结果] 与I/R组比较，SAL中、高剂量组和EGB组大鼠肠组织含水量降低，差异有统计学意义（P<0.05或P<0.01）；肠系膜细胞脱落、固有层分离、炎性细胞浸润等病变和细胞凋亡状况明显改善，Chiu’s氏评分和AI降低，差异有统计学意义（P<0.05或P<0.01）；肠组织CRP、TNF-α、IL-1β水平降低，差异有统计学意义（P<0.05或P<0.01）；GRP78、p-JNK、CHOP、NF-κB、Cleved Caspase-3蛋白表达下调，差异有统计学意义（P<0.05或P<0.01）。与EGB组比较，SAL高剂量组大鼠Chiu’s氏评分、AI降低，差异有统计学意义（P<0.05或P<0.01）；肠组织CRP、TNF-α水平降低，差异有统计学意义（P<0.05或P<0.01）；GRP78、CHOP、NF-κB、Cleved Caspase-3蛋白表达下调差异有统计学意义（P<0.05或P<0.01）。[结论] SAL对大鼠小肠I/R损伤具有保护作用；其机制可能与SAL抑制内质网应激通路，进而抑制炎症反应和细胞凋亡有关。

Abstract:

[Objective] To investigate the protective effect of Salviaolate (SAL) on intestinal tissue after ischemia/reperfusion (I/R) in rats and explore its mechanism.[Methods] 144 rats were randomly divided into sham operation group (Sham group),I/R group,SAL low (10 mg/kg),medium (20 mg/kg),high (40 mg/kg) dose group and Extract of Ginkgo Biloba (EGB) group (positive control group, 3.15 mg/kg);the rat models with intestinal I/R injury were made by clipping superior mesenteric artery for 60 mins. 30 mins before surgery,the drugs were given by intraperitoneal injection. 120mins later,the water content of intestinal tissue was calculated by dry/wet method;the mesentery pathological examination and apoptosis observation were performed by hematoxylin-eosin (HE) staining method and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method respectively,and the injury score (Chiu's score) and apoptosis index (AI) were calculated;the levels of inflammatory factors[C reaction protein (CRP),tumor necrosis factor-α (TNF-α),interleuk-1β (IL-1β)] in intestinal tissue were detected by enzyme linked immunosorbent assay (ELISA) method;the expression of endoplasmic reticulum stress-related proteins[glucose regulated protein 78 (GRP 78),Jun N-terminal kinases (p-JNK),C/Ebp-homologous protein (CHOP)],nuclear factor kappa-B (NF-κB),Cleved Caspase-3 were detected by Western blot method.[Results] Compared with I/R group,the water content in Salvianolate medium,high dose group and EGB group were significantly decreased (P<0.05 or P<0.01);the mesenteric cell detachment,lamina propria separation,inflammatory cell infiltration and other diseases,and cell apoptosis were significantly improved;the Chiu's score and AI were significantly reduced (P<0.05 or P<0.01);the level of CRP,TNF-α,IL-1β were significantly decreased (P<0.01);the expression of RP78,p-JNK,CHOP,NF-κB,Cleved Caspase-3 were significantly down-regulated (P<0.05 or P<0.01). Compared with EGB group,the Chiu's score and AI were significantly reduced (P<0.05);the level of CRP,TNF-α were significantly decreased(P<0.05 or P<0.01);the expression of RP78,CHOP,NF-κB,Cleved Caspase-3 were significantly down-regulated (P<0.05 or P<0.01).[Conclusion] SAL has protective effect on intestine I/R injury in rats;which mechanism may be related to SAL inhibiting the endoplasmic reticulum stress pathway,thereby inhibiting inflammation and apoptosis.