张水军翟文龙, 赵永福, 朱长举, 邓中月, 郑州大学第一附属医院普外科 河南省郑州市 450052 雷公藤内脂醇对大鼠重症急性胰腺炎的治疗作用
Triptolide treatment of severe acute pancreatitis in rats
Shui-Jun Zhang, Wen-Long Zhai, Yong-Fu Zhao, Chang-Ju Zhu, Zhong-Yue Deng
Shui-Jun Zhang, Wen-Long Zhai, Yong-Fu Zhao, Chang-Ju Zhu, Zhong-Yue Deng, Department of General Surgery , The First Affiliated Hospital, Zhengzhou University, Zheng Zhou 450052, Henan Province, China
Correspondence to: Shui-Jun Zhang Department of General Surgery, The First Affiliated Hospital, Zhengzhou University, 1 Jianshe Road, Zhengzhou, Henan Province, 450052, China. zhangshuijun@zzu.edu.cn
Received: 2005-01-17 Accepted: 2005-02-02
Abstract
AIM: To investigate the role of NF-kB activation in the development of severe acute pancreatitis (SAP) and the therapeutic effect of triptolide.
METHODS: Ninety Wistar rats were randomly allocated into three groups: sham operation group (group S), pancreatitis group (group P), and triptolide group (group T). There were 30 rats in each group. Rats in group S only received abdomen opening surgery. SAP model was induced by retrograde injection of 5% sodium taurocholate to the pancreatic duct. After model was established, no treatment was given in group P. Triptolide (0.05 mg/mL) was given by intraperitoneal injection at 0.2 mg/kg in group T. Rats were killed 2 h, 6 h,and 12 h (10 rats for each time point). after operation to determine the serum levels of amylase (AMY), TNF-a and IL-6. The pancreas tissues were obtained to examine the changes with microscope. Blood lymphocyte smear and pancreatic tissues were prepared to detect the activity of NF-kB.
RESULTS: Serum AMY Levels in group P were higher than those in group S after operation (P: 47 961.76±11 196.41, 48 768.09±8 699.41, 64 025.30±10 413.25 nKat/L; S: 11 196.07±2 363.64, 10 156.20±1 780.02, 9 987.50±3 114.79 nKat/L, P<0.01). Serum AMY Levels were significantly lower in group T than those in group P 12 h after operation (64 025.30±10 413.25 vs 47 196.94±9 278.36 nKat/L, P<0.01). At the three time points, the levels of TNF-a and IL-6 in group P and T were significantly increased compared to those in group S. In group T, they were significantly decreased compared to those in group P at the three time points (S: 0.63±0.07, 30.48±9.16, 0.67±0.14, 25.71±5.87, 0.84±0.12, 36.98±12.38 pg/L; P: 1.12±0.14, 69.38±19.32, 1.34±0.14, 106.65±18.97, 1.60±0.41, 168.40±43.59 pg/L; T: 0.88±0.19, 35.64±11.81, 0.82±0.20, 69.38±14.37, 0.92±0.19, 67.04±23.21 pg/L, P<0.01). Activity of NF-kB in pancreatic tissue and blood lymphocyte was increased 2 h and 6 h after operation in group P and group T. In group T, activity of NF-kB was significantly decreased compared to that in group P 2 and 6 h after operation(P: 60.73±3.54, 70.77±3.56, 70.05±3.77, 80.03±3.94%; S: 10.67±2.84, 10.27±2.53, 10.90±2.51, 12.96±2.53%; T: 31.54±3.59, 30.57±3.54, 39.36±3.43, 40.37±3.51%, P<0.01). Pancreatic pathological damages were much milder in group T than those in group P under microscope.
CONCLUSION: NF-kB plays an important role in the development of SAP. Triptolide can inhibit NF-kB activity, decrease inflammatory mediator levels, and reduce the pathological damage of the pancreas.
Key Words: Severe acute pancreatitis; NF-kB; Rats; Triptolide
Zhang SJ, Zhai WL, Zhao YF, Zhu CJ, Deng ZY. Triptolide treatment of severe acute pancreatitis in rats.Shijie Huaren Xiaohua Zazhi 2005;13(8):997-1001
摘要
目的: 探讨NF-kB在重症急性胰腺炎(SAP)发病中的作用及雷公藤内脂醇对SAP的治疗作用.
方法: Wistar大鼠90只，随机分为三组:重症急性胰腺炎组(P组)，雷公藤内脂醇治疗组(T组)，假手术组(S组).每组30只.SAP模型由5%牛磺胆酸钠大鼠胆胰管逆行注射诱发而成.模型成功后，P组不做处理;T组立即行雷公藤内脂醇(0.05 g/L)腹腔注射0.2 mg/kg;S组仅开腹，不做其他处理.各组于术后2、6、12 h三个时间点处死大鼠，每次10只.各时间点测血清淀粉酶(AMY)，TNF-a、IL-6水平;取胰腺组织行病理检查并测胰腺组织和血淋巴细胞NF-kB活性.
结果: AMY P组术后各时间点均明显升高 (P组: 47 961.76± 11 196.41，48 768.09±8 699.41，64 025.30±10 413.25 nKat/L;S组:11 196.07±2 363.64，10 156.20±1 780.02，9 987.50±3 114.79 nKat/L，P<0.01)，在术后12 h点 T组低于P组( 64 025.30±10 413.25 vs 47 196.94±9 278.36 nKat/L, P<0.01)血清TNF-a、IL-6水平随SAP病情的进展而升高，P组及T组术后各时间点均较S组高其水平T组低于P组(S组:0.63±0.07，30.48±9.16，0.67±0.14，25.71±5.87，0.84±0.12，36.98±12.38 pg/L;P组:1.12±0.14，69.38±19.32，1.34±0.14，106.65±18.97，1.60±0.41，168.40±43.59 pg/L;T组0.88±0.19，35.64±11.81，0.82±0.20，69.38±14.37，0.92±0.19，67.04±23.21 pg/L，P<0.01);血淋巴细胞及胰腺组织NF-κB活性术后2、6 h P组及T组均升高(P组:60.73±3.54，70.77±3.56，70.05±3.77，80.03±3.94%;S组:10.67±2.84，10.27±2.53，10.90±2.51，12.96±2.53%;T组:31.54±3.59，30.57±3.54，39.36±3.43，40.37±3.51%，P<0.01);胰腺病理损害光镜及电镜下T组明显轻于P组.
结论: NF-kB可能参与了大鼠SAP发病过程;雷公藤内脂醇可以通过抑制NF-kB活性，减少炎症递质的释放，从而减轻SAP病理损害.
关键词: 重症急性胰腺炎; NF-kB; 大鼠; 雷公藤内脂醇
张水军, 翟文龙, 赵永福, 朱长举, 邓中月. 雷公藤内脂醇对大鼠重症急性胰腺炎的治疗作用. 世界华人消化杂志 2005;13(8):997-1001
http://www.wjgnet.com/1009-3079/13/997.asp
0 引言
重症急性胰腺炎(severe acute pancreatitis，SAP)是一种常见的急危重病，其发病机制尚未完全阐明，研究表明白细胞过度激活后引起机体炎症递质瀑布样释放，导致机体免疫紊乱，可能是SAP发病的基本机制之一.同时研究表明核转录因子-kB(nuclear factor-kB，NF-kB)是调控机体炎症反应的总源头[1].目前有研究认为雷公藤内脂醇可以通过抑制NF-kB的活性抑制炎症反应.我们通过检测NF-kB在 SAP大鼠体内的活性及炎症递质的水平变化，探讨NF-kB在大鼠SAP发病机制中的作用，并对SAP大鼠施以雷公藤内脂醇，观察其治疗作用.
1 材料和方法
1.1 材料 Wistar大鼠90只，雌雄不限，体重300±30 g，购自河南省实验动物中心.随机分为三组:SAP组(P组)、雷公藤内脂醇治疗组(T组)、假手术组(S组).每组30只.P组与T组动物先制成大鼠SAP模型.SAP模型由5%牛磺胆酸钠(Sigma公司)大鼠胆胰管逆行注射制备而成.模型成功后，P组不作其他处理;T组立即行雷公藤内脂醇(福建省医科所植化室，纯度>99.99%)腹腔注射0.2 mg/kg，雷公藤内脂醇无菌蒸馏水配制浓度0.05 g/L.S组只开腹，轻揉胰腺，不作其他处理.术后各组于2、6、12 h三个时间点处死大鼠，每次10只.
1.2 方法
1.2.1 动物模型的建立(参照Aho[2]的方法，并略有改进) Wistar大鼠术前12 h禁食，自由饮水.10%水合氯醛腹腔注射麻醉后固定于手术台上，备皮，消毒，铺无菌巾，上腹正中做2 cm切口进入腹腔，提起十二指肠，显露胆胰管，肝门部无创血管钳阻断胆管.1 mL注射器通过十二指肠侧壁，潜行穿刺入胆胰管，推注5%牛磺胆酸钠1 mL/kg，推注速度0.2 mL/min，压力约20 cm H2O，拨针后棉签压迫穿刺点2 min，以免药液逆流.推注后观察5 min，确认大鼠脾胃之间胰腺组织出血、呈暗红色后，去掉肝门部血管夹.查腹腔无活动出血后关腹.
1.2.2 血清标本的采集与检测 模型成功后，分别按时间点处死大鼠，开腹下腔静脉取血3 mL，2 mL离心留置血清于-30度冰箱内保存，1 mL以备分离血淋巴细胞.自动生化分析仪鉴定血清淀粉酶(AMY);放免法测血清TNF-a、IL-6.(按试剂盒说明进行，试剂盒购自北京北免东雅生物技术研究所).
1.2.3 NF-kB活性测定 取同一部位胰腺组织，40 g/L多聚甲醛固定，石蜡包埋，切片.常规S-P法免疫组化染色，抗p65单克隆抗体，工作浓度1∶100.PBS代替一抗为空白对照.NF -kB试剂盒，购自北京中山公司.
1.2.4 血淋巴细胞涂片制备 使用密度梯度离心法，抽取下腔静脉血1 mL，加入0.5 mL生理盐水肝素液抗凝，缓慢加在2 mL淋巴细胞分离液(购自北京中山公司)上、1 500 r/min、梯度水平离心20 min，移液器小心移出两液面间的白色絮状物，置换试管，以1∶3比例加入0.1 mol/L PBS 吹洗均匀，800 r/min离心5 min，弃上清，反复洗涤2-3次，0.1 mol/L PBS适量重悬至3×106，涂片.涂片后，冷风快速吹干，入40 g/L多聚甲醛中固定30 min，蒸馏水洗冲5 min×3次，冷风吹干，置-20℃贮存备检.
1.2.5 血淋巴细胞SP法免疫组化操作与组织切片大体相同.
1.2.6 免疫组化结果判定 细胞染色为棕黄色者为阳性，定位于胞质与胞核，高倍镜下数1 000个细胞，计算其中阳性细胞数，阳性率=阳性细胞数/总细胞数.
统计学处理 所有数值变异均采用(mean±SD)表示，应用SPSS 10.0分析软件，采用重复数据方差分析、单因素方差分析，进行统计处理，显著性检验水准取a = 0.05.
2 结果
2.1 血清淀粉酶变化 P组、T组血清淀粉酶水平随SAP病情的进展而升高，各时间点淀粉酶较S组均明显升高，T组淀粉酶水平在12 h点则明显较P组降低(表1).
表1 血清AMY组间变化情况(nKat/L)
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