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三七总皂苷对肺缺血/再灌注损伤时细胞凋亡及c-Jun氨基末端激酶的影响

邱晓晓, 宋张娟, 戴雍月, 方周溪, 王万铁^*

温州医学院 病理生理学教研室；电镜室，温州 325035

摘要

本研究旨在探讨三七总皂苷(Panax notoginseng saponins, PNS)对大鼠肺缺血/再灌注(ischemia/reperfusion, I/R)损伤时细胞凋亡及凋亡相关蛋白和c-Jun氨基末端激酶(c-Jun N-terminal kinase, JNK)的影响。实验用健康清洁级Wistar大鼠30只，随机分为假手术对照组(Control组)、缺血/再灌注组(I/R组)与缺血/再灌注 + 三七总皂苷干预组(PNS组)。采用阻断左肺门30 min后松开结扎的方法复制在体大鼠原位单肺缺血/再灌注模型，PNS干预方法为缺血前60 min与再灌注前10 min腹腔注射PNS。实验结束时取部分左肺组织测湿/干重比(wet/dry weight ratio, W/D)；采用蛋白免疫印迹法检测肺组织JNK、磷酸化JNK (p-JNK)蛋白的表达；免疫组化法检测肺组织Bcl-2、Bax、Caspase-3蛋白的表达；原位末端标记法(TUNEL)检测肺组织细胞凋亡指数(apoptotic index, AI)，光镜、电镜观察肺组织形态结构变化，并测定肺泡损伤数(injured alveolus rate, IAR)。结果显示，与Control组相比，I/R组肺组织p-JNK、Bcl-2、Bax、Caspase-3蛋白的表达均显著增加(均P < 0.01)，Bcl-2/Bax的比值明显下降(P < 0.05)，AI、W/D及IAR均显著升高(均P < 0.01)，光镜、电镜示肺组织结构呈明显损伤性变化；与I/R组相比，PNS组肺组织p-JNK、Bax、Caspase-3蛋白的表达均显著下调(均P < 0.01)，Bcl-2蛋白的表达及Bcl-2/Bax的比值显著上调(均P < 0.01)，AI、W/D及IAR也显著降低(均P < 0.01)，肺组织形态学异常改变不同程度减轻。以上结果提示：PNS对缺血/再灌注肺具有保护作用，该作用可能与其抑制JNK信号转导通路、上调Bcl-2/Bax的比值从而减少Caspase-3依赖性的肺细胞凋亡有关。

关键词： 三七总皂苷; 肺; 再灌注损伤; 细胞凋亡; c-Jun氨基末端激酶; Bcl-2/Bax; Caspase-3

分类号：R363

[Effects of Panax notoginseng saponins on pneumocyte apoptosis and c-Jun N-terminal kinase in lung ischemia/reperfusion injury.] [Article in Chinese]

QIU Xiao-Xiao, SONG Zhang-Juan, DAI Yong-Yue, FANG Zhou-Xi, WANG Wan-Tie^*

Department of Pathophysiology； Department of Electron Microscope, Wenzhou Medical College, Wenzhou 325035, China

Abstract

The aim of the present study is to investigate the effects of Panax notoginseng saponins (PNS) on pneumocyte apoptosis and apoptosis-related protein, as well as c-Jun N-terminal kinase (JNK) in lung ischemia/reperfusion (I/R) injury. Thirty Wistar rats were randomly divided into control group, I/R group and PNS group. The unilateral lung I/R model was replicated by obstruction of left lung hilus for 30 min and reperfusion for 120 min in vivo. The rats in PNS group were given intraperitoneal injection of PNS at 60 min before ischemia and 10 min before reperfusion. Some lung tissues sampled at the end of the experiment were assayed for wet/dry weight ratio (W/T). The expressions of phosphorylated JNK (p-JNK) and JNK protein were detected by Western blot. The expressions of Bcl-2, Bax and Caspase-3 protein were detected by immunocytochemistry techniques. The pneumocyte apoptotic index (AI) was detected by terminal deoxynuleotidy1 transferase mediated dUTP nick end labeling (TUNEL). The morphological and ultrastructure changes were observed under light microscope and electron microscope, and the injured alveolus rate (IAR) was counted as well. The results showed that compared to control group, I/R group showed increased expressions of p-JNK, Bcl-2, Bax and Caspase-3 protein (all P < 0.01), decreased ratio of Bcl-2/Bax (P < 0.05), and increased values of AI, W/T and IAR (all P < 0.01). Moreover, light microscope and electron microscope showed serious morphological and ultrastructure injury in I/R group. Compared to I/R group, PNS group showed markedly decreased expressions of p-JNK, Bax and Caspase-3 protein (all P < 0.01), increased expression of Bcl-2 protein and ratio of Bcl-2/Bax (both P < 0.01), and lower values of AI, W/T and IAR (all P < 0.01). Meanwhile, light morphological and ultrastructure injury was found to be alleviated in PNS group. These results suggest that PNS can protect lung tissue from I/R injury, and the mechanism may correlate with suppressing JNK signal pathway, up-regulating the ratio of Bcl-2/Bax which results in inhibition of Caspase-3 dependent apoptosis.

Key words: Panax notoginseng saponins; lung; reperfusion injury; apoptosis; c-Jun N-terminal kinase; Bcl-2/Bax; Caspase-3

收稿日期：2011-08-26　　录用日期：2012-02-17

通讯作者：王万铁　　E-mail: wzwwt@tom.com

引用本文：

邱晓晓, 宋张娟, 戴雍月, 方周溪, 王万铁. 三七总皂苷对肺缺血/再灌注损伤时细胞凋亡及c-Jun氨基末端激酶的影响[J]. 生理学报 2012; 64 (2): 135-141.

QIU Xiao-Xiao, SONG Zhang-Juan, DAI Yong-Yue, FANG Zhou-Xi, WANG Wan-Tie. [Effects of Panax notoginseng saponins on pneumocyte apoptosis and c-Jun N-terminal kinase in lung ischemia/reperfusion injury.] [Article in Chinese]. Acta Physiol Sin 2012; 64 (2): 135-141 (in Chinese with English abstract).