西洋参茎叶总皂苷通过抑制内质网应激减轻毒胡萝卜素诱导的心肌细胞凋亡

doi:10.3969/j.issn.1000-4718.2014.10.002

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目的:研究西洋参茎叶总皂苷 (PQS) 减轻毒胡萝卜素(TG) 诱导的心肌细胞凋亡的分子机制。方法:原代培养的心肌细胞分为：control组、TG组、PQS (40 mg/L、80 mg/L及160 mg/L)+TG组、牛磺酸 (40 mmol/L)+TG组、蛋白激酶R样内质网激酶 (PERK)敲低+TG组及随机双链RNA转染对照 (mock)+TG组。通过向培养液中加入1 μmol/L TG作用24 h诱导离体培养的乳大鼠心肌细胞凋亡。以RNAi方法敲低心肌细胞PERK基因。CCK-8法和流式细胞术检测细胞活性及凋亡率，Western blotting方法检测内质网应激(ERS)标志分子葡萄糖调节蛋白78 (GRP 78)、钙网蛋白 (CRT)、PERK、p-PERK、真核起始因子2α (eIF2α)、p- eIF2α、活化转录因子4 (ATF4)、C/EBP同源蛋白 (CHOP) 及凋亡相关蛋白Bcl-2、Bax的表达。结果:与对照组比较，TG孵育明显诱导细胞凋亡，降低细胞活力，上调PERK和eIF2α磷酸化以及GRP78、CRT、ATF4、CHOP及促凋亡蛋白Bax表达，降低抗凋亡蛋白Bcl-2表达 (P<0.05)；与TG组比较，PQS 160 mg/L及敲低PERK均可显著降低细胞凋亡率，升高细胞活力 (P<0.05)；3种不同浓度的PQS可呈剂量依赖性降低Bax蛋白表达，升高Bcl-2蛋白表达 (P<0.05)，敲低PERK基因及PQS (160 mg/L) 预处理均可降低ERS分子GRP78、CRT、ATF4及CHOP表达，降低PERK及eIF2α的磷酸化水平 (P<0.05)。结论: PQS 160 mg/L 减轻ERS诱导剂TG诱导的心肌细胞凋亡，PQS预处理可以模拟PERK敲低减轻TG致心肌细胞凋亡的效应，提示PERK-eIF2α-ATF4-CHOP途径参与PQS减轻ERS相关凋亡的作用。

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	刘蜜
	王晓礽
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	徐菲菲
	刘秀华
	史大卓

关键词 ：西洋参茎叶总皂苷, 毒胡萝卜素, 心肌细胞凋亡, 内质网应激

Abstract：

AIM:To study the effect of Panax quinquefoliumsaponin (PQS) on cardiomyocyte apoptosis induced by thapsigargin (TG). METHODS:Primary cultured cardiomyocytes from neonatal SD rats were divided into control group, TG group, PQS (40 mg/L, 80 mg/L and 160 mg/L)+TG group, si-PERK+TG group, and mock+TG group. The cells were treated with 1 μmol/L TG for 24 h to induce apoptosis. The PERKgene in the cardiomyocytes was knocked down by RNAi. The cell viability was detected by CCK-8 assay. Apoptosis was analyzed by flow cytometry. Wes-tern blotting was used to determine the expression of ERS molecules GRP78, CRT, ATF4 and CHOP, anti-apoptosis protein Bcl-2 and pro-apoptosis protein Bax. RESULTS:Compared with control group, TG significantly and the apoptosis, reduced the cell viability (P<0.05), increased the phosphorylation of PERK and eIF2α, increased the expression of GRP78, CRT, ATF4, CHOP and pro-apoptosis protein Bax, and decreased the expression of anti-apoptosis protein Bcl-2 (P<0.05). Compared with TG group, PQS treatment (160 mg/L) significantly reduced the apoptosis and increased the cell viability (P<0.05). All the 3 different concentrations of PQS significantly increased the expression of anti-apoptosis protein Bcl-2 and reduced the expression of pro-apoptosis protein Bax (P<0.05) in a dose-dependent manner. PQS pretreatment and knockdown of PERK both reduced the protein levels of GRP78, CRT, PERK, p-PERK, eIF2α, p-eIF2α, ATF4, CHOP and pro-apoptosis protein Bax, and increased the expression of anti-apoptosis protein Bcl-2 (P<0.05). CONCLUSION: PQS at concentration of 160 mg/L attenuated cardiomyocyte apoptosis induced by TG. PQS had the similar effect as PERKknockdown on cardiomyocyte apoptosis. The mechanism may be associated with inhibiting PERK-eIF2α-ATF4-CHOP pathway of ERS-related apoptosis.

Key words： Panax quinquefoliumsaponin Thapsigargin Cardiomyocyte apoptosis Endoplasmic reticulum stress

收稿日期: 2014-02-14 出版日期: 2014-10-15

中图分类号:

R363

基金资助:

国家自然科学基金资助项目 (No. 81170140;No. 81030063)

通讯作者: 刘秀华 E-mail: xiuhualiu98@163.com; heartmail@263.net

引用本文:

刘蜜，王晓礽，陶天琪，徐菲菲，刘秀华，史大卓. 西洋参茎叶总皂苷通过抑制内质网应激减轻毒胡萝卜素诱导的心肌细胞凋亡[J]. 中国病理生理杂志, 2014, 30(10): 1735-1741. LIU Mi, WANG Xiao-reng, TAO Tian-qi, XU Fei-fei, LIU Xiu-hua, SHI Da-zhuo. PQS attenuates cardiomyocyte apoptosis induced by thapsigargin through inhibiting endoplasmic reticulum stress. Chin J Pathophysiol, 2014, 30(10): 1735-1741.

链接本文:

http://www.cjpp.net/CN/10.3969/j.issn.1000-4718.2014.10.002 或 http://www.cjpp.net/CN/Y2014/V30/I10/1735

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