2)预处理1 h,用Western blot检测葡萄糖调节蛋白78(GRP78)判断模型是否建立成功,并探索E2对内质网应激的作用。检测内质网应激的三条主要信号通路蛋白的变化,上调最显著的为内质网应激最主要的信号通路。Western blot检测内质网应激凋亡蛋白C/EBP-同源蛋白(CHOP),Hochest染色检测细胞凋亡率,探索E2对内质网应激凋亡的作用。添加E2受体拮抗剂ICI182780(ERα、ERβ拮抗剂及GPER激动剂)和G15(GPER拮抗剂)后检测内质网应激最主要通路蛋白表达量的变化,探索雌激素受体在其抑制内质网应激中的作用。添加E2受体后信号通路阻断剂,检测雌激素抑制内质网应激的过程中活化其受体后激活的最主要受体后信号通路。结果 TM/DTT 组GRP78的表达量显著上调,内质网应激三条信号通路中蛋白激酶R样内质网激酶(PERK)信号通路上调最明显,而TM/DTT+E2组上调显著回复。TM/DTT组CHOP的表达量显著上调且细胞凋亡率显著增加,而TM/DTT+E2组上调明显回复,凋亡细胞减少。E2有显著抑制p-PERK/PERK上调的作用,而E2的保护作用可分别被ICI182780和G15阻断,同时添加ICI182780和G15时阻断作用最显著。分别添加信号通路阻断剂后,E2抑制p-PERK/PERK上调的作用均减弱,其中以磷脂酰肌醇-3羟基激酶(PI3K)通路阻断剂的作用最显著。结论 E2可抑制TM/DTT诱导的HUVEC内质网应激。p-PERK/PERK通路可能为TM/DTT诱导的HUVEC内质网应激最主要的信号通路。E2可抑制过度内质网应激引起的细胞凋亡。E2受体在E2抑制内质网应激凋亡的作用中起重要作用。E2受体激活包括PI3K-蛋白激酶B(PKB/Akt)、细胞外信号调节激酶1/2(ERK1/2)、c-Jun氨基末端激酶(JNK)和p38-丝裂原活化蛋白激酶(p38-MAPK)在内的信号通路快速起到抑制内质网应激的作用,其中PI3K-Akt通路可能为最主要的通路。雌激素通过抑制PERK信号通路引起的内质网应激凋亡,保护血管内皮细胞,其抑制内质网应激的机制主要为活化的雌激素受体激活PI3K/Akt通路。"/>

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雌激素抑制内质网应激引起的血管内皮细胞凋亡及机制
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(1.北京大学人民医院妇产科,北京市 100044;2.河北医科大学第二医院妇产科,河北省石家庄市 050000;3.北京大学第三医院妇产科,北京市 100000)

作者简介:

王宇,硕士研究生,医师,E-mail为1213315208@qq.com。李晓冬,博士,主任医师,教授,硕士研究生导师,研究方向为妇科内分泌。

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国家自然科学基金(2101000216)


Estrogen Reduces Apoptosis Induced by Endoplasmic Reticulum Stress in Vascular Endothelial Cells and the Mechanism
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1.Department of Obstetrics and Gynecology,People’s Hospital of Peking University,Beijing 100040;2.Department of Obstetrics and Gynecology, the Second Hospital of Hebei Medical University, Shijiazhuang, Hebei 050000;3.Department of Obstetrics and Gynecology,the Third Hospital of Peking University, Beijing 100000)

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    摘要:

    目的 通过建立人脐静脉内皮细胞(HUVEC)内质网应激(ERS)的细胞模型,研究雌激素抑制内质网应激引起的凋亡的信号传导机制,以探讨雌激素对心血管的保护机制。方法 分别用10 μmol/L的衣霉素(TM)或2 mmol/L的二硫苏糖醇(DTT)诱导HUVEC,建立内质网应激细胞模型,提前给予10-8 mol/L的17-β雌二醇(E2)预处理1 h,用Western blot检测葡萄糖调节蛋白78(GRP78)判断模型是否建立成功,并探索E2对内质网应激的作用。检测内质网应激的三条主要信号通路蛋白的变化,上调最显著的为内质网应激最主要的信号通路。Western blot检测内质网应激凋亡蛋白C/EBP-同源蛋白(CHOP),Hochest染色检测细胞凋亡率,探索E2对内质网应激凋亡的作用。添加E2受体拮抗剂ICI182780(ERα、ERβ拮抗剂及GPER激动剂)和G15(GPER拮抗剂)后检测内质网应激最主要通路蛋白表达量的变化,探索雌激素受体在其抑制内质网应激中的作用。添加E2受体后信号通路阻断剂,检测雌激素抑制内质网应激的过程中活化其受体后激活的最主要受体后信号通路。结果 TM/DTT 组GRP78的表达量显著上调,内质网应激三条信号通路中蛋白激酶R样内质网激酶(PERK)信号通路上调最明显,而TM/DTT+E2组上调显著回复。TM/DTT组CHOP的表达量显著上调且细胞凋亡率显著增加,而TM/DTT+E2组上调明显回复,凋亡细胞减少。E2有显著抑制p-PERK/PERK上调的作用,而E2的保护作用可分别被ICI182780和G15阻断,同时添加ICI182780和G15时阻断作用最显著。分别添加信号通路阻断剂后,E2抑制p-PERK/PERK上调的作用均减弱,其中以磷脂酰肌醇-3羟基激酶(PI3K)通路阻断剂的作用最显著。结论 E2可抑制TM/DTT诱导的HUVEC内质网应激。p-PERK/PERK通路可能为TM/DTT诱导的HUVEC内质网应激最主要的信号通路。E2可抑制过度内质网应激引起的细胞凋亡。E2受体在E2抑制内质网应激凋亡的作用中起重要作用。E2受体激活包括PI3K-蛋白激酶B(PKB/Akt)、细胞外信号调节激酶1/2(ERK1/2)、c-Jun氨基末端激酶(JNK)和p38-丝裂原活化蛋白激酶(p38-MAPK)在内的信号通路快速起到抑制内质网应激的作用,其中PI3K-Akt通路可能为最主要的通路。雌激素通过抑制PERK信号通路引起的内质网应激凋亡,保护血管内皮细胞,其抑制内质网应激的机制主要为活化的雌激素受体激活PI3K/Akt通路。

    Abstract:

    Aim To investigate the signaling mechanism of estrogen reducing endoplasmic reticulum stress induced apoptosis, to explore the protective mechanism of the effect on cardiovascular, through establishing the ER stress cell model in human umbilical vein endothelial cell. Methods To establish ERS cell model, HUVECs were incubated in 10 μmol/L tunicamycin (TM) or 2 mmol/L dithiothreitol(DTT) with or without 10-8 mol/L 17β-estradiol(E2) pretreatment. Tested glucose regulated protein (GRP78) to determine whether the model was successfully established and explored the impact of E2 in ERS. The changes of three major signaling pathway proteins of ERS were detected by Western blot, and the most significant increase is the most important signal pathway of ERS. To explore the impact of E2 in apoptosis induced by ERS, the apoptosis protein C/EBP-homologous protein( CHOP) was analyzed by Western blot and the apoptosis rate of cells was tested by Hochest staining. To explore the role of estrogen receptor in its suppression of ERS, we added ICI182780 (ERα, ERβ antagonist and GPER agonist) and G15(GPER antagonist) before adding E2, and analyzed the expression of the main signal pass of ERS by western blot. Added the inhibitors of E2 receptor signaling, to explore the main post-receptor signaling pathway. Results The expression of GRP78 was significantly increased in TM/DTT group,and the protein kinase R-like ER kinase(PERK) was the most significant increase one among the three signaling pathways of ERS. In the presence of E2, the regulations replied. The expression of CHOP and the apoptosis rate of cells were significantly increased in TM/DTT group,and regulations replied when added E2. The inhibition of E2 to the up-regulation of p-PERK/PERK was inhibited by ICI182780 and G15 respectively, the blocking is the most obvious while adding both. The effect of E2 reduced p-PERK/PERK were weakened when added the inhibitors of E2 receptor signaling respectively,and the weakness was the most obvious when added the signaling inhibitor of Akt. Among them, the effect of PI3K-Akt inhibitor was the most significant. Conclusions E2 can protect human endothelial cells from ERS induced by TM and DTT. p-PERK/PERK pathway may be the most important signal path of ERS. E2 plays a role in inhibiting HUVEC apoptosis induced by over ERS. The receptors of E2 play an important role when E2 inhibited apoptosis induced by over ERS. The activated E2 receptor can activate rapid estrogen receptor signaling pathways include PI3K-Akt, ERK1/2, JNK and p38-MAPK to inhibit ERS, and PI3K-Akt pathway may be the most important one. E2 possibly prevent vascular endothelial cells by inhibition of apoptosis induced by ERS, and the mechanism of inhibiting ERS was mainly through the activated E2 receptors activating PI3K-Akt pathway.

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王宇,杨欣,李晓冬,何小静,王彦洁.雌激素抑制内质网应激引起的血管内皮细胞凋亡及机制[J].中国动脉硬化杂志,2016,24(3):217~223.

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  • 收稿日期:2015-06-08
  • 最后修改日期:2015-08-25
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  • 在线发布日期: 2016-04-15
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