三七总皂甙调控内皮Vps34自噬通路减轻慢性间歇性缺氧诱导的内皮-血小板黏附机制研究

Mechanism of Panax Notoginseng Saponins Alleviating Endothelial Cell-Platelet Adhesion Induced by Chronic Intermittent Hypoxia via Modulation of Endothelial Vps34 Autophagy Pathway

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DOI：10.7661/j.cjim.20230403. 041

中文关键词: 三七总皂苷慢性间歇性缺氧血小板内皮细胞自噬中药

英文关键词:Panax notoginseng saponins chronic intermittent hypoxia platelet endothelial cell autophagy Chinese herbal medicine

基金项目:国家自然科学基金青年基金资助项目（No. 81803949）

作者	单位
王铭铭,王彦辉,张红丽,史大卓,李平	1.北京中医药大学第三附属医院心内科（北京 100029） 2.北京市第一中西医结合医院心内科（北京 100026） 3. 中国中医科学院心血管病研究所（北京 100091）

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中文摘要:

目的通过内皮Vps34自噬通路，探讨三七总皂苷（PNS）抑制慢性间歇性缺氧（CIH）诱导内皮-血小板黏附的作用机制。方法采用随机数字表法将90只大鼠分为对照组（n=10）、CIH组（n=20）、阿司匹林组（n=20）、PNS低剂量组（n=20）及PNS高剂量组（n=20）。除对照组外，通过控制动物饲养箱中氧气浓度，建立CIH大鼠模型。造模成功率为51%，CIH组10只，阿司匹林组10只，PNS低剂量组12只，PNS高剂量组9只。阿司匹林组给予阿司匹林肠溶片12 mg/（kg·d），PNS低、高剂量组分别灌胃给予血塞通软胶囊80、160 mg/（kg·d），对照组和CIH组给予等量生理盐水，均持续3周。采用流式细胞术（FCM）检测大鼠主动脉内皮细胞（AEC）凋亡率，FCM及免疫荧光法检测大鼠AEC表面血管血友病因子（VWF）表达，Western Blot法检测AEC中Vps34自噬通路相关蛋白表达水平。细胞研究采取正常大鼠AEC，分为无抑制剂对照组、CIH组、PNS组、SAR405（简称抑制剂）对照组、抑制剂CIH组及抑制剂PNS组。所有抑制剂组细胞采用SAR405预孵育1 h，PNS组细胞加入PNS标准品（160 μg/mL）。除对照组外余组均建立间歇性低氧AEC模型。在AEC中加入正常大鼠血小板，诱导AEC-血小板黏附。采用FCM观察AEC凋亡率、VWF表达率及AEC-血小板黏附程度。结果动物实验部分：与对照组比较，CIH组大鼠AEC凋亡率、VWF表达率及AEC中Beclin-1、Vps34、自噬相关蛋白（Atg）14及微管相关蛋白轻链3Ⅱ（LC3Ⅱ）表达均增加（P<0.05）。与CIH组比较，阿司匹林组、PNS高、低剂量组大鼠AEC凋亡率、VWF表达、Vps34、Atg14及LC3Ⅱ蛋白表达降低（P<0.05）。细胞研究部分：与空白对照组比较，CIH组AEC凋亡率、VWF表达率及血小板黏附均升高（P<0.05）；与CIH组比较，PNS组AEC凋亡率、VWF表达率及血小板黏附均降低（P<0.05）。用特异性抑制剂SAR405抑制Vps34自噬通路后，PNS减少AEC凋亡率、VWF表达及血小板黏附的作用消失，结论 PNS可减轻CIH诱导的内皮-血小板黏附，其作用机制与PNS抑制内皮细胞Vps34自噬通路有关。

英文摘要:

Objective To explore the inhibitory mechanisms of Panax notoginseng saponins（PNS）on endothelial cell-platelet adhesion induced by chronic intermittent hypoxia（CIH） based on the endothelial Vps34 autophagy pathway. Methods Ninety rats were randomly divided into control group（n=10）， CIH group（n=20）， aspirin group（n=20）， low-dose PNS group（n=20）， and high-dose PNS group （n=20） using random number table. The CIH rat model was established by controlling the oxygen levels in the rat feeding box. The overall modelling success rate was 51%：10 in the CIH group， 10 in the aspirin group， 12 in the low-dose PNS group， and 9 in the high-dose PNS group.At the same time，the aspirin group was administered aspirin enteric-coated tablets 12 mg·kg-1·d-1， the low-dose PNS group was administered Xuesaitong soft Capsules 80 mg·kg-1·d-1， and the high-dose PNS group was administered Xuesaitong soft Capsules 160 mg·kg-1·d-1，the control group and CIH group were administered with equal volume saline by gastrogavage. Flow cytometry（FCM）was used to detect the apoptosis rates of aortic endothelial cell（AEC）. The von-Willebrand factor（VWF）expression in AEC was determined by FCM and immunofluorescence method respectively. The levels of protein expression of the Vps34 autophagy pathway were detected by Western Blot. For the in vitro experiments， AEC from normal rats were divided into no inhibitor control， CIH， PNS， SAR405（named inhibitor）control， inhibitor CIH， and inhibitor PNS groups. Cells in all the inhibitor groups were pre-incubated with SAR405 for 1 h. The PNS standards were added to the PNS groups at a final concentration of 160 μg/mL. Except for control groups， all other groups were cultured in an intermittent hypoxia cell incubator. The AEC were co-incubated with platelets from normal rats to induce AEC-platelet adhesion. The apoptosis rates of AEC， VWF expression， and AEC-platelet adhesion were detected using FCM. Results For the in vivo experiment， compared with the control group， AEC apoptosis， VWF expression， and protein expression of Beclin-1， Vps34， autophagy-related gene（Atg）， and microtubule-associated protein light chain 3Ⅱ（LC3Ⅱ） increased in CIH group（P<0.05）. Compared with the CIH group， AEC apoptosis， VWF expression， and protein expression of Vps34， Atg14， and LC3Ⅱ decreased in aspirin group，low- and high-dose PNS groups （P<0.05）. In the in vitro experiment， compared with the vacant control group， AEC apoptosis， VWF expression， and platelet adhesion increased in the CIH group（P<0.05）. Compared with the CIH group， AEC apoptosis，VWF expression， and platelet adhesion decreased in the PNS group （P<0.05）. When Vps34 was inhibited by SAR405， the effects of PNS on AEC apoptosis， VWF expression， and platelet adhesion were abrogated. There was no statistical difference between the inhibitor PNS group and inhibitor CIH group（P>0.05）. Conclusion PNS could alleviate CIH-induced endothelial-platelet adhesion， and its mechanism might be related with inhibiting of the endothelial cell Vps34 autophagy pathway by PNS.

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