通心络对心脏微血管内皮缺血再灌注损伤的保护机制研究
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摘要
通心络通过激活MEK/ERK信号通路诱导细胞自噬而在缺氧/复氧损伤过程中保护心脏微血管内皮细胞
背景与目的：关于缺血再灌注损伤的机制研究中,心肌细胞的自噬发生及调节研究较多,而心脏微血管内皮细胞中自噬的作用尚未被探索。通心络是一种具有血管保护功能的中药复方制剂,目前在中国此药已用于6百万心脑血管疾病患者。因此,本研究的目的即探索缺氧/复氧损伤时自噬在心脏微血管内皮细胞中的作用,以及通心络对其的调节机制。
方法：给予不同药物处理心脏微血管内皮细胞30分钟后,缺氧2小时,复氧2小时。不同浓度的通心络溶液,人参皂甙Rgl,芍药苷以及3-甲基腺嘌呤(3-MA),雷帕霉素,PD98059等药物被用以研究自噬与凋亡的关系以及其在心脏微血管内皮细胞中的调节机制。
结果：缺氧/复氧明显地升高了心脏微血管内皮细胞的自噬水平,表现为单丹磺酰尸胺(MDC)染色阳性的细胞数目增加,透射电镜下自噬体形成增多,微管蛋白轻链3(LC3)-Ⅱ/Ⅰ的比值升高；然而Beclin-1的水平并无变化。自噬抑制剂3-MA显著促进了细胞凋亡,而自噬促进剂雷帕霉素则抑制了细胞凋亡。通心络浓度依赖性地增加了细胞自噬水平和降低了细胞凋亡率,在800μg/mL时达到最佳效应,可将凋亡率从21.04±1.11%降至9.92±0.49%。3-MA明显抑制了通心络的促自噬及抗凋亡的作用,而雷帕霉素并没有进一步增加通心络的促自噬及抗凋亡的作用。通心络促进了MEK和ERK蛋白的磷酸化；加入MEK/ERK抑制剂PD98059后,与单用通心络相比,ERK的磷酸化水平降低,细胞自噬被削弱,并且细胞凋亡率增加。
结论：上述结果表明,自噬诱导在缺血再灌注过程中对心脏微血管内皮细胞中是一种保护机制。通心络可以通过激活MEK/ERK信号通路诱导细胞自噬。
中药通心络可调节缺血再灌注损伤时心脏微血管内皮细胞的分泌功能
背景与目的：心脏微血管内皮细胞在缺血再灌注损伤时可通过近分泌及旁分泌功能调节心肌细胞及血液细胞的功能。通心络是一种由人参和其他11种天然药材构成的中药复方制剂,具有保护内皮和防治心肌缺血再灌注损伤的功能。因此,我们设·计此研究以发现在缺氧/复氧环境下心脏微血管内皮分泌功能的改变,以及通心络对其变化的调节作用。
方法：以不同浓度的通心络处理心脏微血管内皮细胞30分钟后,缺氧12小时,复氧2小时。流式细胞术分析凋亡率以确定通心络作用的最佳浓度。蛋白芯片双抗体夹心法检测心脏微血管内皮细胞在缺氧/复氧后培养基上清中发生变化的细胞因子。基因本体论分析及文献综述发生变化的细胞因子的功能。
结果：通心络在缺氧12小时/复氧2小时的环境下浓度依赖性地抑制了心脏微血管内皮细胞的凋亡,并且仍然在800μg/mL的浓度达到顶峰,可降低凋亡率从35.15±0.78%到14.64±2.23%。缺氧/复氧环境显著改变了细胞分泌的33种细胞因子的水平,其中19个因子下降,14个因子上调。通心络改变了121个细胞因子的变化情况,其中93个下降,28个上调。发生显著变化的细胞因子的功能集中于细胞分化,增殖,信号通路的调节,以及物质转运等方面。在发生显著变化的所有细胞因子中,被缺氧/复氧上调但被通心络下调的有10个,如单核细胞趋化因子-2；被缺氧/复氧下调但被通心络上调的有5个,如滤泡抑素；缺氧/复氧组下降,通心络组进一步下降的有8个,如碱性成纤维细胞生长因子；胰岛素样生长因子结合蛋白在缺氧/复氧组分泌增多且在通心络组进一步上调。
结论：通心络抑制了在缺氧/复氧情况下诱导的心脏微血管内皮细胞凋亡,并且其保护心肌缺血再灌注损伤的作用与调节缺氧/复氧时心脏微血管内皮细胞的旁分泌功能有关。
Induction of autophagy by Tongxinluo via the MEK/ERK pathway protects human cardiac microvascular endothelial cells from hypoxia/reoxygenation injury
Backgrounds and Objectives:In contrast to cardiomyocytes, autophagy in cardiac microvascular endothelial cells (CMECs) during ischemia/reperfusion (I/R) injury has not been fully investigated. Tongxinluo (TXL) is a Traditional Chinese Medicine formulation that is widely used by more than6million patients with cardio-cerebral vascular diseases in China for its vascular-protective effect. We aimed to elucidate the role of autophagy and its regulatory mechanisms by TXL in CMECs subjected to I/R injury.
Methods:CMECs were exposed to different treatments for30min and subjected to hypoxia/reoxygenation (H/R) each for2h. The TXL solution at different concentrations, ginsenoside Rgl, paeoniflorin,3-methyladenine (3-MA), rapamycin and PD98059were used to further investigate the role and the modulatory mechanism of autophagy in CMECs.
Results:The results indicated that H/R significantly induced autophagy, as identified by an increased number of monodansylcadaverine (MDC)-positive CMECs, increased autophagosome formation, and a higher type II/type I of light chain3ratio, but not Beclin-1expression. Autophagy inhibition using3-methyladenine (3-MA) was proapoptotic, but rapamycin-induced autophagy was antiapoptotic. TXL enhanced autophagy and decreased apoptosis rates in a dose-dependent manner, reaching its largest effect at800μg/mL, which decreased apoptosis from21.04±1.11%to9.92±0.49%.3-MA attenuated the TXL-promoted autophagy and antiapoptotic effects, whereas rapamycin had no additional effects compared to TXL alone. TXL upregulated MEK and ERK phosphorylation; however, PD98059abrogated ERK phosphorylation and decreased autophagy and increased apoptosis compared to TXL alone.
Conclusions:These results suggest that autophagy is a protective mechanism in CMECs subjected to I/R injury and that TXL can promote autophagy via activation of the MEK/ERK pathway.
Traditional Chinese Medicine Tongxinluo modulates the secretion of cytokines in vitro by cardiac microvascular endothelial cells in ischemia/reperfusion injury.
Backgrounds and Objectives:Cardiac microvascular endothelial cells (CMECs) regulate the function of cardiomyocytes and blood cells in ischemia/reperfusion injury via autocrine and paracrine methods. Tongxinluo (TXL) is a Traditional Chinese Medicine compound which is constituted of Radix ginseng and other11kinds of natural products. It is proved to improve the function of endothelium and be protective from ischemia/reperfusion injury. Thus, we designed the study to find alterations in paracrine function of CMECs under the hypoxia/reoxygenation (H/R) situation and its modulation by TXL.
Methods:CMECs were exposed to different concentrations of TXL for30min and then subjected to H/R for12h/2h. Apoptotic rates of the cells were measured to determine the optimal concentration. Protein antibody arrays were used to find the alterations of cytokines in conditioned medium (CM) secreted by CMECs. Gene Ontology (GO) project was adopted to describe the functions of changed cytokines.
Results:TXL inhibited apoptosis of CMECs dose-dependently under H/R and reached its peak effect at the dose of800μg/mL, which reduce cell apoptosis from35.15±0.78%to14.64±2.23%. Thirty-three types of cytokines were significantly changed by H/R (19factors decreased and14increased), and TXL at800μg/ml changed121types of cytokines compared to the H/R group (93factors decreased and28increased). The cytokines with significant alterations were involved in cell differentiation and proliferation, regulation of signalling pathway, and transportation. Among these cytokines,10kinds of cytokines were increased by H/R but were decreased by TXL, such as MCP-2;5were decreased by H/R but increased by TXL, such as Follistatin;8were attenuated by H/R and further decreased by TXL, such as bFGF; IGFBP-1were upregulated by H/R and further increased by TXL.
Conclusions:TXL inhibited apoptosis of CMECs and modulated the paracrine function of the cells in ischemia/reperfusion injury.
背景与目的：关于缺血再灌注损伤的机制研究中,心肌细胞的自噬发生及调节研究较多,而心脏微血管内皮细胞中自噬的作用尚未被探索。通心络是一种具有血管保护功能的中药复方制剂,目前在中国此药已用于6百万心脑血管疾病患者。因此,本研究的目的即探索缺氧/复氧损伤时自噬在心脏微血管内皮细胞中的作用,以及通心络对其的调节机制。
方法：给予不同药物处理心脏微血管内皮细胞30分钟后,缺氧2小时,复氧2小时。不同浓度的通心络溶液,人参皂甙Rgl,芍药苷以及3-甲基腺嘌呤(3-MA),雷帕霉素,PD98059等药物被用以研究自噬与凋亡的关系以及其在心脏微血管内皮细胞中的调节机制。
结果：缺氧/复氧明显地升高了心脏微血管内皮细胞的自噬水平,表现为单丹磺酰尸胺(MDC)染色阳性的细胞数目增加,透射电镜下自噬体形成增多,微管蛋白轻链3(LC3)-Ⅱ/Ⅰ的比值升高；然而Beclin-1的水平并无变化。自噬抑制剂3-MA显著促进了细胞凋亡,而自噬促进剂雷帕霉素则抑制了细胞凋亡。通心络浓度依赖性地增加了细胞自噬水平和降低了细胞凋亡率,在800μg/mL时达到最佳效应,可将凋亡率从21.04±1.11%降至9.92±0.49%。3-MA明显抑制了通心络的促自噬及抗凋亡的作用,而雷帕霉素并没有进一步增加通心络的促自噬及抗凋亡的作用。通心络促进了MEK和ERK蛋白的磷酸化；加入MEK/ERK抑制剂PD98059后,与单用通心络相比,ERK的磷酸化水平降低,细胞自噬被削弱,并且细胞凋亡率增加。
结论：上述结果表明,自噬诱导在缺血再灌注过程中对心脏微血管内皮细胞中是一种保护机制。通心络可以通过激活MEK/ERK信号通路诱导细胞自噬。
中药通心络可调节缺血再灌注损伤时心脏微血管内皮细胞的分泌功能
背景与目的：心脏微血管内皮细胞在缺血再灌注损伤时可通过近分泌及旁分泌功能调节心肌细胞及血液细胞的功能。通心络是一种由人参和其他11种天然药材构成的中药复方制剂,具有保护内皮和防治心肌缺血再灌注损伤的功能。因此,我们设·计此研究以发现在缺氧/复氧环境下心脏微血管内皮分泌功能的改变,以及通心络对其变化的调节作用。
方法：以不同浓度的通心络处理心脏微血管内皮细胞30分钟后,缺氧12小时,复氧2小时。流式细胞术分析凋亡率以确定通心络作用的最佳浓度。蛋白芯片双抗体夹心法检测心脏微血管内皮细胞在缺氧/复氧后培养基上清中发生变化的细胞因子。基因本体论分析及文献综述发生变化的细胞因子的功能。
结果：通心络在缺氧12小时/复氧2小时的环境下浓度依赖性地抑制了心脏微血管内皮细胞的凋亡,并且仍然在800μg/mL的浓度达到顶峰,可降低凋亡率从35.15±0.78%到14.64±2.23%。缺氧/复氧环境显著改变了细胞分泌的33种细胞因子的水平,其中19个因子下降,14个因子上调。通心络改变了121个细胞因子的变化情况,其中93个下降,28个上调。发生显著变化的细胞因子的功能集中于细胞分化,增殖,信号通路的调节,以及物质转运等方面。在发生显著变化的所有细胞因子中,被缺氧/复氧上调但被通心络下调的有10个,如单核细胞趋化因子-2；被缺氧/复氧下调但被通心络上调的有5个,如滤泡抑素；缺氧/复氧组下降,通心络组进一步下降的有8个,如碱性成纤维细胞生长因子；胰岛素样生长因子结合蛋白在缺氧/复氧组分泌增多且在通心络组进一步上调。
结论：通心络抑制了在缺氧/复氧情况下诱导的心脏微血管内皮细胞凋亡,并且其保护心肌缺血再灌注损伤的作用与调节缺氧/复氧时心脏微血管内皮细胞的旁分泌功能有关。
Induction of autophagy by Tongxinluo via the MEK/ERK pathway protects human cardiac microvascular endothelial cells from hypoxia/reoxygenation injury
Backgrounds and Objectives:In contrast to cardiomyocytes, autophagy in cardiac microvascular endothelial cells (CMECs) during ischemia/reperfusion (I/R) injury has not been fully investigated. Tongxinluo (TXL) is a Traditional Chinese Medicine formulation that is widely used by more than6million patients with cardio-cerebral vascular diseases in China for its vascular-protective effect. We aimed to elucidate the role of autophagy and its regulatory mechanisms by TXL in CMECs subjected to I/R injury.
Methods:CMECs were exposed to different treatments for30min and subjected to hypoxia/reoxygenation (H/R) each for2h. The TXL solution at different concentrations, ginsenoside Rgl, paeoniflorin,3-methyladenine (3-MA), rapamycin and PD98059were used to further investigate the role and the modulatory mechanism of autophagy in CMECs.
Results:The results indicated that H/R significantly induced autophagy, as identified by an increased number of monodansylcadaverine (MDC)-positive CMECs, increased autophagosome formation, and a higher type II/type I of light chain3ratio, but not Beclin-1expression. Autophagy inhibition using3-methyladenine (3-MA) was proapoptotic, but rapamycin-induced autophagy was antiapoptotic. TXL enhanced autophagy and decreased apoptosis rates in a dose-dependent manner, reaching its largest effect at800μg/mL, which decreased apoptosis from21.04±1.11%to9.92±0.49%.3-MA attenuated the TXL-promoted autophagy and antiapoptotic effects, whereas rapamycin had no additional effects compared to TXL alone. TXL upregulated MEK and ERK phosphorylation; however, PD98059abrogated ERK phosphorylation and decreased autophagy and increased apoptosis compared to TXL alone.
Conclusions:These results suggest that autophagy is a protective mechanism in CMECs subjected to I/R injury and that TXL can promote autophagy via activation of the MEK/ERK pathway.
Traditional Chinese Medicine Tongxinluo modulates the secretion of cytokines in vitro by cardiac microvascular endothelial cells in ischemia/reperfusion injury.
Backgrounds and Objectives:Cardiac microvascular endothelial cells (CMECs) regulate the function of cardiomyocytes and blood cells in ischemia/reperfusion injury via autocrine and paracrine methods. Tongxinluo (TXL) is a Traditional Chinese Medicine compound which is constituted of Radix ginseng and other11kinds of natural products. It is proved to improve the function of endothelium and be protective from ischemia/reperfusion injury. Thus, we designed the study to find alterations in paracrine function of CMECs under the hypoxia/reoxygenation (H/R) situation and its modulation by TXL.
Methods:CMECs were exposed to different concentrations of TXL for30min and then subjected to H/R for12h/2h. Apoptotic rates of the cells were measured to determine the optimal concentration. Protein antibody arrays were used to find the alterations of cytokines in conditioned medium (CM) secreted by CMECs. Gene Ontology (GO) project was adopted to describe the functions of changed cytokines.
Results:TXL inhibited apoptosis of CMECs dose-dependently under H/R and reached its peak effect at the dose of800μg/mL, which reduce cell apoptosis from35.15±0.78%to14.64±2.23%. Thirty-three types of cytokines were significantly changed by H/R (19factors decreased and14increased), and TXL at800μg/ml changed121types of cytokines compared to the H/R group (93factors decreased and28increased). The cytokines with significant alterations were involved in cell differentiation and proliferation, regulation of signalling pathway, and transportation. Among these cytokines,10kinds of cytokines were increased by H/R but were decreased by TXL, such as MCP-2;5were decreased by H/R but increased by TXL, such as Follistatin;8were attenuated by H/R and further decreased by TXL, such as bFGF; IGFBP-1were upregulated by H/R and further increased by TXL.
Conclusions:TXL inhibited apoptosis of CMECs and modulated the paracrine function of the cells in ischemia/reperfusion injury.
引文
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1. Hausenloy DJ, Yellon DM. Myocardial ischemia-reperfusion injury:a neglected therapeutic target. The Journal of clinical investigation.2013 January;123(1):92-100.
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