| 高维伟,盛思琪,曹正宇,谭凡成,田 野.致凋亡的声动力疗法诱导巨噬细胞线粒体钙升高的机制研究[J].现代生物医学进展英文版,2019,19(7):1212-1217. |
| 致凋亡的声动力疗法诱导巨噬细胞线粒体钙升高的机制研究 |
| Mechanism Research on Mitochondrial Calcium Increase in Macrophage induced by Apoptotic Sonodynamic Therapy |
| Received:August 07, 2018 Revised:August 31, 2018 |
| DOI：10.13241/j.cnki.pmb.2019.07.003 |
| 中文关键词: SDT 巨噬细胞 VDAC1 IP3R-III Ca2+ |
| 英文关键词: SDT macrophage VDAC1 IP3R-III Ca2+ |
| 基金项目:国家自然科学基金项目（81371709） |
| Author Name | Affiliation | E-mail | | GAO Wei-wei | Cardiovascular Institute, The 1st Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, 150001, China | jcyxgww@163.com | | SHENG Si-qi | Cardiovascular Institute, The 1st Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, 150001, China | | | CAO Zheng-yu | Department of Cardiology, The 1st Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, 150001, China | | | TAN Fan-cheng | Department of Pathophysiology, Harbin Medical University, Harbin, Heilongjiang, 150081, China | | | TIAN Ye | Department of Cardiology, The 1st Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, 150001, China
Department of Pathophysiology, Harbin Medical University, Harbin, Heilongjiang, 150081, China | |
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| 中文摘要: |
| 摘要 目的：研究致凋亡的声动力疗法诱导巨噬细胞线粒体钙升高的机制。方法：应用佛波酯（PMA）诱导THP-1单核细胞分化为巨噬细胞进行实验研究。选用5-氨基酮戊酸（ALA）作为声敏剂，进行声动力治疗（SDT）。应用流式细胞术证实SDT显著促进了细胞凋亡；应用Rhod 2/AM实时监测线粒体Ca2+水平；通过蛋白质免疫印迹对全细胞蛋白中的Bax、Cleaved-caspase3、电压依赖性阴离子通道1（VDAC1）和三磷酸肌醇III型受体（IP3R-III）进行检测；应用VDAC1抗体进行免疫共沉淀，检测VDAC1和IP3R-III之间的相互作用；实时监测线粒体Ca2+水平，检测VDAC抑制剂DIDS和IP3Rs抑制剂2-ABP对SDT效果的影响。结果：与对照组相比，仅SDT组出现了显著的细胞凋亡（P<0.001）。与对照组相比，ALA对线粒体Ca2+水平无明显影响，超声诱导了线粒体Ca2+水平的明显升高，SDT诱导了线粒体Ca2+水平快速且大幅度的升高，且去除超声后仍维持在较高水平。与对照组相比，超声对Bax、Cleaved-caspase3、VDAC1和IP3R-III的表达无明显影响，ALA诱导了VDAC1（P<0.01）和IP3R-III表达量的增加（P<0.05），SDT诱导了Bax（P<0.001）、Cleaved-caspase3（P<0.001）、VDAC1（P<0.01）和IP3R-III（P<0.05）表达量的增加，VDAC1和IP3R-III的增加幅度与ALA组接近；ALA和SDT均诱导了VDAC1和IP3R-III之间相互作用的显著增强（P<0.05）。DIDS和2-ABP均明显抑制了SDT诱导的线粒体Ca2+增加。结论：在致凋亡的SDT作用于THP-1巨噬细胞的过程中，ALA诱导了线粒体外膜Ca2+转运通道VDAC1和内质网重要Ca2+转运通道IP3R-III的表达量增加与二者间相互作用的增强，在内质网和线粒体之间建立了大量的Ca2+转运通道，超声的作用则在于触发这些Ca2+转运通道的开放，进而引发线粒体钙的迅速增加。这是后续线粒体凋亡通路启动的重要机制之一。 |
| 英文摘要: |
| ABSTRACT Objective: To investigate the mechanism by which sonodynamic therapy (SDT) initiated the mitochondrial calcium in- crease in macrophages. Methods: THP-1 monocytes were differentiated into macrophages by adding PMA. For SDT, ALA was used as sonosensitizer. Flow cytometry was used to certify the SDT-induced apoptosis of macrophages. Real-time detection of mitochondrial Ca2+ indicated by Rhod 2/AM was applied. Whole cell lysates were aquired. Bax, Cleaved caspase3, voltage dependent anion channe1 1(VDAC1) and inositol 1, 4, 5-trisphosphate receptor III (IP3R-III) were separately detected by Western blot. Primary antibodies against VDAC1 were used for immunoprecipitation to analyze interactions between VDAC1 and IP3R-III. 4,4'-diisothiocyanostilbene-2,2'-disul- fonic acid (DIDS) and 2-aminoethoxydiphenyl borate (2-ABP) were separately used to inhibite functions of VDAC1 and IP3R-III, real-time detection of mitochondrial Ca2+ was conducted again. Results: Compared with the control group, only SDT induced obvious increase of cell apoptosis(P<0.001). Compared with the control group, ALA showed no significant effect to mitochondrial Ca2+ level, ultrasound in- duced obvious increase of mitochondrial Ca2+ level, SDT led to rapid and sharp increase of mitochondrial Ca2+ level which sustained at high level even after ultrasound exposure like the ultrasound group. Compared with the control group, ultrasound showed no obvious ef- fect to the proteins, ALA induced marked upregulation of VDAC1 (P<0.01) and IP3R-III (P<0.05), SDT led to significant upregulation of Bax (P<0.001), Cleaved-caspase3 (P<0.001), VDAC1 (P<0.01) as well as IP3R-III (P<0.05). Enhanced interactions between VDAC1 and IP3R-III were observed in ALA and SDT groups(P<0.05). SDT-induced increase of mitochondrial Ca2+ level were markedly weakened by DIDS and 2-ABP. Conclusion: During the course of apoptotic SDT, ALA induced the upregulation of VDAC1 and IP3R-III, as well as the enhanced interactions between the proteins, thus building lots of channels for Ca2+ transiton between endoplasmic reticulum and mito- chondria. While the main function of ultrasound is to trigger the opening of these channels, thus leading to the rapid calcium increase in mitochondria. This may be an important mechanism of the following mitochondrial apoptosis pathway. |
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