斑蝥素阻断核转录因子κB信号通路抑制血管平滑肌细胞增殖和迁移
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摘要
目的:探讨斑蝥素对大鼠血管平滑肌细胞(VSMCs)增殖和迁移的影响,以及从核转录因子κB(NF-κB)信号通路角度探讨其机制。方法:组织块贴壁法获取SD大鼠原代VSMCs,以1 mg/L的脂多糖诱导VSMCs增殖和迁移。实验分4组:对照组、1 mg/L脂多糖刺激组、脂多糖刺激后加用5μmol/L斑蝥素组和脂多糖刺激后加用10μmol/L斑蝥素组。观察不同浓度斑蝥素对VSMCs增殖和迁移的影响及从NF-κB信号通路角度探讨其机制。结果:细胞毒性及增殖检测实验显示,斑蝥素浓度在10μmol/L内细胞活性不受影响,脂多糖使用浓度为1 mg/L时VSMCs增殖能力显著提高,但随着脂多糖浓度的进一步提高其增殖能力增速减慢。流式细胞仪检测结果显示,脂多糖可促进VSMCs由S期向G2期转化,而斑蝥素可明显抑制脂多糖的上述作用(P<0.05)。Transwell结果显示,脂多糖可显著诱导细胞迁移(P<0.01),而斑蝥素可呈浓度依赖性抑制脂多糖诱导的细胞迁移(P<0.01)。蛋白免疫印迹法检测显示,脂多糖显著增加磷酸化NF-κB p65水平,同时明显抑制NF-κB抑制蛋白(IκB-α)的表达,而斑蝥素呈浓度依赖性抑制脂多糖的上述作用(P<0.05)。实时荧光定量聚合酶链式反应法和酶联免疫吸附测定结果均显示,脂多糖显著增加促炎因子肿瘤坏死因子-α、白细胞介素-6和单核细胞趋化蛋白-1的表达(P<0.05),而斑蝥素可呈浓度依赖性抑制其表达(P<0.05)。结论:斑蝥素对脂多糖诱导的VSMCs的增殖和迁移具有显著抑制作用,其机制与抑制NF-κB通路并减轻炎症反应有关。
Objectives: To investigate the effects of cantharidin on proliferation and migration of rat vascular smooth muscle cells(VSMCs)and to explore its mechanism by observing its effect on the NF-κB signaling pathway.Methods: VSMCs were isolated from thoracic aorta of male Sprague-Dawley rats by tissue adherent method, and the proliferation and migration of VSMCs were induced by 1 mg/L lipopolysaccharide in the absence and presence of different concentrations of cantharidin. The experiments included 4 groups: control group(group 1); 1 mg/L lipopolysaccharide stimulation group(group 2); lipopolysaccharide stimulation plus 5 μmol/L cantharidin group(group 3); lipopolysaccharide stimulation plus 10 μmol/L cantharidin group(group 4).Results: The CCK-8 assay results showed that cantharidin at concentrations between 0-10 μmol/L did not affect VSMCs viability and the proliferation ability of VSMCs was significantly increased by LPS at a concentration of 1 mg/L, but the proliferation ability increase slowed down with further increased concentrations of LPS. The results of flow cytometry showed that there was a remarkable increase in the percentage of VSMCs in the G2 phase in LPS-stimulated group, this effect could be significantly attenuated by cantharidin(P<0.05). Transwell assay results showed that LPS could significantly induce cell migration(P<0.05), while cantharidin could inhibit LPS-induced cell migration in a concentration-dependent manner(P<0.05). Western blot results showed that LPS significantly increased the phosphorylation level of NF-κB p65, and inhibited the expression of IκB-α, while above effects could be partly blocked by cantharidin in a concentration-dependent manner(P<0.05). Both q-PCR and ELISA results suggested that LPS significantly increased the expression of pro-inflammatory factors including TNF-α, IL-6 and MCP-1, while the expressions of above pro-inflammatory factors could be significantly downregulated by cantharidin in a concentration-dependent manner.Conclusions: Cantharidin can inhibit the proliferation and migration of VSMCs induced by LPS, and the mechanism might be related to the inhibition of NF-κB pathway and the downregulation of the expression of inflammatory factors.
Objectives: To investigate the effects of cantharidin on proliferation and migration of rat vascular smooth muscle cells(VSMCs)and to explore its mechanism by observing its effect on the NF-κB signaling pathway.Methods: VSMCs were isolated from thoracic aorta of male Sprague-Dawley rats by tissue adherent method, and the proliferation and migration of VSMCs were induced by 1 mg/L lipopolysaccharide in the absence and presence of different concentrations of cantharidin. The experiments included 4 groups: control group(group 1); 1 mg/L lipopolysaccharide stimulation group(group 2); lipopolysaccharide stimulation plus 5 μmol/L cantharidin group(group 3); lipopolysaccharide stimulation plus 10 μmol/L cantharidin group(group 4).Results: The CCK-8 assay results showed that cantharidin at concentrations between 0-10 μmol/L did not affect VSMCs viability and the proliferation ability of VSMCs was significantly increased by LPS at a concentration of 1 mg/L, but the proliferation ability increase slowed down with further increased concentrations of LPS. The results of flow cytometry showed that there was a remarkable increase in the percentage of VSMCs in the G2 phase in LPS-stimulated group, this effect could be significantly attenuated by cantharidin(P<0.05). Transwell assay results showed that LPS could significantly induce cell migration(P<0.05), while cantharidin could inhibit LPS-induced cell migration in a concentration-dependent manner(P<0.05). Western blot results showed that LPS significantly increased the phosphorylation level of NF-κB p65, and inhibited the expression of IκB-α, while above effects could be partly blocked by cantharidin in a concentration-dependent manner(P<0.05). Both q-PCR and ELISA results suggested that LPS significantly increased the expression of pro-inflammatory factors including TNF-α, IL-6 and MCP-1, while the expressions of above pro-inflammatory factors could be significantly downregulated by cantharidin in a concentration-dependent manner.Conclusions: Cantharidin can inhibit the proliferation and migration of VSMCs induced by LPS, and the mechanism might be related to the inhibition of NF-κB pathway and the downregulation of the expression of inflammatory factors.
引文
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[17] Lee MS, David EM, Makkar RR, et al. Molecular and cellular basis of restenosis after percutaneous coronary intervention:the intertwining roles of platelets, leukocytes, and the coagulationfibrinolysis system[J]. J Pathol, 2004, 203(4):861-870. DOI:10.1002/path. 1598.
[18] Wang T, Liu J, Xiao XQ. Cantharidin inhibits angiogenesis by suppressing VEGF-induced JAK1/STAT3, ERK and AKT signaling pathways[J]. Arch Pharm Res, 2015, 38(2):282-289. DOI:10. 1007/s12272-014-0383-8.
[19] Yang D, Okamura H, Morimoto H, et al. Protein phosphatase2A Calpha regulates proliferation, migration, and metastasis of osteosarcoma cells[J]. Lab Invest, 2016, 96(10):1050-1062. DOI:10. 1038/labinvest. 2016. 82.
[20] Hsia TC, Yu CC, Hsiao YT, et al. Cantharidin impairs cell migration and invasion of human lung cancer NCI-H460 cells via UPA and MAPK signaling pathways[J]. Anticancer Res, 2016, 36(11):5989-5997. DOI:10. 21873/anticanres. 11187.
[21] Landry DB, Couper LL, Bryant SR, et al. Activation of the NF-kappa B and I kappa B system in smooth muscle cells after rat arterial injury. Induction of vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1[J]. Am J Pathol, 1997, 151(4):1085-1095.
[22] Kwon JS, Joung H, Kim YS, et al. Sulforaphane inhibits restenosis by suppressing inflammation and the proliferation of vascular smooth muscle cells[J]. Atherosclerosis, 2012, 225(1):41-49. DOI:10. 1016/j.atherosclerosis. 2012. 07. 040.
[2] Raber L, Mintz GS, Koskinas KC, et al. Clinical use of intracoronary imaging. Part 1:guidance and optimization of coronary interventions.An expert consensus document of the European association of percutaneous cardiovascular interventions[J]. Euro Intervention,2018, 14(6):656-677. DOI:10. 4244/EIJY18M06_01.
[3]杨宁,苏斌,寇璐,等.载脂蛋白J在大鼠颈动脉再狭窄模型中表达规律的研究[J].中国循环杂志,2017, 32(12):1222-1226. DOI:10. 3969/j. issn. 1000-3614. 2017. 12. 019.
[4] Qin ZX, Yu P, Qian DH, et al. Hydrogen-rich saline prevents neointima formation after carotid balloon injury by suppressing ROS and the TNF-alpha/NF-kappaB pathway[J]. Atherosclerosis, 2012,220(2):343-350. DOI:10. 1016/j. atherosclerosis. 2011. 11. 002.
[5] Jun MY, Karki R, Paudel KR, et al. Alkaloid rich fraction from Nelumbo nucifera targets VSMC proliferation and migration to suppress restenosis in balloon-injured rat carotid artery[J].Atherosclerosis, 2016, 248:179-189. DOI:10. 1016/j.atherosclerosis. 2016. 03. 020.
[6] Liu YP, Li L, Xu L, et al. Cantharidin suppresses cell growth and migration, and activates autophagy in human non-small cell lung cancer cells[J]. Oncol Lett, 2018, 15(5):6527-6532. DOI:10. 3892/ol. 2018. 8141.
[7] Gu XD, Xu LL, Zhao H, et al. Cantharidin suppressed breast cancer MDA-MB-231 cell growth and migration by inhibiting MAPK signaling pathway[J]. Braz J Med Biol Res, 2017, 50(7):e5920.DOI:10. 1590/1414-431X20175920.
[8] Ji BC, Hsiao YP, Tsai CH, et al. Cantharidin impairs cell migration and invasion of A375. S2 human melanoma cells by suppressing MMP-2 and-9 through PI3K/NF-kappaB signaling pathways[J].Anticancer Res, 2015, 35(2):729-738.
[9] Maekawa Y, Yagi K, Nonomura A, et al. A tetrazolium-based colorimetric assay for metabolic activity of stored blood platelets[J].Thromb Res, 2003, 109(5-6):307-314.
[10] Hu Q, Chen J, Zhang J, et al. IOX1, a JMJD2A inhibitor, suppresses the proliferation and migration of vascular smooth muscle cells induced by angiotensinⅡby regulating the expression of cell cyclerelated proteins[J]. Int J Mol Med, 2016, 37(1):189-196. DOI:10.3892/ijmm. 2015.2393.
[11] Liu Q, Li J, Liang Q, et al. Sparstolonin B suppresses rat vascular smooth muscle cell proliferation, migration, inflammatory response and lipid accumulation[J]. Vascul Pharmacol, 2015, 3:59-66. DOI:10. 1016/j. vph. 2015.03.015.
[12]徐珊珊,阎春兰,刘黎明,等.细胞裂解液对蛋白质定量方法的影响[J].浙江大学学报(医学版),2008, 37(1):45-50.
[13] Hunter DJ, Reddy KS. Noncommunicable diseases[J]. N Engl J Med,2013, 369(14):1336-1343. DOI:10. 1056/NEJMra 1109345.
[14]陈伟伟,高润霖,刘力生,等.《中国心血管病报告2017》概要[J].中国循环杂志,2018, 33(1):1-8. DOI:10. 3969/j. issn. 1000-3614.2018.01.001.
[15] Simon DI. Inflammation and vascular injury:basic discovery to drug development[J]. Circ J, 2012, 76(8):1811-1818.
[16] Collins MJ, Li X, Lv W, et al. Therapeutic strategies to combat neointimal hyperplasia in vascular grafts[J]. Expert Rev Cardiovasc Ther, 2012, 10(5):635-647. DOI:10. 1586/erc. 12. 33.
[17] Lee MS, David EM, Makkar RR, et al. Molecular and cellular basis of restenosis after percutaneous coronary intervention:the intertwining roles of platelets, leukocytes, and the coagulationfibrinolysis system[J]. J Pathol, 2004, 203(4):861-870. DOI:10.1002/path. 1598.
[18] Wang T, Liu J, Xiao XQ. Cantharidin inhibits angiogenesis by suppressing VEGF-induced JAK1/STAT3, ERK and AKT signaling pathways[J]. Arch Pharm Res, 2015, 38(2):282-289. DOI:10. 1007/s12272-014-0383-8.
[19] Yang D, Okamura H, Morimoto H, et al. Protein phosphatase2A Calpha regulates proliferation, migration, and metastasis of osteosarcoma cells[J]. Lab Invest, 2016, 96(10):1050-1062. DOI:10. 1038/labinvest. 2016. 82.
[20] Hsia TC, Yu CC, Hsiao YT, et al. Cantharidin impairs cell migration and invasion of human lung cancer NCI-H460 cells via UPA and MAPK signaling pathways[J]. Anticancer Res, 2016, 36(11):5989-5997. DOI:10. 21873/anticanres. 11187.
[21] Landry DB, Couper LL, Bryant SR, et al. Activation of the NF-kappa B and I kappa B system in smooth muscle cells after rat arterial injury. Induction of vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1[J]. Am J Pathol, 1997, 151(4):1085-1095.
[22] Kwon JS, Joung H, Kim YS, et al. Sulforaphane inhibits restenosis by suppressing inflammation and the proliferation of vascular smooth muscle cells[J]. Atherosclerosis, 2012, 225(1):41-49. DOI:10. 1016/j.atherosclerosis. 2012. 07. 040.