低强度脉冲超声对高糖诱导人脐静脉内皮细胞损伤的保护作用
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摘要
背景:研究表明,人脐静脉内皮细胞参与多种原因引起的血管结构紊乱和功能障碍,有可能成为相关疾病治疗的新靶点。目的:探讨低强度脉冲超声对高糖环境诱导的人脐静脉内皮细胞损伤的保护作用及机制。方法:将人脐静脉内皮细胞随机分为正常对照组、高糖刺激模型组、低强度脉冲超声30,60,90m W/cm~2组。高糖刺激模型组和低强度脉冲超声组人脐静脉内皮细胞给予25 mmol/L葡萄糖高糖刺激处理24 h从而建立高糖模型;低强度脉冲超声组细胞造模后给予30,60,90 mW/cm~2低频脉冲处理,每天20 min,连续7 d。采用MTT法检测各组细胞活力,流式细胞仪检测各组细胞凋亡率,比色法检测各组细胞Caspase-3、9活性,分光光度法检测丙二醛水平、超氧化物歧化酶、过氧化氢酶及谷胱甘肽过氧化物酶活性,Westernblot及RT-PCR检测各组细胞中JNK的表达。结果与结论:①与正常对照组比较,高糖刺激模型组细胞活力明显降低(P <0.01),细胞凋亡率升高(P <0.01),丙二醛水平以及Caspase-3、9活性均显著提高(P <0.01),超氧化物歧化酶、过氧化氢酶及谷胱甘肽过氧化物酶活性降低(P <0.01),JNK表达水平上调(P <0.01);②与高糖刺激模型组比较,低强度脉冲超声组细胞活力提高(P <0.01),细胞凋亡率、丙二醛水平以及Caspase-3、9活性降低(P <0.01),超氧化物歧化酶、过氧化氢酶及谷胱甘肽过氧化物酶活性提高(P <0.01),JNK表达水平下调(P <0.01),照射强度90 mW/cm~2时以上变化最明显;③低强度脉冲超声可能通过提高细胞的抗氧化能力以及调控细胞中的JNK信号转导通路以抵抗细胞凋亡,进而抑制高糖诱导的人脐静脉内皮细胞损伤;照射强度90 mW/cm~2时作用最显著。
BACKGROUND: Studies have shown that human umbilical vein endothelial cells participate in vascular structural disorders and dysfunction due to a series of causes, which may become a new target for the treatment of related diseases.OBJECTIVE: To investigate the protective effect and mechanism of low-intensity pulsed ultrasound on the high glucose-induced injury to human umbilical vein endothelial cells.METHODS: Human umbilical vein endothelial cells were randomly divided into normal control group, high glucose group and 30, 60 and 90 mW/cm~2 low-intensity pulsed ultrasound groups. For the high glucose group and three low-intensity pulsed ultrasound groups, the high-glucose stimulation with 25 mmol/L glucose was given. Additionally, the cells in the three low-intensity pulsed ultrasound groups received 30, 60, and 90 mW/cm~2 low-intensity pulsed ultrasounds, 20 minutes per day for 7 continuous days, respectively. MTT was used to detect cell activity and flow cytometry was used to detect cell apoptosis in each experimental group. Colorimetry was used to detect Caspase-3 and -9 activities in the human umbilical vein endothelial cells. Spectrophotometry was used to detect malonaldehyde content, superoxide dismutase activity, catalase activity, and glutathione peroxidase activity. Expression of JNK in the cells was observed by western blot and RT-PCR.RESULTS AND CONCLUSION: After high glucose treatment, the viability of human umbilical vein endothelial cells was significantly lowered;the apoptosis rate, malonaldehyde content and activities of Caspase 3 and 9 were significantly increased(P < 0.01); the activities of superoxide dismutase, catalase and glutathione peroxidase significantly decreased(P < 0.01); and the JNK expression level was up-regulated(P < 0.01). Compared with the high glucose group, the cell viability of the low-intensity pulsed ultrasound groups(30, 60,90 mW/cm~2 subgroups) was increased(P < 0.01), and the apoptosis rate, malonaldehyde content, and Caspase 3, 9 activities were decreased(P < 0.01), superoxide dismutase, catalase and glutathione peroxidase activities were increased(P < 0.01), and JNK expression level was down-regulated(P < 0.01). The above changes were most obvious when the irradiation intensity was 90 mW/cm~2. In conclusion,low-intensity pulsed ultrasound can inhibit the high glucose-induced apoptosis of human umbilical vein endothelial cells by increasing the antioxidant capacity and regulating the JNK signaling pathway, further inhibiting the high glucose-induced injury to the human umbilical vein endothelial cells. The low-intensity pulsed ultrasound at 90 mW/cm~2 can achieve the best outcomes.
BACKGROUND: Studies have shown that human umbilical vein endothelial cells participate in vascular structural disorders and dysfunction due to a series of causes, which may become a new target for the treatment of related diseases.OBJECTIVE: To investigate the protective effect and mechanism of low-intensity pulsed ultrasound on the high glucose-induced injury to human umbilical vein endothelial cells.METHODS: Human umbilical vein endothelial cells were randomly divided into normal control group, high glucose group and 30, 60 and 90 mW/cm~2 low-intensity pulsed ultrasound groups. For the high glucose group and three low-intensity pulsed ultrasound groups, the high-glucose stimulation with 25 mmol/L glucose was given. Additionally, the cells in the three low-intensity pulsed ultrasound groups received 30, 60, and 90 mW/cm~2 low-intensity pulsed ultrasounds, 20 minutes per day for 7 continuous days, respectively. MTT was used to detect cell activity and flow cytometry was used to detect cell apoptosis in each experimental group. Colorimetry was used to detect Caspase-3 and -9 activities in the human umbilical vein endothelial cells. Spectrophotometry was used to detect malonaldehyde content, superoxide dismutase activity, catalase activity, and glutathione peroxidase activity. Expression of JNK in the cells was observed by western blot and RT-PCR.RESULTS AND CONCLUSION: After high glucose treatment, the viability of human umbilical vein endothelial cells was significantly lowered;the apoptosis rate, malonaldehyde content and activities of Caspase 3 and 9 were significantly increased(P < 0.01); the activities of superoxide dismutase, catalase and glutathione peroxidase significantly decreased(P < 0.01); and the JNK expression level was up-regulated(P < 0.01). Compared with the high glucose group, the cell viability of the low-intensity pulsed ultrasound groups(30, 60,90 mW/cm~2 subgroups) was increased(P < 0.01), and the apoptosis rate, malonaldehyde content, and Caspase 3, 9 activities were decreased(P < 0.01), superoxide dismutase, catalase and glutathione peroxidase activities were increased(P < 0.01), and JNK expression level was down-regulated(P < 0.01). The above changes were most obvious when the irradiation intensity was 90 mW/cm~2. In conclusion,low-intensity pulsed ultrasound can inhibit the high glucose-induced apoptosis of human umbilical vein endothelial cells by increasing the antioxidant capacity and regulating the JNK signaling pathway, further inhibiting the high glucose-induced injury to the human umbilical vein endothelial cells. The low-intensity pulsed ultrasound at 90 mW/cm~2 can achieve the best outcomes.
引文
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[17]王喜欢,张金华,胡亚南,等.紫草素对高糖诱导的血管内皮细胞凋亡和氧化应激的影响[J].中国病理生理杂志,2018,34(7):1222-1227.
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[19]祖海越,易雪婷,赵德伟.低强度脉冲超声促进骨与软骨再生的研究与临床应用[J].中国组织工程研究,2018,22(16):2593-2600.
[20]Rossato DD,Dal Lago P,Hentschke VS,et al.Ultrasound modulates skeletal muscle cytokine levels in rats with heart failure.Ultrasound Med Biol.2015;41(3):797-805.
[21]Chongsatientam A,Yimlamai T.Therapeutic Pulsed Ultrasound Promotes Revascularization and Functional Recovery of Rat Skeletal Muscle after Contusion Injury.Ultrasound Med Biol.2016;42(12):2938-2949.
[22]Miyasaka M,Nakata H,Hao J,et al.Low-Intensity Pulsed Ultrasound Stimulation Enhances Heat-Shock Protein 90 and Mineralized Nodule Formation in Mouse Calvaria-Derived Osteoblasts.Tissue Eng Part A.2015;21(23-24):2829-2839.
[23]Novoselova EG,Khrenov MO,Parfenyuk SB,et al.The NF-κB,IRF3,and SAPK/JNK signaling cascades of animal immune cells and their role in the progress of type 1 diabetes mellitus.Dokl Biol Sci.2014;457(1):255-257.
[24]Barcelona PF,Saragovi HU.A Pro-Nerve Growth Factor(proNGF)and NGF Binding Protein,α2-Macroglobulin,Differentially Regulates p75 and TrkA Receptors and Is Relevant to Neurodegeneration Ex Vivo and In Vivo.Mol Cell Biol.2015;35(19):3396-3408.
[25]侯瑞雪.广西眼镜蛇毒神经生长因子的精制及其对HSC--T6细胞SAPK/JNK信号通路的影响[D].南宁:广西医科大学,2016.
[26]Choudhury S,Ghosh S,Gupta P,et al.Inflammation-induced ROS generation causes pancreatic cell death through modulation of Nrf2/NF-κB and SAPK/JNK pathway.Free Radic Res.2015;49(11):1371-1383.
[27]Glushkova OV,Khrenov MO,Novoselova TV,et al.The role of the NF-κB,SAPK/JNK,and TLR4 signalling pathways in the responses of RAW 264.7 cells to extremely low-intensity microwaves.Int J Radiat Biol.2015;91(4):321-328.
[28]Di Pietro R,Centurione L,Sabatini N,et al.Caspase-3 is dually regulated by apoptogenic factors mitochondrial release and by SAPK/JNK metabolic pathway in leukemic cells exposed to etoposide-ionizing radiation combined treatment.Int J Immunopathol Pharmacol.2004;17(2):181-190.
[2]Xie CL,Pan YB,Hu LQ,et al.Propofol attenuates hydrogenperoxide-induced apoptosis in human umbilical vein endothelial cells via multiple signaling pathways.Korean JAnesthesiol.2015;68(5):488-495.
[3]Chu C,Lu FJ,Yeh RH,et al.Synergistic antioxidant activity of resveratrol with genistein in high-glucose treated Madin-Darby canine kidney epithelial cells.Biomed Rep.2016;4(3):349-354.
[4]Zhu D,Zhang N,Zhou X,et al.Cichoric acid regulates the hepatic glucose homeostasis via AMPK pathway and activates the antioxidant response in high glucose-induced hepatocyte injury.Rsc Advances.2017;7(3):1363-1375.
[5]Nur H,Rao L,Frassanito MA,et al.Stimulation of invariant natural killer T cells byα-Galactosylceramide activates the JAK-STAT pathway in endothelial cells and reduces angiogenesis in the 5T33 multiple myeloma model.Br JHaematol.2014;167(5):651-663.
[6]Kim BH,Lee Y,Yoo H,et al.Anti-angiogenic activity of thienopyridine derivative LCB03-0110 by targeting VEGFR-2and JAK/STAT3 Signalling.Exp Dermatol.2015;24(7):503-509.
[7]Kusuyama J,Bandow K,Shamoto M,et al.Low intensity pulsed ultrasound(LIPUS)influences the multilineage differentiation of mesenchymal stem and progenitor cell lines through ROCK-Cot/Tpl2-MEK-ERK signaling pathway.J Biol Chem.2014;289(15):10330-10344.
[8]冯天明,杨明聪,潘兰兰,等.低频超声联合曲安奈德对金黄地鼠颊黏膜溃疡愈合的影响[J].上海口腔医学,2017,26(4):379-383.
[9]杨少玲.低频超声联合微泡造影剂对前列腺增生的作用[C].重庆:第一届全国暨第二届国际超声分子影像学术会议论文集,2014.
[10]邢宇,宋晓彬,施克新,等.高血糖人群周围血管病变的患病率调查[J].中国冶金工业医学杂志,2017,34(4):432-433.
[11]薛亚,曹洁,陈荣华,等.急性缺血性卒中患者入院高血糖对血管内治疗结局的影响[J].中国脑血管病杂志,2018,15(3):124-128.
[12]黄燕,何婧,向阳,等.持续高血糖模型小鼠大脑皮质血管周围星形胶质细胞足突的变化[J].中国组织工程研究,2018,22(20):3218-3223.
[13]Ku SK,Kwak S,Bae JS.Orientin inhibits high glucose-induced vascular inflammation in vitro and in vivo.Inflammation.2014;37(6):2164-2173.
[14]Pan Y,Wang Y,Zhao Y,et al.Inhibition of JNKphosphorylation by a novel curcumin analog prevents high glucose-induced inflammation and apoptosis in cardiomyocytes and the development of diabetic cardiomyopathy.Diabetes.2014;63(10):3497-3511.
[15]Renaud J,Bournival J,Zottig X,et al.Resveratrol protects DAergic PC12 cells from high glucose-induced oxidative stress and apoptosis:effect on p53 and GRP75 localization.Neurotox Res.2014;25(1):110-123.
[16]Guo S,Yao Q,Ke Z,et al.Resveratrol attenuates high glucose-induced oxidative stress and cardiomyocyte apoptosis through AMPK.Mol Cell Endocrinol.2015;412:85-94.
[17]王喜欢,张金华,胡亚南,等.紫草素对高糖诱导的血管内皮细胞凋亡和氧化应激的影响[J].中国病理生理杂志,2018,34(7):1222-1227.
[18]Peng H,Zhong W,Zhao H,et al.Lack of PGC-1αexacerbates high glucose-induced apoptosis in human umbilical vein endothelial cells through activation of VADC1.Int J Clin Exp Pathol.2015;8(5):4639-4650.
[19]祖海越,易雪婷,赵德伟.低强度脉冲超声促进骨与软骨再生的研究与临床应用[J].中国组织工程研究,2018,22(16):2593-2600.
[20]Rossato DD,Dal Lago P,Hentschke VS,et al.Ultrasound modulates skeletal muscle cytokine levels in rats with heart failure.Ultrasound Med Biol.2015;41(3):797-805.
[21]Chongsatientam A,Yimlamai T.Therapeutic Pulsed Ultrasound Promotes Revascularization and Functional Recovery of Rat Skeletal Muscle after Contusion Injury.Ultrasound Med Biol.2016;42(12):2938-2949.
[22]Miyasaka M,Nakata H,Hao J,et al.Low-Intensity Pulsed Ultrasound Stimulation Enhances Heat-Shock Protein 90 and Mineralized Nodule Formation in Mouse Calvaria-Derived Osteoblasts.Tissue Eng Part A.2015;21(23-24):2829-2839.
[23]Novoselova EG,Khrenov MO,Parfenyuk SB,et al.The NF-κB,IRF3,and SAPK/JNK signaling cascades of animal immune cells and their role in the progress of type 1 diabetes mellitus.Dokl Biol Sci.2014;457(1):255-257.
[24]Barcelona PF,Saragovi HU.A Pro-Nerve Growth Factor(proNGF)and NGF Binding Protein,α2-Macroglobulin,Differentially Regulates p75 and TrkA Receptors and Is Relevant to Neurodegeneration Ex Vivo and In Vivo.Mol Cell Biol.2015;35(19):3396-3408.
[25]侯瑞雪.广西眼镜蛇毒神经生长因子的精制及其对HSC--T6细胞SAPK/JNK信号通路的影响[D].南宁:广西医科大学,2016.
[26]Choudhury S,Ghosh S,Gupta P,et al.Inflammation-induced ROS generation causes pancreatic cell death through modulation of Nrf2/NF-κB and SAPK/JNK pathway.Free Radic Res.2015;49(11):1371-1383.
[27]Glushkova OV,Khrenov MO,Novoselova TV,et al.The role of the NF-κB,SAPK/JNK,and TLR4 signalling pathways in the responses of RAW 264.7 cells to extremely low-intensity microwaves.Int J Radiat Biol.2015;91(4):321-328.
[28]Di Pietro R,Centurione L,Sabatini N,et al.Caspase-3 is dually regulated by apoptogenic factors mitochondrial release and by SAPK/JNK metabolic pathway in leukemic cells exposed to etoposide-ionizing radiation combined treatment.Int J Immunopathol Pharmacol.2004;17(2):181-190.