虾青素通过线粒体抑制高糖诱导的HBZY-1细胞凋亡
|
摘要
为了探究虾青素是否能有效改善高糖诱导的HBZY-1细胞的凋亡及其抗凋亡的机制,本研究采用CCK8法测定了正常糖和高糖环境下,不同浓度的虾青素对HBZY-1细胞活力的影响,并运用相关试剂盒测试了细胞死活比例和细胞凋亡的变化,在光镜下观察了HBZY-1细胞形态的变化,使用JC-1探针在荧光显微镜下观察了HBZY-1细胞线粒体膜电位的变化,运用Western Blot技术检测了HBZY-1细胞中促细胞凋亡蛋白bax和抑制细胞凋亡蛋白bcl-2的表达水平,运用caspase 3/7活细胞荧光实时法检测了HBZY-1细胞中的caspase 3/7活性.结果表明:虾青素可有效逆转高糖引起的细胞活力降低,能抑制高糖引起的HBZY-1细胞凋亡和细胞形态异常,并能改善线粒体膜电位和影响参与调控HBZY-1细胞的凋亡相关蛋白.由此认为:虾青素可以通过影响线粒体膜电位来改善高糖引起的肾小球系膜细胞凋亡,其有可能成为改善糖尿病肾病的潜在药物.
To explore whether astaxanthin can effectively improve the apoptosis and anti-apoptosis mechanism of HBZY-1 cells induced by high glucose. CCK8 method was performed to determine the effects of astaxanthin at the different concentration on the viability of HBZY-1 cells; the relevant kits were used to measure the changes of the death rate and apoptosis; the morphological changes of HBZY-1 cells were observed under light microscope;the changes of the mitochondrial membrane potential of HBZY-1 cells were observed under fluorescence microscopy using JC-1 probes; the activity of caspase 3/7 in HBZY-1 cells was detected by caspase 3/7 fluorescence real-time methods; the expression levels of pro-apoptotic protein bax and anti-apoptotic protein bcl-2 in HBZY-1 cells were detected by Western Blot. The results indicated that astaxanthin effectively reversed the decrease of cells viability induced by high glucose and inhibited the apoptosis of HBZY-1 cells induced by high glucose;astaxanthin improved the mitochondrial membrane potential and participated in the regulation of the apoptotic protein of HBZY-1 cells; astaxanthin can affect mitochondria and improve the apoptosis of HBZY-1 cells induced by high glucose,which suggested that astaxanthin therapy may improving the occurrence and development of diabetic nephropathy.
To explore whether astaxanthin can effectively improve the apoptosis and anti-apoptosis mechanism of HBZY-1 cells induced by high glucose. CCK8 method was performed to determine the effects of astaxanthin at the different concentration on the viability of HBZY-1 cells; the relevant kits were used to measure the changes of the death rate and apoptosis; the morphological changes of HBZY-1 cells were observed under light microscope;the changes of the mitochondrial membrane potential of HBZY-1 cells were observed under fluorescence microscopy using JC-1 probes; the activity of caspase 3/7 in HBZY-1 cells was detected by caspase 3/7 fluorescence real-time methods; the expression levels of pro-apoptotic protein bax and anti-apoptotic protein bcl-2 in HBZY-1 cells were detected by Western Blot. The results indicated that astaxanthin effectively reversed the decrease of cells viability induced by high glucose and inhibited the apoptosis of HBZY-1 cells induced by high glucose;astaxanthin improved the mitochondrial membrane potential and participated in the regulation of the apoptotic protein of HBZY-1 cells; astaxanthin can affect mitochondria and improve the apoptosis of HBZY-1 cells induced by high glucose,which suggested that astaxanthin therapy may improving the occurrence and development of diabetic nephropathy.
引文
[1]Stenvinkel P.Chronic kidney disease:a public health priority and harbinger of premature cardiovascular disease[J].Journal of Internal Medicine,2010,268(5):456.
[2]Karihaloo A.Anti-fibrosis therapy and diabetic nephropathy[J].Current Diabetes Reports,2012,12(4):414-422.
[3]Stanton R C.Oxidative stress and diabetic kidney disease[J].Current Diabetes Reports,2011,11(4):330-336.
[4]Tesch G H,Lim A K.Recent insights into diabetic renal injury from the db/db mouse model of type 2diabetic nephropathy.[J].American Journal of Physiology Renal Physiology,2011,300(2):F301.
[5]Coles H S,Burne J F,Raff M C.Large-scale normal cell death in the developing rat kidney and its reduction by epidermal growth factor[J].Development,1993,118(3):777-784.
[6]Rao A R,Phang S M,Sarada R.Astaxanthin:Sources,Extraction,Stability,Biological Activities and Its Commercial Applications-A Review[J].Marine Drugs,2014,12(1):128-152.
[7]Gal A F,Andrei S,Cernea C,et al.Effects of astaxanthin supplementation on chemically induced tumorigenesis in Wistar rats[J].Acta Veterinaria Scandinavica,2012,54(1):50.
[8]曹秀明,高越,徐德林.虾青素保护活性氧所致线粒体损伤的作用[J].食品与药品,2010,12(11):412-414.
[9]Song X,Wang B,Lin S,et al.Astaxanthin inhibits apoptosis in alveolar epithelial cells type II in vivo and in vitro through the ROS-dependent mitochondrial signalling pathway[J].Journal of Cellular and Molecular Medicine,2014,18(11):15.
[10]Lee D H,Kim C S,Lee Y J.Astaxanthin protects against MPTP/MPP+-induced mitochondrial dysfunction and ROS production in vivo and in vitro[J].Food and Chemical Toxicology,2011,49(1):0-280.
[11]Saw C L L,Yang A Y,Guo Y,et al.Astaxanthin and omega-3 fatty acids individually and in combination protect against oxidative stress via the Nrf2-ARE pathway[J].Food&Chemical Toxicology,2013,62(12):869-875.
[12]Mano C M,Guaratini T,Cardozo K,et al.Astaxanthin restrains nitrative-oxidative peroxidation in mitochondrial-mimetic liposomes:A Pre-apoptosis model[J].Marine Drugs,2018,16(4):126.
[13]Zhang J,Xu P,Wang Y,et al.Astaxanthin prevents pulmonary fibrosis by promoting myofibroblast apoptosis dependent on Drp1-mediated mitochondrial fission[J].Journal of Cellular&Molecular Medicine,2015,19(9):2215-2231.
[14]Wolf A M,Asoh S,Hiranuma H,et al.Astaxanthin protects mitochondrial redox state and functional integrity against oxidative stress[J].Journal of Nutritional Biochemistry,2010,21(5):381.
[15]曹秀明,杨贵群,杨菲菲.虾青素对过氧化氢所致Hep G2细胞线粒体氧化损伤及生存能力下降的保护作用[J].中国海洋药物,2010(5):26-32.
[16]宋晓冬,刘文波,张丽霞,等.虾青素对ECV-304细胞的抗氧化作用及其作用机制[J].滨州医学院学报,2009,32(3):161-163.
[17]Liu X,Shibata T S.Astaxanthin inhibits reactive oxygen species-mediated cellular toxicity in dopaminergic SH-SY5Y cells via mitochondria-targeted protective mechanism.[J].Brain Research,2009,1254(Complete):18-27.
[18]Lee D H,Kim C S,Lee Y J.Astaxanthin protects against MPTP/MPP+-induced mitochondrial dysfunction and ROS production in vivo and in vitro.[J].Food&Chemical Toxicology,2011,49(1):271-80.
[19]詹明,董政,孙林.线粒体损伤与糖尿病肾病[J].中华肾脏病杂志,2011,27(6):460-463.
[20]Khoshjou F,Dadras F.Mitochondrion and its role in diabetic nephropathy[J].Iranian Journal of Kidney Diseases,2014,8(5):355.
[21]Zhang S,Sumei L I.Mitochondria-induced oxidative stress and diabetic nephropathy[J].International Journal of Pathology&Clinical Medicine,2011.
[22]曹延萍,王莎,刘杰,等.线粒体凋亡途径在高糖刺激的小鼠足细胞凋亡中的作用[J].中国应用生理学杂志,2013,29(3):244-246.
[23]董雅洁,高维娟.bcl-2、bax、caspase-3在细胞凋亡中的作用及其关系[J].中国老年学,2012,32(21):4828-4830.
[24]Uchiyama K,Naito Y,Hasegawa,等.虾青素对糖尿病引起的肾脏并发症的影响[C]∥中日国际维生素学术研讨会.2001:230.
[25]孔德贤.冬虫夏草含药血清对高糖培养大鼠肾小球系膜细胞PEDF及VEGF表达的影响[D].石家庄:河北医科大学,2009.
[26]林玉玲.C肽对高糖环境下大鼠肾小球系膜细胞增殖及凋亡影响的研究[D].郑州:郑州大学,2007.
[27]潘晓燕,龚小花,沈飞霞,等.高糖环境对HBZY-1大鼠肾小球系膜细胞IL-18、IL-1β、IL-6、TNF-α表达的影响[J].中国中西医结合肾病杂志,2012,13(6):519-521.
[28]杨丹丽,张迎秋,李媛,等.冬凌草甲素对甲状腺癌BCPAP细胞增殖的抑制作用[J].大连医科大学学报,2016,38(4):330-333.
[29]王碧,邵晓琳,赵雨.姜黄素和Dickkopf-1联用对口腔鳞状细胞癌细胞增殖和凋亡的影响研究[J].口腔医学研究,2017,33(8):877-880.
[30]MPA、MG132对不同PR表达卵巢癌细胞增殖、凋亡影响的实验研究[D].遵义:遵义医学院,2017.
[31]沈会,陶汉华,张诏,等.建中理劳汤含药血清对TGF-β1诱导的大鼠肾小球系膜细胞增殖及基质金属蛋白酶表达的影响[J].世界科学技术-中医药现代化,2015(9).
[32]Xie X,Chen Q,Tao J.Astaxanthin promotes Nrf2/ARE signaling to inhibit HG-induced renal fibrosis in GMCs[J].Marine Drugs,2018,16(4):117.
[33]Zhu X,Chen Y,Chen Q,et al.Astaxanthin promotes Nrf2/ARE signaling to alleviate renal fibronectin and collagen IV accumulation in diabetic rats[J].Journal of Diabetes Research,2018,2018:1-7.
[34]Scabini M,Stellari F,Cappella P,et al.In vivo imaging of early stage apoptosis by measuring real-time caspase-3/7 activation[J].Apoptosis,2011,16(2):198-207.
[35]高春林,赵亚萍,陈小慧,等.高糖对原代培养人脂肪细胞线粒体膜电位及胰岛素敏感性的影响[J].医学研究生学报,2010,23(8):805-808.
[36]鲍丽颖,姜希娟,郑纺,等.高糖高脂诱导脐静脉内皮细胞凋亡及对线粒体膜电位的影响[J].天津中医药,2010,27(6):501-502.
[37]李圣杰.低氧微环境对神经胶质瘤细胞线粒体功能的影响[D].昆明:昆明医科大学,2016.
[38]车晓芳,罗颖,刘云鹏.Bcl-2和Bax调节细胞凋亡的研究[J].国际输血及血液学杂志,2001,24(2):103-105
[39]Bagci E Z,Vodovotz Y,Billiar T R,et al.Bistability in apoptosis:roles of bax,bcl-2,and mitochondrial permeability transition pores[J].Biophysical Journal,2007,92(9):3332-3334.
[40]邓红,王新,徐芳,等.六味地黄汤对糖尿病肾病大鼠肾功能及细胞凋亡的影响[J].中草药,2010,41(10):1679-1682.
[41]罗文娟,喻嵘,唐元,等.左归降糖益肾方对2型糖尿病肾病小鼠足细胞凋亡的影响[J].中医杂志,2015,56(6):511-514.
[42]游娜,李慧敏,丁大法,等.肾上腺髓质素对高糖培养的肾小球系膜细胞增殖和凋亡的影响及其机制的研究[J].中国糖尿病杂志,2013,21(1):66-71.
[2]Karihaloo A.Anti-fibrosis therapy and diabetic nephropathy[J].Current Diabetes Reports,2012,12(4):414-422.
[3]Stanton R C.Oxidative stress and diabetic kidney disease[J].Current Diabetes Reports,2011,11(4):330-336.
[4]Tesch G H,Lim A K.Recent insights into diabetic renal injury from the db/db mouse model of type 2diabetic nephropathy.[J].American Journal of Physiology Renal Physiology,2011,300(2):F301.
[5]Coles H S,Burne J F,Raff M C.Large-scale normal cell death in the developing rat kidney and its reduction by epidermal growth factor[J].Development,1993,118(3):777-784.
[6]Rao A R,Phang S M,Sarada R.Astaxanthin:Sources,Extraction,Stability,Biological Activities and Its Commercial Applications-A Review[J].Marine Drugs,2014,12(1):128-152.
[7]Gal A F,Andrei S,Cernea C,et al.Effects of astaxanthin supplementation on chemically induced tumorigenesis in Wistar rats[J].Acta Veterinaria Scandinavica,2012,54(1):50.
[8]曹秀明,高越,徐德林.虾青素保护活性氧所致线粒体损伤的作用[J].食品与药品,2010,12(11):412-414.
[9]Song X,Wang B,Lin S,et al.Astaxanthin inhibits apoptosis in alveolar epithelial cells type II in vivo and in vitro through the ROS-dependent mitochondrial signalling pathway[J].Journal of Cellular and Molecular Medicine,2014,18(11):15.
[10]Lee D H,Kim C S,Lee Y J.Astaxanthin protects against MPTP/MPP+-induced mitochondrial dysfunction and ROS production in vivo and in vitro[J].Food and Chemical Toxicology,2011,49(1):0-280.
[11]Saw C L L,Yang A Y,Guo Y,et al.Astaxanthin and omega-3 fatty acids individually and in combination protect against oxidative stress via the Nrf2-ARE pathway[J].Food&Chemical Toxicology,2013,62(12):869-875.
[12]Mano C M,Guaratini T,Cardozo K,et al.Astaxanthin restrains nitrative-oxidative peroxidation in mitochondrial-mimetic liposomes:A Pre-apoptosis model[J].Marine Drugs,2018,16(4):126.
[13]Zhang J,Xu P,Wang Y,et al.Astaxanthin prevents pulmonary fibrosis by promoting myofibroblast apoptosis dependent on Drp1-mediated mitochondrial fission[J].Journal of Cellular&Molecular Medicine,2015,19(9):2215-2231.
[14]Wolf A M,Asoh S,Hiranuma H,et al.Astaxanthin protects mitochondrial redox state and functional integrity against oxidative stress[J].Journal of Nutritional Biochemistry,2010,21(5):381.
[15]曹秀明,杨贵群,杨菲菲.虾青素对过氧化氢所致Hep G2细胞线粒体氧化损伤及生存能力下降的保护作用[J].中国海洋药物,2010(5):26-32.
[16]宋晓冬,刘文波,张丽霞,等.虾青素对ECV-304细胞的抗氧化作用及其作用机制[J].滨州医学院学报,2009,32(3):161-163.
[17]Liu X,Shibata T S.Astaxanthin inhibits reactive oxygen species-mediated cellular toxicity in dopaminergic SH-SY5Y cells via mitochondria-targeted protective mechanism.[J].Brain Research,2009,1254(Complete):18-27.
[18]Lee D H,Kim C S,Lee Y J.Astaxanthin protects against MPTP/MPP+-induced mitochondrial dysfunction and ROS production in vivo and in vitro.[J].Food&Chemical Toxicology,2011,49(1):271-80.
[19]詹明,董政,孙林.线粒体损伤与糖尿病肾病[J].中华肾脏病杂志,2011,27(6):460-463.
[20]Khoshjou F,Dadras F.Mitochondrion and its role in diabetic nephropathy[J].Iranian Journal of Kidney Diseases,2014,8(5):355.
[21]Zhang S,Sumei L I.Mitochondria-induced oxidative stress and diabetic nephropathy[J].International Journal of Pathology&Clinical Medicine,2011.
[22]曹延萍,王莎,刘杰,等.线粒体凋亡途径在高糖刺激的小鼠足细胞凋亡中的作用[J].中国应用生理学杂志,2013,29(3):244-246.
[23]董雅洁,高维娟.bcl-2、bax、caspase-3在细胞凋亡中的作用及其关系[J].中国老年学,2012,32(21):4828-4830.
[24]Uchiyama K,Naito Y,Hasegawa,等.虾青素对糖尿病引起的肾脏并发症的影响[C]∥中日国际维生素学术研讨会.2001:230.
[25]孔德贤.冬虫夏草含药血清对高糖培养大鼠肾小球系膜细胞PEDF及VEGF表达的影响[D].石家庄:河北医科大学,2009.
[26]林玉玲.C肽对高糖环境下大鼠肾小球系膜细胞增殖及凋亡影响的研究[D].郑州:郑州大学,2007.
[27]潘晓燕,龚小花,沈飞霞,等.高糖环境对HBZY-1大鼠肾小球系膜细胞IL-18、IL-1β、IL-6、TNF-α表达的影响[J].中国中西医结合肾病杂志,2012,13(6):519-521.
[28]杨丹丽,张迎秋,李媛,等.冬凌草甲素对甲状腺癌BCPAP细胞增殖的抑制作用[J].大连医科大学学报,2016,38(4):330-333.
[29]王碧,邵晓琳,赵雨.姜黄素和Dickkopf-1联用对口腔鳞状细胞癌细胞增殖和凋亡的影响研究[J].口腔医学研究,2017,33(8):877-880.
[30]MPA、MG132对不同PR表达卵巢癌细胞增殖、凋亡影响的实验研究[D].遵义:遵义医学院,2017.
[31]沈会,陶汉华,张诏,等.建中理劳汤含药血清对TGF-β1诱导的大鼠肾小球系膜细胞增殖及基质金属蛋白酶表达的影响[J].世界科学技术-中医药现代化,2015(9).
[32]Xie X,Chen Q,Tao J.Astaxanthin promotes Nrf2/ARE signaling to inhibit HG-induced renal fibrosis in GMCs[J].Marine Drugs,2018,16(4):117.
[33]Zhu X,Chen Y,Chen Q,et al.Astaxanthin promotes Nrf2/ARE signaling to alleviate renal fibronectin and collagen IV accumulation in diabetic rats[J].Journal of Diabetes Research,2018,2018:1-7.
[34]Scabini M,Stellari F,Cappella P,et al.In vivo imaging of early stage apoptosis by measuring real-time caspase-3/7 activation[J].Apoptosis,2011,16(2):198-207.
[35]高春林,赵亚萍,陈小慧,等.高糖对原代培养人脂肪细胞线粒体膜电位及胰岛素敏感性的影响[J].医学研究生学报,2010,23(8):805-808.
[36]鲍丽颖,姜希娟,郑纺,等.高糖高脂诱导脐静脉内皮细胞凋亡及对线粒体膜电位的影响[J].天津中医药,2010,27(6):501-502.
[37]李圣杰.低氧微环境对神经胶质瘤细胞线粒体功能的影响[D].昆明:昆明医科大学,2016.
[38]车晓芳,罗颖,刘云鹏.Bcl-2和Bax调节细胞凋亡的研究[J].国际输血及血液学杂志,2001,24(2):103-105
[39]Bagci E Z,Vodovotz Y,Billiar T R,et al.Bistability in apoptosis:roles of bax,bcl-2,and mitochondrial permeability transition pores[J].Biophysical Journal,2007,92(9):3332-3334.
[40]邓红,王新,徐芳,等.六味地黄汤对糖尿病肾病大鼠肾功能及细胞凋亡的影响[J].中草药,2010,41(10):1679-1682.
[41]罗文娟,喻嵘,唐元,等.左归降糖益肾方对2型糖尿病肾病小鼠足细胞凋亡的影响[J].中医杂志,2015,56(6):511-514.
[42]游娜,李慧敏,丁大法,等.肾上腺髓质素对高糖培养的肾小球系膜细胞增殖和凋亡的影响及其机制的研究[J].中国糖尿病杂志,2013,21(1):66-71.