研究论文 RESEARCH PAPER

根皮素对过氧化氢诱导的牛脂肪源性干细胞氧化应激的缓解作用

  • 蒋恩惠 ,
  • 刘媛 ,
  • 李洁 ,
  • 马思佳 ,
  • 李向臣 ,
  • 蓝贤勇
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  • 1. 西北农林科技大学动物科技学院, 杨凌 712100;
    2. 浙江农林大学动物科技学院·动物医学院, 杭州 311300;
    3. 浙江省畜禽绿色生态健康养殖应用技术研究重点实验室, 杭州 311300
蒋恩惠(1992-),男,吉林延边人,博士研究生,研究方向为动物遗传育种与繁殖。E-mail:jiangenhui@yeah.net

收稿日期: 2021-06-17

  网络出版日期: 2022-01-18

基金资助

国家自然科学基金项目(31872331,31672404);浙江农林大学科研发展基金(2018FR044);国家级大学生创新创业训练计划项目(201910241039)

Alleviation Effect of Phloretin on Oxidative Stress in Cattle Adipose Stem Cells Induced by Hydrogen Peroxide

  • JIANG Enhui ,
  • LIU Yuan ,
  • LI Jie ,
  • MA Sijia ,
  • LI Xiangchen ,
  • LAN Xianyong
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  • 1. College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China;
    2. College of Animal Science and Technology·College of Veterinary Medicine, Zhejiang A&F University, Hangzhou 311300, China;
    3. Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, Hangzhou 311300, China

Received date: 2021-06-17

  Online published: 2022-01-18

摘要

本研究旨在探究根皮素(PT)对过氧化氢(H2O2)诱导的牛脂肪源性干细胞(ADSCs)氧化应激和凋亡的影响。试验选取牛脂肪组织,使用Ⅳ型胶原酶分离牛ADSCs,传代培养后利用免疫荧光对牛ADSCs表面标记蛋白CD29、CD44、CD73及Vimentin进行鉴定。用不同浓度(0、100、200、500、1 000 μmol/L)的过氧化氢(H2O2)处理牛ADSCs 24 h,通过CCK-8法检测细胞存活率,筛选构建牛ADSCs氧化应激模型的适宜H2O2浓度。利用CCK-8法检测不同浓度(0、10、50、100 μmol/L)PT作用4 h后牛ADSCs的存活率,确定PT对牛ADSCs进行预保护的适宜浓度。随后先用不同浓度(0、10、50 μmol/L)PT预处理牛ADSCs 4 h,对牛ADSCs进行预保护;再利用500 μmol/L H2O2处理牛ADSCs 24 h。采用试剂盒检测牛ADSCs内氧化应激相关指标,包括丙二醛(MDA)含量及超氧化物歧化酶(SOD)活性;荧光探针2',7'-二氯二氢荧光素二乙酸酯(DCFH-DA)测定细胞内活性氧(ROS)含量;实时荧光定量PCR(qRT-PCR)检测细胞凋亡相关基因B细胞淋巴瘤/白血病-2相关X蛋白(Bax)、B细胞淋巴瘤/白血病-2(Bcl-2)及半胱天冬酶-3(Caspase-3)mRNA相对表达量。结果显示:500 μmol/L的H2O2能引起牛ADSCs存活率极显著下降(P<0.01),可以用来诱导其发生氧化应激。10和50 μmol/L的PT不会对牛ADSCs的存活率产生显著影响(P>0.05),而100 μmol/L的PT会引起牛ADSCs的存活率显著下降(P<0.05),因此后续试验选用10、50 μmol/L的PT处理牛ADSCs进行4 h的预保护。免疫荧光检测结果显示,牛ADSCs中CD29、CD44、CD73及Vimentin蛋白表达呈阳性;氧化应激相关指标检测结果显示,与空白组(0 μmol/L PT预保护,0 μmol/L H2O2处理)相比,500 μmol/L H2O2极显著增加牛ADSCs内MDA含量(P<0.01),极显著降低牛ADSCs内SOD活性(P<0.01),说明500 μmol/L H2O2处理牛ADSCs引起了氧化应激。与氧化应激组(0 μmol/L PT预保护,500 μmol/L H2O2处理)相比,10或50 μmol/L PT与500 μmol/L H2O2共处理可以显著减少牛ADSCs内MDA含量(P<0.05),50 μmol/L PT与500 μmol/L H2O2共处理可以显著增加牛ADSCs内SOD活性(P<0.05),说明适宜浓度的PT可以缓解H2O2诱导的牛ADSCs氧化应激。ROS染色结果显示,10、50 μmol/L PT与500 μmol/L H2O2共处理均可极显著降低牛ADSCs内ROS含量(P<0.01),同样说明适宜浓度的PT可以缓解H2O2诱导的牛ADSCs氧化应激。此外,qRT-PCR检测结果显示,与空白组相比,500 μmol/L H2O2显著增加牛ADSCs内促凋亡基因BaxCaspase-3 mRNA相对表达量(P<0.05),极显著减少牛ADSCs内抗凋亡基因Bcl-2 mRNA相对表达量(P<0.01),说明500 μmol/L H2O2处理可以引起牛ADSCs凋亡。而10或50 μmol/L PT与500 μmol/L H2O2共处理时,牛ADSCs内凋亡相关基因mRNA相对表达量与氧化应激组没有显著差异(P>0.05),说明在本试验的条件下,PT不会缓解H2O2诱导的牛ADSCs凋亡。综上所述,适量的PT可以有效缓解H2O2诱导的牛ADSCs氧化应激。

本文引用格式

蒋恩惠 , 刘媛 , 李洁 , 马思佳 , 李向臣 , 蓝贤勇 . 根皮素对过氧化氢诱导的牛脂肪源性干细胞氧化应激的缓解作用[J]. 动物营养学报, 2022 , 34(1) : 600 -611 . DOI: 10.3969/j.issn.1006-267x.2022.01.055

Abstract

The aim of this study was to investigate the effects of phloretin (PT) on oxidative stress and apoptosis of cattle adipose-derived stem cells (ADSCs) induced by hydrogen peroxide (H2O2). Bovine adipocytes were selected and collagenase was used to isolate cattle ADSCs. After subculture, the surface marker proteins CD29, CD44, CD73 and Vimentin of cattle ADSCs were analyzed by immunofluorescence technique. Different concentrations (0, 100, 200, 500 and 1 000 μmol/L) of H2O2 were set to treat cattle ADSCs for 24 h, the cell viability was determined by CCK-8 method to screen the optimal concentration of H2O2 to build oxidative stress in cattle ADSCs. Different concentrations (0, 10, 50 and 100 μmol/L) of PT were set to treat cattle ADSCs for 4 h. The cell viability was determined by CCK-8 method to determine the optimal concentration of PT to protect cattle ADSCs. Cattle ADSCs were pretreated with different concentrations (0, 10 and 50 μmol/L) of PT for 4 h, and then treated with 500 μmol/L H2O2 for 24 h. The oxidative stress related indices such as malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in cattle ADSCs were detected by corresponding kit. The intracellular reactive oxygen species (ROS) content was determined by fluorescence probe 2',7'-dichlorodi-hydrofluorescein diacetate (DCFH-DA). The mRNA relative expression levels of apoptosis related genes B-cell lymphoma/leukaemia-2-associated X protein (Bax), B-cell lymphoma/leukaemia-2 (Bcl-2) and cysteinyl aspartate specific proteinase-3 (Caspase-3) were detected by quantitative real-time PCR (qRT-PCR). The results showed as follows:500 μmol/L H2O2 extremely significantly decreased cattle ADSCs viability (P<0.01), which could induce oxidative stress in cattle ADSCs. 10 and 50 μmol/L PT had no significant difference in cattle ADSCs viability (P>0.05), but 100 μmol/L PT significantly decreased cattle ADSCs viability (P<0.05). Therefore, subsequent experiment was selected 10 and 50 μmol/L PT to treat cattle ADSCs for 4 h. Immunofluorescence detection results showed that the expression of CD29, CD44, CD73 and Vimentin were positive in cattle ADSCs. Oxidative stress related indices detection results showed that 500 μmol/L H2O2 extremely significantly increased MDA content (P<0.01), and extremely significantly decreased SOD content in cattle ADSCs compared with the blank group (0 μmol/L PT, 0 μmol/L H2O2) (P<0.01), this demonstrated that 500 μmol/L H2O2 treatment caused oxidative stress in cattle ADSCs. Compared with the oxidative stress group (0 μmol/L PT, 500 μmol/L H2O2), the co-treatment of 10 or 50 μmol/L PT and 500 μmol/L H2O2 could significantly reduce the MDA content in cattle ADSCs (P<0.05), and the co-treatment of 50 μmol/L PT and 500 μmol/L H2O2 could significantly increase the SOD activity in cattle ADSCs (P<0.05), this showed that optimal concentration of PT could relieve the oxidative stress in cattle ADSCs induced by H2O2. ROS staining results showed that the co-treatment of 10 or 50 μmol/L PT and 500 μmol/L H2O2 could extremely significantly reduce the ROS content in cattle ADSCs, that also showed that optimal concentration of PT could relieve the oxidative stress in cattle ADSCs induced by H2O2. In addition, qPCR detection results showed that compared with the blank group, 500 μmol/L H2O2 significantly increased the mRNA relative expression levels of pro-apoptotic gene Bax and Caspase-3, and extremely significantly decreased the mRNA relative expression level of anti-apoptotic gene Bcl-2 (P<0.05). this showed that 500 μmol/L H2O2 treatment could cause the apoptosis of cattle ADSCs. However, compared with 500 μmol/L H2O2 alone treatment, the co-treatment of 10 or 50 μmol/L PT and 500 μmol/L H2O2 had no significant difference in the mRNA relative expression levels of apoptosis-related genes in cattle ADSCs (P>0.05), which showed PT could not relieve the apoptosis of cattle ADSCs induced by H2O2 under this experiment condition. In conclusion, the optimal amount of PT can effectively alleviate the oxidative stress in cattle ADSCs induced by H2O2.

参考文献

[1] 孙睿,解影,方俊.农业畜禽养殖中植物多糖缓解氧化应激的研究进展[J].现代农业科技,2020(24):194-196. SUN R,XIE Y,FANG J.Advances of plant polysaccharides in alleviating oxidative stress in agricultural livestock and poultry breeding[J].Modern Agricultural Science and Technology,2020(24):194-196.(in Chinese)
[2] 陈宝江.植物提取物缓解动物氧化应激应用研究进展[J].饲料工业,2021,42(10):1-5. CHEN B J.Research progress of plant extracts in animal oxidative stress[J].Feed Industry,2020,42(10):1-5.(in Chinese)
[3] ZUK P A,ZHU M,MIZUNO H,et al.Multilineage cells from human adipose tissue:implications for cell-based therapies[J].Tissue Engineering Part A,2001,7(2):211-228.  
[4] ABDIK E A,ABDIK H,TAŞLI P N,et al.Suppressive role of boron on adipogenic differentiation and fat deposition in human mesenchymal stem cells[J].Biological Trace Element Research,2019,188(2):384-392.  
[5] HU L,TIAN K,ZHANG T,et al.Cyanate induces oxidative stress injury and abnormal lipid metabolism in liver through Nrf2/HO-1[J].Molecules,2019,24(18):3231.
[6] 杨琴,漆国栋,伍亚民,等.川芎嗪对过氧化氢诱导星形胶质细胞氧化应激损伤的保护作用[J].第三军医大学学报,2021,43(3):226-233. YANG Q,QI G D,WU Y M,et al.Tetramethylpyrazine protects rat astrocytes against H2O2-induced oxidative stress injury[J].Journal of Third Military Medical University,2021,43(3):226-233.(in Chinese)
[7] WARINHOMHOUN S,MUANGNOI C,BURANASUDJA V,et al.Antioxidant activities and protective effects of dendropachol,a new bisbibenzyl compound from Dendrobium pachyglossum,on hydrogen peroxide-induced oxidative stress in HaCaT keratinocytes[J].Antioxidants,2021,10(2):252.
[8] OROOJAN A A,CHENANI N,AN'AAM M.Antioxidant effects of eugenol on oxidative stress induced by hydrogen peroxide in islets of Langerhans isolated from male mouse[J].International Journal of Hepatology,2020,2020:5890378.
[9] ZHOU K,HU L Y,LI P M,et al.Genome-wide identification of glycosyltransferases converting phloretin to phloridzin in Malus species[J].Plant Science,2017,265:131-145.
[10] BIRRU R L,BEIN K,WELLS H,et al.Phloretin,an apple polyphenol,inhibits pathogen-induced mucin overproduction[J].Molecular Nutrition & Food Research,2021,65(2):e2000658.
[11] 周北斗,曾丽兰,胡栋宝.天然酚类化合物根皮素与根皮苷的抗氧化活性的DFT研究[J].分子科学学报,2018,34(6):517-520. ZHOU B D,ZENG L L,HU D B.Study on antioxidant activity of natural phenolic compounds about phloretin and phlorizin by DFT method[J].Journal of Molecular Science,2018,34(6):517-520.(in Chinese)
[12] 魏丽娜,赵静,罗仓学,等.根皮素的制备、结构修饰及生理活性研究进展[J].食品与发酵工业,2019,45(17):278-285. WEI L N,ZHAO J,LUO C X,et al.Preparation,structural modification and physiological activities of phloretin:a review[J].Food and Fermentation Industries,2019,45(17):278-285.(in Chinese)
[13] 冯甜,王力彬,周楠,等.根皮素的研究进展[J].转化医学杂志,2017,6(1):42-46. FENG T,WANG L B,ZHOU N,et al.Advance in studies on phloretin[J].Translational Medicine Journal,2017,6(1):42-46.(in Chinese)
[14] 李慧灵,周金林,卢宇靖,等.酶法催化柚皮苷制备根皮素及其抗氧化活性分析[J].现代食品科技,2020,36(12):69-76. LI H L,ZHOU J L,LU Y J,et al.Enzymatic preparation of phloretin from naringin and their antioxidant activity analysis[J].Modern Food Science & Technology,2020,36(12):69-76.(in Chinese)
[15] MARIADOSS A V A,VINAYAGAM R,SENTHILKUMAR V,et al.Phloretin loaded chitosan nanoparticles augments the pH-dependent mitochondrial-mediated intrinsic apoptosis in human oral cancer cells[J].International Journal of Biological Macromolecules,2019,130:997-1008.
[16] YANG Q,HAN L,LI J,et al.Activation of Nrf2 by phloretin attenuates palmitic acid-induced endothelial cell oxidative stress via AMPK-dependent signaling[J].Journal of Agricultural and Food Chemistry,2019,67(1):120-131.  
[17] CHOI B Y.Biochemical basis of anti-cancer-effects of phloretin-a natural dihydrochalcone[J].Molecules,2019,24(2):278.
[18] UN H,UGAN R A,GURBUZ M A,et al.Phloretin and phloridzin guard against cisplatin-induced nephrotoxicity in mice through inhibiting oxidative stress and inflammation[J].Life Sciences,2021,266:118869.
[19] LI J,YANG Q,HAN L,et al.C2C12 mouse myoblasts damage induced by oxidative stress is alleviated by the antioxidant capacity of the active substance phloretin[J].Frontiers in Cell and Developmental Biology,2020,8:541260.
[20] LU T F,XIONG H,WANG K F,et al.Isolation and characterization of adipose-derived mesenchymal stem cells (ADSCs) from cattle[J].Applied Biochemistry and Biotechnology,2014,174(2):719-728.  
[21] 王帅帅,张才,孟素丹,等.犊牛脂肪干细胞的分离、培养与鉴定[J].浙江农业学报,2019,31(2):235-241. WANG S S,ZHANG C,MENG S D,et al.Isolation,culture and identification of adipose-derived stem cells from calves[J].Acta Agriculturae Zhejiangensis,2019,31(2):235-241.(in Chinese)
[22] YUE Y L,ZHANG L C,ZHANG X,et al.De novo lipogenesis and desaturation of fatty acids during adipogenesis in bovine adipose-derived mesenchymal stem cells[J].In Vitro Cellular & Developmental Biology:Animal,2018,54(1):23-31.  
[23] 姚莉韵,林琦,陆阳.N-硬脂酰酪氨酸对H2O2诱导PC12细胞氧化应激损伤的影响[J].上海交通大学学报(医学版),2014,34(5):631-634,644. YAO L Y,LIN Q,LU Y.Effects of N-stearoyltyrosine on H2O2-induced oxidative-stress damage of PC12 cells[J].Journal of Shanghai Jiaotong University (Medical Science),2014,34(5):631-634,644.(in Chinese)
[24] MOHAMMADI S,BARZEGARI A,DEHNAD A,et al.Astaxanthin protects mesenchymal stem cells from oxidative stress by direct scavenging of free radicals and modulation of cell signaling[J].Chemico-Biological Interactions,2021,333:109324.
[25] 刘珂娣,段佳林,苏晶,等.紫铆花素对PC12细胞氧化应激损伤的保护作用及对线粒体功能的影响研究[J].中国药房,2020,31(24):2974-2981. LIU K D,DUAN J L,SU J,et al.Study on the protective effects of butein on oxidative stress injury of PC12 cell and its effects on mitochondrial function[J].China Pharmacy,2020,31(24):2974-2981.(in Chinese)
[26] 陈新瑶,张建龙,董星,等.猴头菇多糖对氧化应激的IPEC-J2细胞抗氧化能力及紧密连接蛋白ZO-1的影响[J].畜牧兽医学报,2017,48(9):1769-1776. CHEN X Y,ZHANG J L,DONG X,et al.Effects of Hericium erinaceus polysaccharide on antioxidant ability and ZO-1 expression in IPEC-J2 cells under oxidative stress[J].Acta Veterinaria et Zootechnica Sinica,2017,48(9):1769-1776.(in Chinese)
[27] LIU C H,LU D Y,YOU J R,et al.Efficacy of water fraction from Dioscorea cirrhosa on oxidative stress and apoptosis in H9c2 cardiomyocytes induced by H2O2[J].Journal of Traditional Chinese Medicine,2021,41(1):51-58.
[28] KIM M H,KIM D H,YANG S G,et al.Improved effect of a mitochondria-targeted antioxidant on hydrogen peroxide-induced oxidative stress in human retinal pigment epithelium cells[J].BMC Pharmacology & Toxicology,2021,22(1):7.
[29] CHEN X,ZHANG W J,SUN L R,et al.Tectorigenin protect HUVECs from H2O2-induced oxidative stress injury by regulating PI3K/Akt pathway[J].Tissue & Cell,2021,68:101475.
[30] PRAJAPATI P,GOHEL D,SHINDE A,et al.TRIM32 regulates mitochondrial mediated ROS levels and sensitizes the oxidative stress induced cell death[J].Cellular Signalling,2020,76:109777.
[31] MENG F H,CHENG J,SANG P,et al.Effects of bronchoalveolar lavage with ambroxol hydrochloride on treating pulmonary infection in patients with cerebral infarction and on serum proinflammatory cytokines,MDA and SOD[J].Computational and Mathematical Methods in Medicine,2020,2020:7984565.
[32] 姜丽莹,何剑斌,郑伟萍,等.莱菔硫烷对染镉小鼠睾丸间质细胞抗氧化功能影响的研究[J].中国畜牧兽医,2017,44(2):568-577. JIANG L Y,HE J B,ZHENG W P,et al.Research on the antioxident function of sulforaphane on the leydig cells of cadmium exposured mice[J].China Animal Husbandry & Veterinary Medicine,2017,44(2):568-577.(in Chinese)
[33] EL-MAHDY M A,ALZARIE Y A,HEMANN C,et al.The novel SOD mimetic GC4419 increases cancer cell killing with sensitization to ionizing radiation while protecting normal cells[J].Free Radical Biology and Medicine,2020,160:630-642.
[34] 付远妨,赵尉丹,杨潇,等.鸡血藤总黄酮对猪圆环病毒2型感染小鼠脾脏氧化应激的影响[J].中国畜牧兽医,2017,44(5):1526-1532. FU Y F,ZHAO W D,YANG X,et al.Effect of total flavonoids of Spatholobus suberectus Dunn on oxidative stress in mice spleen induced by PCV2[J].China Animal Husbandry & Veterinary Medicine,2017,44(5):1526-1532.(in Chinese)
[35] BEN-SHAHAR Y,ABASSI Z,POLLAK Y,et al.Cell death induction (extrinsic versus intrinsic apoptotic pathway) by intestinal ischemia-reperfusion injury in rats is time-depended[J].Pediatric Surgery International,2021,37(3):369-376.  
[36] ZHOU X,HUANG N O,CHEN W X,et al.HPLC phenolic profile and induction of apoptosis by Linum usitatissimum extract in LNCaP cells by caspase3 and Bax pathways[J].AMB Express,2020,10(1):203.
[37] KE W W,ZHAO X X,LU Z M.Foeniculum vulgare seed extract induces apoptosis in lung cancer cells partly through the down-regulation of Bcl-2[J].Biomedicine & Pharmacotherapy,2021,135:111213.
[38] SUN Y,WANG Y W,LU W B,et al.A novel surface-enhanced Raman scattering probe based on Au nanoboxes for dynamic monitoring of caspase-3 during cervical cancer cell apoptosis[J].Journal of Materials Chemistry B,2021,9(2):381-391.  
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