蒲公英甾醇对AFB1所致鸡原代肝细胞氧化损伤的保护作用

doi:10.16431/j.cnki.1671-7236.2023.09.010

摘要/Abstract

摘要： 【目的】探讨蒲公英甾醇对黄曲霉毒素B₁(AFB₁)诱导的鸡原代肝细胞氧化损伤的保护作用及机制,为应用蒲公英甾醇防治AFB₁中毒提供理论依据。【方法】采用组织块酶消化法分离鸡原代肝细胞并进行PAS糖原染色鉴定,通过CCK-8法绘制肝细胞生长曲线;通过MTT法测定AFB₁对鸡肝细胞的毒性,结合测定肝细胞上清液中谷丙转氨酶(ALT)和谷草转氨酶(AST)水平,以确定AFB₁的体外建模浓度。通过MTT法确定蒲公英甾醇的安全给药浓度后,将试验分为6组:空白对照组、模型组、蒲公英甾醇剂量组(高、中、低剂量组)、阳性对照组。建立AFB₁诱导的鸡原代肝细胞损伤模型并给予药物,通过试剂盒测定细胞内活性氧(ROS)、丙二醛(MDA)、超氧化物歧化酶(SOD)以及还原型谷胱甘肽(GSH)水平。通过实时荧光定量PCR法测定Keap1/Nrf2信号通路关键基因血红素加氧酶-1(HO-1)、NADPH醌氧化还原酶1(NQO1)、Kelch样环氧氯丙烷相关蛋白1(Keap1)和核转录因子E2相关因子2(Nrf2)表达量。【结果】试验成功分离鉴定鸡原代肝细胞,在培养24~72 h细胞活力较高;确定0.05 μg/mL作为AFB₁体外诱导鸡原代肝细胞损伤的建模浓度;确定20、10、5 μg/mL作为蒲公英甾醇高、中、低剂量组的给药浓度;与模型组相比,蒲公英甾醇可极显著降低AFB₁所致鸡原代肝细胞氧化应激相关指标ROS和MDA含量(P<0.01),极显著提高抗氧化物SOD活性(P<0.01);极显著或显著提高AFB₁所致鸡原代肝细胞中HO-1、NQO1和Nrf2基因(除低剂量组外)表达量(P＜0.01;P＜0.05),降低Keap1基因表达量。【结论】蒲公英甾醇通过调控鸡原代肝细胞Keap1/Nrf2信号通路提高其抗氧化能力,从而对AFB₁诱导的鸡原代肝细胞氧化损伤起到保护作用。

关键词: 黄曲霉毒素B₁(AFB₁); 蒲公英甾醇; 鸡原代肝细胞; 氧化应激; 保护作用

Abstract: 【Objective】 The purpose of this study was to explore the protective effect and mechanism of taraxasterol on oxidative damage of chicken primary hepatocytes induced by aflatoxin B₁ (AFB₁), and provide theoretical basis for the prevention and treatment of AFB₁ poisoning with taraxasterol.【Method】 Chicken primary hepatocytes were isolated by tissue block enzyme digestion and identified by PAS glycogen staining.The growth curve of hepatocytes was drawn by CCK-8 method.The toxicity of AFB₁ on hepatocytes was determined by MTT method,and the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the supernatant of hepatocytes were measured to determine the modeling concentration of AFB₁ in vitro.MTT method was used to find out the safe concentration of taraxasterol.The test was divided into 6 groups: Normal group,AFB₁ group,taraxasterol groups (high,medium and low doses) and Sil group.After the establishment of the primary hepatocyte injury model induced by AFB₁ and drug administration,the contents of reactive oxygen species (ROS) and malondialdehyde (MDA),superoxide dismutase (SOD) and glutathione (GSH) in the cells were determined by reagent kits.The expression of heme oxygenase-1 (HO-1),NADPH quinone oxidoreductase 1 (NQO1),Kelch like ECH associated protein 1 (Keap1),and nuclear factor E2 related factor 2 (Nrf2) of the key genes in Keap1/Nrf2 signal pathway were determined by Real-time quantitative PCR.【Result】 The cells were successfully isolated and identified as chicken primary hepatocytes. The cell viability was higher after 24-72 h culture; 0.05 μg/mL concentration was used as the modeling concentration of AFB₁ which induced primary hepatocyte injury in vitro; 20, 10 and 5 μg/mL concentrations were used as taraxasterol administration concentrations of the high, medium and low dose groups. Compared with AFB₁ group, taraxasterol could extremely significantly reduce the contents of ROS and MDA (P＜0.01), extremely significantly increase the activity of antioxidant SOD in AFB₁-induced chicken primary hepatocytes (P＜0.01), and extremely significantly or significantly increase the expression of HO-1, NQO1 and Nrf2 (except for 5 μg/mL taraxasterol) genes (P＜0.01 or P＜0.05), and reduce the expression of Keap1 gene in chicken primary hepatocytes induced by AFB₁. 【Conclusion】 Taraxasterol could improve the antioxidant capacity of chicken primary hepatocytes by regulating the Keap1/Nrf2 signal pathway,thus protecting against the oxidative damage of chicken primary hepatocytes induced by AFB₁.

Key words: aflatoxin B₁ (AFB₁); taraxasterol; chicken primary hepatocytes; oxidative stress; protective effect

中图分类号:

S852.4⁺4

鲁萍, 王萌, 桑锐, 王巍, 张雪梅. 蒲公英甾醇对AFB₁所致鸡原代肝细胞氧化损伤的保护作用[J]. 中国畜牧兽医, 2023, 50(9): 3541-3549.

LU Ping, WANG Meng, SANG Rui, WANG Wei, ZHANG Xuemei. Protective Effect of Taraxasterol on Oxidative Damage of Chicken Primary Hepatocytes Induced by AFB₁[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(9): 3541-3549.

参考文献

[1] ERDEM V Z,BASEGMEZ H O,PESINT G B.AFB₁ recognition from liver tissue via AFB₁ imprinted magnetic nanoparticles[J].Journal of Chromatography.B,Analytical Technologies in the Biomedical and Life Sciences,2022,1210:123453.
[2] LIU X Q,MISHRA S K,WANG T,et al.AFB₁ induced transcriptional regulation related to apoptosis and lipid metabolism in liver of chicken[J]. Toxins(Basel),2020,12(5):290.
[3] NABI F,TAO W L,YE R L,et al.Penthorum Chinense pursh extract alleviates aflatoxin B₁-induced liver injury and oxidative stress through mitochondrial pathways in broilers[J].Frontiers in Veterinary Science,2022,9:822259.
[4] LI L,WANG H Y.Heterogeneity of liver cancer and personalized therapy[J]. Cancer Letters,2016,379(2):191-197.
[5] SANG R,GE B J,LI H F,et al.Taraxasterol alleviates aflatoxin B₁-induced liver damage in broiler chickens via regulation of oxidative stress,apoptosis and autophagy[J]. Ecotoxicology and Environmental Safety,2023,251:114546.
[6] BELLEZZA I,GIAMBANCO I,MINELLI A,et al.Nrf2-Keap1 signaling in oxidative and reductive stress[J].Biochimica et Biophysica Acta,2018,1865(5):721-723.
[7] TRAN H T T,GIGL M,LE N P K,et al.In vitro effect of Taraxacum officinale leaf aqueous extract on the interaction between ACE2 cell surface receptor and SARS-CoV-2 spike protein D614 and four mutants[J].Pharmaceuticals (Basel,Switzerland),2021,14(10):1055.
[8] LIU J T,XIONG H Z,CHENG Y,et al.Effects of taraxasterol on ovalbumin-induced allergic asthma in mice[J].Journal of Ethnopharmacology,2013,148(3):787-793.
[9] ZHANG X M,XIONG H Z,LI H Y,et al.Protective effect of taraxasterol against LPS-induced endotoxic shock by modulating inflammatory responses in mice[J].Immunopharmacology & Immunotoxicology,2014,36(1):11-16.
[10] WANG S S,WANG Y,LIU X Y,et al.Anti-inflammatory and anti-arthritic effects of taraxasterol on adjuvant-induced arthritis in rats[J].Journal of Ethnopharmacology,2016,187:42-48.
[11] ZHANG X M,XIONG H Z,LIU L B.Effects of taraxasterol on inflammatory responses in lipopolysaccharide-induced RAW264.7 macrophages[J].Journal of Ethnopharmacology,2012,141(1):206-211.
[12] XIONG H Z,CHENG Y,ZHANG X,et al.Effects of taraxasterol on iNOS and COX-2 expression in LPS-induced RAW264.7 macrophages[J]. Journal of Ethnopharmacology,2014,155(1):753-757.
[13] SANG R,YU Y F,GE B J,et al.Taraxasterol from Taraxacum prevents concanavalin A-induced acute hepatic injury in mice via modulating TLRs/NF-κB and Bax/Bc1-2 signalling pathways[J].Artificial Cells,Nanomedicine,and Biotechnology,2019,47(1):3929-3937.
[14] 于逸凡.蒲公英甾醇对APAP诱导小鼠肝损伤的保护作用及机制研究[D].延吉:延边大学,2020. YU Y F.Study on protective effects and mechanisms of taraxasterol on liver injury induced by APAP in mice[D].Yanji:Yanbian University,2020.(in Chinese)
[15] ISHFAQ MUHAMMAD.AA肉鸡肝脏生物活化AFB₁的关键Ⅰ相代谢酶及姜黄素的干预机理[D].哈尔滨:东北农业大学,2018. MUHAMMAD I.Role of key phase-Ⅰ metabolizing enzymes in AFB₁ biotransformation and curcumin interventions in liver of Arbor Acres broiler[D].Harbin:Northeast Agricultural University,2018.(in Chinese)
[16] 郝蓓莉.二氢杨梅素对内毒素诱导鸡肝损伤的保护作用研究[D].哈尔滨:东北农业大学,2020. HAO B L.Protective effects of dihydromyricetin on LPS-induced liver injury in chickens[D].Harbin:Northeast Agricultural University,2020.(in Chinese)
[17] 周静静,郭晓艳,杜瑞雪,等.不同日龄鸡的原代肝细胞分离与鉴定[J].中国兽医科学,2022,52(10):1328-1333. ZHOU J J,GUO X Y,DU R X,et al.Isolation and identification of primary hepatocytes from chickens of different ages[J]. Chinese Veterinary Science,2022,52(10):1328-1333.(in Chinese)
[18] 邓燕.血清转氨酶及血脂检测诊断脂肪肝的意义[J].中国继续医学教育,2021,13(1):100-102. DENG Y.Significance of serum transaminase and lipid determination in diagnosis of fatty liver[J].China Continuing Medical Education,2021,13(1):100-102.(in Chinese)
[19] TAO W L,LI Z Z,NABI F,et al.Penthorum chinense Pursh compound ameliorates AFB₁-induced oxidative stress and apoptosis via modulation of mitochondrial pathways in broiler chicken kidneys[J]. Frontiers in Veterinary Science,2021,8:750937.
[20] AYALA A,MUNOZ M F,ARGUELLES S.Lipid peroxidation:Production,metabolism,and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal[J].Oxidative Medicine and Cellular Longevity,2014,2014:360438.
[21] CHENG Z,NING W,YU X,et al.Molecular mechanisms involved in oxidative stress-associated liver injury induced by Chinese herbal medicine:An experimental evidence-based literature review and network pharmacology study[J].International Journal of Molecular Sciences,2018,19(9):2745.
[22] 袁超,游莎,李鲲,等.红芪醇提物对脓毒症所致大鼠肝损伤的影响[J].广州中医药大学学报,2021,38(7):1447-1453. YUAN C,YOU S,LI K,et al.Effects of Radix hedysari alcohol extract on liver damage in sepsis rats[J].Journal of Guangzhou University of Traditional Chinese Medicine,2021,38(7):1447-1453.(in Chinese)
[23] TONELLI C,CHIO I I C,TUVESON D A.Transcriptional regulation by Nrf2[J].Antioxid Redox Signal,2018,29(17):1727-1745.
[24] JADEJA R N,DEVKAR R V,NAMMI S.Oxidative stress in liver diseases:Pathogenesis,prevention,and therapeutics[J]. Oxidative Medicine and Cellular Longevity,2017,2017:8341286.
[25] SAJADIMAJD S,KHAZAEI M.Oxidative stress and cancer:The role of Nrf2[J].Current Cancer Drug Targets,2018,18(6):538-557.
[26] TANG W,JIANG Y F,PONNUSAMY M,et al.Role of Nrf2 in chronic liver disease[J]. World Journal of Gastroenterology,2014,20(36):13079-13087.
[27] TOROK N J.Dysregulation of redox pathways in liver fibrosis[J].American Journal of Physiology-Gastrointestinal and Liver Physiology,2016,311(4):G667-G674.
[28] YANG F,LIAO J,PEI R,et al.Autophagy attenuates copper-induced mitochondrial dysfunction by regulating oxidative stress in chicken hepatocytes[J]. Chemosphere,2018,204:36-43.
[29] WANG Y,YANG C,EISHEIKH N A H,et al.HO-1 reduces heat stress-induced apoptosis in bovine granulosa cells by suppressing oxidative stress[J].Aging (Albany NY),2019,11(15):5535-5547.
[30] SIEGEL D,GUSTAFSON D L,DEHN D L,et al.NAD(P)H:Quinone oxidoreductase 1:Role as a superoxide scavenger[J]. Molecular Pharmacology,2004,65:1238-1247.
[31] 赵燕婷,舒祥兵,杨志新,等.当飞利肝宁胶囊调控肝脏氧化应激改善非酒精性脂肪性肝病小鼠的机制研究[J].上海中医药大学学报,2021,35(5):37-43. ZHAO Y T,SHU X B,YANG Z X,et al.Mechanism of Dangfei Liganning capsule on non-alcoholic fatty liver disease via inhibiting liver oxidative stress in mice[J].Academic Journal of Shanghai University of Traditional Chinese Medicine,2021,35(5):37-43.(in Chinese)
[32] RAJPUT S A,SUN L H,ZHANG N Y,et al.Grape seed proanthocyanidin extract alleviates aflatoxin B₁-induced immunotoxicity and oxidative stress via modulation of NF-κB and Nrf2 signaling pathways in broilers[J].Toxins(Basel),2020,11(1):23.
[33] LI S H,MUHAMMAD I,YU H X,et al.Detection of aflatoxin adducts as potential markers and the role of curcumin in alleviating AFB₁-induced liver damage in chickens[J].Ecotoxicology and Environmental Safety,2019,176:137-145.

蒲公英甾醇对AFB₁所致鸡原代肝细胞氧化损伤的保护作用

Protective Effect of Taraxasterol on Oxidative Damage of Chicken Primary Hepatocytes Induced by AFB₁

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