肌肽是一种新型的食品补充剂,具有多种生物活性功能,已广泛应用于保健品、功能饮料、美容等领域,但肌肽对肝损伤修复的研究与应用鲜有报道。该文旨在研究肌肽对肝损伤的修复作用并探究其作用机制。肌肽预处理正常人肝细胞(normal human hepatocyte, L-02)12 h,400 mmol/mL叔丁基过氧化氢(tert butyl hydrogen peroxide, TBHP)处理L-02细胞4 h,细胞计数试剂盒-8检测细胞存活率,试剂盒检测谷丙转氨酶(alanine transaminase, ALT)、谷草转氨酶(aspartate transaminase, AST)、活性氧(reactive oxygen species, ROS)、谷胱甘肽(glutathione, GSH)水平,流氏细胞术检测细胞凋亡,蛋白印迹定量分析氧化信号通路与凋亡信号通路的相关蛋白表达。结果表明,肌肽预处理显著降低了L-02细胞外液ALT、AST活性,抑制ROS形成,提高细胞内GSH含量,显著抑制L-02细胞凋亡,上调核红细胞2相关因子2(nuclear factor-erythroid 2-related factor-2, Nrf2)、醌氧化还原酶1(quinone oxidoreductase 1, NQO1)、血红素氧合酶1(heme oxygenase-1, HO-1)、B细胞淋巴瘤-2(B-cell lymphoma 2, Bcl-2)的蛋白表达,下调Kelch样ECH关联蛋白1(Kelch like ECH associated protein 1, Keap1)、磷酸化氨基末端激酶(amino-terminal kinases phosphorylation, p-JNK)、胱天蛋白酶3(caspase-3,Cas-3)的蛋白表达。肌肽通过调控Keap1-Nrf2与JNK-Cas-3信号通路有效修复TBHP诱导的肝细胞损伤。
Carnosine is a new type of food supplement with various bioactive functions, which has been widely used in fields such as health products, functional beverages, cosmetology, etc.However, there are few studies on the research and application of carnosine in the repair of liver injury.This study aimed to verify the repair effect of carnosine on liver injury and explore its mechanism.L-02 cells were pretreated with carnosine for 12 hours and then were treated with 400 mmol/ml tert butyl hydrogen peroxide (TBHP) for 4 hours.The cell viability was assessed using the cell count kit-8 (CCK-8), the levels of alanine transaminase (ALT), aspartate transaminase (AST), reactive oxygen species (ROS), and glutathione (GSH) were detected using their respective activity assay kits, the cell apoptosis was measured by flow cytometry, the expression of Keap1-Nrf2 and JNK-Cas-3 signaling pathway-related proteins were quantified by western blot.Results showed that after carnosine pretreatment, the activities of ALT and AST were significantly reduced in the L-02 cell culture medium, the formation of ROS and cell apoptosis were significantly inhibited, and the contents of GSH were significantly increased.The protein expression of nuclear factor-erythroid 2-related factor-2(Nrf2), quinone oxidoreductase 1(NQO1), heme oxygenase-1(HO-1), and B-cell lymphoma 2(Bcl-2) were upregulated by anserine, with the downregulation of Kelch like ECH associated protein 1(Keap1), amino-terminal kinases phosphorylation(p-JNK), and caspase-3(Cas-3).Carnosine effectively alleviated TBHP-induced hepatocyte injury by regulating the Keap1-Nrf2 and JNK-Cas-3 signaling pathways.
[1] 王春月, 李艳荣, 潘晨, 等.舟山海域常见经济物种组氨酸二肽的提取和鉴定[J].浙江海洋大学学报(自然科学版), 2020, 39(3):200-208.
WANG C Y, LI Y R, PAN C, et al.Extraction and quantification of histidine-containing dipeptides from economic species in Zhoushan[J].Journal of Zhejiang Ocean University (Natural Science), 2020, 39(3):200-208.
[2] BOLDYREV A A, ALDINI G, DERAVE W.Physiology and pathophysiology of carnosine[J].Physiological Reviews, 2013, 93(4):1803-1845.
[3] 钱雯, 骆丹, 周炳荣.肌肽抗衰老机制的研究进展[J].实用皮肤病学杂志, 2018, 11(6):360-363.
QIAN W, LUO D, ZHOU B R.Research progress of the anti-ageing mechanism of carnosine[J].Journal of Practical Dermatology, 2018, 11(6):360-363.
[4] SOLANA-MANRIQUE C, SANZ F J, MARTÍNEZ-CARRIÓN G, et al.Antioxidant and neuroprotective effects of carnosine:Therapeutic implications in neurodegenerative diseases[J].Antioxidants, 2022, 11(5):848.
[5] ALDINI G, DE COURTEN B, REGAZZONI L, et al.Understanding the antioxidant and carbonyl sequestering activity of carnosine:Direct and indirect mechanisms[J].Free Radical Research, 2021, 55(4):321-330.
[6] HORVATITS T, DROLZ A, TRAUNER M, et al.Liver injury and failure in critical illness[J].Hepatology, 2019, 70(6):2204-2215.
[7] BARDALLO R G, PANISELLO-ROSELLÓ A, SANCHEZ-NUNO S, et al.Nrf2 and oxidative stress in liver ischemia/reperfusion injury[J].The FEBS Journal, 2022, 289(18):5463-5479.
[8] SIES H, BERNDT C, JONES D P.Oxidative stress[J].Annual Review of Biochemistry, 2017, 86:715-748.
[9] CUADRADO A, ROJO A I, WELLS G, et al.Therapeutic targeting of the NRF2 and KEAP1 partnership in chronic diseases[J].Nature Reviews.Drug Discovery, 2019, 18(4):295-317.
[10] EL-KASHEF D H, SEWILAM H M.Empagliflozin mitigates methotrexate-induced hepatotoxicity:Targeting ASK-1/JNK/Caspase-3 pathway[J].International Immunopharmacology, 2023, 114:109494.
[11] WEDEL S, MARTIC I, HRAPOVIC N, et al.tBHP treatment as a model for cellular senescence and pollution-induced skin aging[J].Mechanisms of Ageing and Development, 2020, 190:111318.
[12] 洪玥, 陈友霞, 刘珍珍, 等.咖啡酸对CCl4诱导BRL大鼠肝细胞损伤的保护作用[J].食品工业科技, 2021, 42(23):356-361.
HONG Y, CHEN Y X, LIU Z Z, et al.Protective effect of caffeic acid on CCl4-induced injury of BRL hepatocyte[J].Science and Technology of Food Industry, 2021, 42(23):356-361.
[13] 张淑霞, 张雪涟, 卢珊, 等.柚皮苷对酒精诱导急性肝损伤的保护作用[J].中国实验方剂学杂志, 2023, 9(1):61-66.
ZHANG S X, ZHANG X L, LU S, et al.Protective effect of naringin on alcohol-induced acute liver injury[J].Chinese Journal of Experimental Traditional Medical Formulae, 2023, 9(1):61-66.
[14] SIES H, BELOUSOV V V, CHANDEL N S, et al.Defining roles of specific reactive oxygen species (ROS) in cell biology and physiology[J].Nature Reviews.Molecular Cell Biology, 2022, 23(7):499-515.
[15] 郑碧丹, 王晓婉, 王思玉, 等.雷公藤内酯三醇通过Nrf2/Keap1信号通路抑制氧化应激和炎症减轻雷公藤甲素诱导的肝损伤[J].中药新药与临床药理, 2023, 34(4):509-519.
ZHENG B D, WANG X W, WANG S Y, et al.Triptriolide alleviates triptolide-induced liver injury through reducing oxidative stress and inflammation via Nrf2/Keap1 signaling pathway[J].Traditional Chinese Drug Research and Clinical Pharmacology, 2023, 34(4):509-519.
[16] 吕莹, 郝尧坤, 张照兰.槲皮苷通过Nrf2/HO-1通路抑制H2O2诱导人肝细胞L-02凋亡和损伤[J].中南药学, 2021, 19(1):44-50.
LYU Y, HAO Y K, ZHANG Z L.Quercetin inhibits H2O2-induced apoptosis and injury of human hepatocytes L-02 via the Nrf2/HO-1 pathway[J].Central South Pharmacy, 2021, 19(1):44-50.
[17] LIU T, SUN L, ZHANG Y B, et al.Imbalanced GSH/ROS and sequential cell death[J].Journal of Biochemical and Molecular Toxicology, 2022, 36(1):e22942.
[18] WANG X, ZHOU T J, YANG X, et al.DDRGK1 enhances osteosarcoma chemoresistance via inhibiting KEAP1-mediated NRF2 ubiquitination[J].Advanced Science, 2023, 10(14):e2204438.
[19] XU H Y, FENG X H, ZHAO P F, et al.Procyanidin A2 penetrates L-02 cells and protects against tert-butyl hydroperoxide-induced oxidative stress by activating Nrf2 through JNK and p38 phosphorylation[J].Journal of Functional Foods, 2019, 62:103562.
[20] NI B R, LIU Y, GAO X, et al.Isoliquiritigenin attenuates emodin-induced hepatotoxicity in vivo and in vitro through Nrf2 pathway[J].Comparative Biochemistry and Physiology.Toxicology & Pharmacology:CBP, 2022, 261:109430.
[21] WANG Y, JIN R, CHEN J B, et al.Tangeretin maintains antioxidant activity by reducing CUL3 mediated NRF2 ubiquitination[J].Food Chemistry, 2021, 365:130470.
[22] NAGATA S.Apoptosis and clearance of apoptotic cells[J].Annual Review of Immunology, 2018, 36:489-517.
[23] LAI M C, LIU W Y, LIOU S S, et al.P-hydroxybenzyl alcohol antagonized the ROS-dependent JNK/Jun/caspase-3 pathway to produce neuroprotection in a cellular model of Parkinson’s disease[J].Nutrients, 2022, 14(23):5002.
[24] YANG C F, ZHONG Y J, MA Z H, et al.NOX4/ROS mediate ethanol-induced apoptosis via MAPK signal pathway in L-02 cells[J].International Journal of Molecular Medicine, 2018, 41(4):2306-2316.
[25] SHI C, CHEN X R, LIU Z J, et al.Oleuropein protects L-02 cells against H2O2-induced oxidative stress by increasing SOD1, GPx1 and CAT expression[J].Biomedicine & Pharmacotherapy=Biomedecine & Pharmacotherapie, 2017, 85:740-748.
