目的 初步探讨葛根(Puerariae Lobatae Radix,PLR)与粉葛(Puerariae Thomsonii Radix,PTR)抗心肌细胞损伤的物质基础、作用及机制的异同;比较二者不同含药体系中有效成分相对含量及抗心肌(H9c2)细胞氧-糖剥夺/复氧(oxygen-glucose deprivation/reoxygenation,OGD/R)损伤效应的差异。方法 应用网络药理学方法找出PLR和PTR抗H9c2细胞OGD/R损伤的有效成分,结合超高效液相色谱-四极杆飞行时间串联质谱(UPLC-Q-TOF-MS)检测鉴定二者含药肠吸收液(intestinal absorption fluid,IAF)和含药血清(medicated serum,MS)中的主要有效成分及相对含量;建立H9c2细胞OGD/R损伤模型,PLR与PTR不同含药体系干预后,CCK-8检测细胞活力,生化试剂盒检测细胞内超氧化物歧化酶(superoxide dismutase,SOD)、乳酸脱氢酶(lactate dehydrogenase,LDH)活性,蛋白免疫印迹法(Western blot)检测细胞丝裂原活化蛋白激酶p38(MAPKp38)和核转录因子-κBp65(NFκBp65)蛋白表达。结果 研究发现PLR和PTR抗H9c2细胞OGD/R损伤的有效成分为葛根素、滨蒿内酯、大豆素、芒柄花素、染料木素,且MS中各有效成分相对含量均远低于IAF;与正常组比较,模型组SOD活性降低(P<0.01),LDH活性升高(P<0.01),MAPKp38和NFκBp65蛋白表达增加(P<0.05);与模型组比较,各给药组均可以升高SOD活性(P<0.01),降低LDH活性(P<0.01),下调MAPKp38和NFκBp65蛋白表达(P<0.05)。比较发现,PLR抗H9c2细胞损伤的作用优于PTR;IAF对各药效指标的影响优于MS。结论 PLR和PTR均可不同程度的保护OGD/R引起的H9c2细胞损伤,而PLR的作用较强,其作用机制可能是通过调控MAPKp38和NFκBp65蛋白表达发挥作用;IAF中有效成分相对含量较高,对各药效指标影响的较强而重现性较好,适合于中药体外药理学研究。
Abstract
OBJECTIVE To explore the similarities and differences of material basis, action, and mechanism of Puerariae Lobatae Radix(PLR) and Puerariae Thomsonii Radix(PTR) against myocardial cell injury, and compare the differences of relative amount of active ingredients and the effect against H9c2 cells OGD/R injury in different medicated systems. METHODS By means of network pharmacology, the active components of PLR and PTR against cardiomyocyte OGD/R injury were identified. The main active components and relative contents in medicated intestinal absorption fluid(IAF) and medicated serum(MS) of PLR and PTR were determined by UPLC-Q-TOF-MS. The OGD/R injury model in H9c2 cells was established and intervened with different drug-containing systems of PLR and PTR. The cell viability was detected by CCK-8,the intracellular superoxide dismutase(SOD) and lactate dehydrogenase(LDH) activities were detected by a biochemical kit, and the protein expressions of MAPKp38 and NFκBp65 in cells were detected by Western Blot. RESULTS The effective components of PLR and PTR against cardiomyocyte OGD/R injury were puerarin, scoparone, daidzein, formononetin, and genistein, and the relative content of each effective component in MS was much lower than that in IAF.Compared with the normal group, the SOD activity in the model group was decreased(P<0.01), the LDH activity was increased(P<0.01), and the protein expressions of MAPKp38 and NFκBp65 were increased(P<0.05). Compared with the model group, each administration group could increase SOD activity(P<0.01), reduce LDH activity(P<0.01), and down-regulate the expression of MAPKp38 and NFκBp65 proteins(P<0.05). The comparison showed that the anti-H9c2 cell injury effect of PLR was superior to that of PTR, and the effect of IAF on each pharmacodynamic index was better than that of MS. CONCLUSION PLR and PTR can protect H9c2 cells against injury induced by OGD/R in different degrees, while PLR playing a stronger role, which might be through the regulation of MAPKp38 and NFκBp65 protein expressions. The relative content of effective components in IAF is high, and it has a strong effect on each pharmacodynamic index with good repeatability. Therefore, IAF is suitable for pharmacology research in vitro of Chinese medicine.
关键词
葛根 /
粉葛 /
含药肠吸收液 /
氧-糖剥夺/复氧 /
心肌细胞
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Key words
Puerariae Lobatae Radix /
Puerariae Thomsonii Radix /
medicine intestinal absorption fluid /
oxygen-glucose deprivation/reoxy genation /
cardiac muscle cell
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参考文献
[1] ROTH G, MENSAH G, JOHNSON C, et al.Global burden of cardiovascular diseases and risk factors, 1990-2019: update from the GBD 2019 study[J]. J Am Coll Cardiol, 2020,76(25):2982-3021.
[2] GONG D F, FANG L H, DU G H. Research progress of natural drugs against ischemic heart disease targeting mitochondrial autophagy[J]. Acta Pharm Sin(药学学报), 2021,56(7): 1872-1879.
[3] KHUAN J T, PALEE S, KERdDPHOO S, et al. Donepezil attenuated cardiac ischemia/reperfusion injury through balancing mitochondrial dynamics, mitophagy, and autophagy[J]. Transl Res, 2021,230:82-97. Doi: 10.1016/j.trsl.2020.10.010.
[4] LI Q S. To explore the effect and mechanism of Yimai Granule on myocardial injury in rats with hyperlipidemia and Miri based on nrf2/ho-1 pathway[D]. Shenyang:Liaoning University of traditional Chinese medicine, 2019.
[5] QI J, YU J, WANG L, et al. Tongguan capsule protects against myocardial ischemia and reperfusion injury in mice[J]. Evid-Based Compl Alt, 2013,2013. Doi: 10.1155/2013/159237.
[6] VAHABZADEH G, SOLTANI H, BARATI M, et al. Noscapine protects the H9c2 cardiomyocytes of rats against oxygen-glucose deprivation/reperfusion injury[J]. Mol Biol Rep, 2020,47(8):5711-5719.
[7] DING S, WANG L, GUO L, et al. Syringic acid inhibits apoptosis pathways via downregulation of p38MAPK and JNK signaling pathways in H9c2 cardiomyocytes following hypoxia/reoxygenation injury[J]. Mol Med Rep,2017,16(2):2290-2294.
[8] KUZNETSOV A V, JAVADOV S, SICKINGER S, et al. H9c2 and HL-1 cells demonstrate distinct features of energy metabolism, mitochondrial function and sensitivity to hypoxia-reoxygenation[J]. BBA Mol Cell Res, 2015,1853(2):276-284.
[9] ZHU W F, LI J L, MENG X W, et al. Research progress on chemical constituents and pharmacological activities of Pueraria[J]. China J Chin Mater Med(中国中药杂志), 2021,46(6): 1311-1331.
[10] HUANG Z Q, WANG T T, MA S Y, et al. Study on quality evaluation of Pueraria lobata based on multi index component content and antioxidant activity[J]. Chin Tradit Herb Drugs(中草药), 2018,49(7): 1667-1676.
[11] YU H H, MENG X W, LI J R, et al. Comparative study on serum pharmacochemistry of Pueraria lobata and pueraria lobata in rats based on UPLC-Q-TOF-MS[J]. China J Chin Mater Med(中国中药杂志), 2022,47(2): 528-536.
[12] LI W H, XU G L, PENG C X, et al Preliminary study on the effect and mechanism of compound salvia miltiorrhiza tablets based on drug containing intestinal absorption solution on rat thoracic aortic rings in vitro[J]. Lishizhen Med Mater Med Res(时珍国医国药), 2014,25(3): 570-573.
[13] XU M, WANG Y, HIRAI K, et al. Calcium preconditioning inhibits mitochondrial permeability transition and apoptosis[J]. Am J Physiol Heart Circ Physiol, 2001,280(2):H899-H908.
[14] KOYAMA T, TEMMA K, AKERA T. Reperfusion-induced contracture develops with a decreasing[Ca2+]i in single heart cells[J]. Am J Physiol, 1991,261(Pt 2):H1115-H1122. Doi: 10.1152/ajpheart.1991.261.4.H1115.
[15] HUNDAHL L A, TFELT-HANSEN J, JESPERSEN T. Rat Models of Ventricular Fibrillation Following Acute Myocardial Infarction[J]. J Cardiovasc Pharmacol Ther, 2017,22(6):514-528.
[16] ZHENG K, ZHANG Q, LIN G, et al. Activation of Akt by SC79 protects myocardiocytes from oxygen and glucose deprivation(OGD)/re-oxygenation[J]. Oncotarget, 2017,8(9):14978-14987.
[17] WANG Z H. Study on the protective effect and mechanism of 25 hydroxy protopanaxatriol on experimental myocardial ischemia / reperfusion injury in rats[D] Changchun:Jilin University, 2015.
[18] SUGIYAMA A, SHIMIZU Y, OKADA M, et al. Preventive Effect of Canstatin against Ventricular Arrhythmia Induced by Ischemia/Reperfusion Injury: A Pilot Study[J]. Int J Mol Sci, 2021,22(3):1004. Doi: 10.3390/ijms22031004.
[19] JOHNSON DM, ANTOONS G. Arrhythmogenic mechanisms in heart failure: linking β-adrenergic stimulation, stretch, and calcium[J]. Front Physiol, 2018,9:1453. Doi:10.3389/fphys.2018.01453.
[20] ZENG L. Ion change characteristics of calcium overload in masseter muscle mitochondria of rats with occlusion interference and its correlation with calmodulin kinase Ⅱ[D].Jinan: Jinan University, 2018.
[21] ZHONG P. Study on the role and mechanism of CaMKII in obesity induced myocardial remodeling and arrhythmia[D] Wuhan:Wuhan University, 2018.
[22] LIU Z, TAO B, FAN S, et al. MicroRNA-145 protects against myocardial ischemia reperfusion injury via CaMKII-mediated antiapoptotic and anti-inflammatory pathways[J]. Oxid Med Cell Longev, 2019,2019. Doi: 10.1155/2019/8948657.
[23] SWAMINATHAN P D, PUROHIT A, HUND T J, et al. Calmodulin-dependent protein kinase II: linking heart failure and arrhythmias[J]. Circ Res, 2012,110(12):1661-1677.
[24] FAN H, DING D Q. Preliminary experimental study on the mechanism of Pueraria lobata extract against myocardial ischemia[J]. China Pharm(中国药业), 2013,22(19): 25-26.
[25] GUO B Q, XU J B, XIAO M, et al. Puerarin reduces ischemia/reperfusion-induced myocardial injury in diabetic rats via upregulation of vascular endothelial growth factor A/angiotensin-1 and suppression of apoptosis[J]. Mol Med Rep, 2018,17(5):7421-7427.
[26] WANG X, HUANG H, MA X, et al. Anti-inflammatory effects and mechanism of the total flavonoids from Artemisia scoparia Waldst. et Kit. in vitro and in vivo[J]. Biomed Pharmacother, 2018, 104:390-403.
[27] ZENG J, WEI G L, ZHONG X G, et al. Screening of in vitro antitumor activity of 3′-daidzein sulfonate sodium[J]. J Gannan Med Coll(赣南医学院学报), 2017,37(3): 351-353.
[28] ZENG J, HUANG Z H, YE H, et al. Protective effect of sodium 3′-daidzein sulfonate on myocardial ischemia-reperfusion injury in rats[J]. Chin Tradit Pat Med(中成药), 2009,31(3): 478-479.
[29] BAO C, LI T C. Protective effect of sodium formononetin sulfonate on myocardial ischemia-reperfusion injury in rats[J]. Guangdong Med J(广东医学), 2020,41(24): 2531-2534.
[30] LI H L, CHENG Q L, ZENG J. Study on the protective effect and mechanism of genistein on myocardial ischemia-reperfusion injury[J] Shandong Med J(山东医药), 2010,50(40): 38-39.
[31] HUANG J, ZHANG X, QIN F, et al. Protective effects of Millettia pulchra flavonoids on myocardial ischemia in vitro and in vivo[J]. Cell Physiol Biochem, 2015,35(2):516-528.
[32] ZHANG N, CHEN L Y, LUO Y, et al. Study on the anti hypoxia reoxygenation injury effect of Dalbergia odorifera new flavone latifolin through nrf2/ho-1 pathway[J]. J Chin Med Mater(中药材), 2019,42(7): 1629-1634.
[33] WANG D, CHEN T, LIU F. Betulinic acid alleviates myocardial hypoxia/reoxygenation injury via inducing Nrf2/HO-1 and inhibiting p38 and JNK pathways[J]. Eur J Pharmacol, 2018,838:53-59. Doi: 10.1016/j.ejphar.2018.08.024.
[34] LI W H, PAN L N, XU G L, et al.Study on relaxing vascular smooth muscle with compound angelica decoction containing intestinal absorption solution[J]. Chin J Exp Tradit Med Form(中国实验方剂学杂志), 2012,18(11): 205-208.
[35] LEI T, YU R Y, LI W H. Research methods and improvement of in vitro pharmacology of traditional Chinese medicine[J]. Chin Pharm J(中国药学杂志), 2022,57(8): 595-598.
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脚注
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基金
国家重点研发计划项目资助(2017YFC1702902)
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