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.
谢金根, 刘荣华, 马嘉鑫, 樊学程, 雷婷, 陈林林, 肖雄, 高文军, 李文宏. 葛根与粉葛抗H9c2细胞OGD/R损伤的有效成分及作用机制初步研究[J]. 中国药学杂志, 2023, 58(3): 243-253.
XIE Jin-gen, LIU Rong-hua, MA Jia-xin, FAN Xue-cheng, LEI Ting, CHEN Lin-lin, XIAO Xiong, GAO Wen-jun, LI Wen-hong. Preliminary Study on Differential Components and Mechanism of Puerariae Lobatae Radix and Puerariae Thomsonii Radix against OGD/R Injury in H9c2 Cells. Chinese Pharmaceutical Journal, 2023, 58(3): 243-253.
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.
2023, Vol. 58 
