Molecular mechanism of Puerariae Lobatae Radix in treatment of hepatocellular carcinoma based on network pharmacology
(2.Jiangsu Cancer Hospital & Jiangsu Institute of Cancer Research & the Affiliated Cancer Hospital of Nanjing Medical University, Nanjing, Jiangsu, China 210009)
(3.Department of General Surgery, the Affiliated Sir Run Run Shaw Hospital of School of Medicine of Zhejiang University, Hangzhou, Zhejiang, China 310016)
【Abstract】To investigate the potential mechanism of Puerariae Lobatae Radix in the treatment of hepatocellular carcinoma (HCC) by network pharmacology and in vitro cell experiment. The main active components of Puerariae Lobatae Radix and their predicted targets were obtained from TCMSP, and the disease targets were obtained from GeneCards database. The disease and drug prediction targets were intersected to select the common potential therapeutic targets. The “compound-target-disease” network diagram was constructed in Cytoscape 3.7.1, and the common targets were input into the STRING database to build the PPI network. GO function and KEGG pathway enrichment analysis on effective targets were performed by R software. Autodock vina 1.1.2 was used for molecular docking. Finally, the core targets and pathways were preliminarily verified by in vitro experiments. The proliferation of human HCC cells was detected by CCK-8 and EdU enzyme staining, and the expressions of PTEN, PDK1, Akt and GSK3 were detected by Western blot. In this study, 10 components of Puerariae Lobatae Radix (9 components involved in HCC-related targets and signaling pathways), 149 HCC-related targets, and 156 signaling pathways were screened out. The results of network analysis indicated that Puerariae Lobatae Radix may play an anti-HCC effect on key targets, such as Akt, IL6, MAPK3, EGFR, and key pathways, such as PI3K-Akt. The results of molecular docking indicated that puerarin, genistein and daidzein had good binding abilities with the key targets such as AKT1, MAPK3, MAPK1 and CASP3, and puerarin had the lowest Vina score with AKT1 and MAPK3 and also similar to them. In vitro cell experiments confirmed that puerarin had a significantly inhibitory effect on the proliferation of human HCC cells. Western blot results showed that puerarin could increase the phosphorylation of PTEN in human HCC cells through the PTEN/Akt/GSK3β signaling pathway, and the phosphorylation level of its downstream Akt decreased. This series of studies confirm that puerarin can treat HCC by blocking PTEN/Akt/GSK3β cellular signaling pathway, so as to provide ideas for subsequent studies for the molecular mechanism of puerarin in the treatment of liver cancer.
【Keywords】 network pharmacology; Puerariae Lobatae Radix; puerarin; hepatocellular carcinoma; in vitro cell experiment;
 HE S, LIAO C X. 中药治疗肝癌机制的研究进展 [J]. Chinese Traditional Patent Medicine, 2017, 39 (1): 155 (in Chinese).
 XING Z H, MA Y C, LI X P, et al. Research progress of puerarin and its derivatives on anti-inflammatory and anti-gout activities [J]. China Journal of Chinese Materia Medica, 2017, 42 (19): 3703 (in Chinese).
 LI Y, LUO X X, YAN F D. Puerariae Lobatae Radix elevated expression levels of OB-R, IRS2, GLUT1 and GLUT2 to regulate glucose metabolism in insulin-resistance HepG2 cells [J]. China Journal of Chinese Materia Medica, 2017, 42 (10): 1939 (in Chinese).
 LI K, QIN R, SHAO J L, et al. Preventive effect and mechanism of puerarin on rat models of disuse osteoporosis [J]. China Journal of Chinese Materia Medica, 2019, 44 (3): 535 (in Chinese).
 ZHANG X D, DU W, ZHANG C, et al. Effect of puerarin on hypoxic pulmonary hypertension and accompanying pulmonary fibrosis [J]. China Journal of Chinese Materia Medica, 2018, 43 (12): 2618 (in Chinese).
 GUO H, WANG X L. Effects of puerarin on oxidative stress and hemorheology of patients with diabetes mellitus combined with carotid atherosclerosis [J]. World Chinese Medicine, 2018, 13 (10): 2526 (in Chinese).
 LI B T, ZHAI X Y, LI J, et al. Network pharmacology-based study on mechanisms of antipyretic action of Pueraria radix [J]. Acta Pharmaceutica Sinica, 2019, 54 (8): 1409 (in Chinese).
 DAI Z Y, DONG Y S, DING P J, et al. Effect of puerarin in reducing insulin resistance in HepG2 cells via PI3K/Akt/GSK-3β signal transduction pathway [J]. Chinese Journal of Experimental Traditional Medical Formulae, 2019, 25 (12): 77 (in Chinese).
 QU Y, ZHANG S X, ZHOU L, et al. Network pharmacological study of Schizonepetae Herba and Saposhnikoviae Radix in treatment of ulcerative colitis [J]. China Journal of Chinese Materia Medica, 2019, 44 (24): 5465 (in Chinese).
 JIANG Y P, CHEN Z Y, MO F, et al. Pharmacological mechanism of traditional sedative effect of alkaloids in Nelumbinis Plumula based on network pharmacology [J]. China Journal of Chinese Materia Medica, 2019, 44 (19): 4225 (in Chinese).
 ZHAO S, WU D X, CHEN Q, et al. Study on mechanism for treating ischemic stroke of Siegesbeckiae Herba based on network pharmacology [J]. China Journal of Chinese Materia Medica, 2019, 44 (13): 2727 (in Chinese).
 WANG Q, YUAN L L, ZHANG Y L, et al. Research on network pharmacology of Acori Tatarinowii Rhizoma combined with Curcumae Radix in treating epilepsy [J]. China Journal of Chinese Materia Medica, 2019, 44 (13): 2701 (in Chinese).
 HU Y L. Inhibitory effect of Puerarin on the growth of hepatoma cells and its mechanism [D]. Shijiazhuang: Hebei Medical University, 2018 (in Chinese).
 YANG J J, LIU S F, LI W L. Inhibitive effect of daidzein on growth of H22 tumor cells in mice [J]. Chinese Journal of Public Health, 2012, 28 (2): 197 (in Chinese).
 CHEN X. Study on the effect of Gegen Qinlian decoction and its main metabolites on P38MAPK signaling pathway in Huh7 cells and the differentially expressed proteins after Gegen Qinlian decoction [D]. Guangzhou: Guangdong Pharmaceutical University, 2015 (in Chinese).
 LIU B. Mechanism of genistein reversing drug resistance in Bel-7402/5Fu cells by inhibiting autophagy [J]. Chinese Journal of Current Advances in General Surgery, 2019, 22 (2): 90 (in Chinese).
 ZENG J Q, LIN Y, LIU T T, et al. Effects of daucosterol on proliferation and apoptosis of human hepatocellular carcinoma cell line HepG2 [J]. Shandong Medical Journal, 2018, 58 (23): 5 (in Chinese).
 LIU H R. Antitumor activities and mechanisms of scoparone [D]. Shenyang: China Medical University, 2005 (in Chinese).
 PAN Z Y, BAO Z S, WU Z M, et al. The myocardial protective effects of puerarin on STZ induced diabetic rats [J]. J Mol Cell Biol, 2009, 42 (2): 137.
 ZHAO M, DU Y Q, YUAN L, et al. Protective effect of puerarin on acute alcoholic liver injury [J]. Am J Chin Med, 2010, 38 (2): 241.
 LIU X, LI S, LI Y, et al. Puerarin inhibits proliferation and induces apoptosis by upregulation of miR-16 in bladder cancer cell line T24 [J]. Oncol Res, 2018, 26 (8): 1227.
 JIANG K, CHEN H, TANG K, et al. Puerarin inhibits bladder cancer cell proliferation through the mTOR/p70S6K signaling pathway [J]. Oncol Lett, 2018, 15 (1): 167.
 HU Y, LI X, LIN L, et al. Puerarin inhibits non-small cell lung cancer cell growth via the induction of apoptosis [J]. Oncol Rep, 2018, 39 (4): 1731.
 LIU X, ZHAO W, WANG W, et al. Puerarin suppresses LPS-induced breast cancer cell migration, invasion and adhesion by blockage NF-kappaB and Erk pathway [J]. Biomed Pharmaco-ther, 2017, 92: 429.
 ZHANG W G, YIN X C, LIU X F, et al. Puerarin induces hepatocellular carcinoma cell apoptosis modulated by MAPK signaling pathways in a dose-dependent manner [J]. Anticancer Res, 2017, 37 (8): 4425.
 KANG H, ZHANG J, WANG B, et al. Puerarin inhibits M2 polarization and metastasis of tumor-associated macrophages from NSCLC xenograft model via inactivating MEK/ERK 1/2 pathway [J]. Int J Oncol, 2017, 50 (2): 545.
 CHEN T, CHEN H, WANG Y, et al. In vitro and in vivo antitumour activities of puerarin 6″-O-xyloside on human lung carcinoma A549 cell line via the induction of the mitochondria-mediated apoptosis pathway [J]. Pharm Biol, 2016, 4 (9): 1793.
 HU Y, LIU K, BO S, et al. Inhibitory effect of puerarin on vascular smooth muscle cells proliferation induced by oxidised low-density lipoprotein via suppressing ERK 1/2 phosphorylation and PCNA expression [J]. Pharmazie, 2016, 71 (2): 89.
 TAO L, JI C G, ZHANG X X. MiR-202-5p/PTEN mediates doxorubicin-resistance of breast cancer cells via PI3K/Akt signaling pathway [J]. Cancer Biol Ther, 2019, 20 (7): 1.
 LI J J, YIN H K, GUAN D X, et al. Chemerin suppresses hepatocellular carcinoma metastasis through CMKLR1-PTEN-Akt axis [J]. Br J Cancer, 2018, 118 (10): 1337.
 WANG P, GUAN Q, ZHOU D, et al. miR-21 Inhibitors modulate biological functions of gastric cancer cells via PTEN/PI3K/mTOR pathway [J]. DNA Cell Biol, 2018, 37 (1): 38.
 CHENG D Y, WEN B Y, XIAO Y, et al. Circular RNA circ-ITCH inhibits bladder cancer progression by sponging miR-17/miR-224 and regulating p21, PTEN expression [J]. Mol Cancer, 2018, 17 (1): 19.
 JIANG L, QIAO Y, WANG Z, et al. Inhibition of microRNA-103 attenuates inflammation and endoplasmic reticulum stress in atherosclerosis through disrupting the PTEN-mediated MAPK signaling [J]. J Cell Physiol, 2020, 235 (1): 380.
 BERNS K, HORLINGS H M, HENNESSY B T, et al. A functional genetic approach identifies the PI3K pathway as a major determinant of trastuzumab resistance in breast cancer [J]. Can-cer Cell, 2007, 12 (4): 395.
 YANG J Y, WANG R, JIN T, et al. Material basis and molecular mechanism of Dachengqi Decoction in treatment of acute pancreatitis based on network pharmacology [J]. China Journal of Chinese Materia Medica, 2020, 45 (6): 1423 (in Chinese).
 LI L, CHEN J, WANG H Z, et al. Study on mechanism of Guilu Erxianjiao in treatment of post-traumatic stress disorder based on network pharmacology [J]. China Journal of Chinese Materia Medica, 2020, 45 (8): 1816 (in Chinese).