Mechanistic investigation of the ameliorative effect of liquiritin on hypoxia/reoxygenation‑induced cardiomyocyte injury based on network pharmacology and <em>in</em> <em>vitro</em> validation

Li,Haoying; Bu,Linlin; Sun,Xiaoqi; Chu,Xi; Xue,Yucong; Zhang,Muqing; Shi,Jing; Liu,Yanshuang; Guan,Shengjiang; Han,Xue; Wang,Hongfang

doi:10.3892/etm.2024.12405

Mechanistic investigation of the ameliorative effect of liquiritin on hypoxia/reoxygenation‑induced cardiomyocyte injury based on network pharmacology and in vitro validation

Authors:
- Haoying Li
- Linlin Bu
- Xiaoqi Sun
- Xi Chu
- Yucong Xue
- Muqing Zhang
- Jing Shi
- Yanshuang Liu
- Shengjiang Guan
- Xue Han
- Hongfang Wang
View Affiliations

Affiliations: School of Pharmacy, Hebei University of Chinese Medicine, Shijiazhuang, Hebei 050200, P.R. China, Department of Pharmacy, The Fourth Hospital of Hebei Medical University, Shijiazhuang, Hebei 050011, P.R. China, Affiliated Hospital, Hebei University of Chinese Medicine, Shijiazhuang, Hebei 050000, P.R. China, College of Integrative Medicine, Hebei University of Chinese Medicine, Shijiazhuang, Hebei 050200, P.R. China
Published online on: January 29, 2024 https://doi.org/10.3892/etm.2024.12405
Article Number: 117
Copyright: © Li et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Liquiritin (LIQ) is a flavonoid known for its cardioprotective properties, extracted from Glycyrrhiza uralensis Fisch. The purpose of the present study was to investigate the protective mechanism of LIQ against hypoxia/reoxygenation (H/R) injury through in vitro experiments, with the goal of enhancing its pharmacological effects. Initially, network pharmacology was employed to explore the targets and mechanisms of LIQ. Subsequently, an in vitro H/R model was established using H9c2 cells. Potential targets for LIQ and myocardial ischemia‑reperfusion injury (MIRI) were identified through online databases. The STRING, Cytoscape and DAVID databases were used to extract intersecting targets and mechanisms. In vitro experiments were conducted to validate these findings, assessing cardiac enzymes, oxidative stress indicators, mitochondrial fluorescence, apoptotic fluorescence, inflammation and related protein expression. The network pharmacological analysis revealed that the protective effects of LIQ on MIRI involve oxidative stress, inflammation and apoptosis. The results of in vitro experimental validation demonstrated that LIQ significantly reduced the activities of lactated dehydrogenase and creatine kinase isoenzyme‑MB (P<0.05 or 0.01), as well as the level of malondialdehyde (P<0.01). It also inhibited the production of reactive oxygen species (P<0.01), the release of inflammatory factors (P<0.05 or 0.01) and apoptosis (P<0.01). By contrast, the LIQ pre‑treatment group exhibited a significant increase in mitochondrial membrane potential level (P<0.05 or 0.01) and the activities of antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase (P<0.05 or 0.01). Furthermore, LIQ reduced the protein expressions of TNF‑α receptor type 1 (TNFR1) and MMP9, along with the level of NF‑κB phosphorylation (P<0.05 or 0.01). In conclusion, LIQ mitigated H/R‑induced cardiomyocyte injury through mechanisms that may involve antioxidants, anti‑apoptotic effects, protection against mitochondrial damage and suppression of inflammatory levels. These effects are achieved via inhibition of the TNFR1/NF‑κB/MMP9 pathway.

View Figures

View References

1	Pollard TJ: The acute myocardial infarction. Prim Care. 27:631–649. 2000.PubMed/NCBI View Article : Google Scholar
2	Eltzschig HK, Bonney SK and Eckle T: Attenuating myocardial ischemia by targeting A2B adenosine receptors. Trends Mol Med. 19:345–354. 2013.PubMed/NCBI View Article : Google Scholar
3	Yang H, Wang C, Zhang L, Lv J and Ni H: Rutin alleviates hypoxia/reoxygenation-induced injury in myocardial cells by up-regulating SIRT1 expression. Chem Biol Interact. 297:44–49. 2019.PubMed/NCBI View Article : Google Scholar
4	Zhang T, Zhang Y, Cui M, Jin L, Wang Y, Lv F, Liu Y, Zheng W, Shang H, Zhang J, et al: CaMKII is a RIP3 substrate mediating ischemia- and oxidative stress-induced myocardial necroptosis. Nat Med. 22:175–182. 2016.PubMed/NCBI View Article : Google Scholar
5	Murphy E and Steenbergen C: Mechanisms underlying acute protection from cardiac ischemia-reperfusion injury. Physiol Rev. 88:581–609. 2008.PubMed/NCBI View Article : Google Scholar
6	Du L, Shen T, Liu B, Zhang Y, Zhao C, Jia N, Wang Q and He Q: Shock Wave Therapy Promotes Cardiomyocyte Autophagy and Survival during Hypoxia. Cell Physiol Biochem. 42:673–684. 2017.PubMed/NCBI View Article : Google Scholar
7	Gao X, Zhang H, Zhuang W, Yuan G, Sun T, Jiang X, Zhou Z, Yuan H, Zhang Z and Dong H: PEDF and PEDF-derived peptide 44mer protect cardiomyocytes against hypoxia-induced apoptosis and necroptosis via anti-oxidative effect. Sci Rep. 4(5637)2014.PubMed/NCBI View Article : Google Scholar
8	Yao H, Xie Q, He Q, Zeng L, Long J, Gong Y, Li X, Li X, Liu W, Xu Z, et al: Pretreatment with panaxatriol saponin attenuates mitochondrial apoptosis and oxidative stress to facilitate treatment of myocardial ischemia-reperfusion injury via the regulation of Keap1/Nrf2 activity. Oxid Med Cell Longev. 2022(9626703)2022.PubMed/NCBI View Article : Google Scholar
9	Pastorino G, Cornara L, Soares S, Rodrigues F and Oliveira M: Liquorice (Glycyrrhiza glabra): A phytochemical and pharmacological review. Phytother Res. 32:2323–2339. 2018.PubMed/NCBI View Article : Google Scholar
10	Fan S, Gu K, Wu Y, Luo H, Wang Y, Zhang T, Wang X, Zhang Y and Li Y: Liquiritinapioside-A mineralocorticoid-like substance from liquorice. Food Chem. 289:419–425. 2019.PubMed/NCBI View Article : Google Scholar
11	Hou Y and Jiang JG: Origin and concept of medicine food homology and its application in modern functional foods. Food Funct. 4:1727–1741. 2013.PubMed/NCBI View Article : Google Scholar
12	Zhang Y, Zhang L, Zhang Y, Xu JJ, Sun LL and Li SZ: The protective role of liquiritin in high fructose-induced myocardial fibrosis via inhibiting NF-κB and MAPK signaling pathway. Biomed Pharmacother. 84:1337–1349. 2016.PubMed/NCBI View Article : Google Scholar
13	Mou SQ, Zhou ZY, Feng H, Zhang N, Lin Z, Aiyasiding X, Li WJ, Ding W, Liao HH, Bian ZY and Tang QZ: Liquiritin attenuates lipopolysaccharides-induced cardiomyocyte injury via an AMP-Activated protein kinase-dependent signaling pathway. Front Pharmacol. 12(648688)2021.PubMed/NCBI View Article : Google Scholar
14	Thu VT, Yen NTH and Ly NTH: Liquiritin from radix glycyrrhizae protects cardiac mitochondria from Hypoxia/Reoxygenation damage. J Anal Methods Chem. 2021(1857464)2021.PubMed/NCBI View Article : Google Scholar
15	Han X, Yang Y, Zhang M, Li L, Xue Y, Jia Q, Wang X and Guan S: Liquiritin Protects against cardiac fibrosis after myocardial infarction by inhibiting CCL5 expression and the NF-kappaB signaling pathway. Drug Des Devel Ther. 16:4111–4125. 2022.PubMed/NCBI View Article : Google Scholar
16	Boezio B, Audouze K, Ducrot P and Taboureau O: Network-based approaches in pharmacology. Mol Inform: 36, 2017 doi: 10.1002/minf.201700048.
17	Otasek D, Morris JH, Bouças J, Pico AR and Demchak B: Cytoscape Automation: Empowering workflow-based network analysis. Genome biology. 20(185)2019.PubMed/NCBI View Article : Google Scholar
18	Hu X, Jiao R, Li H, Wang X, Lyu H, Gao X, Xu F, Li Z, Hua H and Li D: Antiproliferative hydrogen sulfide releasing evodiamine derivatives and their apoptosis inducing properties. Eur J Med Chem. 151:376–388. 2018.PubMed/NCBI View Article : Google Scholar
19	Fang Z, Luo W and Luo Y: Protective effect of α-mangostin against CoCl₂-induced apoptosis by suppressing oxidative stress in H9c2 rat cardiomyoblasts. Mol Med Rep. 17:6697–6704. 2018.PubMed/NCBI View Article : Google Scholar
20	Munoz-Sanchez J and Chanez-Cardenas ME: The use of cobalt chloride as a chemical hypoxia model. J Appl Toxicol. 39:556–570. 2019.PubMed/NCBI View Article : Google Scholar
21	Amani M, Jeddi S, Ahmadiasl N, Usefzade N and Zaman J: Effect of HEMADO on level of CK-MB and LDH enzymes after ischemia/reperfusion injury in isolated rat heart. Bioimpacts. 3:101–104. 2013.PubMed/NCBI View Article : Google Scholar
22	Raedschelders K, Ansley DM and Chen DD: The cellular and molecular origin of reactive oxygen species generation during myocardial ischemia and reperfusion. Pharmacol Ther. 133:230–255. 2012.PubMed/NCBI View Article : Google Scholar
23	Farias JG, Molina VM, Carrasco RA, Zepeda AB, Figueroa E, Letelier P and Castillo RL: Antioxidant therapeutic strategies for cardiovascular conditions associated with oxidative stress. Nutrients. 9(996)2017.PubMed/NCBI View Article : Google Scholar
24	Rodrigo R: Prevention of postoperative atrial fibrillation: Novel and safe strategy based on the modulation of the antioxidant system. Front Physiol. 3(93)2012.PubMed/NCBI View Article : Google Scholar
25	Xiang M, Lu Y, Xin L, Gao J, Shang C, Jiang Z, Lin H, Fang X, Qu Y, Wang Y, et al: Role of oxidative stress in reperfusion following myocardial ischemia and its treatments. Oxid Med Cell Longev. 2021(6614009)2021.PubMed/NCBI View Article : Google Scholar
26	Marin W, Marin D, Ao X and Liu Y: Mitochondria as a therapeutic target for cardiac ischemia-reperfusion injury (Review). Int J Mol Med. 47:485–499. 2021.PubMed/NCBI View Article : Google Scholar
27	Carreira RS, Lee P and Gottlieb RA: Mitochondrial therapeutics for cardioprotection. Curr Pharm Des. 17:2017–2035. 2011.PubMed/NCBI View Article : Google Scholar
28	Su X, Zhou M, Li Y, An N, Yang F, Zhang G, Xu L, Chen H, Wu H and Xing Y: Mitochondrial damage in myocardial Ischemia/Reperfusion injury and application of natural plant products. Oxid Med Cell Longev. 2022(8726564)2022.PubMed/NCBI View Article : Google Scholar
29	Lesnefsky EJ, Chen Q, Tandler B and Hoppel CL: Mitochondrial dysfunction and myocardial Ischemia-Reperfusion: Implications for novel therapies. Annu Rev Pharmacol Toxicol. 57:535–565. 2017.PubMed/NCBI View Article : Google Scholar
30	Anzell AR, Maizy R, Przyklenk K and Sanderson TH: Mitochondrial quality control and disease: Insights into Ischemia-Reperfusion injury. Mol Neurobiol. 55:2547–2564. 2018.PubMed/NCBI View Article : Google Scholar
31	Chazotte B: Labeling mitochondria with rhodamine 123. Cold Spring Harb Protoc. 2011:892–894. 2011.PubMed/NCBI View Article : Google Scholar
32	Zhou H, Ma Q, Zhu P, Ren J, Reiter RJ and Chen Y: Protective role of melatonin in cardiac ischemia-reperfusion injury: From pathogenesis to targeted therapy. J Pineal Res: 64, 2018 doi: 10.1111/jpi.12471.
33	Yang J, Guo X, Yang J, Ding JW, Li S, Yang R, Fan ZX and Yang CJ: RP105 Protects against apoptosis in Ischemia/Reperfusion-Induced myocardial damage in rats by suppressing TLR4-Mediated signaling pathways. Cell Physiol Biochem. 36:2137–2148. 2015.PubMed/NCBI View Article : Google Scholar
34	Li X, Hu X, Wang J, Xu W, Yi C, Ma R and Jiang H: Short-Term hesperidin pretreatment attenuates rat myocardial Ischemia/Reperfusion injury by inhibiting high mobility group box 1 protein expression via the PI3K/Akt Pathway. Cell Physiol Biochem. 39:1850–1862. 2016.PubMed/NCBI View Article : Google Scholar
35	Zhang BF, Jiang H, Chen J, Guo X, Li Y, Hu Q and Yang S: Nobiletin ameliorates myocardial ischemia and reperfusion injury by attenuating endoplasmic reticulum stress-associated apoptosis through regulation of the PI3K/AKT signal pathway. Int Immunopharmacol. 73:98–107. 2019.PubMed/NCBI View Article : Google Scholar
36	Yang Y, Lv J, Jiang S, Ma Z, Wang D, Hu W, Deng C, Fan C, Di S, Sun Y and Yi W: The emerging role of Toll-like receptor 4 in myocardial inflammation. Cell Death Dis. 7(e2234)2016.PubMed/NCBI View Article : Google Scholar
37	Naidu BV, Farivar AS, Woolley SM, Grainger D, Verrier ED and Mulligan MS: Novel broad-spectrum chemokine inhibitor protects against lung ischemia-reperfusion injury. J Heart Lung Transplant. 23:128–134. 2004.PubMed/NCBI View Article : Google Scholar
38	Chen L, Liu P, Feng X and Ma C: Salidroside suppressing LPS-induced myocardial injury by inhibiting ROS-mediated PI3K/Akt/mTOR pathway in vitro and in vivo. J Cell Mol Med. 21:3178–3189. 2017.PubMed/NCBI View Article : Google Scholar
39	Ting AT and Bertrand MJM: More to Life than NF-κB in TNFR1 Signaling. Trends Immunol. 37:535–545. 2016.PubMed/NCBI View Article : Google Scholar
40	Pasparakis M and Vandenabeele P: Necroptosis and its role in inflammation. Nature. 517:311–320. 2015.PubMed/NCBI View Article : Google Scholar
41	Van der Heiden K, Cuhlmann S, Luong le A, Zakkar M and Evans PC: Role of nuclear factor kappaB in cardiovascular health and disease. Clin Sci (Lond). 118:593–605. 2010.PubMed/NCBI View Article : Google Scholar
42	Newby AC: Dual role of matrix metalloproteinases (matrixins) in intimal thickening and atherosclerotic plaque rupture. Physiol Rev. 85:1–31. 2005.PubMed/NCBI View Article : Google Scholar
43	Spinale FG, Coker ML, Heung LJ, Bond BR, Gunasinghe HR, Etoh T, Goldberg AT, Zellner JL and Crumbley AJ: A matrix metalloproteinase induction/activation system exists in the human left ventricular myocardium and is upregulated in heart failure. Circulation. 102:1944–1949. 2000.PubMed/NCBI View Article : Google Scholar
44	Spinale FG: Matrix metalloproteinase gene polymorphisms in heart failure: New pieces to the myocardial matrix puzzle. Eur Heart J. 25:631–633. 2004.PubMed/NCBI View Article : Google Scholar
45	Luo X, Wu J and Wu G: PPARγ activation suppresses the expression of MMP9 by downregulating NF-κB post intracerebral hemorrhage. Neurosci Lett. 752(135770)2021.PubMed/NCBI View Article : Google Scholar
46	Kleinbongard P, Heusch G and Schulz R: TNFalpha in atherosclerosis, myocardial ischemia/reperfusion and heart failure. Pharmacol Ther. 127:295–314. 2010.PubMed/NCBI View Article : Google Scholar
47	Hayden MS and Ghosh S: Regulation of NF-κB by TNF family cytokines. Semin Immunol. 26:253–266. 2014.PubMed/NCBI View Article : Google Scholar
48	Hayden MS and Ghosh S: NF-κB, the first quarter-century: Remarkable progress and outstanding questions. Genes Dev. 26:203–234. 2012.PubMed/NCBI View Article : Google Scholar
49	Blackwell K, Zhang L, Thomas GS, Sun S, Nakano H and Habelhah H: TRAF2 phosphorylation modulates tumor necrosis factor alpha-induced gene expression and cell resistance to apoptosis. Mol Cell Biol. 29:303–314. 2009.PubMed/NCBI View Article : Google Scholar
50	Devin A, Cook A, Lin Y, Rodriguez Y, Kelliher M and Liu Z: The distinct roles of TRAF2 and RIP in IKK activation by TNF-R1: TRAF2 recruits IKK to TNF-R1 while RIP mediates IKK activation. Immunity. 12:419–429. 2000.PubMed/NCBI View Article : Google Scholar
51	Wei Q, Tu Y, Zuo L, Zhao J, Chang Z, Zou Y and Qiu J: MiR-345-3p attenuates apoptosis and inflammation caused by oxidized low-density lipoprotein by targeting TRAF6 via TAK1/p38/NF-kB signaling in endothelial cells. Life Sci. 241(117142)2020.PubMed/NCBI View Article : Google Scholar
52	Howell JA and Bidwell GL III: Targeting the NF-κB pathway for therapy of ischemic stroke. Ther Deliv. 11:113–123. 2020.PubMed/NCBI View Article : Google Scholar
53	Gum R, Lengyel E, Juarez J, Chen JH, Sato H, Seiki M and Boyd D: Stimulation of 92-kDa gelatinase B promoter activity by ras is mitogen-activated protein kinase kinase 1-independent and requires multiple transcription factor binding sites including closely spaced PEA3/ets and AP-1 sequences. J Biol Chem. 271:10672–10680. 1996.PubMed/NCBI View Article : Google Scholar
54	Wu HT, Sie SS, Kuan TC and Lin CS: Identifying the regulative role of NF-κB binding sites within promoter region of human matrix metalloproteinase 9 (mmp-9) by TNF-α induction. Appl Biochem Biotechnol. 169:438–449. 2013.PubMed/NCBI View Article : Google Scholar
55	Neri M, Riezzo I, Pascale N, Pomara C and Turillazzi E: Ischemia/Reperfusion Injury following Acute Myocardial Infarction: A critical issue for clinicians and forensic pathologists. Mediators Inflamm. 2017(7018393)2017.PubMed/NCBI View Article : Google Scholar
56	Hescheler J, Meyer R, Plant S, Krautwurst D, Rosenthal W and Schultz G: Morphological, biochemical, and electrophysiological characterization of a clonal cell (H9c2) line from rat heart. Circ Res. 69:1476–1486. 1991.PubMed/NCBI View Article : Google Scholar
57	Zhu A, Wei X, Zhang Y, You T, Yao S, Yuan S, Xu H, Li F and Mao W: Propofol provides cardiac protection by suppressing the proteasome degradation of caveolin-3 in ischemic/reperfused rat hearts. J Cardiovasc Pharmacol. 69:170–177. 2017.PubMed/NCBI View Article : Google Scholar