Abstract
To investigate the protective effects of the combined concentrated liquid extract of Pueraria lobata (Willd.) Ohwi root (P. lobata, Ge Gen) and Hovenia dulcis Thunb. (H. dulcis, Zhi Ju Zi) against ethanol-induced liver damage in vitro, using a human hepatoma cell line G2 (HepG2) cell model.
HepG2 cells were cultured in medium containing 4% ethanol to establish a model of alcoholic liver damage. The cells were then treated with the combined extract obtained via cryogenic extraction. Biochemical assays and Western blot analyses were performed to assess the levels of oxidative stress markers, antioxidant enzymes, and inflammatory cytokines. In addition, activation of the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathway was examined to elucidate the mechanisms underlying the effects of the extract.
Treatment with the extract contributed to a significant reduction in the release of nitric oxide and reactive oxygen species in the ethanol-treated HepG2 cells; promoted the elevated expression of superoxide dismutase, catalase, and glutathione, indicating enhanced antioxidant defenses; and showed strong free radical-scavenging activity against 1,1-diphenyl-2-picrylhydrazyl radicals. In addition, by activating the PI3K/AKT pathway, treatment promoted increases in the expression of nuclear factor erythroid 2-related factor 2 and its downstream targets, subsequently inhibiting apoptosis. Moreover. inflammatory responses were mitigated, as indicated by reductions in the expression of tumor necrosis factor-alpha and interleukin-6, and we detected reduction in the levels of alanine aminotransferase and aspartate aminotransferase, thereby indicating hepatoprotective effects.
The combined P. lobata root and H. dulcis extract was established to have notable antioxidative and anti-inflammatory properties, effectively alleviating ethanol-induced liver damage in vitro. These findings highlight the potential applicability of this extract as a candidate for treating alcoholic liver disease.
References
1
Meroni M, Longo M, Dongiovanni P. Alcohol or gut microbiota: who is the guilty? Int J Mol Sci. 2019;20(18):4568.
2
Wu XQ, Fan XD, Miyata T, et al. Recent advances in understanding of pathogenesis of alcohol-associated liver disease. Annu Rev Pathol. 2023;18:411-438.
3
Seitz HK, Bataller R, Cortez-Pinto H, et al. Alcoholic liver disease. Nat Rev Dis Prim. 2018;4(1):16.
4
Singal AK, Bataller R, Ahn J, Kamath PS, Shah VH. ACG clinical guideline: alcoholic liver disease. Am J Gastroenterol. 2018;113(2):175-194.
5
Salete-Granado D, Carbonell C, Puertas-Miranda D, et al. Autophagy, oxidative stress, and alcoholic liver disease: a systematic review and potential clinical applications. Antioxidants. 2023;12(7):1425.
6
Li JD, Wang TQ, Liu PP, et al. Hesperetin ameliorates hepatic oxidative stress and inflammation via the PI3K/AKT-Nrf2-ARE pathway in oleic acid-induced HepG2 cells and a rat model of high-fat diet-induced NAFLD. Food Funct. 2021;12(9):3898-3918.
7
Liu X, Wang YN, Wu D, et al. Magnolol prevents acute alcoholic liver damage by activating PI3K/Nrf2/PPARγ and inhibiting NLRP3 signaling pathway. Front Pharmacol. 2019;10:1459.
8
Liao ZZ, Zhang JY, Liu B, et al. Polysaccharide from Okra (Abelmoschus esculentus (L.) Moench) improves antioxidant capacity via PI3K/AKT pathways and Nrf2 translocation in a type 2 diabetes model. Molecules. 2019;24(10):1906.
9
Wang SG, Zhang SM, Wang SP, Gao P, Dai L. A comprehensive review on Pueraria: insights on its chemistry and medicinal value. Biomed Pharmacother. 2020;131:110734.
10
Liu YS, Yuan MH, Zhang CY, et al. Puerariae Lobatae Radix flavonoids and puerarin alleviate alcoholic liver injury in zebrafish by regulating alcohol and lipid metabolism. Biomed Pharmacother. 2021;134:111121.
11
Sferrazza G, Brusotti G, Zonfrillo M, et al. Hovenia dulcis thumberg: phytochemistry, pharmacology, toxicology and regulatory framework for its use in the European Union. Molecules. 2021;26(4):903.
12
He YX, Liu MN, Wang YY, et al. Hovenia dulcis: a Chinese medicine that plays an essential role in alcohol-associated liver disease. Front Pharmacol. 2024;15:1337633.
13
Meng X, Tang GY, Zhao CN, et al. Hepatoprotective effects of Hovenia dulcis seeds against alcoholic liver injury and related mechanisms investigated via network pharmacology. World J Gastroenterol. 2020;26(24):3432-3446.
14
Zhang HT, Wei ZY, Tong YC, et al. Spectrum-effect relationship study to reveal the pharmacodynamic substances in Flos Puerariae-Semen Hoveniae medicine pair for the treatment of alcohol-induced liver damage. J Ethnopharmacol. 2023;314:116628.
15
Zhang Z, Yu A, Hu W, et al. A review on extraction, purification, structural characteristics, biological activities, applications of polysaccharides from Hovenia dulcis Thunb. (Guai Zao). Int J Biol Macromol. 2024;265(Pt 2):131097.
16
Du J, He D, Sun LN, et al. Semen Hoveniae extract protects against acute alcohol-induced liver injury in mice. Pharm Biol. 2010;48(8):953-958.
17
Zhao L, Zhang H, Li N, et al. Network pharmacology, a promising approach to reveal the pharmacology mechanism of Chinese medicine formula. J Ethnopharmacol. 2023;309:116306.
18
Shang LR, Wang YC, Li JX, et al. Mechanism of Sijunzi Decoction in the treatment of colorectal cancer based on network pharmacology and experimental validation. J Ethnopharmacol. 2023;302(Pt A):115876.
19
Liu YQ, Li HL, Wang XW, et al. Anti-alzheimers molecular mechanism of icariin: insights from gut microbiota, metabolomics, and network pharmacology. J Transl Med. 2023;21(1):277.
20
Jiang WH, Zhu HK, Xu WQ, et al. Echinacea purpurea polysaccharide prepared by fractional precipitation prevents alcoholic liver injury in mice by protecting the intestinal barrier and regulating liver-related pathways. Int J Biol Macromol. 2021;187:143-156.
22
Fang XY, Cao JM, Tao Z, Yang ZQ, Dai Y, Zhao LG. Hydroxytyrosol attenuates ethanol-induced liver injury by ameliorating steatosis, oxidative stress and hepatic inflammation by interfering STAT3/iNOS pathway. Redox Rep. 2023;28(1):2187564.
23
Zhou BJ, Liu PP, Yao XG, et al. Hepatoprotective effects of peach gum polysaccharides against alcoholic liver injury: moderation of oxidative stress and promotion of lipid metabolism. Front Nutr. 2024;10:1325450.
24
Jeong YH, Oh YC, Cho WK, Yim NH, Ma JY. Hoveniae Semen seu fructus ethanol extract exhibits anti-inflammatory activity via MAPK, AP-1, and STAT signaling pathways in LPS-stimulated RAW 264.7 and mouse peritoneal macrophages. Mediat Inflamm. 2019;2019:9184769.
25
Xi C, Zhang MY, Li BT, et al. Metabolomics of the anti-inflammatory effect of Pueraria lobata and Pueraria lobata var. thomsonii in rats. J Ethnopharmacol. 2023;306:116144.
26
Reddy BS, Reddy RKK, Reddy BP, Ramakrishna S, Diwan PV. Potential in vitro antioxidant and protective effects of Soymida febrifuga on ethanol induced oxidative damage in HepG2 cells. Food Chem Toxicol. 2008;46(11):3429-3442.
27
Nagy LE, Ding WX, Cresci G, Saikia P, Shah VH. Linking pathogenic mechanisms of alcoholic liver disease with clinical phenotypes. Gastroenterology. 2016;150(8):1756-1768.
28
Wan X, Na L, Haiyan L, et al. Flos Puerariae, Hoveniae Semen and their combinations treat oxidative stress in mice with acute alcoholic liver injury via Keap1/Nrf2/ARE signaling pathway. Chin J Exp Tradit Med Formulae. 2023;29(1):37-44.
29
Yiyu N, Daqing Z, Weifeng N, et al. Fermentation technology of Pueraria lobata-Hovenia dulcis and its anti-alcoholic effect. Food Ferment Ind. 2022;48(16):209-215.
30
Lieber CS. Metabolic effects of acetaldehyde. Biochem Soc Trans. 1988;16(3):241-247.
31
Teschke R. Alcoholic liver disease: current mechanistic aspects with focus on their clinical relevance. Biomedicines. 2019;7(3):68.
32
Yan CY, Hu WT, Tu JQ, et al. Pathogenic mechanisms and regulatory factors involved in alcoholic liver disease. J Transl Med. 2023;21(1):300.
33
Jung E, Baek EB, Hong EJ, et al. TXNIP in liver sinusoidal endothelial cells ameliorates alcohol-associated liver disease via nitric oxide production. Int J Biol Sci. 2024;20(2):606-620.
34
Zhang QQ, Yao M, Qi JJ, et al. Puerarin inhibited oxidative stress and alleviated cerebral ischemia-reperfusion injury through PI3K/Akt/Nrf2 signaling pathway. Front Pharmacol. 2023;14:1134380.
35
Wang JM, Lu YF, Zhang CY, et al. Qinggan Huoxue Recipe attenuates Alcoholic Liver Disease by suppressing PI3K/AKT signaling pathway based on network pharmacology. Int J Med Sci. 2023;20(3):346-358.
36
Liu Q, Jin ZQ, Xu ZL, et al. Antioxidant effects of ginkgolides and bilobalide against cerebral ischemia injury by activating the Akt/Nrf2 pathway in vitro and in vivo. Cell Stress Chaperones. 2019;24(2):441-452.
37
Chiang SK, Chen SE, Chang LC. The role of HO-1 and its crosstalk with oxidative stress in cancer cell survival. Cells. 2021;10(9):2401.
38
Mohamed ME, Elmorsy MA, Younis NS. Renal ischemia/reperfusion mitigation via geraniol: the role of nrf-2/HO-1/NQO-1 and TLR2, 4/MYD88/NFκB pathway. Antioxidants. 2022;11(8):1568.
39
Lopetuso LR, Mocci G, Marzo M, et al. Harmful effects and potential benefits of anti-tumor necrosis factor (TNF)-α on the liver. Int J Mol Sci. 2018;19(8):2199.
40
Zhang ZJ, Li S, Jiang J, et al. Preventive effects of Flos Perariae (Gehua) water extract and its active ingredient puerarin in rodent alcoholism models. Chin Med. 2010;5:36.
41
Jegal KH, Park HR, Choi BR, Kim JK, Ku SK. Synergistic protective effect of fermented schizandrae fructus pomace and hoveniae Semen cum fructus extracts mixture in the ethanol-induced hepatotoxicity. Antioxidants. 2023;12(8):1602.