Alantolactone mitigates thioacetamide-induced liver fibrosis in mice by inhibiting TLR4/MyD88/NF-κB axis

Abstract

Objective: To explore the effect of alantolactone on thioacetamide-induced liver fibrosis in mice as well as elucidate its underlying mechanism.

Methods: Animals were divided into 5 groups: the control, the thioacetamide group (150 mg/kg/twice weekly), the thioacetamide groups treated with alantolactone (5 and 10 mg/kg) or silymarin (50 mg/kg), respectively. All treatments were continued for 6 successive weeks, followed by collection of sera and tissue samples. Biochemical, histological, and immunohistochemical analyses were performed to examine the hepatoprotective effects of alantolactone.

Results: Alantolactone ameliorated thioacetamide-induced hepatic impairment and prevented the rise of serum activities of liver enzymes. Its hepatoprotective effect was further confirmed by histological examinations. Moreover, alantolactone lowered the expression of transforming growth factor-beta 1 and alpha-smooth muscle actin, hydroxyproline content as well as COL1A1 mRNA expression. It restored antioxidant balance and inhibited thioacetamide-induced upregulated expression of interleukin-1 beta, interleukin-6, and tumor necrosis factor-alpha, Toll-like receptor 4 (TLR4), myeloid differentiation primary response 88 (MyD88), and nuclear factor kappa B (NF-κB).

Conclusions: Alantolactone protects against thioacetamide-induced liver fibrosis in mice by reducing collagen deposition, oxidative stress, and inflammation. These effects are mediated, at least partly, by the inhibition of TLR4/MyD88/NF-κB axis.

Keywords

Liver fibrosis / Alantolactone / TLR4 / MyD88 / NF-κB

Conflict of interest statement

The authors declare no conflict of interest.

Funding

The Deanship of Scientific Research at King Abdulaziz University, Jeddah, Saudi Arabia has funded this Project under grant no (G: 525-249-1443).

Data availability statement

The data supporting the findings of this study are available from the corresponding author upon request.

Authors’ contributions

HA, ABA, AE and BGE developed the theoretical formalism. HA, NNA and RAS performed the analytic methods and calculations. ABA and BGE supervised the project. HA, NNA, RAS, ABA, AE and BGE contributed to the final version of the manuscript.

Publisher’s note

The Publisher of the Journal remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Acknowledgments

The authors acknowledge with thanks the Deanship of Scientific Research for technical and financial support. The authors are grateful to Dr. Gamal Abdulaziz, Department of Clinical Anatomy, Faculty of Medicine, King Abdulaziz University for histopathological examination.

References

[1] Kisseleva T, Brenner D. Molecular and cellular mechanisms of liver fibrosis and its regression. Nat Rev Gastroenterol Hepatol 2021; 18(3): 151166.

[2] Tan Z, Sun H, Xue T, Gan C, Liu H, Xie Y, et al. Liver fibrosis: Therapeutic targets and advances in drug therapy. Front Cell Dev Biol 2021; 9. doi: 10.3389/fcell.2021.730176.

[3] Puche JE, Saiman Y, Friedman SL. Hepatic stellate cells and liver fibrosis. Compr Physiol 2013; 3(4): 1473-1492.

[4] Seif el-Din SH, Hammam OA, Ezzat SM, Saleh S, Safar MM, El-Maadawy WH, et al. Hydroxysafflor yellow A protects against thioacetamide-induced liver fibrosis in rats via suppressing proinflammatory/fibrogenic mediators and promoting hepatic stellate cell senescence and apoptosis. Asian Pac J Trop Biomed 2023; 13(8): 348-358.

[5] Ezhilarasan D, Sokal E, Najimi M. Hepatic fibrosis: It is time to go with hepatic stellate cell-specific therapeutic targets. Hepatobiliary Pancreat Dis Int 2018; 17(3): 192-197.

[6] Dhar D, Baglieri J, Kisseleva T, Brenner DA. Mechanisms of liver fibrosis and its role in liver cancer. Exp Biol Med Maywood NJ 2020; 245(2): 96-108.

[7] Elpek GÖ. Cellular and molecular mechanisms in the pathogenesis of liver fibrosis: An update. World J Gastroenterol 2014; 20(23): 7260-7276.

[8] El-Kashef DH, Serrya MS. Sitagliptin ameliorates thioacetamide-induced acute liver injury via modulating TLR4/NF-:B signaling pathway in mice. Life Sci 2019; 228: 266-273.

[9] Liu X, Bian L, Duan X, Zhuang X, Sui Y, Yang L. Alantolactone: A sesquiterpene lactone with diverse pharmacological effects. Chem Biol Drug Des 2021; 98(6): 1131-1145.

[10] Pei W, Huang X, Ni B, Zhang R, Niu G, You H. Selective STAT3 inhibitor alantolactone ameliorates osteoarthritis via regulating chondrocyte autophagy and cartilage homeostasis. Front Pharmacol 2021; 12. doi: 10.3389/fphar.2021.730312.

[11] Kim M, Song K, Kim YS. Alantolactone improves prolonged exposure of interleukin-6-induced skeletal muscle inflammation associated glucose intolerance and insulin resistance. Front Pharmacol 2017; 8:405.

[12] Kim M, Song K, Kim YS. Alantolactone improves palmitate-induced glucose intolerance and inflammation in both lean and obese states in vitro: Adipocyte and adipocyte-macrophage co-culture system. Int Immunopharmacol 2017; 49: 187-194.

[13] Chun J, Choi RJ, Khan S, Lee DS, Kim YC, Nam YJ, et al. Alantolactone suppresses inducible nitric oxide synthase and cyclooxygenase-2 expression by down-regulating NF-κB, MAPK and AP-1 via the MyD88 signaling pathway in LPS-activated RAW 264.7 cells. Int Immunopharmacol 2012; 14(4): 375-383.

[14] Lee KM, Hwang YJ, Jung GS. Alantolactone attenuates renal fibrosis via inhibition of transforming growth factor β/Smad3 signaling pathway. Diabetes Metab J 2024; 48(1): 72-82.

[15] Hwang YJ, Jung GS, Lee KM. Alantolactone alleviates epithelial-mesenchymal transition by regulating the TGF-β/STAT3 signaling pathway in renal fibrosis. Heliyon 2024; 10(16): e36253.

[16] Wang J, Jiang Y, Jin L, Qian C, Zuo W, Lin J, et al. Alantolactone attenuates high-fat diet-induced inflammation and oxidative stress in non-alcoholic fatty liver disease. Nutr Diabetes 2024; 14(1): 41.

[17] Li X, Lu C, Liu S, Liu S, Su C, Xiao T, et al. Synthesis and discovery of a drug candidate for treatment of idiopathic pulmonary fibrosis through inhibition of TGF-β1 pathway. Eur J Med Chem 2018; 157: 229-247.

[18] Long X, Wang P, Zhou Y, Wang Q, Ren L, Li Q, et al. Preventive effect of Lactobacillus plantarum HFY 15 on carbon tetrachloride (CC1₄)-induced acute liver injury in mice. J Food Sci 2022; 87(6): 2626-2639.

[19] Luca C, Salvatore F, Vincenzo DP, Giovanni C, Attilio ILM. Anesthesia protocols in laboratory animals used for scientific purposes. Acta Bio Medica Atenei Parm 2018; 89(3): 337-342.

[20] He Z, Feng L, Zhang X, Geng Y, Parodi DA, Suarez-Quian C, et al. Expression of Col1a1, Col1a2 and procollagen I in germ cells of immature and adult mouse testis. Reprod Camb Engl 2005; 130(3): 333-341.

[21] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods San Diego Calif 2001; 25(4): 402-408.

[22] Wallace MC, Hamesch K, Lunova M, Kim Y, Weiskirchen R, Strnad P, et al. Standard operating procedures in experimental liver research: Thioacetamide model in mice and rats. Lab Anim 2015; 49(1 Suppl): 21-29.

[23] Krishna M. Role of special stains in diagnostic liver pathology. Clin Liver Dis 2013; 2(Suppl 1): S8-S10.

[24] Gressner AM, Weiskirchen R, Breitkopf K, Dooley S. Roles of TGF-beta in hepatic fibrosis. Front Biosci J Virtual Libr 2002; 7: d793-807.

[25] Yu B, Jin XQ, Yu WY, Dong YY, Ying HZ, Yu CH. 1β-Hydroxyalantolactone from Inulae Flos alleviated the progression of pulmonary fibrosis via inhibiting JNK/FOXO1/NF-κB pathway. Int Immunopharmacol 2021; 101(Pt A). doi: 10.1016/j.intimp.2021.108339.

[26] Akkiz H, Gieseler RK, Canbay A. Liver fibrosis: From basic science towards clinical progress, focusing on the central role of hepatic stellate cells. Int J Mol Sci 2024; 25(14): 7873.

[27] Kisseleva T, Brenner D. Molecular and cellular mechanisms of liver fibrosis and its regression. Nat Rev Gastroenterol Hepatol 2021; 18(3): 151-166.

[28] Bolarin DM, Azinge EC. Biochemical markers, extracellular components in liver fibrosis and cirrhosis. Niger Q J Hosp Med 2007; 17(1): 42-52.

[29] Sánchez-Valle V, Chávez-Tapia NC, Uribe M, Méndez-Sánchez N. Role of oxidative stress and molecular changes in liver fibrosis: A review. Curr Med Chem 2012; 19(28): 4850-4860.

[30] Liu X, Bian L, Duan X, Zhuang X, Sui Y, Yang L. Alantolactone: A sesquiterpene lactone with diverse pharmacological effects. Chem Biol Drug Des 2021; 98(6): 1131-1145.

[31] Mir-Babaev NF, Sereda NP Antioxidant activity of alantolactone and isoalantolactone. Chem Nat Compd 1987; 23(5): 629-630.

[32] Alegre F, Pelegrin P, Feldstein AE. Inflammasomes in liver fibrosis. Semin Liver Dis 2017; 37(2): 119-127.

[33] Roehlen N, Crouchet E, Baumert TF. Liver fibrosis: Mechanistic concepts and therapeutic perspectives. Cells 2020; 9(4): 875.

[34] Zhang H, Xu J. Unveiling thioacetamide-induced toxicity: Multi-organ damage and omitted bone toxicity. Hum Exp Toxicol 2024; 43. doi: 10.1177/09603271241241807.

[35] Zhu Y, Ling Y, Wang X. Alantolactone mitigates renal injury induced by diabetes via inhibition of high glucose-mediated inflammatory response and macrophage infiltration. Immunopharmacol Immunotoxicol 2020; 42(2): 84-92.

[36] Dang X, He B, Ning Q, Liu Y, Guo J, Niu G, et al. Alantolactone suppresses inflammation, apoptosis and oxidative stress in cigarette smoke-induced human bronchial epithelial cells through activation of Nrf2/HO-1 and inhibition of the NF-κB pathways. Respir Res 2020; 21(1): 95.

[37] Tang YL, Zhu L, Tao Y, Lu W, Cheng H. Role of targeting TLR4 signaling axis in liver-related diseases. Pathol Res Pract 2023; 244. doi: 10.1016/j.prp.2023.154410.

[38] Guo J, Friedman SL. Toll-like receptor 4 signaling in liver injury and hepatic fibrogenesis. Fibrogenesis Tissue Repair 2010; 3:21.

[39] Ge S, Yang W, Chen H, Yuan Q, Liu S, Zhao Y, et al. MyD 88 in macrophages enhances liver fibrosis by activation of NLRP3 inflammasome in HSCs. Int J Mol Sci 2021; 22(22). doi: 10.3390/ijms222212413.

[40] Thapa M, Chinnadurai R, Velazquez VM, Tedesco D, Elrod E, Han JH, et al. Liver fibrosis occurs through dysregulation of MyD88-dependent innate B-cell activity. Hepatol Baltim Md 2015; 61(6): 2067-2079.

[41] Ahmed H, Khan MA, Kahlert UD, Niemelä M, Hänggi D, Chaudhry SR, et al. Role of adaptor protein myeloid differentiation 88 (MyD88) in post-subarachnoid hemorrhage inflammation: A systematic review. Int J Mol Sci 2021; 22(8): 4185.

[42] Liu J, Liu M, Wang S, He Y, Huo Y, Yang Z, et al. Alantolactone induces apoptosis and suppresses migration in MCF-7 human breast cancer cells via the p 38 MAPK, NF-κB and Nrf2 signaling pathways. Int J Mol Med 2018; 42(4): 1847-1856.

[43] Liu M, Xu Y, Han X, Yin L, Xu L, Qi Y, et al. Dioscin alleviates alcoholic liver fibrosis by attenuating hepatic stellate cell activation via the TLR4/MyD88/NF-κB signaling pathway. Sci Rep 2015; 5. doi: 10.1038/srep18038.

[44] Hu N, Guo C, Dai X, Wang C, Gong L, Yu L, et al. Forsythiae Fructuse water extract attenuates liver fibrosis via TLR4/MyD88/NF-κB and TGF-β/smads signaling pathways. J Ethnopharmacol 2020; 262. doi: 10.1016/j.jep.2020.113275.