pp 1–14 | Cite as

Umbelliferone Ameliorates CCl4-Induced Liver Fibrosis in Rats by Upregulating PPARγ and Attenuating Oxidative Stress, Inflammation, and TGF-β1/Smad3 Signaling

  • Ayman M. MahmoudEmail author
  • Walaa G. Hozayen
  • Iman H. Hasan
  • Eman Shaban
  • May Bin-Jumah


Umbelliferone (UMB) is a natural coumarin that has diverse biological activities. However, its potential to protect against liver fibrosis has not been reported yet. This study aimed to investigate the protective effect of UMB against carbon tetrachloride (CCl4)-induced liver fibrosis in rats. Rats received CCl4 and UMB for 8 weeks and samples were collected for analyses. CCl4 induced a significant increase in serum levels of liver function markers and pro-inflammatory cytokines. Treatment with UMB significantly ameliorated liver function markers and pro-inflammatory cytokines and prevented CCl4-induced histological alterations. CCl4 promoted significant upregulation of α-smooth muscle actin (SMA), collagen I, collagen III, NF-κB p65, TGF-β1, and p-Smad3. Masson’s trichrome staining revealed a significant fibrogenesis in CCl4-induced rats. Treatment with UMB suppressed TGF-β1/Smad3 signaling and downregulated α-SMA, collagen I, collagen III, and NF-κB p65. In addition, UMB diminished malondialdehyde and nitric oxide levels, boosted reduced glutathione and antioxidant enzymes, and upregulated the expression of PPARγ. In conclusion, our results demonstrated that UMB prevented CCl4-induced liver fibrosis by attenuating oxidative stress, inflammation, and TGF-β1/Smad3 signaling, and upregulating PPARγ. Therefore, UMB may be a promising candidate for preventing hepatic fibrogenesis, given that further research is needed to delineate the exact molecular mechanisms underlying its antifibrotic efficacy.


fibrosis 7-hydroxycoumarin oxidative stress inflammation TGF-β1 PPARγ 


Authors’ Contribution

AMM, WGH, and IHH conceived the study and designed the experiments. AMM and ES performed the experiments. AMM analyzed the data, prepared the figures, and wrote the manuscript. All the authors participated in the assays, revised the manuscript, and approved the submission.

Compliance with Ethical Standards

The experimental protocol and all animal procedures were approved by the Institutional Animal Ethics Committee of Beni-Suef University (Egypt).

Conflict of Interest

The authors declare that they have no conflict of interest.


  1. 1.
    Kim, W.R., R.S. Brown Jr., N.A. Terrault, and H. El-Serag. 2002. Burden of liver disease in the United States: summary of a workshop. Hepatology (Baltimore, Md.) 36: 227–242.CrossRefGoogle Scholar
  2. 2.
    Zhang, C.Y., W.G. Yuan, P. He, J.H. Lei, and C.X. Wang. 2016. Liver fibrosis and hepatic stellate cells: etiology, pathological hallmarks and therapeutic targets. World Journal of Gastroenterology 22: 10512–10522.CrossRefGoogle Scholar
  3. 3.
    Tacke, F., and C. Trautwein. 2015. Mechanisms of liver fibrosis resolution. Journal of Hepatology 63: 1038–1039.CrossRefGoogle Scholar
  4. 4.
    Josan, S., K. Billingsley, J. Orduna, J.M. Park, R. Luong, L. Yu, R. Hurd, A. Pfefferbaum, D. Spielman, and D. Mayer. 2015. Assessing inflammatory liver injury in an acute CCl4 model using dynamic 3D metabolic imaging of hyperpolarized [1-(13)C] pyruvate. NMR in Biomedicine 28: 1671–1677.CrossRefGoogle Scholar
  5. 5.
    Friedman, S.L. 2008. Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver. Physiological Reviews 88: 125–172.CrossRefGoogle Scholar
  6. 6.
    Cheng, Q., C. Li, C.-f. Yang, Y.-j. Zhong, D. Wu, L. Shi, L. Chen, Y.-w. Li, and L. Li. 2019. Methyl ferulic acid attenuates liver fibrosis and hepatic stellate cell activation through the TGF-β1/Smad and NOX4/ROS pathways. Chemico-Biological Interactions 299: 131–139.CrossRefGoogle Scholar
  7. 7.
    Gandhi, C.R. 2012. Oxidative stress and hepatic stellate cells: a paradoxical relationship. Trends in Cell & Molecular Biology 7: 1–10.Google Scholar
  8. 8.
    Duan, W.J., X. Yu, X.R. Huang, J.W. Yu, and H.Y. Lan. 2014. Opposing roles for Smad2 and Smad3 in peritoneal fibrosis in vivo and in vitro. American Journal of Pathology 184: 2275–2284.CrossRefGoogle Scholar
  9. 9.
    Gressner, A.M., R. Weiskirchen, K. Breitkopf, and S. Dooley. 2002. Roles of TGF-beta in hepatic fibrosis. Frontiers in Bioscience : a Journal and Virtual Library 7: d793–d807.CrossRefGoogle Scholar
  10. 10.
    Rosen, E.D., and B.M. Spiegelman. 2001. PPARgamma: a nuclear regulator of metabolism, differentiation, and cell growth. The Journal of Biological Chemistry 276: 37731–37734.CrossRefGoogle Scholar
  11. 11.
    Ricote, M., J.T. Huang, J.S. Welch, and C.K. Glass. 1999. The peroxisome proliferator-activated receptor (PPARgamma) as a regulator of monocyte/macrophage function. Journal of Leukocyte Biology 66: 733–739.CrossRefGoogle Scholar
  12. 12.
    Yu, Y., Y. Wu, G. Wen, and W. Yang. 2014. Effect of pioglitazone on the expression of TLR4 in renal tissue of diabetic rats. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi 30 (8): 793–797.Google Scholar
  13. 13.
    Lin, L.C., S.L. Hsu, C.L. Wu, W.C. Liu, and C.M. Hsueh. 2011. Peroxisome proliferator-activated receptor gamma (PPARgamma) plays a critical role in the development of TGFbeta resistance of H460 cell. Cellular Signalling 23: 1640–1650.CrossRefGoogle Scholar
  14. 14.
    Mahmoud, A.M., H.M. Mohammed, S.M. Khadrawy, and S.R. Galaly. 2017. Hesperidin protects against chemically induced hepatocarcinogenesis via modulation of Nrf2/ARE/HO-1, PPARgamma and TGF-beta1/Smad3 signaling, and amelioration of oxidative stress and inflammation. Chemico-Biological Interactions 277: 146–158.CrossRefGoogle Scholar
  15. 15.
    Mahmoud, A.M. 2014. Hesperidin protects against cyclophosphamide-induced hepatotoxicity by upregulation of PPARγ and abrogation of oxidative stress and inflammation. Canadian Journal of Physiology and Pharmacology 92: 717–724.CrossRefGoogle Scholar
  16. 16.
    Mahmoud, A.M., and H.S. Al Dera. 2015. 18β-Glycyrrhetinic acid exerts protective effects against cyclophosphamide-induced hepatotoxicity: potential role of PPARγ and Nrf2 upregulation. Genes & Nutrition 10: 1–13.CrossRefGoogle Scholar
  17. 17.
    Mahmoud, A.M., M.O. Germoush, M.F. Alotaibi, and O.E. Hussein. 2017. Possible involvement of Nrf2 and PPARgamma up-regulation in the protective effect of umbelliferone against cyclophosphamide-induced hepatotoxicity. Biomedicine & Pharmacotherapy 86: 297–306.CrossRefGoogle Scholar
  18. 18.
    Alqahtani, S., and A.M. Mahmoud. 2016. Gamma-glutamylcysteine ethyl ester protects against cyclophosphamide-induced liver injury and hematologic alterations via upregulation of PPARgamma and attenuation of oxidative stress, inflammation, and apoptosis. Oxidative Medicine and Cellular Longevity 2016: 4016209.CrossRefGoogle Scholar
  19. 19.
    Mahmoud, A.M., O.E. Hussein, W.G. Hozayen, and S.M. Abd El-Twab. 2017. Methotrexate hepatotoxicity is associated with oxidative stress, and down-regulation of PPARgamma and Nrf2: protective effect of 18beta-glycyrrhetinic acid. Chemico-Biological Interactions 270: 59–72.CrossRefGoogle Scholar
  20. 20.
    Mahmoud, A.M., W.G. Hozayen, and S.M. Ramadan. 2017. Berberine ameliorates methotrexate-induced liver injury by activating Nrf2/HO-1 pathway and PPARgamma, and suppressing oxidative stress and apoptosis in rats. Biomedicine & Pharmacotherapy 94: 280–291.CrossRefGoogle Scholar
  21. 21.
    Abdella, E., A. Mahmoud, and A. El-Derby. 2016. Brown seaweeds protect against azoxymethane-induced hepatic repercussions through up-regulation of peroxisome proliferator activated receptor gamma and attenuation of oxidative stress. Pharmaceutical Biology 54: 2496–2504.CrossRefGoogle Scholar
  22. 22.
    Yang, L., C.-C. Chan, O.-S. Kwon, S. Liu, J. McGhee, S.A. Stimpson, L.Z. Chen, W.W. Harrington, W.T. Symonds, and D.C. Rockey. 2006. Regulation of peroxisome proliferator-activated receptor-γ in liver fibrosis. American Journal of Physiology - Gastrointestinal and Liver Physiology 291: G902–G911.CrossRefGoogle Scholar
  23. 23.
    Yang, L., S.A. Stimpson, L. Chen, W.W. Harrington, and D.C. Rockey. 2010. Effectiveness of the PPARγ agonist, GW570, in liver fibrosis. Inflammation Research 59: 1061–1071.CrossRefGoogle Scholar
  24. 24.
    Mazimba, O. 2017. Umbelliferone: sources, chemistry and bioactivities review. Bulletin of Faculty of Pharmacy, Cairo University 55: 223–232.CrossRefGoogle Scholar
  25. 25.
    Ramesh, B., and K.V. Pugalendi. 2005. Antihyperlipidemic and antidiabetic effects of umbelliferone in streptozotocin diabetic rats. The Yale Journal of Biology and Medicine 78: 189–196.Google Scholar
  26. 26.
    Germoush, M.O., S.I. Othman, M.A. Al-Qaraawi, H.M. Al-Harbi, O.E. Hussein, G. Al-Basher, M.F. Alotaibi, H.A. Elgebaly, M.A. Sandhu, A.A. Allam, and A.M. Mahmoud. 2018. Umbelliferone prevents oxidative stress, inflammation and hematological alterations, and modulates glutamate-nitric oxide-cGMP signaling in hyperammonemic rats. Biomedicine & Pharmacotherapy 102: 392–402.CrossRefGoogle Scholar
  27. 27.
    Yin, J., H. Wang, and G. Lu. 2018. Umbelliferone alleviates hepatic injury in diabetic db/db mice via inhibiting inflammatory response and activating Nrf2-mediated antioxidant. Bioscience Reports 38: BSR20180444.CrossRefGoogle Scholar
  28. 28.
    Al-Sayed, E., O. Martiskainen, S.H. Seif el-Din, A.-N.A. Sabra, O.A. Hammam, N.M. El-Lakkany, and M.M. Abdel-Daim. 2014. Hepatoprotective and antioxidant effect of Bauhinia Hookeri extract against carbon tetrachloride-induced hepatotoxicity in mice and characterization of its bioactive compounds by HPLC-PDA-ESI-MS/MS. BioMed Research International 2014: 9.CrossRefGoogle Scholar
  29. 29.
    Al-Rasheed, N., L. Faddah, N. Al-Rasheed, Y.A. Bassiouni, I.H. Hasan, A.M. Mahmoud, R.A. Mohamad, and H.I. Yacoub. 2016. Protective effects of silymarin, alone or in combination with chlorogenic acid and/or melatonin, against carbon tetrachloride-induced hepatotoxicity. Pharmacognosy Magazine 12: S337–S345.CrossRefGoogle Scholar
  30. 30.
    Al-Sayed, E., and M.M. Abdel-Daim. 2014. Protective role of cupressuflavone from Cupressus macrocarpa against carbon tetrachloride-induced hepato- and nephrotoxicity in mice. Planta Medica 80: 1665–1671.CrossRefGoogle Scholar
  31. 31.
    Fahmy, N.M., E. Al-Sayed, M.M. Abdel-Daim, M. Karonen, and A.N. Singab. 2016. Protective effect of Terminalia muelleri against carbon tetrachloride-induced hepato and nephro-toxicity in mice and characterization of its bioactive constituents. Pharmaceutical Biology 54: 303–313.CrossRefGoogle Scholar
  32. 32.
    Li, X., L. Wang, and C. Chen. 2017. Effects of exogenous thymosin β4 on carbon tetrachloride-induced liver injury and fibrosis. Scientific Reports 7: 5872.CrossRefGoogle Scholar
  33. 33.
    Hardjo, M., M. Miyazaki, M. Sakaguchi, T. Masaka, S. Ibrahim, K. Kataoka, and N.H. Huh. 2009. Suppression of carbon tetrachloride-induced liver fibrosis by transplantation of a clonal mesenchymal stem cell line derived from rat bone marrow. Cell Transplantation 18: 89–99.CrossRefGoogle Scholar
  34. 34.
    Ohkawa, H., N. Ohishi, and K. Yagi. 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry 95: 351–358.CrossRefGoogle Scholar
  35. 35.
    Green, L.C., D.A. Wagner, J. Glogowski, P.L. Skipper, J.S. Wishnok, and S.R. Tannenbaum. 1982. Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Analytical Biochemistry 126: 131–138.CrossRefGoogle Scholar
  36. 36.
    Beutler, E., O. Duron, and B.M. Kelly. 1963. Improved method for the determination of blood glutathione. The Journal of Laboratory and Clinical Medicine 61: 882–888.Google Scholar
  37. 37.
    Marklund, S., and G. Marklund. 1974. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase, FEBS. European Journal of Biochemistry 47: 469–474.CrossRefGoogle Scholar
  38. 38.
    Aebi, H. 1984. [13] Catalase in vitro. Methods in Enzymology 105: 121–126.CrossRefGoogle Scholar
  39. 39.
    Matkovics, B., L. Szabo, and I.S. Varga. 1998. Determination of enzyme activities in lipid peroxidation and glutathione pathways (in Hungarian). Laboratoriumi Diagnosztika 15: 248–249.Google Scholar
  40. 40.
    Livak, K.J., and T.D. Schmittgen. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−delta delta c(t)) method. Methods 25 (4): 402–408.CrossRefGoogle Scholar
  41. 41.
    Schuppan, D. 2015. Liver fibrosis: common mechanisms and antifibrotic therapies. Clinics and Research in Hepatology and Gastroenterology 39 (Suppl 1): S51–S59.CrossRefGoogle Scholar
  42. 42.
    Lang, Q., Q. Liu, N. Xu, K.L. Qian, J.H. Qi, Y.C. Sun, L. Xiao, and X.F. Shi. 2011. The antifibrotic effects of TGF-β1 siRNA on hepatic fibrosis in rats. Biochemical and Biophysical Research Communications 409: 448–453.CrossRefGoogle Scholar
  43. 43.
    Sun, J., Y. Wu, C. Long, P. He, J. Gu, L. Yang, Y. Liang, and Y. Wang. 2018. Anthocyanins isolated from blueberry ameliorates CCl4 induced liver fibrosis by modulation of oxidative stress, inflammation and stellate cell activation in mice. Food and Chemical Toxicology 120: 491–499.CrossRefGoogle Scholar
  44. 44.
    Weber, L.W., M. Boll, and A. Stampfl. 2003. Hepatotoxicity and mechanism of action of haloalkanes: carbon tetrachloride as a toxicological model. Critical Reviews in Toxicology 33: 105–136.CrossRefGoogle Scholar
  45. 45.
    Czaja, A.J., and H.A. Carpenter. 2004. Progressive fibrosis during corticosteroid therapy of autoimmune hepatitis. Hepatology (Baltimore, Md.) 39: 1631–1638.CrossRefGoogle Scholar
  46. 46.
    Cohen-Naftaly, M., and S.L. Friedman. 2011. Current status of novel antifibrotic therapies in patients with chronic liver disease. Therapeutic Advances in Gastroenterology 4: 391–417.CrossRefGoogle Scholar
  47. 47.
    Wheeler, M.D., H. Kono, M. Yin, M. Nakagami, T. Uesugi, G.E. Arteel, E. Gabele, I. Rusyn, S. Yamashina, M. Froh, Y. Adachi, Y. Iimuro, B.U. Bradford, O.M. Smutney, H.D. Connor, R.P. Mason, S.M. Goyert, J.M. Peters, F.J. Gonzalez, R.J. Samulski, and R.G. Thurman. 2001. The role of Kupffer cell oxidant production in early ethanol-induced liver disease. Free Radical Biology & Medicine 31: 1544–1549.CrossRefGoogle Scholar
  48. 48.
    Satta, S., A.M. Mahmoud, F.L. Wilkinson, M. Yvonne Alexander, and S.J. White. 2017. The role of Nrf2 in cardiovascular function and disease. Oxidative Medicine and Cellular Longevity 2017: 18.CrossRefGoogle Scholar
  49. 49.
    Jia, S., X. Liu, W. Li, J. Xie, L. Yang, and L. Li. 2015. Peroxisome proliferator-activated receptor gamma negatively regulates the differentiation of bone marrow-derived mesenchymal stem cells toward myofibroblasts in liver fibrogenesis. Cellular Physiology and Biochemistry 37: 2085–2100.CrossRefGoogle Scholar
  50. 50.
    Wu, M., D.S. Melichian, E. Chang, M. Warner-Blankenship, A.K. Ghosh, and J. Varga. 2009. Rosiglitazone abrogates bleomycin-induced scleroderma and blocks profibrotic responses through peroxisome proliferator-activated receptor-gamma. The American Journal of Pathology 174: 519–533.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Physiology Division, Zoology Department, Faculty of ScienceBeni-Suef UniversityBeni-SuefEgypt
  2. 2.Biochemistry Division, Chemistry Department, Faculty of ScienceBeni-Suef UniversityBeni-SuefEgypt
  3. 3.Biotechnology and Life Sciences Department, Faculty of Postgraduate Studies for Advanced Sciences (PSAS)Beni-Suef UniversityBeni-SuefEgypt
  4. 4.Department of Pharmacology and Toxicology, College of PharmacyKing Saud UniversityRiyadhSaudi Arabia
  5. 5.Department of Biology, College of SciencePrincess Nourah bint Abdulrahman UniversityRiyadhSaudi Arabia

Personalised recommendations