Histological Evaluation of the Effect of Lactoferrin on Experimentally Induced Liver Fibrosis in Adult Male Albino Rat

Document Type : Original articles


1 Lecturer of Histology and Cell Biology Department, Faculty of Medicine, Tanta University, Tanta, Egypt.

2 Lecturer of Medical Biochemistry and Molecular Biology


Background: Carbon tetrachloride (CCl4)-induced liver fibrosis is a well-established model of hepatotoxicity. Lactoferrin (LF) is an iron-binding glycoprotein that has anti-inflammatory and antioxidant effects. Aim of the study: This study was made to demonstrate the ameliorating effect of lactoferrin versus CCL4 induced liver fibrosis in adult male albino rat using various biochemical and histological techniques. Materials & Methods: This study involved forty adult male albino rats that were randomly divided into 4 groups: group I represented the control group, group II received LF (100mg/kg/day) orally for 5 weeks, group III was intraperitoneally injected with CCL4 (1ml/kg/once a week, for five weeks) for induction of liver fibrosis, and group IV received LF and CCL4 concomitantly in the same dose and manner as groups II & III respectively. The liver specimens were processed for different biochemical, histological and immunohistochemical techniques. Morphometrical and statistical studies were also performed. Results:  CCL4 group showed disorganization of liver architecture that was evidenced by light & electron microscopic examination. A significant increase of the mean area percentage of collagen fibers by Masson’s Trichrome, immunopositive reaction of αSMA and CD133 along the sinusoids were also observed. Lactoferrin administration in group IV showed improvement of all the previous results and oxidative stress biomarkers.  Conclusion: Liver fibrosis could be ameliorated by lactoferrin. 


Main Subjects

  1. Bouhrim M, Quassou H, Choukri M, Mekhfi H, Ziyyat A, Legssyer A, et al. Hepatoprotective effect of Opuntia dellenii seed oil on CCL4 induced acute liver damage in rat. Asian Pac J Trop Biomed. 2018;8:254-60. doi: 10.4103/2221-1691.233006.
  2. Huang YH, Shi MN, Zheng WD, Zhang LJ, Chen ZX, Wang XZ. Therapeutic effect of interleukin-10 on CCl4-induced hepatic fibrosis in rats. World J Gastroenterol. 2006;9:1386-1391. doi: 10.3748/wjg.v12.i9.1386.
  3. Lee GP, Jeong WI, Jeong DH, Do SH, Kim TH, Jeong KS. Diagnostic evaluation of carbon tetrachloride-induced rat hepatic cirrhosis model. Anticancer Res. 2005;25:1029-1038. PMID: 15868943.
  4. Chen X, Gao G, Luo W, Wang G, Zheng W, Jia2 Q, et al. Inhibitory effect of Dioscorea panthaica on CCl4-induced liver fibrosis in rats. Journal of Medicinal Plants Research. 2012; 6(9):1532-1538. doi: 10.5897/JMPR11.676
  5. Cosgun, BE, Erdemli, ME, Gul M, Gul S, Bag GH, Zeynep E, et al. Crocin (active constituent of saffron) improves CCl4-induced liver damage by modulating oxidative stress in rats. Turkish Journal of Biochemistry. 2019; 44:370-378. doi: 10.1515/tjb-2017-0173.
  6. El Sayed H, Morsy L, Abo Emara T, Galhom R. Effect of Carbon Tetrachloride (CCL4) on Liver in Adult Albino Rats: Histological study. The Egyptian Journal of Hospital Medicine. 2019;76(60):4254-4261. doi: 10.21608/ejhm.2019.43809.
  7. Aoyama Y, Naiki-Ito A, Xiaochen K, Komura M, Kato H, Nagayasu Y, et al. Lactoferrin Prevents Hepatic Injury and Fibrosis via the Inhibition of NF-κB Signaling in a Rat Non-Alcoholic Steatohepatitis Model. 2021;23;14(1):42. doi: 10.3390/nu14010042. PMID: 35010924; PMCID: PMC8746867.
  8. Safaeian L, Zabolian H. Antioxidant effects of bovine lactoferrin on dexamethasone-induced hypertension in rat. ISRN Pharmacol. 2014; 22;2014:943523. doi: 10.1155/2014/943523. PMID: 24587916; PMCID: PMC3920649.
  9. Singh D, Arya P, Singh V, Arya D, Bhagour K, Gupta R. Role of Cressa Cretica on CCl4- Induced Liver Damage in Experimental Rats. Journal of Pharmacognosy and Phytochemistry. 2018; 7(1): 1368-1373.
  10. Tung YT, Tang TY, Chen HL, Yang SH, Chong KY, Cheng WT, et al. Lactoferrin protects against chemical-induced rat liver fibrosis by inhibiting stellate cell activation. J Dairy Sci. 2014;97(6):3281-3291. doi: 10.3168/jds.2013-7505. Epub 2014 Apr 14. PMID: 24731632.
  11. Ghasi S, Umana I, Ogbonna A, Nwokike M, Ufelle S. Cardioprotective effects of animal grade piperazine citrate on isoproterenol-induced myocardial infarction in Wistar rats: biochemical and histopathological evaluation. African Journal of Pharmacy and Pharmacology. 2020; 14(8): 285-293. doi: 10.5897/AJPP.
  12. Suvarna S, Layton Ch. Bancroft J. Bancroft’s Theory and Practice of Histological Techniques. Chapters 10 and 12. 8th ed. China, Elsevier; 2019; 126:166. https://doi. org/10.1016/B978-0-7020-6864-5.01001-X.
  13. Yin S, Li J, Hu C, Chen X, Yao M ,Yan M, et al. CD133 positive hepatocellular carcinoma cells possess high capacity for tumorigenicity. International journal of cancer. 2007; 120(7):1444-1450. doi: 10.1002/ijc.22476.
  14. Buchwalow IB, Böcker W. Immunohistochemistry: Basics and Methods. Working with Antibodies. London, Springer Dordrecht Heidelberg; 2010: 31–39. doi: 10.1007/978-3-642-04609-4.
  15. Moustafa A. Age-related changes in the immunohistochemical localization pattern of α-smooth muscle actin and vimentin in rat testis. The Egyptian Journal of Histology. 2012;35. 412-423. doi: 10.1097/01.EHX.0000418134.14989.85.
  16. Nallagangula KS, Nagaraj SK, Venkataswamy L, Chandrappa M. Liver fibrosis: a compilation on the biomarkers status and their significance during disease progression. Future Sci OA. 2017;4(1):FSO250. doi: 10.4155/fsoa-2017-0083. PMID: 29255622; PMCID: PMC5729599.
  17. Zaghloul D, Gad-El-Rab WM, Bushra RR, Farahat AA. The possible protective role of methionine against sodium fluoride-induced pancreatic changes in the adult male albino rat: a histological, immunohistochemical and morphometric study. Egyptian Journal of Histology. 2019;42(2):285-296. doi: 21608/EJH.2019.6198.1040.
  18. Abu-Dief E, Mohammed D, Abd El Haliem N, Mohammed S, El-Badry A. Impact of omega-3 fatty acids on evaluation of carbon tetrachloride- (CCL4) induced liver cirrhosis in mice: A histological and immunohistochemical study. Egyptian Journal of Histology. 2018;41(1):61-72. doi: 10.21608/EJH.2018.7522.
  19. Claus Kordes, Iris Sawitza, Alexis Müller-Marbach, Niloofar Ale-Agha, Verena Keitel, Hanne Klonowski-Stumpe, Dieter Häussinger, CD133+ hepatic stellate cells are progenitor cells. Biochemical and Biophysical Research Communications. 2007; 352(2): 410-417.
  20. Carpino G, Morini S, Ginanni Corradini S, Franchitto A, Merli M, Siciliano M, Gentili F, Onetti Muda A, Berloco P, Rossi M, Attili AF, Gaudio E. Alpha-SMA expression in hepatic stellate cells and quantitative analysis of hepatic fibrosis in cirrhosis and in recurrent chronic hepatitis after liver transplantation. Dig Liver Dis. 2005 May;37(5):349-56. doi: 10.1016/j.dld.2004.11.009. PMID: 15843085.
  21. Dawson B, Trapp RG. Basic & Clinical Biostatistics. In: Basic & Clinical Biostatistics. 4th ed. New York, Lange Medical Books / McGraw-Hill Medical Publishing Division.; 2004:162–189.
  22. Ukairo DI, Ojiako OA, Nwaoguikpe R, Ibegbulem CO, Igwe CU, Iheme CI. Ameliorative effects of Nigerian recipes on histopathological and immunohistochemical changes of CCL4-induced hepatic fibrosis in male Wistar rats. J Pharmacogn Phytochem. 2017;6(6):1624-1631.
  23. Guang HY, Yun HC. Sparassis crispa Attenuates Carbon Tetrachloride-Induced Hepatic Injury in Rats. Korean J Phys Anthropo. 2014; 27(3): 113-122. doi.org/10.11637/kjpa.
  24. Ahmed KA, Saikat ASM, Moni A, Kakon SAM, Islam R, Uddin J. Lactoferrin: potential functions, pharmacological insights, and therapeutic promises. J Adv Biotechnol Exp Ther. 2021;4(2):223-237. doi: 10.5455/jabet.2021.d123.
  25. SH A, Alattar HA, Farid AS, Farah KM. Influence of lactoferrin on immune response in rats intoxicated by diazinon. Benha Veterinary Medical Journal. 2018; 34(2):169-181. doi: 10.21608/bvmj.2018.29426.
  26. Frank D, Savir S, Gruenbaum BF, Melamed I, Grinshpun J, Kuts R, Knyazer B, Zlotnik A, Vinokur M, Boyko M. Inducing Acute Liver Injury in Rats via Carbon Tetrachloride (CCl4) Exposure Through an Orogastric Tube. J Vis Exp. 2020; 28(158):10.3791/60695. doi: 10.3791/60695. PMID: 32420997; PMCID: PMC7859859.
  27. Lotowska JM, Sobaniec-Lotowska ME, Lebensztejn DM, Daniluk U, Sobaniec P, Sendrowski K, et al. Ultrastructural Characteristics of Rat Hepatic Oval Cells and Their Intercellular Contacts in the Model of Biliary Fibrosis: New Insights into Experimental Liver Fibrogenesis. Gastroenterol Res Pract. 2017;2017:2721547. doi: 10.1155/2017/2721547. Epub 2017 Jul 9. PMID: 28769978; PMCID: PMC5523291.
  28. Emam MA, Farouk SM, Abdo M. The ameliorative potential of probiotics and/or silymarin on thioacetamide induced hepatotoxicity in rats: histological and immunohistochemical study. J. Morphol. 2018;36(2):661-669. doi: 10.4067/S0717-95022018000200661.
  29. Omara E, El-Toumy S, Shabanaa M, Farraga A, Nadac S, Shafeea N. The Antifibrotic Effect of Zilla Spinosa Extracts Targeting Apoptosis in CCl4-Induced Liver Damage in Rats. Journal of The Arab Society for Medical Research. 2018; 13:129– doi: 10.4103/jasmr.jasmr_29_18.
  30. Elufioye T, Habtemariam S. Hepatoprotective Effects of Rosmarinic Acid: Insight into Its Mechanisms of Action. Biomedicine & Pharmacotherapy. 2019; 112:108600-108611. doi: 10.1016/j.biopha.2019.108600.
  31. Farid AS, El Shemy MA, Nafie E, Hegazy AM, Abdelhiee EY. Anti-inflammatory, anti-oxidant and hepatoprotective effects of lactoferrin in rats. Drug Chem. Toxicol. 2021;44:286–293. doi: 10.1080/01480545.2019.1585868.
  32. Tanaka H, Gunasekaran S, Saleh DM, Alexander WT, Alexander DB, Ohara H, et al. Effects of oral bovine lactoferrin on a mouse model of inflammation associated colon cancer. Cell Biol. 2021;99:159–165. doi: 10.1139/bcb-2020-0087.
  33. Venkatanarayana G, Sudhakara G, Rajeswaramma K, Indira P. Combined Effect of Curcumin and Vitamin E against CCl4 Induced Liver Injury in Rats. American Journal of Life Sciences. 2013; 1, (3):117-124. doi: 10.11648/j.ajls.20130103.17.
  34. Naz S, Irshad F, Mawani, H. CCl4 Induced Liver Injury; Aqueous Extract of Ginkgo Biloba (Gkbe) Ameliorates Cytoplasmic and Mitochondrial Enzymes. The professional medical journal.2017;24,739-744. doi: https://doi.org/10.29309/TPMJ/2017.24.05.1298.
  35. Hassan N, Soliman G, Okasha E, Shalaby A. Histological, Immunohistochemical, and Biochemical Study of Experimentally Induced Fatty Liver in Adult Male Albino Rat and The Possible Protective Role of Pomegranate. Journal of Microscopy and Ultrastructure. 2018;6(1):44-55. doi: 10.4103/JMAU.JMAU_5_18. PMID: 30023266; PMCID: PMC6014250.
  36. Abdeen M, Zaghloul D, Saleh R, Abdelmalek A. Histomorphometric Evidence of Hepatic Recovery of Rats Fed Repeatedly Heated Palm Oil. Egyptian Journal of Histology. 2022;45(1),115-124. doi: 10.21608/ejh.2021.57674.1415.
  37. Kumar V, Abbas A, Aster J. Robbin Basic Pathology. Chapter (16) Liver and Gallbladder. 10th Canada. Philadelphia. 2017.
  38. Ogaly HA, Aldulmani SAA, Al-Zahrani FAM, Abd-Elsalam RM. D-Carvone Attenuates CCl4-Induced Liver Fibrosis in Rats by Inhibiting Oxidative Stress and TGF-ß 1/SMAD3 Signaling Pathway. Biology (Basel). 2022;12;11(5):739. doi: 10.3390/biology11050739. PMID: 35625467; PMCID: PMC9138456.
  39. Moreno MG, Chávez E, Aldaba-Muruato LR, Segovia J, Vergara P, Tsutsumi V, et al. Coffee prevents CCl(4)-induced liver cirrhosis in the rat. Hepatol Int. 2011;5(3):857-863. doi: 10.1007/s12072-010-9247-6. Epub 2011 Jan 25. PMID: 21484136.
  40. Abd Elzaher F, Moussa M, Raafat M, Emara M. Histological Effect of Platelet Rich Plasma on CCL4 Induced Liver Fibrosis in Adult Albino Rat. Egyptian Journal of Histology. 2021;44(4),932-940. doi: 10.21608/ejh.2020.51054.1390.
  41. Khedr NF, and Khedr Antioxidant and Anti-inflammatory Effects of Curcumin on CCl4 – induced Liver Fibrosis in Rats. Am. J. Biomed. Sci. 2014, 6(3), 191-200. doi:10.5099/aj140300191.
  42. Fujii T, Fuchs BC, Yamada S, Lauwers GY, Kulu Y, Goodwin JM, et al. Mouse model of carbon tetrachloride induced liver fibrosis: Histopathological changes and expression of CD133 and epidermal growth factor. BMC Gastroenterol. 2010;10:79. doi: 10.1186/1471-230X-10-79. PMID: 20618941; PMCID.
  43. Lee Y, Cho I, Kim J, Lee M, Ku S, Choi J, et al. Hepatoprotective Effects of Blue Honeysuckle on CCl4-Induced Acute Liver Damaged Mice. Food Science & Nutrition. 2019;7(1):322– doi: 10.1002/fsn3.893.
  44. Eldesouky NA, Elshopakey GE, Yusuf MS, Abdelhamid FM, Risha EF. Association between vitamin d deficiency and ccl4 mediated hepatic inflammation in male albino rats; evaluation of some biochemical and antioxidant marker. Anim. Vet. Sci. 2021;9(7):994-1003. doi:10.17582/journal.aavs/2021/9.7.994.1003
  45. Zowail M, Khater E, Waer H, Eltahawy N, Abd El-Hady A. Curative Effect of Bone Marrow Cells Transplantation and/or Low Dose Gamma Irradiation on Liver Injuries Induced by Carbon Tetrachloride. The Egyptian Journal of Hospital Medicine. 2012;46: 96-114. doi: 21608/EJHM.2012.16362.
  46. Amer MA, Othman AI, El-Missiry MA, Farag AA, Amer ME. Proanthocyanidins attenuated liver damage and suppressed fibrosis in CCl4-treated rats. Environ Sci Pollut Res Int. 2022;29(60):91127-91138. doi: 10.1007/s11356-022-22051-7. PMID: 35881285; PMCID: PMC9722827.
  47. Fathy M, Okabe M, Saad Eldien H, Yoshida T. AT-MSCs Antifibrotic Activity Is Improved by Eugenol Through Modulation of TGF-Β/Smad Signaling Pathway in Rats. Molecules. 2020;25(2):348-365. doi: 3390/molecules25020348.
  48. Chen HA, Chiu CC, Huang CY, Chen LJ, Tsai CC, Hsu TC, et al. Lactoferrin Increases Antioxidant Activities and Ameliorates Hepatic Fibrosis in Lupus-Prone Mice Fed with a High-Cholesterol Diet. Med. Food. 2016;19:670–677. doi: 10.1089/jmf.2015.3634.