Effects of exosomes of mesenchymal stem cells on cholesterol-induced hepatic fibrogenesis

Document Type : Original Article


1 Cellular and Molecular Research Center, Medical Basic Sciences Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

2 Cellular and Molecular Research Center, Medical Basic Science Research Institute, Department of Clinical Biochemistry, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran


Objective(s): Free cholesterol in the diet can cause liver fibrosis by accumulating in Hepatic stellate cells (HSCs). The rate of mortality of this disease is high worldwide and there is no definite remedy for it, but might be treated by anti-fibrotic therapies. MSCs-derived exosomes are known as the new mechanism of cell-to-cell communication, showing that exosomes can be used as a new treatment. In this study, we investigated the ability of exosomes of WJ-MSCs as a new remedy to reduce cholesterol-induced liver fibrosis in the LX2 cell line.
Materials and Methods: MSCs were isolated from Wharton’s jelly of the umbilical cord and the exosomes were extracted. The LX2 cell line was cultured in DMEM medium with 10% FBS, then cells were treated with 75 and 100 μM concentrations of cholesterol for 24 hr. The mRNA expression of TGF-β, αSMA, and collagen1α genes, and the level of Smad3 protein were measured to assess liver fibrosis. 
Results: Cholesterol increased the expression of TGF-β, αand -SMA, and collagen1α genes by increasing the phosphorylation of the Smad3 protein. Treatment with Exosomes significantly reduced the expression of TGF-β, α-SMA, and collagen1α genes (fibrosis genes). Treatment with exosomes prevented the activation of HSCs by inhibiting the phosphorylation of the Smad3 protein. 
Conclusion: The exosomes of WJ-MSCs can inhibit the TGFβ/Smad3 signaling pathway preventing further activation of HSCs and progression of liver fibrosis. So, the exosomes of WJ-MSCs s could be introduced as a treatment for liver failure.


Main Subjects

1.    Afarin R, Rezaei HB, Yaghooti H, Mohammadtaghvaei N. Fibroblast Growth Factor 21 Reduces Cholesterol-Induced Hepatic Fibrogenesis by Inhibiting TGF-β/Smad3C Signaling Pathway in LX2 Cells. Hepat Mon 2021; 21:e113321.
2.    Shakerian E, Akbari R, Mohammadtaghvaei N, Gahrooie MM, Afarin R. Quercetin Reduces Hepatic Fibrogenesis by Inhibiting TGF-β/Smad3 Signaling Pathway in LX-2 Cell Line. Jundishapur J Nat Pharm Prod 2022; 17:e113484.
3.    Wang PP, Xie DY, Liang XJ, Peng L, Zhang GL, Ye YN, et al. direct mesenchymal stem cells contact synergize to inhibit hepatic stellate cells activation through TLR4/NF-kB pathway. PLoS One 2012 ;7:e43408.
4.    Afarin R, Babaahmadi RH, Yaghouti SH, Mohammad TN. The effect of cholesterol on the activation of TGF-β/Smad3C signaling pathway in hepatic stellate cells and its role in the progression of liver fibrogenesis. Isfahan J Med School 2021; 212-218.
5.    Wu J, Zern MA. Hepatic stellate cells: a target for the treatment of liver fibrosis. J Gastroenterol 2000; 35:665-672.
6.    Kawaratani H, Tsujimoto T, Douhara A, Takaya H, Moriya K, Namisaki T, et al. The effect of inflammatory cytokines in alcoholic liver disease. Mediators Inflamm 2013; 2013:495156.
7.    Pinzani M, Marra F. Cytokine receptors and signaling in hepatic stellate cells. Semin Liver Dis 2001;21:397-416.
8.    Cong M, Iwaisako K, Jiang C, Kisseleva T. Cell signals influencing hepatic fibrosis. Int J Hepatol 2012 ;2012:158547.
9.    Yoshida K, Murata M, Yamaguchi T, Matsuzaki K. TGF-β/Smad signaling during hepatic fibro-carcinogenesis (review). Int J Oncol 2014; 45:1363-1371.
10.    Mohammadzadeh G, Afarin R, Bavarsad SS, Aslani F, Zadeh SA, Shakerian E. Comparison of the effects of cholesterol, palmitic acid, and glucose on activation of human hepatic stellate cells to induce liver fibrosis. J Diabetes Metab Disord 2022; 21:1531-1538.
11.    Teratani T, Tomita K, Suzuki T, Oshikawa T, Yokoyama H, Shimamura K, et al. A high-cholesterol diet exacerbates liver fibrosis in mice via accumulation of free cholesterol in hepatic stellate cells. Gastroenterology 2012; 142:152-164.e10.
12.    Gressner AM, Weiskirchen R. Modern pathogenetic concepts of liver fibrosis suggest stellate cells and TGF-beta as major players and therapeutic targets. J Cell Mol Med 2006; 10:76-99.
13.    Liu X, Hu H, Yin JQ. Therapeutic strategies against TGF-beta signaling pathway in hepatic fibrosis. Liver Int 2006; 26:8-22.
14.    Hammam OA, Elkhafif N, Attia YM, Mansour MT, Elmazar MM, Abdelsalam RM, et al. Wharton’s jelly-derived mesenchymal stem cells combined with praziquantel as a potential therapy for Schistosoma mansoni-induced liver fibrosis. Sci Rep 2016; 6:21005.
15.    Li T, Yan Y, Wang B, Qian H, Zhang X, Shen L, et al. Exosomes derived from human umbilical cord mesenchymal stem cells alleviate liver fibrosis. Stem Cells Dev 2013; 22:845-854.
16.    Tögel F, Weiss K, Yang Y, Hu Z, Zhang P, Westenfelder C. Vasculotropic, paracrine actions of infused mesenchymal stem cells are important to the recovery from acute kidney injury. Am J Physiol Renal Physiol 2007; 292:F1626-1635.
17.    Qu Y, Zhang Q, Cai X, Li F, Ma Z, Xu M, et al.  Exosomes derived from miR-181-5p-modified adipose-derived mesenchymal stem cells prevent liver fibrosis via autophagy activation. J Cell Mol Med 2017; 21:2491-2502.
18.    Tang Y, Zhou Y, Li HJ. Advances in mesenchymal stem cell exosomes: A review. Stem Cell Res Ther 2021; 12:71.
19.    Shibata N, Watanabe T, Okitsu T, Sakaguchi M, Takesue M, Kunieda T, et al. Establishment of an immortalized human hepatic stellate cell line to develop antifibrotic therapies. Cell Transplant 2003;12:499-507.
20.    van Dijk F, Hazelhoff CM, Post E, Prins GGH, Rombouts K, Poelstra K, et al. Design of a gene panel to expose the versatile role of hepatic stellate cells in human liver fibrosis. Pharmaceutics 2020; 12:278.
21.    Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 2006; 8:315-317.
22.    Twu YC, Lee TS, Lin YL, Hsu SM, Wang YH, Liao CY, et al. Niemann-Pick Type C2 Protein Mediates Hepatic Stellate Cells Activation by Regulating Free Cholesterol Accumulation. Int J Mol Sci 2016; 17:1122.
23.    Svegliati-Baroni G, De Minicis S, Marzioni M. Hepatic fibrogenesis in response to chronic liver injury: Novel insights on the role of cell-to-cell interaction and transition. Liver Int. 2008; 28:1052-1064.
24.    Friedman SL. Mechanisms of hepatic fibrogenesis. Gastroenterology. 2008; 134:1655-1669.
25.    Li JH, Huang XR, Zhu HJ, Johnson R, Lan HY. Role of TGF-beta signaling in extracellular matrix production under high glucose conditions. Kidney Int 2003; 63:2010-2019.
26.    Moreira RK. Hepatic stellate cells and liver fibrosis. Arch Pathol Lab Med 2007; 131:1728-1734.
27.    Rengasamy M, Singh G, Fakharuzi NA, Siddikuzzaman, Balasubramanian S, Swamynathan P, et al. Transplantation of human bone marrow mesenchymal stromal cells reduces liver fibrosis more effectively than Wharton’s jelly mesenchymal stromal cells. Stem Cell Res Ther 2017; 8:143.
28.    Csonka C, Baranyai T, Tiszlavicz L, Fébel H, Szűcs G, Varga ZV, et al. Isolated hypercholesterolemia leads to steatosis in the liver without affecting the pancreas. Lipids Health Dis 2017; 16:144.
29.    Hoekstra M, Out R, Kruijt JK, Van Eck M, Van Berkel TJ. Diet induced regulation of genes involved in cholesterol metabolism in rat liver parenchymal and Kupffer cells. J Hepatol 2005; 42:400-407.
30.    Matsuda A, Wang Z, Takahashi S, Tokuda T, Miura N, Hasegawa J. Upregulation of mRNA of retinoid binding protein and fatty acid binding protein by cholesterol enriched-diet and effect of ginger on lipid metabolism. Life Sci 2009; 84:903-907.
31.    Damania A, Jaiman D, Teotia AK, Kumar A. Mesenchymal stromal cell-derived exosome-rich fractionated secretome confers a hepatoprotective effect in liver injury. Stem Cell Res Ther 2018; 9:31.
32.    Berardis S, Dwisthi Sattwika P, Najimi M, Sokal EM. Use of mesenchymal stem cells to treat liver fibrosis: current situation and future prospects. World J Gastroenterol 2015; 21:742-758.
33.    Kharaziha P, Hellström PM, Noorinayer B, Farzaneh F, Aghajani K, Jafari F, et al. Improvement of liver function in liver cirrhosis patients after autologous mesenchymal stem cell injection: a phase I-II clinical trial. Eur J Gastroenterol Hepatol 2009; 21:1199-1205.
34.    Tomita K, Teratani T, Suzuki T, Shimizu M, Sato H, Narimatsu K, et al. Free cholesterol accumulation in hepatic stellate cells: mechanism of liver fibrosis aggravation in nonalcoholic steatohepatitis in mice. Hepatology 2014; 59:154-169.
35.    Seki E, De Minicis S, Osterreicher CH, Kluwe J, Osawa Y, Brenner DA, et al. TLR4 enhances TGF-beta signaling and hepatic fibrosis. Nat Med 2007; 13:1324-1332.
36.    Schwartz YSh, Dushkin MI, Komarova NI, Vorontsova EV, Kuznetsova IS. Cholesterol-induced stimulation of postinflammatory liver fibrosis. Bull Exp Biol Med 2008; 145:692-695.
37.    Nojehdehi S, Hashemi SM, Hesampour A. Isolation and characterization of exosomes separated from stem cells by ultra-centrifuge method. Res Med 2017; 73:244-250.
38.    Lou G, Chen Z, Zheng M, Liu Y. Mesenchymal stem cell-derived exosomes as a new therapeutic strategy for liver diseases. Exp Mol Med 2017; 49:e346.