In Vitro Effects of Curcumin on Transforming Growth Factor-β-mediated Non-Smad Signaling Pathway, Oxidative Stress, and Pro‐inflammatory Cytokines Production with Human Vascular Smooth Muscle Cells

  • Amirhooman Asadi Veterinary Medicine, Faculty of Veterinary Medicine, Karaj Branch, Islamic Azad University, Karaj, Iran
  • Davood Yaghobi Nezhad Department of Agriculture Biotechnology, Faculty of Agriculture, Payame Noor University, Tehran, Iran
  • Amirreza Rafie Javazm Student Research Committee, Faculty of Nursing and Midwifery, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
  • Parisa Khanicheragh Department of Clinical Biochemistry, Lorestan University of Medical Sciences, Khorram abad, Iran
  • Ladan Mashouri Department of Genetics, Faculty of Science, Shahrekord University, Shahrekord, Iran
  • Fatemeh Shakeri Jahrom University of Medical Sciences, Jahrom, Iran
  • Mojtaba Abbasi Veterinary Medicine, Faculty of Veterinary Medicine, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran
  • Mahya Sadat Afrazian Department of Biology, Faculty of Basic Sciences, University of Shiraz, Shiraz, Iran
  • Zahra Niknam Student Research Committee, Department of Clinical Biochemistry, Faculty of Medicine, Ahvaz Jundishapor University of Medical Science, Ahvaz, Iran
  • Omid Abazari Mail Department of Biochemistry, School of Medicine, Shahid Sadoughi University of Medical Sciences and Health Services, Yazd, Iran
Curcumin, Interleukin-6, Smad2 protein, Transforming growth factor beta, Tumor necrosis factor-alpha


Transforming growth factor-β (TGF-β) induces pro-inflammatory cytokines expression including interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) and these cytokines are associated with the development of atherosclerosis. Curcumin has anti-atherogenic effects and anti-inflammatory properties in the vascular wall, but the relative mechanisms are almost unknown. In the present study, we investigate the effect of curcumin on modulating the pro-inflammatory action of TGF-β in human vascular smooth muscle cells (VSMCs) and its molecular mechanisms. Cultured VSMCs were seeded into several groups: a control group (untreated group), a group treated with TGF-β, and several groups treated with TGF-β plus inhibitors. The cells were pre-treated with diphenyleneiodonium chloride, DPI, (20 μM), curcumin (5, 10 and 20 μM) and N-Acetyl-L-Cysteine, NAC, (10 mM) and then TGF-β (5 ng/mL) was added to the culture medium. The mRNA levels of IL-6 and TNF-α were detected by quantitative Real-Time Polymerase Chain Reaction. For monitoring the Smad2 linker region phosphorylation (pSmad2L), the western-blotting technique was applied and reactive oxygen species (ROS) generation was measured by utilizing 2′,7′-dichlorofluorescein diacetate-based assay. TGF-β increased the mRNA expression of IL-6 (p=0.02 and p=0.001) and TNF-α (p =0.014 and p = 0.001) in a time-dependent manner, ROS production (p=0.03) and Smad2L phosphorylation (p=0.015). Pre-treatment with curcumin, DPI and NAC inhibited TGF-β–induced IL-6 (p=0.04) and TNF-α (p=0.001) mRNA expression, Smad2L phosphorylation (p=0.02) and ROS production (0.03). Pharmacological inhibition by Curcumin blocks TGF-β–induced ROS production, Smad2L phosphorylation, and IL-6 and TNF-α mRNA expression in human VSMCs.


1. MEMBERS WG, Benjamin EJ, Blaha MJ, Chiuve SE, Cushman M, Das SR, et al. Heart disease and stroke statistics—2017 update: a report from the American Heart Association. Circulation 2017;135(10):e146.
2. Moss JW, Ramji DP. Cytokines: roles in atherosclerosis disease progression and potential therapeutic targets. Future Med Chem 2016;8(11):1317-30.
3. Ramji DP, Davies TS. Cytokines in atherosclerosis: Key players in all stages of disease and promising therapeutic targets. Cytokine Growth Factor Rev 2015;26(6):673-85.
4. Tousoulis D, Oikonomou E, Economou EK, Crea F, Kaski JC. Inflammatory cytokines in atherosclerosis: current therapeutic approaches. Eur Heart J 2016;37(22):1723-32.
5. Hashizume M, Mihara M. Atherogenic effects of TNF-α and IL-6 via up-regulation of scavenger receptors. Cytokine 2012;58(3):424-30.
6. Bernberg E, Ulleryd MA, Johansson ME, Bergström GM. Social disruption stress increases IL-6 levels and accelerates atherosclerosis in ApoE−/− mice. Atherosclerosis 2012;221(2):359-65.
7. Libby P, Lichtman AH, Hansson GK. Immune effector mechanisms implicated in atherosclerosis: from mice to humans. Immunity 2013;38(6):1092-104.
8. Xie C, Kang J, Ferguson ME, Nagarajan S, Badger TM, Wu X. Blueberries reduce pro‐inflammatory cytokine TNF‐α and IL‐6 production in mouse macrophages by inhibiting NF‐κB activation and the MAPK pathway. Mol Nutr Food Res 2011;55(10):1587-91.
9. Michaeloudes C, Sukkar MB, Khorasani NM, et al. TGF-β regulates Nox4, MnSOD and catalase expression, and IL-6 release in airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 2010;300(2):L295-L304.
10. Weiss A, Attisano L. The TGFbeta superfamily signaling pathway. Wiley Interdiscip Rev Dev Biol 2013;2(1):47-63.
11. Yoshimura A, Wakabayashi Y, Mori T. Cellular and molecular basis for the regulation of inflammation by TGF-β. J Biochem 2010;147(6):781-92.
12. Mu Y, Gudey SK, Landstrom M. Non-Smad signaling pathways. Cell Tissue Res 2012;347(1):11-20.
13. Zhang YE. Non-Smad Signaling Pathways of the TGF-beta Family. Cold Spring Harb Perspect Biol 2017;9(2).
14. Boudreau HE, Casterline BW, Rada B, Korzeniowska A, Leto TL. Nox4 involvement in TGF-beta and SMAD3-driven induction of the epithelial-to-mesenchymal transition and migration of breast epithelial cells. Free Radic Biol Med 2012;53(7):1489-99.
15. Bryk D, Olejarz W, Zapolska-Downar D. The role of oxidative stress and NADPH oxidase in the pathogenesis of atherosclerosis. Postepy Hig Med Dosw (Online) 2017;71(10):57-68.
16. Jiang F, Zhang Y, Dusting GJ. NADPH oxidase-mediated redox signaling: roles in cellular stress response, stress tolerance, and tissue repair. Pharmacol Rev 2011;63(1):218-42.
17. Lozhkin A, Vendrov AE, Pan H, Wickline SA, Madamanchi NR, Runge MS. NADPH oxidase 4 regulates vascular inflammation in aging and atherosclerosis. J Mol Cell Cardiol 2017;102:10-21.
18. Drummond GR, Selemidis S, Griendling KK, Sobey CG. Combating oxidative stress in vascular disease: NADPH oxidases as therapeutic targets. Nat Rev Drug Discov 2011;10(6):453-71.
19. Rahimi HR, Kazemi Oskuee R. Curcumin from traditional Iranian medicine to molecular medicine. Razavi Int J Med 2014;2(2):e19982.
20. Mirzaei H, Naseri G, Rezaee R, Mohammadi M, Banikazemi Z, Mirzaei HR, et al. Curcumin: A new candidate for melanoma therapy? Int J Cancer 2016;139(8):1683-95.
21. Shafabakhsh R, Pourhanifeh MH, Mirzaei HR, Sahebkar A, Asemi Z, Mirzaei H. Targeting regulatory T cells by Curcumin: A potential for cancer immunotherapy. Pharmacol Res 2019:104353.
22. Zou J, Zhang S, Li P, Zheng X, Feng D. Supplementation with curcumin inhibits intestinal cholesterol absorption and prevents atherosclerosis in high-fat diet–fed apolipoprotein E knockout mice. Nutr Res 2018;56:32-40.
23. Zhang S, Zou J, Li P, Zheng X, Feng D. Curcumin protects against atherosclerosis in apolipoprotein e-knockout mice by inhibiting toll-like receptor 4 expression. J Agric Food Chem 2018;66(2):449-56.
24. Epstein J, Sanderson IR, MacDonald TT. Curcumin as a therapeutic agent: the evidence from in vitro, animal and human studies. Br J Nutr 2010;103(11):1545-57.
25. Hu Y, Liang H, Du Y, Zhu Y, Wang X. Curcumin inhibits transforming growth factor-β activity via inhibition of Smad signaling in HK-2 cells. Am J Nephrol 2010;31(4):332-41.
26. Zhong S, Zhao L, Wang Y, Zhang C, Liu J, Wang P, et al. CD36 deficiency aggravates macrophage infiltration and hepatic inflammation by up-regulating MCP-1 expression of hepatocytes through HDAC2-dependant pathway. Antioxidants & Redox Signaling. 2017.
27. Pyla R, Poulose N, Jun JY, Segar L. Expression of conventional and novel glucose transporters, GLUT1,-9,-10, and-12, in vascular smooth muscle cells. Am J Physiol Cell Physiol 2013;304(6):C574-C89.
28. Zhang YE. Non-Smad signaling pathways of the TGF-β family. ColdSpringHarbPerspectBiol 2017;9(2):a022129.
29. Yang Y, Wang Z, Yang H, Wang L, Gillespie SR, Wolosin JM, et al. TRPV1 potentiates TGFβ-induction of corneal myofibroblast development through an oxidative stress-mediated p38-SMAD2 signaling loop. PLoS One 2013;8(10):e77300.
30. Cho S, Yu S-L, Kang J, Jeong BY, Lee HY, Park CG, et al. NADPH oxidase 4 mediates TGF-β1/Smad signaling pathway induced acute kidney injury in hypoxia. PLoS One 2019;14(7):e0219483.
31. Cavalla F, Osorio C, Paredes R, Valenzuela MA, García-Sesnich J, Sorsa T, et al. Matrix metalloproteinases regulate extracellular levels of SDF-1/CXCL12, IL-6 and VEGF in hydrogen peroxide-stimulated human periodontal ligament fibroblasts. Cytokine 2015;73(1):114-21.
32. Zhang H, Jiang Z, Chang J, Li X, Zhu H, Lan HY, et al. Role of NAD (P) H oxidase in transforming growth factor‐β1‐induced monocyte chemoattractant protein‐1 and interleukin‐6 expression in rat renal tubular epithelial cells. Nephrology 2009;14(3):302-10.
33. Meng Z, Yan C, Deng Q, Gao D-f, Niu X-l. Curcumin inhibits LPS-induced inflammation in rat vascular smooth muscle cells in vitro via ROS-relative TLR4-MAPK/NF-κB pathways. Acta Pharmacol Sin 2013;34(7):901.
34. Ghasemi F, Shafiee M, Banikazemi Z, Pourhanifeh MH, Khanbabaei H, Shamshirian A, et al. Curcumin inhibits NF-kB and Wnt/β-catenin pathways in cervical cancer cells. Pathol Res Pract 2019:152556.
35.Jiang S, Han J, Li T, Xin Z, Ma Z, Di W, et al. Curcumin as a potential protective compound against cardiac diseases. Pharmacol Res. 2017;119:373-83.
36. Bavarsad K, Barreto GE, Sahebkar A. Protective effects of curcumin against ischemia-reperfusion injury in the nervous system. Mol Neurobiol 2019;56(2):1391-404.
37. Kelany ME, Hakami TM, Omar AH. Curcumin improves the metabolic syndrome in high-fructose-diet-fed rats: role of TNF-α, NF-κB, and oxidative stress. Can J Physiol Pharmacol 2016;95(2):140-50.
How to Cite
Asadi A, Yaghobi Nezhad D, Rafie Javazm A, Khanicheragh P, Mashouri L, Shakeri F, Abbasi M, Afrazian MS, Niknam Z, Abazari O. In Vitro Effects of Curcumin on Transforming Growth Factor-β-mediated Non-Smad Signaling Pathway, Oxidative Stress, and Pro‐inflammatory Cytokines Production with Human Vascular Smooth Muscle Cells. Iran J Allergy Asthma Immunol. 19(1):84-93.
Original Article(s)