Itaconic Acid as A Differential Transcription Regulator of Apoptosis and Autophagy Pathways Genes: A Rat Adipose Mesenchymal Stem Cells Model

Document Type : Original Article


1 Department of Basic Sciences, Division of Biochemistry and Molecular Biology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran

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


Objective: Itaconate, a novel regulatory immunometabolite, is synthesized by inflammatory macrophage. It acts as an anti-inflammatory mediator and regulates several metabolic and signaling pathways particularly Nrf2 pathway. The immunometabolites can affect the stemness potency, differentiation ability and viability of stem cells, but little is known about the critical function of Itaconate on the stem cell fate. The objective of the present study was to determine the regulatory effects of Itaconic acid on the cell viability and transcription of apoptosis and autophagy pathways genes in the rat adipose derived mesenchymal stem cells (ADMSCs).
Materials and Methods: In this experimental study, the ADMSCs were incubated with 125 μM and 250 μM dimethyl
itaconate (DMI) for 24 hours or 48 hours. The expression of apoptosis pathway genes (Bax, Bcl2, Caspase 3, Fas, Fadd and Caspase 8) and autophagy pathway genes (Atg12, Atg5, Beclin, Lc3b and P62) were determined using real time polymerase chain reaction (PCR) assay. Using the ELISA method, cellular level of phospho-NRF2 protein was measured.
Results: The results indicated that DMI increased the expression of NRF2 protein, altered the expression of some
apoptosis genes (Fadd, Bax and Bcl2), and changed the expression of some autophagy related genes (Lc3b, Becline and P62) in ADMSCs. DMI had no obvious effect on the transcription of caspases enzymes.
Conclusion: Because autophagy activation and apoptosis suppression can protect stem cells against environmental stress, it seems Itaconate can affect the functions and viability of ADMSCs via converse regulation of these pathways.


  1. Argentati C, Morena F, Bazzucchi M, Armentano I, Emiliani C, Martino S. Adipose stem cell translational applications: from bench-tobedside. Int J Mol Sci. 2018; 19(11): 3475.
  2. Han Y, Li X, Zhang Y, Han Y, Chang F, Ding J. Mesenchymal stem cells for regenerative medicine. Cells. 2019; 8(8): 886.
  3. Kizil C, Kyritsis N, Brand M. Effects of inflammation on stem cells: together they strive? EMBO Rep. 2015; 16(4): 416-426.
  4. Weiss AR, Dahlke MH. Immunomodulation by mesenchymal stem cells (MSCs): mechanisms of action of living, apoptotic, and dead MSCs. Front Immun. 2019; 10: 1191.
  5. Wang M, Yuan Q, Xie L. Mesenchymal stem cell-based immunomodulation:properties and clinical application. Stem Cells Int. 2018; 2018.
  6. Planat-Benard V, Varin A, Casteilla L. MSCs and inflammatory cells crosstalk in regenerative medicine: concerted actions for optimized resolution driven by energy metabolism. Front Immunol. 2021; 12: 626755.
  7. McKee C, Chaudhry GR. Advances and challenges in stem cell culture. Colloids Surf B Biointerfaces. 2017; 159: 62-77.
  8. Cruciani S, Santaniello S, Montella A, Ventura C, Maioli M. Orchestrating stem cell fate: novel tools for regenerative medicine. World J Stem Cells. 2019; 11(8): 464.
  9. Chen X, He Y, Lu F. Autophagy in stem cell biology: a perspective on stem cell self-renewal and differentiation. Stem Cells Int. 2018; 21; 2018.
  10. Fairlie WD, Tran S, Lee EF. Crosstalk between apoptosis and autophagy signaling pathways. Int Rev Cell Mol Biol. 2020; 352: 115- 158.
  11. Liu WJ, Ye L, Huang WF, Guo LJ, Xu ZG, Wu HL, et al. p62 links the autophagy pathway and the ubiqutin–proteasome system upon ubiquitinated protein degradation. Cell Mol Biol Lett. 2016; 21(1): 1-4.
  12. Dang S, Yu ZM, Zhang CY, Zheng J, Li KL, Wu Y, et al. Autophagy promotes apoptosis of mesenchymal stem cells under inflammatory microenvironment. Stem Cell Res Ther. 2015; 6(1): 1-9.
  13. Wang J, Xia J, Huang R, Hu Y, Fan J, Shu Q, et al. Mesenchymal stem cell-derived extracellular vesicles alter disease outcomes via endorsement of macrophage polarization. Stem Cell Res Therap. 2020: 11(1): 1-2.
  14. Zhao H, Shang Q, Pan Z, Bai Y, Li Z, Zhang H, et al. Exosomes from adipose-derived stem cells attenuate adipose inflammation and obesity through polarizing M2 macrophages and beiging in white adipose tissue. Diabetes. 2018; 67(2): 235-247.
  15. Michelucci A, Cordes T, Ghelfi J, Pailot A, Reiling N, Goldmann O, et al. Immune- responsive gene 1 protein links metabolism to immunity by catalyzing itaconic acid production. Proc Nat Acad Sci USA. 2013; 110: 7820-7825.
  16. Strelko CL, Lu W, Dufort FJ, Seyfried TN, Chiles TC, Rabinowitz JD, et al. Itaconic acid is a mammalian metabolite induced during macrophage activation. J Am Chem Soc. 2011; 133(41): 16386- 16389.
  17. Lampropoulou V, Sergushichev A, Bambouskova M, Nair S, Vincent EE, Loginicheva E, et al. Itaconate links inhibition of succinate dehydrogenase with macrophage metabolic remodeling and regulation of inflammation. Cell Metab. 2016; 24(1): 158-166.
  18. O’Neill LA, Artyomov MN. Itaconate: the poster child of metabolic reprogramming in macrophage function. Nat Rev Immun. 2019; 19(5): 273-281.
  19. Mills EL, Ryan DG, Prag HA, Dikovskaya D, Menon D, Zaslona Z, et al. Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1. Nature. 2018; 556(7699): 113-117.
  20. Németh B, Doczi J, Csete D, Kacso G, Ravasz D, Adams D, et al. Abolition of mitochondrial substratelevel phosphorylation by itaconic acid produced by LPS- induced Irg1 expression in cells of murine macrophage lineage. FASEB J. 2016; 30(1): 286-300.
  21. Hooftman A, Angiari S, Hester S, Corcoran SE, Runtsch MC, Ling C, et al. The immunomodulatory metabolite itaconate modifies NLRP3 and inhibits inflammasome activation. Cell Metab. 2020; 32(3): 468-478.
  22. Hashemi Tabar M, Tabandeh MR, Moghimipour E, Dayer D, Ghadiri AA, Allah Bakhshi E, et al. The combined effect of Pdx1 overexpression and Shh manipulation on the function of insulin-producing cells derived from adipose-tissue stem cells. FEBS Open Bio. 2018; 8(3): 372-382.
  23. Bambouskova M, Gorvel L, Lampropoulou V, Sergushichev A, Loginicheva E, Johnson K, et al. Electrophilic properties of itaconate and derivatives regulate the IkappaBzeta-ATF3 inflammatory axis. Nature. 2018; 556(7702): 501-504.
  24. Orellana EA, Kasinski AL. Sulforhodamine B (SRB) assay in cell culture to investigate cell proliferation. Bio Protoc. 2016; 6(21): e1984.
  25. Sun KA, Li Y, Meliton AY, Woods PS, Kimmig LM, Cetin-Atalay R, et al. Endogenous itaconate is not required for particulate matterinduced NRF2 expression or inflammatory response. eLife. 2020; 9: e54877.
  26. Muri J, Wolleb H, Broz P, Carreira EM, Kopf M. Electrophilic Nrf2 activators and itaconate inhibit inflammation at low dose and promote IL-1β production and inflammatory apoptosis at high dose. Redox Biol. 2020; 36: 101647.
  27. Yi Z, Deng M, Scott MJ, Fu G, Loughran PA, Lei Z, et al. Immuneresponsive gene 1/Itaconate activates nuclear factor erythroid 2– related factor 2 in hepatocytes to protect against liver ischemia– reperfusion injury. Hepatology. 2020; 72(4): 1394-1411.
  28. Chang NC. Autophagy and stem cells: self-eating for self-renewal. Front Cell Dev Biol. 2020; 8: 138.
  29. Liu WJ, Ye L, Huang WF, Guo LJ, Xu ZG, Wu HL, et al. p62 links the autophagy pathway and the ubiqutin–proteasome system upon ubiquitinated protein degradation. Cell Mol Biol Lett. 2016; 21(1): 1-4.
  30. Yan XY, Zhong XR, Yu SH, Zhang LC, Liu YN, Zhang Y, et al. p62 aggregates mediated Caspase 8 activation is responsible for progression of ovarian cancer. J Cell Mol Med. 2019; 23(6): 4030- 4042.
  31. Chen Y, Zhang W, Guo X, Ren J, Gao A. The crosstalk between autophagy and apoptosis was mediated by phosphorylation of Bcl- 2 and beclin1 in benzene-induced hematotoxicity. Cell Death Dis. 2019; 10(10): 1-5.
  32. Zhu Y, Zhao L, Liu L, Gao P, Tian W, Wang X, et al. Beclin 1 cleavage by caspase-3 inactivates autophagy and promotes apoptosis. Protein Cell. 2010; 1(5): 468-477.
  33. Mnich K, Koryga I, Pakos-Zebrucka K, Thomas M, Logue SE, Eriksson LA, et al. The stressosome, a caspase-8-activating signalling complex assembled in response to cell stress in an ATG5- mediated manner. J Cell Mol Med. 2021; 25(18): 8809-8820.
  34. Katsuragi Y, Ichimura Y, Komatsu M. Regulation of the Keap1-Nrf2 pathway by p62/SQSTM1. Curr Opin Toxicol. 2016; 1: 54-61.
  35. Dai X, Yan X, Wintergerst KA, Cai L, Keller BB, Tan Y. Nrf2: redox and metabolic regulator of stem cell state and function. Tren Mol Med. 2020; 26(2): 185-200.
  36. Jiang T, Harder B, De La Vega MR, Wong PK, Chapman E, Zhang DD. p62 links autophagy and Nrf2 signaling. Free Radic Biol Med. 2015; 88: 199-204.
  37. Tao J, Wang H, Zhai Y, Park H, Wang J, Ji F, et al. Downregulation of Nrf2 promotes autophagy-dependent osteoblastic differentiation of adipose-derived mesenchymal stem cells. Exp Cell Res. 2016; 349(2): 221-229.