Carbenoxolone Could Deteriorate Streptozotocin-induced Diabetes through Induction of Heat Shock Protein 70 and IFN-γ in C57BL/6 Mice
Type 1 diabetes (T1D), a spontaneous autoimmune disease, is associated with destruction of insulin-producing β-cells in the pancreas. Since some heat shock proteins (HSP), such as HSP70 exert a protective effect in both tissues and cells, the present study was conducted to elucidate the effects of carbenoxolone (CBX) as an HSP70 inducer on T1D. The disease was induced in male C57BL/6 mice using streptozotocin (STZ) and subjects were allocated to therapeutic 1 and therapeutic 2 groups, as well as negative and positive control groups. The treated mice (therapeutic 1 and therapeutic 2 groups) received 50 mg/kg CBX intraperitoneally every 24 hours, in the therapeutic 1 group the drug was injected before and after disease induction whereas in the therapeutic 2 group the drug was injected only after disease induction. Serum fasting blood sugar (FBS) level, cytokines production (Interferon-gamma (IFN-γ), Interleukin 10 (IL-10), and IL-17), serum HSP70 level and CD4+CD25+Foxp3+ regulatory T cell (Treg) frequency measurements were outperformed 14 days after the last STZ injection. Our results showed that in the treated groups, serum HSP70, IFN-γ, and IL-17 levels were increased in contrast to the untreated groups. The IL-10 level was markedly decreased in comparison to untreated diabetic mice (p<0.05). Moreover, it was found that the frequency of Tregs in treated mice was lower in comparison to the untreated mice but the difference was not significant (p>0.05). Our results confirm that CBX might through HSP70 induction, followed by increasing IFN-γ level leads to suppression of IL-10 production in diabetic mice resulted in toxic effects on pancreatic islet beta cells and deteriorating of disease.
1. Lukic ML, Pejnovic N, Lukic A. New Insight Into Early Events in Type 1 Diabetes: Role for Islet Stem Cell Exosomes. Diabetes 2014; 63(3):835-7.
2. Bradley LM, Asensio VC, Schioetz L-K, Harbertson J, Krahl T, Patstone G, et al. Islet-specific Th1, but not Th2, cells secrete multiple chemokines and promote rapid induction of autoimmune diabetes. The Journal of Immunology 1999; 162(5):2511-20.
3. Martin‐Orozco N, Chung Y, Chang SH, Wang YH, Dong C. Th17 cells promote pancreatic inflammation but only induce diabetes efficiently in lymphopenic hosts after conversion into Th1 cells. European journal of immunology 2009; 39(1):216-24.
4. Gomez-Tourino I, Arif S, Eichmann M, Peakman M. T cells in type 1 diabetes: instructors, regulators and effectors: a comprehensive review. Journal of autoimmunity 2016; 66:7-16.
5. Raz I, Eldor R, Naparstek Y. Immune modulation for prevention of type 1 diabetes mellitus. Trends in biotechnology 2005; 23(3):128-34.
6. Walker LS, von Herrath M. CD4 T cell differentiation in type 1 diabetes. Clinical & Experimental Immunology 2016; 183(1):16-29.
7. Shi B, Wang Z, Jin H, Chen YW, Wang Q, Qian Y. Immunoregulatory Cordyceps sinensis increases regulatory T cells to Th17 cell ratio and delays diabetes in NOD mice. International immunopharmacology 2009; 9(5):582-6.
8. Zdravkovic N, Shahin A, Arsenijevic N, Lukic ML, Mensah-Brown EP. Regulatory T cells and ST2 signaling control diabetes induction with multiple low doses of streptozotocin. Molecular immunology 2009; 47(1):28-36.
9. Pop SM, Wong CP, He Q, Wang Y, Wallet MA, Goudy KS, et al. The type and frequency of immunoregulatory CD4+ T-cells govern the efficacy of antigen-specific immunotherapy in nonobese diabetic mice. Diabetes 2007; 56(5):1395-402.
10. Van Eden W, Wick G, Albani S, Cohen I. Stress, heat shock proteins, and autoimmunity. Annals of the New York Academy of Sciences 2007; 1113(1):217-37.
11. Atalay M, Oksala NK, Laaksonen DE, Khanna S, Nakao C, Lappalainen J, et al. Exercise training modulates heat shock protein response in diabetic rats. Journal of Applied Physiology 2004; 97(2):605-11.
12. Thakur P, Nehru B. Long-term heat shock proteins (HSPs) induction by carbenoxolone improves hallmark features of Parkinson's disease in a rotenone-based model. Neuropharmacology 2014; 79:190-200.
13. Pockley AG, Muthana M, Calderwood SK. The dual immunoregulatory roles of stress proteins. Trends in biochemical sciences 2008; 33(2):71-9.
14. Millar DG, Garza KM, Odermatt B, Elford AR, Ono N, Li Z, et al. Hsp70 promotes antigen-presenting cell function and converts T-cell tolerance to autoimmunity in vivo. Nature medicine 2003; 9(12):1469-76.
15. Wieten L, Berlo SE, Corlinda B, van Kooten PJ, Singh M, van der Zee R, et al. Il-10 is critically involved in mycobacterial hsp70 induced suppression of proteoglycan-induced arthritis. PLOS one 2009; 4(1):e4186.
16. Moosavi MA, Asadi M, Asvadi Kermani I. Inhibition of survivin and its anti-apoptotic splice variant sur-∆ Ex3 genes expression followed by apoptosis through carbenoxolone in K562 cells. Arak Medical University Journal 2011; 14(4):86-96.
17. Moosavi M, Moasses GS, Asvadi KI, Hamzeiy H, Rahmati M, Ahmadi A, et al. carbenoxolone induces apoptosis and inhibits survivin and survivin-ΔEx3 genes expression in human leukemia K562 cells. Daru 2011 19(6): 455-61.
18. Kilpatrick K, Novoa JA, Hancock T, Guerriero CJ, Wipf P, Brodsky JL, et al. Chemical induction of Hsp70 reduces α-synuclein aggregation in neuroglioma cells. ACS chemical biology 2013; 8(7):1460-8.
19. Nagayama S-i, Jono H, Suzaki H, Sakai K, Tsuruya E, Yamatsu I, et al. Carbenoxolone, a new inducer of heat shock protein 70. Life sciences 2001; 69(24):2867-73.
20. Kawashima D, Asai M, Katagiri K, Takeuchi R, Ohtsuka K. Reinvestigation of the effect of carbenoxolone on the induction of heat shock proteins. Cell Stress and Chaperones 2009; 14(5):535-43.
21. Leshchenko Y, Likhodii S, Yue W, Burnham WM, Velazquez JLP. Carbenoxolone does not cross the blood brain barrier: an HPLC study. BMC neuroscience 2006; 7(1):3.
22. Dhanesha N, Joharapurkar A, Shah G, Kshirsagar S, Dhote V, Sharma A, et al. Inhibition of 11β‐hydroxysteroid dehydrogenase 1 by carbenoxolone affects glucose homeostasis and obesity in db/db mice. Clinical and Experimental Pharmacology and Physiology 2012; 39(1):69-77.
23. Choi J, Uchino H, Azuma K, Iwashita N, Tanaka Y, Mochizuki H, et al. Little evidence of transdifferentiation of bone marrow-derived cells into pancreatic beta cells. Diabetologia 2003; 46(10):1366-74.
24. Sabz FTK, Farokhi F, Delirezh N, Javadi S, Chapari H. In-vitro differentiation of rat peripheral blood monocytes into insulin-producing cells by rat pancreatic extract. Tehran University Medical Journal 2011; 69(4).
25. Mosayebi G, Ghazaavi, A., and Payani, M. . Effect of sesame oil on production of IFN-γ and IL-10 from TH1 and TH2 cells in autoimmune encephalomyelitis on mice. medical journal of tabriz University of Medical Sciences 2007; 29(3):99-104.
26. Hosseini Jazani N, Mehdi Shishavan M, Shahabi S, Ilkhanizadeh B, Mansourafshar B. Investigation Of The Effect Of Carbenoxolone In Reduction Of Inflammation Severity Induced By Salmonella Enterica Lps In A Mouse Model. BIOLOGICAL JOURNAL OF MICROORGANISM 2012; 1:29-40.
27. Li W, Li J, Sama AE, Wang H. Carbenoxolone blocks endotoxin-induced protein kinase R (PKR) activation and high mobility group box 1 (HMGB1) release. Molecular medicine 2013; 19(1):203.
28. Isbrucker R, Burdock G. Risk and safety assessment on the consumption of Licorice root (Glycyrrhiza sp.), its extract and powder as a food ingredient, with emphasis on the pharmacology and toxicology of glycyrrhizin. Regulatory Toxicology and Pharmacology 2006; 46(3):167-92.
29. Minoda M, Funauchi M, Horiuchi A. Effect of interferon-γ on the abnormality of T cell activation in NZB mice. Clinical immunology and immunopathology 1988; 49(2):283-91.
30. Schroder K, Hertzog PJ, Ravasi T, Hume DA. Interferon-γ: an overview of signals, mechanisms and functions. Journal of leukocyte biology 2004; 75(2):163-89.
31. Roth AD, Hornicek F, Gerstner C, Kirkwood J. Effects of interferon‐gamma and tumour necrosis factor‐alpha on the development of cytotoxic T lymphocytes in autologous mixed lymphocyte tumour cultures with human melanoma. Clinical & Experimental Immunology 1991; 86(1):163-72.
32. Hu X, Ivashkiv LB. Cross-regulation of signaling pathways by interferon-γ: implications for immune responses and autoimmune diseases. Immunity 2009; 31(4):539-50.
33. Conzelmann M, Wagner AH, Hildebrandt A, Rodionova E, Hess M, Zota A, et al. IFN-γ activated JAK1 shifts CD40-induced cytokine profiles in human antigen-presenting cells toward high IL-12p70 and low IL-10 production. Biochemical pharmacology 2010; 80(12):2074-86.
34. Hu X, Paik PK, Chen J, Yarilina A, Kockeritz L, Lu TT, et al. IFN-γ suppresses IL-10 production and synergizes with TLR2 by regulating GSK3 and CREB/AP-1 proteins. Immunity 2006; 24(5):563-74.
35. Liu TF, Jones BM. Impaired production of IL-12 in system lupus erythematosus. II: IL-12 production in vitro is correlated negatively with serum IL-10, positively with serum IFN-γ and negatively with disease activity in SLE. Cytokine 1998; 10(2):148-53.
36. Schloot N, Hanifi‐Moghaddam P, Goebel C, Shatavi S, Flohe S, Kolb H, et al. Serum IFN‐γ and IL‐10 levels are associated with disease progression in non‐obese diabetic mice. Diabetes/metabolism research and reviews 2002; 18(1):64-70.
37. Emamaullee JA, Davis J, Merani S, Toso C, Elliott JF, Thiesen A, et al. Inhibition of Th17 cells regulates autoimmune diabetes in NOD mice. Diabetes 2009; 58(6):1302-11.
38. Ablamunits V, Quintana F, Reshef T, Elias D, Cohen IR. Acceleration of autoimmune diabetes by cyclophosphamide is associated with an enhanced IFN-γ secretion pathway. Journal of autoimmunity 1999; 13(4):383-92.
39. Hull CM, Peakman M, Tree TI. Regulatory T cell dysfunction in type 1 diabetes: what’s broken and how can we fix it? Diabetologia 2017; 60:1839-50.
40. Kleinewietfeld M, Hafler DA. The plasticity of human Treg and Th17 cells and its role in autoimmunity. Seminars in immunology 2013; 25(4):305-12.
41. Ryba-Stanisławowska M, Skrzypkowska M, Myśliwska J, Myśliwiec M. The serum IL-6 profile and Treg/Th17 peripheral cell populations in patients with type 1 diabetes. Mediators of inflammation 2013; 2013:1-7.
42. Garbers C, Rose‐John S. The balance between Treg and TH17 cells: CD11b and interleukin‐6. European Journal of Immunology 2017; 47(4):629-32.
43. Nimmanapalli R, Gerbino E, Dalton WS, Alsina M. HSP70 Induces IL-6 in Stromal Cells and Stat-3 Activation in Myeloma Cells. ASH Annual Meeting Abstracts 2004; 104(11):3353-.
44. Asea A, Kraeft S-K, Kurt-Jones EA, Stevenson MA, Chen LB, Finberg RW, et al. HSP70 stimulates cytokine production through a CD14-dependant pathway, demonstrating its dual role as a chaperone and cytokine. Nature medicine 2000; 6(4):435-42.
45. Dardalhon V, Korn T, Kuchroo VK, Anderson AC. Role of Th1 and Th17 cells in organ-specific autoimmunity. Journal of autoimmunity 2008; 31(3):252-6.