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LPS-induced response in J774A.1 macrophage cell culture – indicative markers for stimulated antioxidant defense, inflammation and phagocytosis

Miglena Todorova, Milena Pasheva, Oskan Tasinov, Yoana Kiselova


Introduction: Treatment with bacterial lipopolysaccharides (LPS) is a convenient model used for stimulation of inflammatory response in cell cultures, which is also suggested to be associated with provoked antioxidant defense. Well defined markers are needed to verify pronounced cellular response in this model.

Aim: The aim of current study was to measure changes in expression levels of selected genes in order to identify indicative markers for verification of induced cellular response in a model of LPS-treated J744A.1 macrophages.

Materials and Methods: In order to determine most appropriate LPS treatment concentration, an MTT test was performed. LPS was applied in different concentrations (50 - 300 ng/mL) and their effect on the cell viability of J744A.1 macrophages was measured.

Results and Discussion: Analysis of the results of the MTT test showed a statistically significant (p <0.001) and equal effect of the three LPS concentrations (100, 200 and 300 ng/mL) applied. Cell viability was decreased with 20%. The effect of 100 ng/mL LPS treatment on the following genes was evaluated: antioxidant defense-related (GCLc, GPx1, GSS, GR and SOD2); inflammation-related (IL1β, IL6, MCP1, TNFα, IL1RN, NOS2, CRP, COX2); phagocytosis-related (NOX1 and MPO), and LPS/TLR4 signaling cascade-related (TLR4, IKK2, NRF1, NQO1). All of the studied genes were significantly induced upon LPS treatment for 24h determining it is sufficient to provoke pronounced cellular response. However, the following genes appear to be most affected by 24h LPS treatment: GCLc, COX2, NOS2, IL6, IL1β, CRP, NOX, TLR4 and IKK2.

Conclusion: In conclusion, studied genes may serve as suitable indicative markers for triggered cellular response were LPS stimulation of J774A.1 cells is about to be used as a model of oxidative and inflammatory provocation.


LPS; inflammation; phagocytosis; antioxidant defense; macrophages

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Takashiba S, Van Dyke TE, Amar S, Murayama Y, Soskolne AW, Shapira L. Differentiation of monocytes to macrophages primes cells for lipopolysaccharide stimulation via accumulation of cytoplasmic nuclear factor kB. Infect Immun. 1999;67(1):5573–8. doi: 10.1128/IAI.67.11.5573-5578.1999.

Kim MS, Park SB, Suk K, Kim IK, Kim SY, Kim JA, et al. Gallotannin isolated from Euphorbia species, 1,2,6-tri-O-galloyl-beta-D-allose, decreases nitric oxide production through inhibition of nuclear factor-kappa>B and downstream inducible nitric oxide synthase expression in macrophages. Biol Pharm Bull. 2009; 32(6):1053—6. doi: 10.1248/bpb.32.1053.

Calder PC, Ahluwalia N, Brouns F, Buetler T, Clement K, Cunningham K, et al. Dietary factors and low-grade inflammation in relation to overweight and obesity. Br J Nutr. 2011; 106 Suppl 3:S5-78. doi: 10.1017/S0007114511005460.

Anan F, Masaki T, Umeno Y, Iwao T, Yonemochi H, Eshima N, et al. Correlations of high-sensitivity C-reactive protein and atherosclerosis in Japanese type 2 diabetic patients. Eur J Endocrinol. 2007; 157(3):311-7. doi: 10.1530/EJE-07-0388.

Virág L, Jaén R, Regdon Z, Boscá L, Prieto P. Self-defense of macrophages against oxidative injury: Fighting for their own survival. Redox Biol. 2019; 26:101261. doi: 10.1016/j.redox.2019.101261.

Kiselova-Kaneva Y, Tasinov O, Vankova D, Ivanova D. Ethanol induces IL6 and TNFα cytokine and NOS2 and COX2 enzyme gene expression in 3T3-L1 preadipocytes. Scr Sci Med. 2012; 44(2):31-5. doi: 10.14748/ssm.v44i2.354.

Maraslioglu M, Oppermann E, Blattner C, Weber R, Henrich D, Jobin C, et al. Chronic ethanol feeding modulates inflammatory mediators, activation of nuclear factor-κB, and responsiveness to endotoxin in murine Kupffer cells and circulating leukocytes. Mediators Inflamm. 2014:808695. doi: 10.1155/2014/808695.

Tan KS, Qian L, Rosado R, Flood PM, Cooper LF. The role of titanium surface topogra-phy on J774A.1 macrophage inflammatory cytokines and nitric oxide production. Bio-materials. 2006; 27(30):5170-7. doi: 10.1016/j.biomaterials.2006.05.002.

Yang, J, Della-Fera, S, Rayalam, S, Ambati, Hartzell D, Park, Baile C. Enhanced inhibition of adipogenesis and induction of apoptosis in 3T3-L1 adipocytes with combinations of resveratrol and quercetin. J Life Sciences. 2008; 82(19-20):1032-9. doi: 10.1016/j.lfs.2008.03.003.

Fujiwara N, Kobayashi K. Macrophages in inflammation. Curr Drug Targets Inflamm Allergy. 2005; 4(3):281-6. doi: 10.2174/1568010054022024.

Singh PP, Goyal A. Interleukin-6: a potent biomarker of mycobacterial infection. Springerplus. 2013; 2:686. doi: 10.1186/2193-1801-2-686.

Gabay C. Interleukin-6 and chronic inflammation. Arthritis Res Ther. 2006;8 Suppl 2(Suppl 2):S3. doi: 10.1186/ar1917.

Valko M, Leibfritz D, Moncol J, Cronin M, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol. 2007;39(1):44-84. doi: 10.1016/j.biocel.2006.07.001.

Nauseef WM. Biological roles for the NOX family NADPH oxidases. J Biol Chem. 2008;283(25):16961-5. doi: 10.1074/jbc.R700045200.

Lu YC, Yeh WC, Ohashi P. LPS/TLR4 signal transduction pathway. Cytokine. 2008; 42(2):145-51. doi: 10.1016/j.cyto.2008.01.006.

Rogero MM, Borelli P, Fock RA, Borges MC, Vinolo MA, Curi R, et al. Effects of glutamine on the nuclear factor-kappaB signaling pathway of murine peritoneal macrophages. Amino Acids. 2010 Jul;39(2):435-41. doi: 10.1007/s00726-009-0459-9.



About The Authors

Miglena Todorova
Medical University of Varna

Department of Biochemistry, Molecular Мedicine and Nutrigenomics, Faculty of Pharmacy

Milena Pasheva
Medical University of Varna

Department of Biochemistry, Molecular Мedicine and Nutrigenomics, Faculty of Pharmacy

Oskan Tasinov
Medical University of Varna

Department of Biochemistry, Molecular Мedicine and Nutrigenomics, Faculty of Pharmacy

Yoana Kiselova
Medical University of Varna

Department of Biochemistry, Molecular Мedicine and Nutrigenomics, Faculty of Pharmacy

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