Guggulipid and nimesulide differentially regulated inflammatory genes mRNA expressions via inhibition of NF-kB and CHOP activation in LPS-stimulated rat astrocytoma cells, C6

Abstract

Neuroinflammation is an integral part of neurodegenerative diseases. Lipo-polysacharide (LPS) induces reactive astrogliosis, the cellular manifestation of neuroinflammation, in various models of neurological diseases, but its mechanism of action is still not properly known. The effect of guggulipid and nimesulide on LPS-induced neuroinflammatory changes is also not properly understood. This work demonstrated the mechanism of actions of guggulipid and nimesulide on inflammatory genes expressions in LPS-stimulated rat astrocytoma cells, C6. We observed that LPS (10 μg/ml) treatment of rat astrocytoma cells, C6, for 24 h significantly increased intracellular Ca(2+) ion and expression of inducible nitric oxide synthase (iNOS), nuclear factor kappa-B (NF-kB), C/EBP homologous protein 10 (CHOP), c-fos, and c-jun proteins. At transcriptional stage, LPS upregulated mRNA levels of cyclooxygenase-2 and IL-6 with downregulation in IL-1α, IL-1β, and microsomal prostaglandin E synthase-1 (mPGES-1) through activating NF-kB translocation. Treatment with guggulipid reversed these LPS-induced changes in rat astrocytoma cells. Treatment with nimesulide also attenuated LPS-induced Ca(2+) ion, iNOS, NF-kB, and c-fos expressions, but does not significantly influence CHOP, c-jun protein expressions, and mRNA levels of IL-6, IL-1α, IL-1β, and mPGES-1 genes. In conclusion, our findings elucidated the molecular mechanism of neuroinflammation in response to LPS and its modulation by guggulipid and nimesulide in rat astrocytoma cells (C6), which suggest the use of these drugs in the treatment of neuroinflammation-associated disorders.

Fig. 1

Effect of guggulipid and nimesulide on the LPS (10 μg/ml) induced Ca⁺⁺ ion level and iNOS expression in C6 cells. All given concentrations are in μg/ml. a Left panel: Lanes 1 = control, 2 = LPS, 3 = LPS + GL 3.12, 4 = LPS + GL 6.25, and 5 = GL 6.25. Right panel: Lanes 1 = control, 2 = LPS, 3 = LPS + Ne 0.75, 4 = LPS + Ne 1.5, and 5 = Ne 1.5. b Left panel: Lanes 1 = control, 2 = LPS, 3 = LPS + GL 0.78, 4 = LPS + GL 1.5, 5 = LPS + GL 3.12, and 6 = LPS + GL 6.25. Right panel: Lanes 1 = control, 2 = LPS, 3 = LPS + Ne 0.75, and 4 = LPS + Ne 1.5. #P < 0.001 significant with control group, *P < 0.05, ** P < 0.01, and *** P < 0.001 significant with LPS group. β-actin was taken as an internal control

Fig. 2

Result of RT–PCR of inhibitory effect of guggulipid and nimesulide on LPS (10 μg/ml) induced COX-2 and mPGES-1 transcription. All concentrations are given in μg/ml. a COX-2 mRNA expression; Left panel = LPS + GL; Lanes 1 = control, 2 = LPS, 3 = LPS + GL 0.78, 4 = LPS + GL 1.5, 5 = LPS + GL 3.12, and 6 = LPS + GL 6.25. Right panel: Lanes 1 = control, 2 = LPS, and 3 = LPS + Ne 1.5. b mPGES-1 mRNA expression; Lanes 1 = control, 2 = LPS, 3 = LPS + GL 3.12, 4 = LPS + GL 6.25, 5 = LPS + Ne 0.75, and 6 = LPS + Ne 1.5. #P < 0.001 significant with control group, **P < 0.001 and ***P < 0.001 significant with LPS group. GAPDH was taken as an internal control

Fig. 3

Result of RT–PCR of modulatory effect of guggulipid and nimesulide on LPS (10 μg/ml) induced IL-1α, IL-1β, and IL-6 expression in C6 cells. All concentrations are given in μg/ml. a LPS + GL; Lanes 1 = control, 2 = LPS, and 3 = LPS + GL 6.25. IL-1β, #P < 0.01 significant with control group, **P < 0.01 significant with LPS group, IL-1α, #P < 0.01 significant with control group, **P < 0.01 significant with LPS group, IL-6, #P < 0.05 significant with control group, *P < 0.05 significant with LPS group. GAPDH was taken as internal control. b LPS + Ne; Lanes 1 = control, 2 = LPS, and 3 = LPS + Ne 1.5. IL-1β, *P < 0.05 significant with control group, IL-1α, *P < 0.05 significant with control group, IL-6, **P < 0.01 significant with control group

Fig. 4

Effect of guggulipid and nimesulide on LPS (10 μg/ml) induced (a) CHOP and (b) NF-kB expressions and (c) NF-kB translocation in C6 cells. All concentrations are given in μg/ml. In (a) and (b) A control, B LPS, C LPS + GL 3.12, D LPS + GL 6.25, E LPS + Ne 0.75, and F LPS + Ne 1.5. a #P < 0.05 significant with control, *P < 0.05 and **P < 0.01 significant with LPS group. b #P < 0.001 significant with control, ***P < 0.001 significant with LPS group. (c) NF-kB nuclear translocation: Arrow shows NF-kB (p-65) localization in cytosol and in nucleus. A control, B LPS, C LPS + GL-6.2, and D LPS + Ne 1.5

Fig. 5

Effect of guggulipid and nimesulide on LPS (10 μg/ml) induced c-fos and c-jun expressions in C6 cells. All concentrations are given in μg/ml. A control, B LPS, C LPS + GL 3.12, D LPS + GL 6.25, E LPS + Ne 0.75, and F LPS + Ne 1.5. a c-fos expression: #P < 0.01 significant with control, *P < 0.05 significant with LPS group. b c-jun: #P < 0.05 significant with control, **P < 0.05 significant with LPS group, **P < 0.01 significant with LPS group