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PPARγ ameliorated LPS induced inflammation of HEK cell line expressing both human Toll-like receptor 4 (TLR4) and MD2

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Abstract

TLR4 is transmembrane pattern-recognition receptor that initiates signals in response to diverse pathogen-associated molecular patterns especially LPS. Recently, there have been an increasing number of studies about the role of TLRs in the pathogenesis of several disorders as well as the therapeutic potential of TLR intervention in such diseases. Peroxisome proliferator-activated receptor-gamma (PPARγ) is a ligand-activated transcription factor with numerous biological effects. PPARγ has been shown to exert a potential anti-inflammatory effect through suppression of TLR4-mediated inflammation. Therefore, PPARγ agonists may have a potential to combat inflammatory conditions in pathologic states. The current study aims to show the decrease of inflammation by overexpression of PPARγ in a cell reporter model. To reach this goal, recombinant pBudCE4.1 (+) containing encoding sequences of human TLR4 and MD2 was constructed and used to transfect HEK cells. Subsequently, inflammation was induced by LPS treatment as control group. In the treatment group, overexpression of PPARγ prior to inflammation was performed and the expression of inflammatory markers was assessed in this condition. The expression of inflammatory markers (TNFα and iNOS) was defined by quantitative real time PCR and the amount of phosphorylated NF-κB was measured by western blot. Data indicated expression of TNFα and iNOS increased in LPS induced inflammation of stably transformed HEK cells with MD2 and TLR4. In this cell reporter model overexpression of PPARγ dramatically prevented LPS-induced inflammation through the blocking of TLR4/NF-κB signaling. PPARγ was shown to negatively regulate TLR4 activity and therefore exerts its anti-inflammatory action against LPS induced inflammation.

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Abbreviations

BSA:

Bovine serum albumin

CDS:

Coding sequence

DAPI:

4,6-Diamidino-2-phenylindole

DMEM:

Dulbecco’s modified Eagle’s medium

EF-1α:

Elongation factor 1α-subunit

FBS:

Fetal bovine serum

GAPDH:

Glyceraldehyde 3-phosphate dehydrogenase

HEK:

Human embryonic kidney

IL:

Interleukin

LPS:

Lipopolysaccharide

LRR:

Leucine-rich repeat

MD2:

Myeloid differentiation protein 2

MTS/PMS:

3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)/phenazine methosulfate

OPC:

Oligodendrocyte precursor cell

PAMP:

Pathogen-associated molecular patterns

PBS:

Phosphate buffered saline

PRR:

Pattern-recognition receptor

PVDF:

Polyvinylidenedifluoride

TLR:

Toll-like receptor

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Authors and Affiliations

  1. Department of Medical Genetics, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Velenjak, 1985717443, Tehran, Iran

    Reyhaneh Darehgazani, Mir Davood Omrani & Abolfazl Movafagh

  2. Department of Cellular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, Royan Street, Salman Street, Khorsagan, 816513-1378, Isfahan, Iran

    Reyhaneh Darehgazani, Maryam Peymani, Motahare-Sadat Hashemi, Kamran Ghaedi & Mohammad Hossein Nasr-Esfahani

  3. Department of Biology, Faculty of Basic Sciences, Shahrekord Branch, Islamic Azad University, Sahrekord, Iran

    Maryam Peymani

  4. Biology Department, School of Sciences, University of Isfahan, Isfahan, Iran

    Kamran Ghaedi

Authors
  1. Reyhaneh Darehgazani
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  2. Maryam Peymani
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  3. Motahare-Sadat Hashemi
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  4. Mir Davood Omrani
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  7. Mohammad Hossein Nasr-Esfahani
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Correspondence to Mir Davood Omrani, Kamran Ghaedi or Mohammad Hossein Nasr-Esfahani.

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Darehgazani, R., Peymani, M., Hashemi, MS. et al. PPARγ ameliorated LPS induced inflammation of HEK cell line expressing both human Toll-like receptor 4 (TLR4) and MD2. Cytotechnology 68, 1337–1348 (2016). https://doi.org/10.1007/s10616-015-9893-6

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  • DOI: https://doi.org/10.1007/s10616-015-9893-6

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