P2X7 Receptor Antagonist A804598 Inhibits Inflammation in Brain and Liver in C57BL/6J Mice Exposed to Chronic Ethanol and High Fat Diet

Abstract

Chronic low-grade neuroinflammation is increasingly implicated in organ damage caused by alcohol abuse. Purinergic P2X7 receptors (P2X7Rs) play an important role in the generation of inflammatory responses during a number of CNS pathologies as evidenced from studies using pharmacological inhibition approach. P2X7Rs antagonism has not been tested during chronic alcohol abuse. In the present study, we tested the potential of P2X7R antagonist A804598 to reduce/abolish alcohol-induced neuroinflammation using chronic intragastric ethanol infusion and high-fat diet (Hybrid) in C57BL/6J mice. We have previously demonstrated an increase in neuroinflammatory response in 8 weeks of Hybrid paradigm. In the present study, we found neuroinflammatory response to 4 weeks of Hybrid exposure. A804598 treatment reversed the changes in microglia and astrocytes, reduced/abolished increases in mRNA levels of number of inflammatory markers, including IL-1β, iNOS, CXCR2, and components of inflammatory signaling pathways, such as TLR2, CASP1, NF-kB1 and CREB1, as well in the protein levels of pro-IL-1β and Nf-kB1. The P2X7R antagonist did not affect the increase in mRNA levels of fraktalkine (CX3CL1) and its receptor CX3CR1, an interaction that plays a neuroprotective role in neuron-glia communication. P2X7R antagonism also resulted in reduction of the inflammatory markers but did not alter steatosis in the liver. Taken together, these findings demonstrate how P2X7R antagonism suppresses inflammatory response in brain and liver but does not alter the neuroprotective response caused by Hybrid exposure. Overall, these findings support an important role of P2X7Rs in inflammation in brain and liver caused by combined chronic alcohol and high-fat diet.

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Abbreviations

P2X7R:

purinergic P2X7 receptor

BEC:

blood ethanol concentration

iG:

intragastric

HFD:

high fat diet

HCFD:

high cholesterol high fat diet

RT-qPCR:

reverse transcriptase – quantitative polymerase chain reaction

Gapdh:

glyceraldehyde 3-phosphate dehydrogenase

GFAP:

glial fibrillary acidic protein

Iba1:

ionized calcium binding adaptor molecule 1

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Acknowledgements

We would like to thank the Animal Core of the Southern California Research Center for ALPD and Cirrhosis (Director Dr. H. Tsukamoto) for providing us with the experimental animals, Elliott Cheung for Western blots and Lisa Walter for proof reading the manuscript and valuable recommendations. This work was conducted as partial fulfilment of the requirements for the MS. degree in Pharmaceutical Sciences, University of Southern California (D.F.). Sources of support: NIH/NIAAA AA017243 and Zumberge Individual Research Fund (L.A.), NIH/NIAAA A022448 (D.L.D.), NIH P50AA011999 (H.T.).

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Correspondence to Liana Asatryan.

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Freire, D., Reyes, R.E., Baghram, A. et al. P2X7 Receptor Antagonist A804598 Inhibits Inflammation in Brain and Liver in C57BL/6J Mice Exposed to Chronic Ethanol and High Fat Diet. J Neuroimmune Pharmacol 14, 263–277 (2019). https://doi.org/10.1007/s11481-018-9816-3

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Keywords

  • Intragastric ethanol and high fat diet exposure
  • Purinergic P2X7 receptor pharmacological inhibition
  • Neuroinflammation
  • Liver inflammation and steatosis
  • Immunofluorescence
  • Taqman custom gene expression