Angiotensin II induces RAW264.7 macrophage polarization to the M1‑type through the connexin 43/NF‑κB pathway

Mol Med Rep. 2020 May;21(5):2103-2112. doi: 10.3892/mmr.2020.11023. Epub 2020 Mar 12.

Abstract

Angiotensin II (AngII) serves an important inflammatory role in cardiovascular disease; it can induce macrophages to differentiate into the M1‑type, produce inflammatory cytokines and resist pathogen invasion, and can cause a certain degree of damage to the body. Previous studies have reported that connexin 43 (Cx43) and NF‑κB (p65) are involved in the AngII‑induced inflammatory pathways of macrophages; however, the mechanisms underlying the effects of Cx43 and NF‑κB (p65) on AngII‑induced macrophage polarization have not been determined. Thus, the present study aimed to investigate the effects of Cx43 and NF‑κB (p65) on the polarization process of AngII‑induced macrophages. The macrophage polarization‑related proteins and mRNAs were examined by flow cytometry, western blotting, immunofluorescence, ELISA and reverse transcription‑quantitative PCR analyses. RAW264.7 macrophages were treated with AngII to simulate chronic inflammation and it was subsequently found that AngII promoted RAW 264.7 macrophage polarization towards the M1‑type by such effects as the release of inducible nitric oxide synthase (iNOS), tumour necrosis factor (TNF)‑α, IL‑1β, the secretion of IL‑6, and the expression of M1‑type indicators, such as CD86. Simultaneously, compared with the control group, the protein expression levels of Cx43 and phosphorylated (p)‑p65 were significantly increased following AngII treatment. The M1‑related phenotypic indicators, iNOS, TNF‑α, IL‑1β, IL‑6 and CD86, were inhibited by the NF‑κB (p65) signalling pathway inhibitor BAY117082. Similarly, the Cx43 inhibitors, Gap26 and Gap19, also inhibited the expression of M1‑related factors, and the protein expression levels of p‑p65 in the Gap26/Gap19 groups were significantly decreased compared with the AngII group. Altogether, these findings suggested that AngII may induce the polarization of RAW264.7 macrophages to the M1‑type through the Cx43/NF‑κB (p65) signalling pathway.

Keywords: angiotensin II; macrophage; polarization; connexin 43; NF‑κB signalling pathway.

MeSH terms

  • Angiotensin II / pharmacology*
  • Animals
  • Cell Polarity / drug effects
  • Connexin 43 / metabolism*
  • Cytokines / metabolism
  • Interleukin-1beta / metabolism
  • Interleukin-6 / metabolism
  • Macrophage Activation / drug effects*
  • Macrophages / metabolism*
  • Mice
  • NF-kappa B / metabolism*
  • Nitric Oxide Synthase Type II / metabolism
  • RAW 264.7 Cells / drug effects*
  • Signal Transduction / drug effects
  • Transcription Factor RelA / metabolism
  • Tumor Necrosis Factor-alpha / metabolism

Substances

  • Connexin 43
  • Cytokines
  • GJA1 protein, mouse
  • IL1B protein, mouse
  • Interleukin-1beta
  • Interleukin-6
  • NF-kappa B
  • Transcription Factor RelA
  • Tumor Necrosis Factor-alpha
  • Angiotensin II
  • Nitric Oxide Synthase Type II